bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunization with the N-Terminal Portion ofTreponema pallidumRepeat Protein K Attenuates Syphilitic Lesion Development in the Rabbit Model
Authors: Morgan Cecilia A. , Lukehart Sheila A. , Van Voorhis Wesley C.
Published: November 2002

When used as an immunogen,Treponema pallidumrepeat protein K (TprK) has been shown to attenuate syphilitic lesions upon homologous intradermal challenge in the rabbit model. To further explore this protein as a potential vaccine component, we sought to identify the immunogenic regions of TprK. The abilities of three recombinant peptides encompassing TprK to elicit T- and B-cell responses and to protect against challenge were examined. All three fragments elicited proliferative responses from splenocytes taken from infected rabbits. However, enzyme-linked immunosorbent assays indicated that only fragments 1 and 3 were consistently recognized by antisera from infected rabbits. Each fragment was also used to immunize rabbits that were subsequently challenged intradermally with infectiousT. pallidum. All lesions on unimmunized control rabbits ulcerated and contained treponemes, while the lesions on rabbits immunized with fragment 1 were the least likely to have detectable treponemes (25%) and the least likely to ulcerate (37.5%). The lesions on rabbits immunized with fragment 3 showed intermediate results, and rabbits immunized with fragment 2 were the most likely of all those on immunized rabbits to have detectable treponemes (91.7%) and to ulcerate (66.7%). These results demonstrate that epitopes in fragment 1 are recognized by T cells and antibodies during infection and that immunization with this portion of TprK most effectively attenuates syphilitic lesion development.
Search for Potential Vaccine Candidate Open Reading Frames in theBacillus anthracisVirulence Plasmid pXO1: In Silico and In Vitro Screening
Authors: Ariel N. , Zvi A. , Grosfeld H. , Gat O. , Inbar Y. , Velan B. , Cohen S. , Shafferman A.
Published: November 2002

A genomic analysis of theBacillus anthracisvirulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol.181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes ofBacillus subtilis,Bacillus halodurans, andBacillus cereusand the pBtoxis plasmid ofBacillus thuringiensisvar. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from ∼30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracisantisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.
Leishmania donovaniSuppresses Activated Protein 1 and NF-κB Activation in Host Macrophages via Ceramide Generation: Involvement of Extracellular Signal-Regulated Kinase
Authors: Ghosh Sanjukta , Bhattacharyya Sandip , Sirkar Madhumita , Sa Gouri Shankar , Das Tanya , Majumdar Debashis , Roy Syamal , Majumdar Subrata
Published: November 2002

In vitro infection of murine peritoneal macrophages with the protozoanLeishmania donovanihas been found to alter the signaling parameters of the host. The present study indicates that the enhancement of intracellular ceramide level in macrophages after infection is a major event relating to macrophage dysfunction. We have previously demonstrated that increased ceramide synthesis in host macrophages was involved in the dephosphorylation of extracellular signal-regulated kinase (ERK). In the present study, we further show that downregulation of ERK by ceramide was found to be associated with the inhibition of activated protein 1 (AP-1) and NF-κB transactivation. Pharmacological inhibition of ceramide synthesis by Fumonisin B1 restored the induction of AP-1 and NF-κB DNA-binding activities in infected BALB/c macrophages. On the contrary, in the case of macrophages from the leishmaniasis-resistant C.D2 mice,L. donovanifailed to induce sustained ceramide synthesis. Enhanced mitogen-activated protein kinase phosphorylation, AP-1 and NF-κB DNA-binding activity, and the generation of nitric oxide (NO) were observed inL. donovani-infected C.D2 macrophages. ERK activation was necessary for the activation of transcription factors AP-1 and NF-κB, NO generation, and restriction of the parasite burden in the resistant murine host macrophages. Hence, the induction of ceramide synthesis in host macrophages appears to be instrumental and one of the turning points leading to silencing of the macrophage antileishmanial responses.
GapA and CrmA Coexpression Is Essential for
Mycoplasma gallisepticum
Cytadherence and Virulence
Authors: Papazisi L. , Frasca S. , Gladd M. , Liao X. , Yogev D. , Geary S. J.
Published: November 2002

It was previously demonstrated that avirulent
Mycoplasma gallisepticum
strain R
high
(passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain R
low
(passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of R
high
with wild-type
gapA
restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the
M. gallisepticum
cytadherence process. CrmA is encoded by the second gene in the
gapA
operon and shares significant sequence homology to the
ORF6
gene of
Mycoplasma pneumoniae
, which has been shown to play an accessory role in the cytadherence process. Complementation of R
high
with wild-type
crmA
resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in R
high
and GT5. In contrast, complementation of R
high
with the entire wild-type
gapA
operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type R
low
. In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for
M. gallisepticum
cytadherence and pathogenesis.
Invasion Activity of aMycobacterium tuberculosisPeptide Presented by theEscherichia coliAIDA Autotransporter
Authors: Casali Nicola , Konieczny Marc , Schmidt M. Alexander , Riley Lee W.
Published: November 2002

Themce1Agene ofMycobacterium tuberculosiswas initially identified by its ability to promote uptake ofEscherichia coliinto HeLa cells. It was subsequently shown that this activity was confined to a 58-amino-acid region of the protein. A 72-amino-acid fragment (InvX) incorporating this active peptide was expressed inE. colias a fusion to the AIDA (adhesin involved in diffuse adherence) autotransporter translocator, and its stable expression on the surface of the bacterium was demonstrated. RecombinantE. coliexpressing InvX-AIDA showed extensive association with HeLa cells, and InvX was shown to be sufficient for internalization. Uptake was found to be both microtubule and microfilament dependent and required the Rho family of GTPases. Thus, theE. coliAIDA system facilitated both the qualitative and quantitative analysis of the functional domain of a heterologous protein.
Molecular Characterization of a Serotype O121:H19 Clone, a Distinct Shiga Toxin-Producing Clone of PathogenicEscherichia coli
Authors: Tarr Cheryl L. , Large Teresa M. , Moeller Chris L. , Lacher David W. , Tarr Phillip I. , Acheson David W. , Whittam Thomas S.
Published: November 2002

Most illnesses caused by Shiga toxin-producingEscherichia coli(STEC) have been attributed toE. coliserotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int-ε) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagicE. coli(EHEC) clones ofE. coli: it included a Shiga toxin 2 gene (stx2), two genes on the EHEC plasmid (toxBandehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands—large chromosomal DNA segments present in the O157:H7 genome—were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenicE. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenicE. coligroups but instead was closely related to twoeae-negative STEC strains.
A Modified Hepatitis B Virus Core Particle Containing Multiple Epitopes of thePlasmodium falciparumCircumsporozoite Protein Provides a Highly Immunogenic Malaria Vaccine in Preclinical Analyses in Rodent and Primate Hosts
Authors: Birkett A. , Lyons K. , Schmidt A. , Boyd D. , Oliveira G. A. , Siddique A. , Nussenzweig R. , Calvo-Calle J. M. , Nardin E.
Published: November 2002

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to thePlasmodiumparasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes ofPlasmodium falciparumCS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimalP. falciparumHBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and inMacaca fascicularis(cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4+-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in naïve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed withP. falciparumsporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.
Genomic Scale Analysis of
Pasteurella multocida
Gene Expression during Growth within the Natural Chicken Host
Authors: Boyce John D. , Wilkie Ian , Harper Marina , Paustian Mike L. , Kapur Vivek , Adler Ben
Published: November 2002

Little is known about the genomic-scale transcriptional responses of bacteria during natural infections. We used whole-genome microarray analysis to assess the transcriptional state of the gram-negative pathogen
Pasteurella multocida
, the causative agent of fowl cholera, during infection in the natural chicken host. We compared the expression profiles of bacteria harvested from the blood of septicemic chickens experiencing late-stage fowl cholera with those from bacteria grown in rich medium. Independent analysis of bacterial expression profiles from the infection of three individual chickens indicated that 40 genes were differentially expressed in all three individuals, 126 were differentially expressed in two of the three individuals, and another 372 were differentially expressed in one individual. Real-time reverse transcription-PCR assays were used to confirm the expression ratios for a number of genes. Of the 40 genes differentially expressed in all three individuals, 17 were up-regulated and 23 were down-regulated in the host compared with those grown in rich medium. The majority (10 of 17) of the up-regulated genes were involved in amino acid transport and metabolism and energy production and conversion, clearly indicating how
P. multocida
alters its biosynthetic and energy production pathways to cope with the host environment. In contrast, the majority (15 of 23) of down-regulated genes were of unknown or poorly characterized functions. There were clear differences in gene expression between the bacteria isolated from each of the three chickens, a finding consistent with individual host variation being an important factor in determining pathogen gene expression. Interestingly, bacteria from only two of the three infected animals had a gene expression profile highly similar to that observed during growth under iron-limiting conditions, suggesting that severe iron starvation may not always occur during
P. multocida
infection.
Opsonophagocytosis-Inhibiting Mac Protein of Group A
Streptococcus
: Identification and Characteristics of Two Genetic Complexes
Authors: Lei Benfang , DeLeo Frank R. , Reid Sean D. , Voyich Jovanka M. , Magoun Loranne , Liu Mengyao , Braughton Kevin R. , Ricklefs Stacy , Hoe Nancy P. , Cole Robert L. , Leong John M. , Musser James M.
Published: November 2002

Recently, it was reported that a streptococcal Mac protein (designated Mac
5005
) made by serotype M1 group A
Streptococcus
(GAS) is a homologue of human CD11b that inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear leukocytes (PMNs) (B. Lei, F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, J. R. Scott, and J. M. Musser, Nat. Med.
7:
1298-1305, 2001). To study
mac
variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 ± 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Complex I Mac variants have greater homology with CD11b than complex II variants. GAS strains belonging to serotypes M1 and M3, the most abundant M protein serotypes responsible for human infections in many case series, have complex I Mac variants. The
mac
gene was cloned from representative strains assigned to complexes I and II, and the Mac proteins were purified to apparent homogeneity. Both Mac variants had immunoglobulin G (IgG)-endopeptidase activity. In contrast to Mac
5005
(complex I), Mac
8345
(complex II) underwent autooxidation of its cysteine residues, resulting in the loss of IgG-endopeptidase activity. A Mac
5005
Cys94Ala site-specific mutant protein was unable to cleave IgG but retained the ability to inhibit IgG-mediated phagocytosis by human PMNs. Thus, the IgG-endopeptidase activity was not essential for the key biological function of Mac
5005
. Although Mac
5005
and Mac
8345
each have an Arg-Gly-Asp (RGD) motif, the proteins differed in their interactions with human integrins α
v
β
3
and α
IIb
β
3
. Binding of Mac
5005
to integrins α
v
β
3
and α
IIb
β
3
was mediated primarily by the RGD motif in Mac
5005
, whereas binding of Mac
8345
involved the RGD motif and a region in the middle one-third of the molecule whose sequence is different in Mac
8345
and Mac
5005
. Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.
Purification and Partial Characterization of a 32-Kilodalton Sialic Acid-Specific Lectin from
Aspergillus fumigatus
Authors: Tronchin Guy , Esnault Karine , Sanchez Myriam , Larcher Gerald , Marot-Leblond Agnes , Bouchara Jean-Philippe
Published: November 2002

Adherence of the opportunistic fungus
Aspergillus fumigatus
to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in
A. fumigatus
was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of
A. fumigatus
. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.
Role of Caspase 1 in Murine Antibacterial Host Defenses and Lethal Endotoxemia
Authors: Joshi Vishwas D. , Kalvakolanu Dhananjaya V. , Hebel John R. , Hasday Jeffrey D. , Cross Alan S.
Published: November 2002

Sepsis is thought to result from an exaggerated innate immune response to microbial components such as lipopolysaccharide (LPS), but the involvement of a specific mechanism(s) has not been identified. We studied the role of caspase 1 (Cas-1) in the murine innate immune response to infection with gram-negative bacteria and to nonlethal and lethal doses of LPS.cas-1−/−and Cas-1 inhibitor (Ac-YVAD-CHO)-treatedcas-1+/+mice were two- to threefold more susceptible to lethalEscherichia coliinfection thancas-1+/+mice. Administration of Cas-1 products, interleukin-18 (IL-18) or IL-1β, protected three of three and six of seven mice, respectively, from lethal infection withE. colicompared to none of six of untreated mice (P= 0.0082). Therefore,cas-1is essential for antibacterial host defense. Nonlethal (75 μg) and lethal (500 μg) doses of LPS induce different patterns of gamma interferon, IL-1β, and IL-18 expression. Consequently, the role of Cas-1, which cleaves pro-IL-18 and pro-IL-1β to their active forms, was investigated in these disparate conditions by using enzymatic assay and reverse transcription-PCR. At 75 μg, LPS induced a transient increase in IL-1β and IL-18 levels in serum, whereas at 500 μg it induced a 1.5-fold-higher IL-18 level in serum, which increased till death. At 75 μg of LPS, spleniccas-1mRNA expression remained unchanged at all time points, but activity increased transiently at 3 h. In lethally treated mice, Cas-1 activity remained elevated until death; however,cas-1mRNA levels increased at 3 h and decreased to basal levels by 8 h. Treatment with Cas-1 inhibitor protected mice from lethal endotoxemia. Thus, Cas-1 is essential for innate antibacterial host defenses and may represent a mechanism of innate immunity that upon excessive stimulation by microbial components may lead to endotoxic shock.
Arginine-Rich Cationic Polypeptides Amplify Lipopolysaccharide-Induced Monocyte Activation
Authors: Bosshart Herbert , Heinzelmann Michael
Published: November 2002

The human neutrophil-derived cationic protein CAP37, also known as azurocidin or heparin-binding protein, enhances the lipopolysaccharide (LPS)-induced release of tumor necrosis factor alpha (TNF-α) in isolated human monocytes. We measured the release of the proinflammatory cytokine interleukin-8 (IL-8) in human whole blood and found that in addition to CAP37, other arginine-rich cationic polypeptides, such as the small structurally related protamines, enhance LPS-induced monocyte activation. As CAP37 and protamines share high levels of arginine content, we tested different synthetic poly-l-amino acids and found that poly-l-arginine, and to a lesser extent poly-l-lysine, increased IL-8 production in LPS-stimulated human whole blood. Protamine-enhanced LPS responses remained unaffected by the presence of freel-arginine orl-lysine, indicating that basic polypeptides but not basic amino acids act synergistically with LPS. In agreement with observations previously reported for CAP37, the LPS-enhancing effect of poly-l-arginine was completely abolished upon antibody blockade of the human LPS receptor, CD14. Protamines, either immobilized or in solution, bound LPS specifically. Poly-l-arginines, protamines, and CAP37 were equally effective in inhibiting binding of LPS to immobilizedl-arginines. Taken together, our results suggest a CD14-dependent mechanism by which arginine-rich cationic proteins modulate LPS-induced monocyte activation and support the prediction that other strongly basic proteins could act as amplifiers of LPS responses.
B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model ofChlamydophila abortusInfection
Authors: Buendía Antonio J. , Del Río Laura , Ortega Nieves , Sánchez Joaquín , Gallego María C. , Caro María R. , Navarro Jose A. , Cuello Francisco , Salinas Jesús
Published: November 2002

The resolution ofChlamydophila abortus(Chlamydia psittaciserotype 1) infection is dependent on gamma interferon and CD8+T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains (μMT). In the absence of B cells, infection withC. abortusleads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. μMT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in μMT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both μMT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor β in the sera of μMT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events ofC. abortusprimary infection that can protect mice against an exaggerated inflammatory response.
Association between the Tumor Necrosis Factor Locus and the Clinical Outcome ofLeishmania chagasiInfection
Authors: Karplus Theresa M. , Jeronimo Selma M. B. , Chang Haeok , Helms Bethany K. , Burns Trudy L. , Murray Jeffrey C. , Mitchell Adele A. , Pugh Elizabeth W. , Braz Regina F. S. , Bezerra Fabiana L. , Wilson Mary E.
Published: November 2002

A periurban outbreak of visceral leishmaniasis (VL) caused by the protozoanLeishmania chagasiis ongoing outside Natal, northeast Brazil. Manifestations range from asymptomatic infection to disseminated visceral disease. Literature reports suggest that both genetic and environmental factors influence the outcome of infection. Due to the association of the tumor necrosis factor (TNF) locus with other infectious diseases, we examined whether polymorphic alleles at this locus are associated with the outcome ofL. chagasiinfection. Neighborhoods with ongoing transmission were identified through patients admitted to local hospitals. Altogether, 1,024 individuals from 183 families were classified with the following disease phenotypes: (i) symptomatic VL, (ii) asymptomatic infection (positive delayed-type hypersensitivity [DTH+]), or (iii) no evidence of infection (DTH−). Genotypes were determined at a microsatellite marker (MSM) upstream of theTNFBgene encoding TNF-β and at a restriction fragment length polymorphism (RFLP) at position −307 in the promoter of theTNFAgene encoding TNF-α. Analyses showed that the distribution ofTNFARFLP alleles (TNF1 and TNF2) and the TNF MSM alleles (TNFa1 to TNFa15) differed between individuals with VL and those with DTH+ phenotypes. TNF1 was transmitted more frequently than expected from heterozygous parents to DTH+ offspring (P= 0.0006), and haplotypes containing TNF2 were associated with symptomatic VL (P= 0.0265, transmission disequilibrium test). Resting serum TNF-α levels were higher in TNF1/2 heterozygotes than in TNF1/1 homozygotes (P< 0.05). These data led us to hypothesize that an individual's genotype at the TNF locus may be associated with whether he or she develops asymptomatic or symptomatic disease afterL. chagasiinfection. The results preliminarily suggest that this may be the case, and follow-up with larger populations is needed for verification.
Rapid Translocation of Polarized MDCK Cell Monolayers by
Leptospira interrogans
, an Invasive but Nonintracellular Pathogen
Authors: Barocchi Michele A. , Ko Albert I. , Reis Mitermayer Galvão , McDonald Kent L. , Riley Lee W.
Published: November 2002

Pathogenic spirochetes of the genus
Leptospira
are a major cause of human zoonotic infectious disease worldwide. After gaining entry through the skin, the organism causes disease by hematogenously disseminating to multiple organs. The mechanism by which it penetrates the mammalian cell barriers to disseminate is not well understood. In this study, we used a low-passage-number isolate of
Leptospira interrogans
to elucidate the invasive potential of this spirochete. Quantification of bacteria by dark-field microscopy revealed that pathogenic spirochetes were able to translocate through polarized MDCK cell monolayers at a rate significantly greater than that of nonpathogenic
Leptospira
or a recognized invasive bacterial pathogen,
Salmonella
. In contrast to
Salmonella
,
L. interrogans
did not alter transepithelial electrical resistance during cell translocation. Both transmission and scanning electron microscopy revealed tight association of the extracellular spirochetes with the host cell plasma membrane, without membrane perturbations suggestive of cytoskeletal rearrangement. Spirochetes were not observed within intercellular junctions or membrane-bound compartments inside cells. They were found within the cytoplasm of only 8% of the counted cells. These results indicate that
Leptospira
is an invasive but not a facultative intracellular organism. We propose that the rapid translocation of mammalian cells by pathogenic
Leptospira
is a mechanism designed to evade killing by host cells that permits the organism to quickly reach the bloodstream and disseminate to multiple organs.
The Function of Gamma Interferon-Inducible GTP-Binding Protein IGTP in Host Resistance toToxoplasma gondiiIs Stat1 Dependent and Requires Expression in Both Hematopoietic and Nonhematopoietic Cellular Compartments
Authors: Collazo Carmen M. , Yap George S. , Hieny Sara , Caspar Patricia , Feng Carl G. , Taylor Gregory A. , Sher Alan
Published: November 2002

IGTP is a member of the 47-kDa family of gamma interferon (IFN-γ)-induced GTPases. We have previously shown that IGTP is critical for host resistance toToxoplasma gondiiinfection. In the present study, we demonstrate thatT. gondii-induced IGTP expression in vivo and IFN-γ-driven synthesis of the protein in vitro are dependent on Stat1. Consistent with this observation, Stat1-deficient animals succumbed toT. gondiiinfection with the same rapid kinetics as IGTP−/−mice. To ascertain the cellular levels at which IGTP functions in host control of acute infection, we constructed reciprocal bone marrow chimeras between IGTP-deficient and wild-type mice. Resistance to infection was observed only when IGTP was present in both hematopoietic and nonhematopoietic compartments. To assess the possible contribution of IGTP to the maintenance of parasite latency, partial chemotherapy was used to allow the establishment of chronic infection in IGTP-deficient animals. Upon cessation of drug treatment, these animals showed delayed mortality compared with similarly infected and treated IFN-γ-deficient or inducible nitric oxide synthase-deficient mice, which succumbed rapidly. Parallel experiments performed with drug-treated bone marrow chimeras supported a role for the hematopoietic compartment in this NO-dependent, IGTP-independent control of chronic infection. Taken together, our findings demonstrate that host resistance mediated by IGTP is a Stat1-induced function which in the case ofT. gondiiacts predominantly to restrict acute as opposed to chronic infection. This effector mechanism requires expression of IGTP in cells of both hematopoietic and nonhematopoietic origin. In contrast, in latent infection, hematopoietically derived cells mediate resistance by means of a largely NO-dependent pathway.
Contribution of Interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 Ligand Pathways to the Development of a Pathological T-Cell Response in IL-10-Deficient Mice
Authors: Wille Ulrike , Villegas Eric N. , Craig Linden , Peach Robert , Hunter Christopher A.
Published: November 2002

The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasiteToxoplasma gondii, IL-10 knockout (KO) mice develop a CD4+-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-γ) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-γ. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-γ and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with αIL-12 plus αB7.1 or αB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-γ and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-γ and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.
In Vitro Studies with RecombinantPlasmodium falciparumApical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response
Authors: Kennedy Michael C. , Wang Jin , Zhang Yanling , Miles Aaron P. , Chitsaz Farideh , Saul Allan , Long Carole A. , Miller Louis H. , Stowers Anthony W.
Published: November 2002

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved inPlasmodiumspp., numerous AMA1 allelic variants ofP. falciparumhave been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by differentP. falciparumstrains is unknown. We characterize two allelic forms of AMA1 that were both produced inPichia pastorisat a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of differentP. falciparumclones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.
Vaccination of Monkeys with RecombinantPlasmodium falciparumApical Membrane Antigen 1 Confers Protection against Blood-Stage Malaria
Authors: Stowers Anthony W. , Kennedy Michael C. , Keegan Brian P. , Saul Allan , Long Carole A. , Miller Louis H.
Published: November 2002

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form ofPlasmodium falciparumAMA1 would have efficacy. We evaluated the efficacy of a form ofP. falciparumAMA1, produced inPichia pastoris, by vaccinatingAotus vociferansmonkeys and then challenging them withP. falciparumparasites. Significant protection from this otherwise lethal challenge withP. falciparumwas observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.
Attenuation of the Virulence of
Porphyromonas gingivalis
by Using a Specific Synthetic Kgp Protease Inhibitor
Authors: Curtis M. A. , Aduse Opoku J. , Rangarajan M. , Gallagher A. , Sterne J. A. C. , Reid C. R. , Evans H. E. A. , Samuelsson B.
Published: November 2002

The Arg- and Lys-gingipains of
Porphyromonas gingivalis
are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in
rgpA
and
rgpB
(encoding Arg-gingipains) and in
kgp
(encoding Lys-gingipain) and a double mutant with mutations in
rgpA
and
rgpB
were prepared by using
P. gingivalis
W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar

K
i

was designed and synthesized. Growth of
P. gingivalis
W50 in the presence of this compound resembled the phenotype of the
kgp
isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of
P. gingivalis
and that inhibitors of this enzyme may have therapeutic potential for the control of
P. gingivalis
infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.
Expression of a Novel
Leishmania
Gene Encoding a Histone H1-Like Protein in
Leishmania major
Modulates Parasite Infectivity In Vitro
Authors: Papageorgiou Fotini T. , Soteriadou Ketty P.
Published: November 2002

We describe identification and characterization of a novel two-copy gene of the parasitic protozoan
Leishmania
that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genus
Leishmania
, and it is developmentally regulated. It is an alanine- and lysine-rich protein with potential bipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of
Leishmania major
. The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet. Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in
Leishmania
infection of macrophages in vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates
Leishmania
infectivity, as assessed in vitro.
Cryptosporidium parvum
Genes Containing Thrombospondin Type 1 Domains
Authors: Deng Mingqi , Templeton Thomas J. , London Nicole R. , Bauer Carrey , Schroeder Alison A. , Abrahamsen Mitchell S.
Published: November 2002

Cryptosporidium parvum
is recognized as an enteropathogen of great worldwide medical and veterinary importance, yet understanding of its pathogenesis has been hampered in part by limited knowledge of the invasion machinery of this parasite. Recently, genes containing thrombospondin type 1 (TSP1) domains have been identified in several genera of apicomplexans, including thrombospondin-related adhesive proteins (TRAPs) that have been implicated as key molecules for parasite motility and adhesion onto host cell surfaces. Previously, a large-scale random survey of the
C. parvum
genome conducted in our laboratory revealed the presence of multiple genomic DNA sequences with a high degree of similarity to known apicomplexan TRAP genes. In the present study, TBLASTN screening of available
C. parvum
genomic sequences by using TSP1 domains as queries identified a total of 12 genes possessing TSP1-like domains. All genes have putative signal peptide sequences, one or more TSP1-like domains, plus additional extracellular protein modules such as Kringle, epidermal growth factor, and Apple domains. Two genes, putative paralogs
Cp
TSP8 and
Cp
TSP9, contain predicted introns near their amino termini, which were verified by comparing PCR products from cDNA versus genomic DNA templates. Reverse transcription-PCR analysis of transcript levels reveals that
C. parvum
TSP genes were developmentally regulated with distinct patterns of expression during in vitro infection. TRAPC1,
Cp
TSP3, and
Cp
TSP11 were expressed at high levels during both early and late stages of infection, whereas
Cp
TSP2,
Cp
TSP5,
Cp
TSP6,
Cp
TSP8, and
Cp
TSP9 were maximally expressed during the late stages of infection. Only
Cp
TSP4 was highly expressed solely at an early stage of infection.
Identification of Novel
Mycobacterium tuberculosis
Antigens with Potential as Diagnostic Reagents or Subunit Vaccine Candidates by Comparative Genomics
Authors: Cockle P. J. , Gordon S. V. , Lalvani A. , Buddle B. M. , Hewinson R. G. , Vordermeier H. M.
Published: November 2002

An independent review for the British government has concluded that the development of a cattle vaccine against
Mycobacterium bovis
holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with
M. bovis
bacillus Calmette-Guérin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify
M. bovis-Mycobacterium tuberculosis
antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the
M. tuberculosis
genome were selected. Pools of overlapping peptides spanning these ORFs were tested in
M. bovis
-infected (
n
= 22), BCG-vaccinated (
n
= 6), and unvaccinated (
n
= 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).
Virulence Criteria forBrucella abortusStrains as Determined by Interferon Regulatory Factor 1-Deficient Mice
Authors: Ko Jinkyung , Gendron-Fitzpatrick Annette , Ficht Thomas A. , Splitter Gary A.
Published: November 2002

Interferon regulatory factor 1-deficient (IRF-1−/−) mice infected with virulentBrucella abortus2308 at 5 × 105CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1−/−mice were unable to resolve infection and died. In contrast, IRF-1−/−mice survived when infected at 5 × 105CFU with several attenuatedBrucellastrains (S19, RB51, cbp, and cyd). The survival of infected IRF-1−/−mice is likely a function of the level of virulence of eachBrucellastrain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1−/−mice since attenuatedBrucellastrains can protect IRF-1−/−mice against lethal challenge with virulentBrucella. Using the IRF-1−/−mouse model, the following set of criteria were identified to defineBrucellavirulence: (i) the day of death for 50% of mice infected with 5 × 105CFU ofBrucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose ofBrucellato protect against challenge with virulent S2308. Thus, IRF-1−/−mice are important to determining the level ofBrucellavirulence, to evaluatingBrucellamutants for attenuation, and to investigating adaptive immunity in brucellosis.
Recognition of Variant Rifin Antigens by Human Antibodies Induced during Natural
Plasmodium falciparum
Infections
Authors: Abdel-Latif Mohamed S. , Khattab Ayman , Lindenthal Christoph , Kremsner Peter G. , Klinkert Mo-Quen
Published: November 2002

Antibodies from individuals living in areas where malaria is endemic are known to react with parasite-derived erythrocyte surface proteins. The major immunogenic and clonally variant surface antigen described to date is
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP-1), which is encoded by members of the multicopy
var
gene family. We report here that rifin proteins (RIF proteins), belonging to the largest known family of variable infected erythrocyte surface-expressed proteins, are also naturally immunogenic. Recombinant RIF proteins were used to analyze the antibody responses of individuals living in an area of intense malaria transmission. Elevated anti-rifin antibody levels were detected in the majority of the adult population tested, whereas the prevalence of such antibodies was much lower in malaria-exposed children. Despite the high degree of diversity between
rif
sequences and the high gene copy number, it appears that
P. falciparum
infections can induce antibodies that cross-react with several variant rifin molecules in many parasite isolates in a given community, and the immune response is most likely to be stable over time in a hyperendemic area. The protein was localized by fluorescence microscopy on the membrane of ring and young trophozoite-infected erythrocytes with antibodies from human immune sera with specificities for recombinant RIF protein.