bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeat Sequences in Block 2 ofPlasmodium falciparumMerozoite Surface Protein 1 Are Targets of Antibodies Associated with Protection from Malaria
Authors: Polley Spencer D. , Tetteh Kevin K. A. , Cavanagh David R. , Pearce Richard J. , Lloyd Jennifer M. , Bojang Kalifa A. , Okenu Daniel M. N. , Greenwood Brian M. , McBride Jana S. , Conway David J.
Published: March 2003

Human antibodies to the block 2 region ofPlasmodium falciparummerozoite surface protein 1 (MSP1) are associated with a reduced prospective risk of clinical malaria. Block 2 is highly polymorphic, but all known alleles can be grouped into three major types. Two of these types (the K1-like and MAD20-like types) contain type-specific sequences (found in all alleles of a particular type) that flank polymorphic tripeptide repeats. These repeats contain both type-specific and subtype-specific sequences. To evaluate the antibody recognition of these parts of block 2, a new panel of six recombinant proteins was used (fused type-specific flanking sequences and two representative repeat sequences for each of the K1-like and MAD20-like types separately). Extensive testing of these antigens and full-length block 2 antigens showed that human serum immunoglobulin G antibodies induced by infection can recognize (i) type-specific epitopes in the repeats, (ii) subtype-specific epitopes in the repeats, or (iii) type-specific epitopes in flanking sequences. A large prospective study in The Gambia showed that antibodies to the repeats are strongly associated with protection from clinical malaria. The results are important for design of a vaccine to induce protective antibodies, and they address hypotheses about repeat sequences in malaria antigens.
Inactivation of the
Moraxella catarrhalis
7169 Ferric Uptake Regulator Increases Susceptibility to the Bactericidal Activity of Normal Human Sera
Authors: Furano Kristin , Campagnari Anthony A.
Published: March 2003

Moraxella catarrhalis
is a strict human pathogen and a significant cause of respiratory disease and otitis media. In direct response to these infections, research efforts have focused primarily on the identification of potential vaccine targets. The general biology of
M. catarrhalis
, however, including the mechanisms utilized to survive in the human host, remains poorly understood. Previous work has demonstrated that
M. catarrhalis
expresses iron-repressible proteins, suggesting the presence of iron acquisition systems under the control of a ferric uptake regulator (Fur). In this study
M. catarrhalis fur
has been cloned and sequenced from strain 7169. A deletion-insertion mutation of 7169
fur
resulted in upregulation of iron-repressible outer membrane proteins in the absence and presence of iron. This mutant strain, 7169
fur
1, was significantly more sensitive to the bactericidal activity of normal human serum than the resistant wild-type strain. These data suggest that constitutive expression of iron-regulated proteins may provide multiple targets for human antibodies. In addition, the 7169
fur
mutant provides an important tool for further investigation of the iron acquisition mechanisms utilized by
M. catarrhalis
.
Development and Evaluation of an Improved Mouse Model of Meningococcal Colonization
Authors: Yi Kyungcheol , Stephens David S. , Stojiljkovic Igor
Published: March 2003

Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model ofNeisseria meningitidiscolonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 107CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO2-lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.
Erythrocyte Invasion Phenotypes of
Plasmodium
falciparum
in The Gambia
Authors: Baum Jake , Pinder Margaret , Conway David J.
Published: March 2003

In vitro experimentation with
Plasmodium
falciparum
has determined that a number of different receptor-ligand interactions are involved in the invasion of erythrocytes. Most culture-adapted parasite isolates use a mechanism of invasion that depends primarily on the erythrocyte sialoglycoprotein glycophorin A (GYPA) and erythrocyte-binding antigen 175 (EBA-175) of the parasite blood-stage merozoite. However, a minority of culture-adapted parasites and a majority of Indian field isolates can apparently invade by other means. Here, erythrocyte invasion phenotypes of
P
.
falciparum
field isolates in Africa were studied. For 38 Gambian isolates, invasion of neuraminidase-treated and trypsin-treated erythrocytes was inhibited, on average, by more than 60 and 85%, respectively, indicating a high level of dependence on sialic acid and trypsin-sensitive proteins on the erythrocyte surface. These results support the hypothesis that African
P
.
falciparum
parasites use GYPA as a primary receptor for invasion. However, the considerable variation among isolates confirms the idea that alternative receptors are also used by many parasites. Three amino acid polymorphisms in the GYPA-binding region of EBA-175 (region II) were not significantly associated with invasion phenotype. There was variation among isolates in the selectivity index (i.e., a statistical tendency toward aggregation or multiple invasions of host erythrocytes), but this variation did not correlate with enzyme-determined invasion phenotype or with
eba-175
alleles. Overall, these invasion phenotypes in Africa support a vaccine strategy of inhibiting EBA-175 binding to GYPA but suggest that parasites with alternative phenotypes would be selected for if this strategy were used alone.
Role of Glutathione in Macrophage Control of Mycobacteria
Authors: Venketaraman Vishwanath , Dayaram Yaswant K. , Amin Amol G. , Ngo Richard , Green Renee M. , Talaue Meliza T. , Mann Jessica , Connell Nancy D.
Published: March 2003

Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form ofS-nitrosoglutathione. Previous work from this laboratory has shown that glutathione andS-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain ofMycobacterium bovisBCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione andS-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione andS-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.
Novel Virulence-Associated Type II Secretion System Unique to High-PathogenicityYersinia enterocolitica
Authors: Iwobi A. , Heesemann J. , Garcia E. , Igwe E. , Noelting C. , Rakin A.
Published: March 2003

Yersinia enterocoliticastrains comprise an important group of bacterial enteropathogens that cause a broad range of gastrointestinal syndromes. Three groups are distinguishable within this bacterial species, namely, the nonpathogenic group (biotype 1A strains), the low-pathogenicity, non-mouse-lethal group (biotypes 2 to 5), and the high-pathogenicity, mouse-lethal group (biotype 1B). To date, the presence of the high-pathogenicity island (HPI), a chromosomal locus that encodes the yersiniabactin system (involved in iron uptake), defines essentially the difference between low-pathogenicity and high-pathogenicityY. enterocoliticastrains, with the low-pathogenicity strains lacking the HPI. Using the powerful tool of representational difference analysis between the nonpathogenic 1A strain, NF-O, and its high-pathogenicity 1B counterpart, WA-314, we have identified a novel type II secretion gene cluster (yts1C-S) occurring exclusively in the high-pathogenicity group. The encoded secreton, designated Yts1 (forYersiniatype II secretion 1) was shown to be important for virulence in mice. A close examination of the almost completed genome sequence of another high-pathogenicity representative,Y. enterocolitica8081, revealed a second putative type II secretion cluster uniformly distributed among allY. enterocoliticaisolates. This putative species-specific cluster (designatedyts2) differed significantly fromyts1, while resembling more closely the putative type II cluster present on the genome ofY. pestis. The Yts1 secreton thus appears to have been additionally acquired by the high-pathogenicity assemblage for a virulence-associated function.
Oral Transfer of Adult
Ancylostoma ceylanicum
Hookworms into Permissive and Nonpermissive Host Species
Authors: Bungiro Richard D. , Anderson Brett R. , Cappello Michael
Published: March 2003

Syrian hamsters become anemic and exhibit delayed growth following oral infection with third-stage
Ancylostoma ceylanicum
hookworm larvae. Here we describe experiments designed to determine the feasibility of adult worm transfer (AWT) between hosts, a technique that would facilitate the specific study of bloodfeeding hookworms in vivo without prior exposure of the host to larva-specific antigens, permit the ex vivo manipulation of adult parasites prior to reimplantation, and also allow for cross-species transfer of worms. Weanling hamsters given an oral AWT of 40 or 60 mixed-sex
A. ceylanicum
worms rapidly developed anemia; in the higher-dose group, hemoglobin levels declined from prechallenge levels by 44% within 4 days following AWT. Long-term survival of transferred worms was demonstrated by recovery of parasites from the intestines 42 days after AWT. AWT hamsters acquired humoral immune responses against soluble adult hookworm extracts and excretory-secretory products that were comparable in magnitude to those of animals given a typical infection with larvae. In AWT experiments employing the nonpermissive murine model, C57BL/6 mice given adult worms rapidly became anemic and lost weight in a manner similar to AWT hamsters. Infection of additional mouse strains demonstrated that while C57BL/10 and CD-1 mice also developed anemia following AWT, BALB/c mice were resistant. The technique of AWT to mice may further our understanding of hookworm pathogenesis by allowing the study of adult hookworm infections in a species with well-characterized genetics and an abundance of available reagents.
Intranasal Immunization with RecombinantLactococcus lactisSecreting Murine Interleukin-12 Enhances Antigen-Specific Th1 Cytokine Production
Authors: Bermúdez-Humarán Luis G. , Langella Philippe , Cortes-Perez Naima G. , Gruss Alexandra , Tamez-Guerra Reyes S. , Oliveira Sergio C. , Cardenas Odila Saucedo- , Montes de Oca-Luna Roberto , Le Loir Yves
Published: March 2003

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacteriumLactococcus lactis.IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinantL. lactisstrain was confirmed in vitro by its ability to induce gamma interferon (IFN-γ) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-γ production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-γ) elicited by a recombinantL. lactisstrain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with anL. lactisstrain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form byL. lactisand that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.
Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses
Authors: Marinaro Mariarosaria , Fasano Alessio , De Magistris Maria Teresa
Published: March 2003

Zonula occludens toxin (Zot) is produced byVibrio choleraeand has the ability to increase mucosal permeability by reversibly affecting the structure of tight junctions. Because of this property, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. Here we show that Zot acts as a mucosal adjuvant to induce long-lasting and protective immune responses upon mucosal immunization of mice. Indeed, the intranasal delivery of ovalbumin with two different recombinant forms of Zot in BALB/c mice resulted in high Ag-specific serum immunoglobulin G titers that were maintained over the course of a year. Moreover, His-Zot induced humoral and cell-mediated responses to tetanus toxoid in C57BL/6 mice and protected the mice against a systemic challenge with tetanus toxin. In addition, we found that Zot also acts as an adjuvant through the intrarectal route and that it has very low immunogenicity compared to the adjuvantEscherichia coliheat-labile enterotoxin. Finally, by using an octapeptide representing the putative binding site of Zot and of its endogenous analogue zonulin, we provide evidence that Zot may bind a mucosal receptor on nasal mucosa and may mimic an endogenous regulator of tight junctions to deliver Ags in the submucosa. In conclusion, Zot is a novel and effective mucosal adjuvant that may be useful for the development of mucosal vaccines.
M Type 1 and 3 Group A Streptococci Stimulate Tissue Factor-Mediated Procoagulant Activity in Human Monocytes and Endothelial Cells
Authors: Bryant A. E. , Hayes-Schroer S. M. , Stevens D. L.
Published: March 2003

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.
P-Selectin Contributes to Severe Experimental Malaria but Is Not Required for Leukocyte Adhesion to Brain Microvasculature
Authors: Chang Wun-Ling , Li Jie , Sun Guang , Chen Hong-Li , Specian Robert D. , Berney Seth Mark , Granger D. Neil , van der Heyde Henri C.
Published: March 2003

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit many of the hallmarks of a systemic inflammatory response, with organ damage in brain, lung, and kidneys. Identification of the molecules mediating pathogenesis of the inflammatory response, such as leukocyte adhesion, may lead to new therapies. Indeed, mice lacking the cell adhesion molecule P-selectin were significantly (P= 0.005) protected from death due toP. bergheimalaria compared with C57BL/6 controls despite similar parasitemia (P= 0.6) being found in both groups of mice. P-selectin levels assessed by the quantitative dual radiolabeled monoclonal antibody technique increased significantly (P< 0.05) in several organs in C57BL/6 mice infected withP. berghei, supporting the concept of a systemic inflammatory response mediating malarial pathogenesis. Intravital microscopic analysis of the brain microvasculature demonstrated significant (P< 0.001) leukocyte rolling and adhesion in brain venules ofP. berghei-infected mice compared with those found in uninfected controls. The maximum leukocyte adhesion occurred on day 6 ofP. bergheiinfection, when the mice become moribund and exhibit marked vascular leakage into the brain, lung, and heart. However, P-selectin levels were significantly (P< 0.005) increased in brain, lung, and kidneys duringP. bergheimalaria in ECM-resistant BALB/c mice compared with those found in uninfected BALB/c controls, indicating that increased P-selectin alone is not sufficient to mediate malarial pathogenesis. Leukocyte adhesion to brain microvessels of P-selectin-deficient mice withP. bergheimalaria was similar to that observed in control mice. Collectively, these results indicate that P-selectin is important for the development of malarial pathogenesis but is not required for leukocyte adhesion in brain.
Contribution of the
Shigella flexneri
Sit, Iuc, and Feo Iron Acquisition Systems to Iron Acquisition In Vitro and in Cultured Cells
Authors: Runyen-Janecky L. J. , Reeves S. A. , Gonzales E. G. , Payne S. M.
Published: March 2003

Shigella flexneri
possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional
S
.
flexneri
putative iron transport gene,
sitA
, in a screen for
S
.
flexneri
genes that are induced in the eukaryotic intracellular environment.
sitA
was present in all
Shigella
species and in most enteroinvasive
Escherichia coli
strains but not in any other
E
.
coli
isolates tested. The
sit
locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the
S
.
flexneri sit
locus was homologous to the
Salmonella enterica
serovar Typhimurium Sit and
Yersinia pestis
Yfe systems, which mediate both manganese and iron transport. The
S
.
flexneri sit
promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A
sitA
::
cam
mutation was constructed in
S
.
flexneri
. The
sitA
mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the
sitA
mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (
sitA iucD
,
sitA feoB
, and
feoB iucD
) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in
sitA
,
feoB
, and
iucD
did not form plaques on Henle cell monolayers.
The Apa Protein ofMycobacterium tuberculosisStimulates Gamma Interferon-Secreting CD4+and CD8+T Cells from Purified Protein Derivative-Positive Individuals and Affords Protection in a Guinea Pig Model
Authors: Kumar Priti , Amara Rama Rao , Challu Vijay Kumar , Chadda Vineet Kumar , Satchidanandam Vijaya
Published: March 2003

The search to identifyMycobacterium tuberculosisantigens capable of conferring protective immunity against tuberculosis has received a boost owing to the resurgence of tuberculosis over the past two decades. It has long been recognized that lymphoid cells are required for protection againstM. tuberculosis. While traditionally the CD4+populations of T cells were believed to predominantly serve this protective function, a pivotal role for CD8+T cells in this task has been increasingly appreciated. We show that the 50- to 55-kDa Apa protein, specified by the Rv1860 gene ofM. tuberculosis, can elicit both lymphoproliferative response and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells (PBMC) of purified protein derivative (PPD)-positive individuals, with significant differences recorded in the levels of responsiveness between PPD-positive healthy controls and pulmonary tuberculosis patients. Flow cytometric analysis of whole blood stimulated with the recombinant Apa protein revealed a sizeable proportion of CD8+T cells in addition to CD4+T cells contributing to IFN-γ secretion. PBMC responding to the Apa protein produced no interleukin-4, revealing a Th1 phenotype. A DNA vaccine and a poxvirus recombinant expressing the Apa protein were constructed and tested for their ability to protect immunized guinea pigs against a challenge dose of virulentM. tuberculosis. Although the DNA vaccine afforded little protection, the poxvirus recombinant boost after DNA vaccine priming conferred a significant level of protective immunity, bringing about a considerable reduction in mycobacterial counts from the challenge bacilli in spleens of immunized guinea pigs, a result comparable to that achieved by BCG vaccination.
The Stress-Responsive
dgk
Gene from
Streptococcus mutans
Encodes a Putative Undecaprenol Kinase Activity
Authors: Lis Maciej , Kuramitsu Howard K.
Published: March 2003

We analyzed a previously constructed stress-sensitive
Streptococcus mutans
mutant Tn-1 strain resulting from disruption by transposon Tn
916
of a gene encoding a protein exhibiting amino acid sequence similarity to the
Escherichia coli
diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the
dgk
gene in
S. mutans
predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the
dgk
gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.
Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants inSalmonellaVaccine Strains
Authors: Kramer Uwe , Rizos Konstantin , Apfel Heiko , Autenrieth Ingo B. , Lattemann Claus T.
Published: March 2003

To optimize antigen delivery bySalmonellavaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, theEscherichia coliadhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of theYersinia enterocoliticaheat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface ofE. coliandSalmonella entericaserovar Typhimurium. Colonization studies in mice vaccinated withSalmonellastrains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response againstYersiniaHsp6074-86was induced in mice vaccinated with aSalmonellavaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoringYersiniaHsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuatedSalmonellavaccine strains.
Synthetic Peptides Identify Promiscuous Human Th1 Cell Epitopes of the Secreted Mycobacterial Antigen MPB70
Authors: Al-Attiyah Raja , Shaban Fatema A. , Wiker Harald G. , Oftung Fredrik , Mustafa Abu S.
Published: March 2003

MPB70 is a secreted protein ofMycobacterium bovisandMycobacterium tuberculosiswhich stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cellM. tuberculosisandM. tuberculosisculture filtrates), with MPB70 purified from the culture filtrate ofM. bovisBCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-γ) assays. Testing of antigen-specific CD4+T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-γ assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-γ-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.
Signaling through the T1/ST2 Molecule Is Not Necessary for Th2 Differentiation but Is Important for the Regulation of Type 1 Responses in NonhealingLeishmaniamajorInfection
Authors: Kropf P. , Herath S. , Klemenz R. , Müller I.
Published: March 2003

T1/ST2 is a stable cell surface marker selectively expressed on type 2 T helper (Th2) effector cells. Since nonhealingLeishmaniamajorinfections in susceptible BALB/c mice have been ascribed to a polarized Th2 response, we used an anti-T1/ST2 monoclonal antibody (MAb) or a T1-Fc fusion protein to investigate the role of CD4+T1/ST2+Th2 cells in experimental leishmaniasis. We show that interfering with T1/ST2 signaling had no effect on lesion development or parasite replication; however, it induced a significantly higher type 1 response and an enhanced capacity of CD4+T cells to respond to interleukin 12 (IL-12). Surprisingly, even in the presence of an elevated Th1 response, the production of antigen-specific type 2 cytokines was not altered in the group of mice treated with the anti-T1/ST2 MAb or the T1-Fc fusion protein. To characterize further this Th2 response, we assessed the cytokine profile of CD4+T cells and found that interfering with T1/ST2 signaling did not alter the cytokine profile of CD4+T1/ST2+T cells. These results show that T1/ST2 signaling is not necessary for the differentiation of naive CD4+T cells into antigen-specific CD4+T1/ST2+Th2 cells. In addition to CD4+T1/ST2+T cells, we detected another subpopulation of CD4+Th2 cells, negative for the expression of T1/ST2, that could differentiate in vivo in response toL.majorinfection. Taken together, our results suggest that CD4+T1/ST2+Th2 cells but not CD4+T1/ST2−Th2 cells can downregulate the Th1 response during the course of a nonhealingL.majorinfection through a mechanism that is independent of IL-4 or IL-10.
Mutation ofluxSAffects Biofilm Formation inStreptococcus mutans
Authors: Merritt Justin , Qi Fengxia , Goodman Steven D. , Anderson Maxwell H. , Shi Wenyuan
Published: March 2003

Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing andluxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role ofluxSin biofilm formation. In this study, we report for the first time thatluxS-dependent quorum sensing is involved in biofilm formation ofStreptococcus mutans. S. mutansis a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog ofluxSforS. mutanswas identified using the data available in theS. mutansgenome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement theluxSnegative phenotype ofEscherichia coliDH5α. It was also shown that AI-2 is indeed produced byS. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of theluxSgene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and theluxSmutants. Microscopic analysis of in vitro-grown biofilm structure revealed that theluxSmutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation ofS. mutans.
Recognition of Mycobacterial Epitopes by T Cells across Mammalian Species and Use of a Program That Predicts Human HLA-DR Binding Peptides To Predict Bovine Epitopes
Authors: Vordermeier Martin , Whelan Adam O. , Hewinson R. Glyn
Published: March 2003

Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. The aim of this study was to assess whether in silico analysis could be combined with in vitro screening methods to rapidly identify peptides that are immunogenic duringMycobacterium bovisinfection of cattle. In the first instance theM. bovis-derived protein ESAT-6 was used as a model antigen to describe peptides containing T-cell epitopes that were frequently recognized across mammalian species, including natural hosts for tuberculosis (humans and cattle) and small-animal models of tuberculosis (mice and guinea pigs). Having demonstrated that some peptides could be recognized by T cells from a number ofM. bovis-infected hosts, we tested whether a virtual-matrix-based human prediction program (ProPred) could identify peptides that were recognized by T cells fromM. bovis-infected cattle. In this study, 73% of the experimentally defined peptides from 10M. bovisantigens that were recognized by bovine T cells contained motifs predicted by ProPred. Finally, in validating this observation, we showed that three of five peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR-restricted epitopes were recognized by T cells fromM. bovis-infected cattle. The results obtained in this study support the approach of using bioinformatics to increase the efficiency of epitope screening and selection.
Cryptococcus neoformans
with a Mutation in the Tetratricopeptide Repeat-Containing Gene,
CCN1
, Causes Subcutaneous Lesions but Fails To Cause Systemic Infection
Authors: Chung Seyung , Mondon Philippe , Chang Yun C. , Kwon-Chung Kyung J.
Published: March 2003

We studied a
Cryptococcus neoformans
strain that caused feline chronic nasal granuloma without disseminated disease. This strain, B-4551, grows at temperatures up to 35°C and fails to cause systemic infection in mice. Many cells of B-4551 formed short hyphal elements in feline nasal tissue and occasionally at 35°C in vitro. A complementation and sequence analysis revealed that the temperature-sensitive (Ts) phenotype of B-4551 was due to deletion of a lysine residue in the cryptococcal
CCN1
gene. B-4551 complemented with the wild type
CCN1
gene grew at 37°C and caused fatal systemic infection in mice. The
CCN1
gene encodes a protein containing 16 copies of a tetratricopeptide repeat.
CCN1
is homologous to the
Saccharomyces cerevisiae CLF1
gene, which is required for pre-mRNA splicing, cell cycle progression, and DNA replication, and to the
Drosophila melanogaster crn
gene, which is involved in neurogenesis.
CLF1
complemented the Ts phenotype of B-4551.
CCN1
, however, failed to rescue the
clf1
mutant in
S. cerevisiae
. These results indicate that the Ccn1p may not be as functionally diverse as Clf1p in yeast.
Anaplasma phagocytophilumReduces Neutrophil Apoptosis In Vivo
Authors: Scaife Helena , Woldehiwet Zerai , Hart C. Anthony , Edwards Steven W.
Published: March 2003

Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces. The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not. Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis. Neutrophils isolated from sheep experimentally infected withAnaplasma phagocytophilumshowed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection. The ability of neutrophils fromA. phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep. These data are the first demonstration that in vivo infection withA. phagocytophilumresults in changes in rates of apoptosis of infected immune cells. This may help explain how these bacteria replicate in these normally short-lived cells.
Resistance to Acute Babesiosis Is Associated with Interleukin-12- and Gamma Interferon-Mediated Responses and Requires Macrophages and Natural Killer Cells
Authors: Aguilar-Delfin Irma , Wettstein Peter J. , Persing David H.
Published: March 2003

We examined the role of the cytokines gamma interferon (IFN-γ) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1Babesia. Mice genetically deficient in IFN-γ-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-γ and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-γ-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenicBabesiaWA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-γ, and induction of macrophage-derived effector molecules like NO.
Effects of Zinc Deficiency and Pneumococcal Surface Protein A Immunization on Zinc Status and the Risk of Severe Infection in Mice
Authors: Strand Tor A. , Hollingshead Susan K. , Julshamn Kåre , Briles David E. , Blomberg Bjørn , Sommerfelt Halvor
Published: March 2003

Streptococcus pneumoniae
is a major cause of illness and death in children in developing countries. In these children, zinc deficiency is associated with an increased risk of acute respiratory tract infections, which can be reduced by daily zinc administration. Severe infections decrease zinc levels in plasma and may thereby move individuals with preexisting low zinc stores into a vicious cycle of infection and unavailable zinc. Pneumococcal surface protein A (PspA) has emerged as a promising vaccine candidate, and immunization with this antigen protects animals from pneumococcal infection. In an animal experiment, we measured the effect of zinc depletion on the immune response to parenterally administrated PspA and assessed the effect of this PspA vaccination and zinc depletion on the severity of pneumococcal infection and on zinc status. Mice were kept on different diets for 5 weeks, immunized twice 14 days apart, and challenged intranasally with
S. pneumoniae
. Mice on the zinc-deficient diet showed substantially reduced immune responses to PspA, more extensive pneumococcal colonization in the nasal mucosa, more severe infections, and an increased risk of death. PspA immunization reduced the risk of severe disease, and the reduction in severity was reflected in substantially reduced zinc depletion from bones.
Pathogenic
Yersinia enterocolitica
Strains Increase the Outer Membrane Permeability in Response to Environmental Stimuli by Modulating Lipopolysaccharide Fluidity and Lipid A Structure
Authors: Bengoechea J. A. , Brandenburg K. , Arraiza M. D. , Seydel U. , Skurnik M. , Moriyón I.
Published: March 2003

Pathogenic biotypes of
Yersinia enterocolitica
(serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca
2+
-restricted) media at 37°C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using
Y. enterocolitica
serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more
N
-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37°C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic
Y. enterocolitica
strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic
Y. enterocolitica
strains approximate the characteristics in
Yersinia pseudotuberculosis
and
Yersinia pestis
and suggest that there is a common outer membrane pattern associated with pathogenicity.
Helicobacter pylori
Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa
Authors: Loughlin Michael F. , Barnard Faye M. , Jenkins David , Sharples Gary J. , Jenks Peter J.
Published: March 2003

Homologous recombination contributes to the extraordinary genetic diversity of
Helicobacter pylori
and may be critical for surface antigen expression and adaptation to environmental challenges within the stomach. We generated isogenic, nonpolar
H. pylori ruvC
mutants to investigate the function of RuvC, a Holliday junction endonuclease that resolves recombinant joints into nicked duplex products. Inactivation of
ruvC
reduced the frequency of homologous recombination of
H. pylori
between 17- and 45-fold and increased sensitivity to DNA-damaging agents and the antimicrobial agents levofloxacin and metronidazole. The
H. pylori ruvC
mutants were more susceptible to oxidative stress and exhibited reduced survival within macrophages. Experiments with the
H. pylori
SS1 mouse model revealed that the 50% infective dose of the
ruvC
mutant was approximately 100-fold higher than that of the wild-type SS1 strain. Although the
ruvC
mutant was able to establish colonization with bacterial loads that were initially similar to those of the parental SS1 strain, infection was spontaneously cleared from the murine gastric mucosa over periods that varied from 36 to 67 days. These results demonstrate that, in this infection model, RuvC is essential for continued survival of
H. pylori
in vivo and raises the possibility that inactivation of
ruvC
might be of value in an attenuated vaccine strain.
Induction of Mosquitocidal Activity in Mice Immunized withAnopheles gambiaeMidgut cDNA
Authors: Foy B. D. , Magalhaes T. , Injera W. E. , Sutherland I. , Devenport M. , Thanawastien A. , Ripley D. , Cárdenas-Freytag L. , Beier J. C.
Published: March 2003

Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquitoAnopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individualA. gambiaemidgut cDNAs,Ag-Aper1(a secreted peritrophic matrix protein) andAgMuc1(a midgut-bound mucin), and anA. gambiaemidgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either theAgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed onAg-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undo mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.
Different Genetic Control of Cutaneous and Visceral Disease after
Leishmania major
Infection in Mice
Authors: Vladimirov Vladimir , Badalová Jana , Svobodová Milena , Havelková Helena , Hart Augustinus A. M. , Blažková Hana , Demant Peter , Lipoldová Marie
Published: March 2003

The mouse strains BALB/cHeA (BALB/c) and STS/A (STS) are susceptible and resistant to
Leishmania major-
induced disease, respectively. We analyzed this difference using recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% genes of the strain STS in a BALB/c background. Previously, testing the resistant strain CcS-5, we found five novel
Lmr
(
Leishmania major
response) loci, each associated with a different combination of pathological and immunological reactions. Here we analyze the response of RC strain CcS-16, which is even more susceptible to
L. major
than BALB/c. In the (CcS-16 × BALB/c)F
2
hybrids we mapped three novel loci that influence cutaneous or visceral pathology
. Lmr14
(chromosome 2) controls splenomegaly and hepatomegaly. On the other hand
Lmr15
(chromosome 11) determines hepatomegaly only, and
Lmr13
(chromosome 18) determines skin lesions only. These data confirm the complex control of
L. major-
induced pathology, where cutaneous and visceral pathology are controlled by different combinations of genes. It indicates organ-specific control of antiparasite responses. The definition of genes controlling these responses will permit a better understanding of pathways and genetic diversity underlying the different disease phenotypes.