bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic
Escherichia coli
K88ac+ Fimbriae
Authors: Fang Lin , Gan Zhibo , Marquardt Ronald R.
Published: July 2002

An affinity chromatography technique was utilized to isolate and purify the receptors of
Escherichia coli
K88ac
+
fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound receptor was washed exhaustively with a buffer containing a high concentration of salt and a detergent. The receptors were eluted as a single component at a low pH. The isolated proteins were then subjected to enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot (immunoblot) analyses. The two proteins were of high purity, were responsible for nearly all of the fimbrial binding capacity of the crude mucus, and had molecular masses of 26 and 41 kDa. The method for isolation of
E. coli
binding proteins is simple and yields purified intestinal receptors in a single chromatographic run. The intestinal mucus of different piglets has different proportions of the two receptor proteins.
Cytokine Expression and Production by PurifiedHelicobacter pyloriUrease in Human Gastric Epithelial Cells
Authors: Tanahashi Toshihito , Kita Masakazu , Kodama Tadashi , Yamaoka Yoshio , Sawai Naoki , Ohno Tomoyuki , Mitsufuji Shoji , Wei Ya-Ping , Kashima Kei , Imanishi Jiro
Published: July 2002

Cytokines have been proposed to play an important role inHelicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear.H. pyloriurease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced inH. pyloriinfection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whetherH. pyloriurease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purifiedH. pyloriurease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation ofH. pyloriurease, indicating that the epithelial cells were involved in the mucosal inflammation that accompaniedH. pyloriinfection.
Streptococcus sobrinus
Antigens That React to Salivary Antibodies Induced by Tonsillar Application of Formalin-Killed
S. sobrinus
in Rabbits
Authors: Fukuizumi Takaki , Inoue Hiromasa , Tsujisawa Toshiyuki , Uchiyama Choji
Published: July 2002

We previously found that tonsillar application of antigen induces a strong antibody response to
Streptococcus sobrinus
in saliva and blood plasma. Rabbits immunized against
S. sobrinus
by tonsillar application were highly resistant to experimental dental caries triggered by oral inoculation of living
S. sobrinus
organisms with sucrose. In the present study, we examined the reaction of
S. sobrinus
antigens to the antibodies induced by the tonsillar application of
S. sobrinus
AHT-k in rabbits and compared them to those antibodies induced by intramuscular injection. In an enzyme-linked immunosorbent assay using ultrasonic fragments from mutans group streptococci, the saliva and blood plasma selectively reacted to
S. sobrinus
AHT-k (serotype g) and serologically related streptococci (serotypes a, d, and h) in the sixth week after tonsillar application, whereas the blood plasma in the sixth week after intramuscular injection reacted to the unrelated streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci. The antibody reactivity induced after tonsillar application was not lost after treatment of the antigen with heat or proteinase digestion, whereas these treatments resulted in a 70% decrease of the antibody reactivity induced by intramuscular injection. The inhibition by haptenic sugars and the decrease in immunoreactivity by heat treatment and proteinase digestion suggested that 80% of the antibodies induced by tonsillar application reacted to saccharides. These saccharide antigens appeared to be involved in a specific reaction with
S. sobrinus
-specific streptococci and a selective reaction with serologically related streptococci. These antigens are probably involved in anticaries reactions in experimental dental caries.
Absence of SpeB Production in Virulent Large Capsular Forms of Group A Streptococcal Strain 64
Authors: Raeder Roberta , Harokopakis Evlambia , Hollingshead Susan , Boyle Michael D. P.
Published: July 2002

Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties.
Use of Defined Mutants To Assess the Role of theCampylobacter rectusS-Layer in Bacterium-Epithelial Cell Interactions
Authors: Wang Beinan , Kraig Ellen , Kolodrubetz David
Published: July 2002

Campylobacter rectusis a periodontal pathogen with a 150-kDa protein on its cell surface. This protein forms a paracrystalline lattice, called the S-layer, surrounding the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the possible role of the S-layer in the initial interaction ofC. rectuswith host epithelial cells,C. rectusstrains lacking the S-layer protein gene (crsA) were constructed by allelic exchange mutagenesis. Surprisingly, the lack of the S-layer had only a minor effect on the interaction ofC. rectuswith HEp-2 epithelial cells; CrsA+cells were 30 to 50% more adherent than were CrsA−bacteria. Since the host cell expression of cytokines appears to play an important role in the pathogenesis of periodontal diseases, the effect of the S-layer on the epithelial cell cytokine response was also examined by quantitative reverse transcriptase PCR and enzyme-linked immunosorbent assay. Although there were no changes in the mRNA levels for the anti-inflammatory cytokines interleukin-1 receptor agonist (IL-1ra), IL-13, and transforming growth factor β, the expression and secretion of the proinflammatory cytokines IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) were significantly induced by both wild-typeC. rectusand CrsA−bacteria. Interestingly, the kinetics of cytokine induction differed for the CrsA+and CrsA−bacteria. At early time points, the HEp-2 cells challenged with CrsA−bacteria produced higher levels of IL-6, IL-8, and TNF-α mRNA and protein than did cells challenged with CrsA+bacteria. We conclude thatC. rectusmay help initiate periodontitis by increasing the expression of proinflammatory cytokines and that the S-layer may temper this response to facilitate the survival ofC. rectusat the site of infection.
Immune Responses to Specific Antigens of
Streptococcus pneumoniae
and
Moraxella catarrhalis
in the Respiratory Tract
Authors: Samukawa Takao , Yamanaka Noboru , Hollingshead Susan , Klingman Karin , Faden Howard
Published: July 2002

Streptococcus pneumoniae
and
Moraxella catarrhalis
are two common respiratory pathogens, colonizing as many as 54 and 72% of children, respectively, by 1 year of age. The immune responses to surface protein A of
S. pneumoniae
(PspA) and the high-molecular-weight outer membrane protein of
M. catarrhalis
(UspA) in the sera of various age groups in the general population and in the nasopharynges of 30 children monitored from birth through 1 year of age were evaluated. Immunoglobulin G (IgG) was the dominant serum antibody to PspA and UspA. Whereas the serum antibody response to PspA peaked in childhood, the antibody response to UspA peaked in adulthood. In the first 2 years of life, comparable amounts of IgM and IgG antibodies to both proteins were observed. In older persons, IgG antibodies to both antigens predominated over IgM antibodies. The levels of IgA antibody to these antigens in serum remained low during the first 2 years of life. The levels of IgM antibody to the two antigens in serum exceeded the levels of IgA antibody to the same two antigens throughout life. Although IgA was the dominant antibody to PspA and UspA in airway secretions, it was detected in a minority of the children (3 of 15 for PspA and 0 of 15 for UspA). Even the majority of the children previously colonized with these pathogens lacked antibody to them in their secretions.
Expression of Polymorphic
msp1
β Genes during Acute
Anaplasma marginale
Rickettsemia
Authors: Camacho-Nuez Minerva , de Lourdes Muñoz Maria , Suarez Carlos E. , McGuire Travis C. , Brown Wendy C. , Palmer Guy H.
Published: July 2002

Immunization of cattle with native MSP1 induces protection against
Anaplasma marginale
. The native immunogen is composed of a single MSP1a protein and multiple, undefined MSP1b polypeptides. In addition to the originally sequenced gene, designated
msp1β(F1)
, we identified three complete
msp1
β genes in the Florida strain:
msp1β(F2)
,
msp1β(F3)
, and
msp1β(F4)
. Each of these polymorphic genes encodes a structurally unique MSP1b protein, and unique transcripts can be identified during acute
A. marginale
rickettsemia. The structural polymorphism is clustered in discrete variable regions, and each MSP1b protein results from a unique mosaic of five variable regions. Although each of the MSP1b proteins in the Florida strain contains epitopes recognized by serum antibody induced by protective immunization with the native MSP1 complex, the variable regions also include epitopes expressed by some but not all of the MSP1b proteins. These data support testing recombinant vaccines composed of the multiple antigenically and structurally unique MSP1b proteins combined with MSP1a in order to mimic the efficacy of native MSP1 immunization.
Molecular Epidemiologic Approaches to Urinary Tract Infection Gene Discovery in Uropathogenic
Escherichia coli
Authors: Zhang Lixin , Foxman Betsy , Manning Shannon D. , Tallman Patricia , Marrs Carl F.
Published: July 2002

Urinary tract infection (UTI) is one of the most frequently acquired bacterial infections. The vast majority of UTIs are caused by a large, genetically heterogeneous group of
Escherichia coli
. This genetic diversity has hampered identification of UTI-related genes. A three-step experimental strategy was used to identify genes potentially involved in
E. coli
UTI transmission or virulence: epidemiologic pairing of a UTI-specific strain with a fecal control, differential cloning to isolated UTI strain-specific DNA, and epidemiologic screening to identify sequences among isolated DNAs that are associated with UTI. The 37 DNA sequences initially isolated were physically located all over the tester strain genome. Only two hybridized to the total DNA of the sequenced
E. coli
K-12 strain; eight sequences were present significantly more frequently in UTI isolates than in fecal isolates. Three of the eight sequences matched to genes for multidrug efflux proteins, usher proteins, and pathogenicity island insertion sites, respectively. Using population characteristics to direct gene discovery and evaluation is a productive strategy applicable to any system.
Staphylococcus aureusProtein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets
Authors: Nguyen Truc , Ghebrehiwet Berhane , Peerschke Ellinor I. B.
Published: July 2002

The adhesion ofStaphylococcus aureusto platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediatingS. aureusinteractions with platelets, however, are incompletely understood. The present study describes the interaction betweenS. aureusprotein A and gC1qR/p33, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of C1q. Suspensions of fixedS. aureusor purified protein A, chemically cross-linked to agarose support beads, were found to capture native gC1qR from whole platelets. Moreover, biotinylated protein A bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled protein A, soluble recombinant gC1qR (rgC1qR), or anti-gC1qR antibody F(ab′)2fragments. The interaction between protein A and platelet gC1qR was underscored by studies illustrating preferential recognition of the protein A-bearingS. aureusCowan I strain by gC1qR compared to recognition of the protein A-deficient Wood 46 strain, as well as inhibition ofS. aureusCowan I strain adhesion to immobilized platelets by soluble protein A. Further characterization of the protein A-gC1qR interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated gC1qR binding to immobilized protein A revealed specific binding that was inhibited by soluble protein A with a 50% inhibitory concentration of (3.3 ± 0.7) × 10−7M (mean ± standard deviation;n= 3). Rabbit immunoglobulin G (IgG) also prevented gC1qR-protein A interactions, and inactivation of protein A tyrosil residues by hyperiodination, previously reported to prevent the binding of IgG Fc, but not Fab, domains to protein A, abrogated gC1qR binding. These results suggest similar protein A structural requirements for gC1qR and IgG Fc binding. Further studies of structure and function using a truncated gC1qR mutant lacking amino acids 74 to 95 demonstrated that the protein A binding domain lies outside of the gC1qR amino-terminal alpha helix, which contains binding sites for the globular heads of C1q. In conclusion, the data implicate the platelet gC1qR as a novel cellular binding site for staphylococcal protein A and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.
A Large Toxin from Pathogenic
Escherichia coli
Strains That Inhibits Lymphocyte Activation
Authors: Klapproth Jan-Michael A. , Scaletsky Isabel C. A. , McNamara Barry P. , Lai Li-Ching , Malstrom Carol , James Stephen P. , Donnenberg Michael S.
Published: July 2002

The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of
Escherichia coli
(EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic
E. coli
strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of
E. coli
is associated with gain of this activity. Among 89 strains of
E. coli
and related bacteria tested,
lifA
sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.
Intracellular Trafficking and Killing ofStreptococcus pneumoniaeby Human Alveolar Macrophages Are Influenced by Opsonins
Authors: Gordon Stephen B. , Irving Glen R. B. , Lawson Roderick A. , Lee Margaret E. , Read Robert C.
Published: July 2002

Human alveolar macrophages (HAM) are the major resident phagocytic cells of the gas-exchanging areas of the lung. Following contact with macrophages, bacteria enter phagosomes, which gradually acquire the characteristics of terminal phagolysosomes, with incorporation of lysosome-associated membrane protein (LAMP). We measured the binding of type 1Streptococcus pneumoniaeto the surface of HAM and then measured subsequent internalization and phagosomal incorporation of LAMP-1 under various opsonic conditions. Opsonization with serum containing immunoglobulin resulted in significantly greater binding of pneumococci to HAM compared with opsonization with immunoglobulin G (IgG)-depleted serum containing complement, which in turn resulted in marginally increased binding over that observed in the absence of opsonization. Internalization of opsonizedS. pneumoniaegradually increased to a maximum of 20% of bound bacteria by 120 min of warm incubation, with 20% of internalized pneumococci being localized within LAMP-containing compartments by 80 min. Internalization of opsonizedS. pneumoniaeby HAM correlated with a reduction of bacterial viability. When inocula were adjusted so that pneumococcal binding under different conditions was equalized, subsequent internalization, trafficking to LAMP-containing compartments, and reduction of bacterial viability were less efficient in the absence of opsonization than that observed following opsonization with adsorbed or IgG-replete adsorbed serum. Once bound to the surface of HAM, pneumococci opsonized with adsorbed serum with or without IgG were internalized, processed, and killed equally well. In conclusion, binding, intracellular trafficking, and killing ofS. pneumoniaeby HAM are each significantly increased by opsonization with serum containing immunogloblin and/or complement.
Bactericidal/Permeability-Increasing Protein Prevents Mucosal Damage in an Experimental Rat Model of Chronic Otitis Media with Effusion
Authors: Nell Marja J. , Koerten Henk K. , Grote Jan J.
Published: July 2002

In this study, the efficacy of bactericidal/permeability-increasing protein (BPI) was assessed in a rat model of chronic otitis media with effusion. BPI injection prevented disturbance of the mucociliary clearance system of the middle ear. Hence, it is postulated that BPI can be a new therapy for chronic otitis media with effusion.
Apoptosis Induced byStaphylococcus aureusin Epithelial Cells Utilizes a Mechanism Involving Caspases 8 and 3
Authors: Wesson Carla A. , Deringer James , Liou Linda E. , Bayles Kenneth W. , Bohach Gregory A. , Trumble William R.
Published: July 2002

In this study, we demonstrate that the mechanism by whichStaphylococcus aureusinduces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalizedS. aureusinduces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1β by MAC-T cells. These data suggest that the internalization ofS. aureuscould induce specific cellular responses in vivo that may ultimately impact the course of infection.
Streptococcal Erythrogenic Toxin B Abrogates Fibronectin-Dependent Internalization of
Streptococcus pyogenes
by Cultured Mammalian Cells
Authors: Chaussee Michael S. , Cole Robert L. , van Putten Jos P. M.
Published: July 2002

Streptococcus pyogenes
secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between
S. pyogenes
strain NZ131 (serotype M49) and mammalian cells. Inactivation of the
speB
gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131
speB
mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-
pgs
745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131
speB
mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-
pgs
745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.
The Relapsing Fever Spirochete
Borrelia hermsii
Contains Multiple, Antigen-Encoding Circular Plasmids That Are Homologous to the cp32 Plasmids of Lyme Disease Spirochetes
Authors: Stevenson Brian , Porcella Stephen F. , Oie Katrina L. , Fitzpatrick Cecily A. , Raffel Sandra J. , Lubke Lori , Schrumpf Merry E. , Schwan Tom G.
Published: July 2002

Borrelia hermsii
, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete,
B. burgdorferi
. Analysis of another relapsing fever bacterium,
B. parkeri
, indicated that it contains linear homologs of the
B. hermsii
and
B. burgdorferi
cp32 plasmids. The
B. hermsii
cp32 plasmids encode homologs of the
B. burgdorferi
Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of
B. burgdorferi erp
genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different
Borrelia
species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.
Allelic Diversity of the Two Transferrin Binding Protein B Gene Isotypes among a Collection of
Neisseria meningitidis
Strains Representative of Serogroup B Disease: Implication for the Composition of a Recombinant TbpB-Based Vaccine
Authors: Rokbi Bachra , Renauld-Mongenie Geneviève , Mignon Michèle , Danve B. , Poncet David , Chabanel Christophe , Caugant Dominique A. , Quentin-Millet Marie-José
Published: July 2002

The distribution of the two isotypes of
tbpB
in a collection of 108 serogroup B meningococcal strains belonging to the four major clonal groups associated with epidemic and hyperendemic disease (the ET-37 complex, the ET-5 complex, lineage III, and cluster A4) was determined. Isotype I strains (with a 1.8-kb
tbpB
gene) was less represented than isotype II strains (19.4 versus 80.6%). Isotype I was restricted to the ET-37 complex strains, while isotype II was found in all four clonal complexes. The extent of the allelic diversity of
tbpB
in these two groups was studied by PCR restriction analysis and sequencing of 10 new
tbpB
genes. Four major
tbpB
gene variants were characterized: B16B6 (representative of isotype I) and M982, BZ83, and 8680 (representative of isotype II). The relevance of these variants was assessed at the antigenic level by the determination of cross-bactericidal activity of purified immunoglobulin G preparations raised to the corresponding recombinant TbpB (rTbpB) protein against a panel of 27 strains (5 of isotype I and 22 of isotype II). The results indicated that rTbpB corresponding to each variant was able to induce cross-bactericidal antibodies. However, the number of strains killed with an anti-rTbpB serum was slightly lower than that obtained with an anti-TbpA
+
B complex. None of the sera tested raised against an isotype I strain was able to kill an isotype II strain and vice versa. None of the specific antisera tested (anti-rTbpB or anti-TbpA
+
B complex) was able to kill all of the 22 isotype II strains tested. Moreover, using sera raised against the C-terminus domain of TbpB M982 (amino acids 352 to 691) or BZ83 (amino acids 329 to 669) fused to the maltose-binding protein, cross-bactericidal activity was detected against 12 and 7 isotype II strains, respectively, of the 22 tested. These results suggest surface accessibility of the C-terminal end of TbpB. Altogether, these results show that although more than one rTbpB will be required in the composition of a TbpB-based vaccine to achieve a fully cross-bactericidal activity, rTbpB and its C terminus were able by themselves to induce cross-bactericidal antibodies.
Fecal Antibodies toCryptosporidium parvumin Healthy Volunteers
Authors: Dann Sara M. , Okhuysen Pablo C. , Salameh Bassam M. , DuPont Herbert L. , Chappell Cynthia L.
Published: July 2002

This study examined the intestinal antibody response in 26 healthy volunteers challenged withCryptosporidium parvumoocysts. Fecal extracts were assayed for total secretory immunoglobulin A (IgA) andC. parvum-specific IgA reactivity. Specific IgA reactivity was standardized to IgA concentration and expressed as a reactivity index (RI). Anti-C. parvumfecal IgA (fIgA) increased significantly in 17 of 26 (65.4%) following oocyst ingestion. Of those with detectable responses, 59, 76.5, and 94.1% were positive by days 7, 14, and 30, respectively. Volunteers receiving high challenge doses (>1,000 and 300 to 500 oocysts) had higher RIs (RI = 5.57 [P= 0.027] and RI = 1.68 [P= 0.039], respectively) than those ingesting low doses (30 to 100 oocysts; RI = 0.146). Subjects shedding oocysts and experiencing a diarrheal illness had the highest fIgA reactivity. When evaluated separately, oocyst excretion was associated with an increased fIgA response compared to nonshedders (RI = 1.679 versus 0.024, respectively;P= 0.003). However, in subjects experiencing diarrhea with or without oocyst shedding, a trend toward a higher RI (P= 0.065) was seen. Extracts positive for fecal IgA were further examined for IgA subclass. The majority of stools contained both IgA1 and IgA2, and the relative proportions did not change following challenge. Also, noC. parvum-specific IgM or IgG was detected in fecal extracts. Thus, fecal IgA toC. parvumantigens was highly associated with infection in subjects who had no evidence of previous exposure and may provide a useful tool in detecting recent infections.
Induction of Colony-Stimulating Factor Expression followingStaphylococcusorSalmonellaInteraction with Mouse or Human Osteoblasts
Authors: Bost Kenneth L. , Bento Jennifer L. , Ellington John K. , Marriott Ian , Hudson Michael C.
Published: July 2002

Staphylococcus aureusandSalmonellaspp. are common causes of bone diseases; however, the immune response during such infections is not well understood. Colony-stimulating factors (CSF) have a profound influence on osteoclastogenesis, as well as the development of immune responses following infection. Therefore, we questioned whether interaction of osteoblasts with two very different bacterial pathogens could affect CSF expression by these cells. Cultured mouse and human osteoblasts were exposed to various numbers ofS. aureusorSalmonella dublinbacteria, and a comprehensive analysis of granulocyte-macrophage (GM)-CSF, granulocyte (G)-CSF, macrophage (M)-CSF, and interleukin-3 (IL-3) mRNA expression and cytokine secretion was performed. Expression of M-CSF and IL-3 mRNAs by mouse osteoblasts was constitutive and did not increase significantly following bacterial exposure. In contrast, GM-CSF and G-CSF mRNA expression by mouse osteoblasts was dramatically upregulated following interaction with either viableS. aureusorSalmonella. This increased mRNA expression also translated into high levels of GM-CSF and G-CSF secretion by mouse and human osteoblasts following bacterial exposure. ViableS. aureusandSalmonellainduced maximal levels of CSF mRNA expression and cytokine secretion compared to UV-killed bacteria. Furthermore, GM-CSF and G-CSF mRNA expression could be induced in unexposed osteoblasts separated by a permeable Transwell membrane from bacterially exposed osteoblasts. M-CSF secretion was increased in cultures of exposed human osteoblasts but not in exposed mouse osteoblast cultures. Together, these studies are the first to define CSF expression and suggest that, following bacterial exposure, osteoblasts may influence osteoclastogenesis, as well as the development of an immune response, via the production of these cytokines.
Uptake and Killing of
Leptospira interrogans
and
Borrelia burgdorferi
, Spirochetes Pathogenic to Humans, by Reticuloendothelial Cells in Perfused Rat Liver
Authors: Marangoni Antonella , Aldini Rita , Sambri Vittorio , Montagnani Marco , Ballardini Giorgio , Storni Elisa , Cevenini Roberto
Published: July 2002

In situ-perfused rat livers were infused with a single dose of 1.5 × 10
7
radiolabeled cells of
Leptospira interrogans
serovar icterohaemorrhagiae, the agent of leptospirosis, or with
Borrelia burgdorferi
IRS, the agent of Lyme disease. Significant (
P
< 0.0001) differences in the liver uptake of
L. interrogans
and of
B. burgdorferi
were observed, the uptakes being 37.4% ± 2.3% for
L. interrogans
and 60.5% ± 3.1% for
B. burgdorferi
. Leptospires, in contrast to borreliae, were recovered from the livers when liver samples were cultured in growth medium. Leptospires but not borreliae were recovered in bile within 30 min of infusion. The association of leptospires and borreliae with reticuloendothelial cells of the liver was demonstrated by immunohistochemistry. Leptospires and borreliae were found to be associated with vimentin-positive cells and not with desmin-positive cells. Few leptospires but no borreliae were also seen associated with vimentin- and desmin-negative cells, suggesting the presence of leptospires outside the sinusoidal spaces, in the liver parenchyma.
Mapping of Binding Domains of Nontypeable
Haemophilus influenzae
HMW1 and HMW2 Adhesins
Authors: Dawid Suzanne , Grass Susan , St. Geme Joseph W.
Published: July 2002

Nontypeable
Haemophilus influenzae
is an important cause of localized respiratory tract disease, which begins with colonization of the upper respiratory mucosa. In previous work we reported that the nontypeable
H. influenzae
HMW1 and HMW2 proteins are high-molecular-weight nonpilus adhesins responsible for attachment to human epithelial cells, an essential step in the process of colonization. Interestingly, although HMW1 and HMW2 share significant sequence similarity, they display distinct cellular binding specificities. In order to map the HMW1 and HMW2 binding domains, we generated a series of complementary HMW1-HMW2 chimeric proteins and examined the ability of these proteins to promote in vitro adherence by
Escherichia coli
DH5α. Using this approach, we localized the HMW1 and HMW2 binding domains to an ∼360-amino-acid region near the N terminus of the mature HMW1 and HMW2 proteins. Experiments with maltose-binding protein fusion proteins containing segments of either HMW1 or HMW2 confirmed these results and suggested that the fully functional binding domains may be conformational structures that require relatively long stretches of sequence. Of note, the HMW1 and HMW2 binding domains correspond to areas of maximal sequence dissimilarity, suggesting that selective advantage associated with broader adhesive potential has been a major driving force during
H. influenzae
evolution. These findings should facilitate efforts to develop a subcomponent vaccine effective against nontypeable
H. influenzae
disease.
Comparison of CXC Chemokines ENA-78 and Interleukin-8 Expression inHelicobacter pylori-Associated Gastritis
Authors: Rieder Gabriele , Einsiedl Wolfgang , Hatz Rudolf A. , Stolte Manfred , Enders Georg A. , Walz Alfred
Published: July 2002

Colonization of the gastric mucosa withHelicobacter pyloriis associated with a dense infiltration of granulocytes into the lamina propria in the active phase of gastritis. In this study, we investigated the involvement of epithelial cell-derived neutrophil-activating protein 78 (ENA-78) in development ofH. pylori-associated gastritis. Antral biopsies from 27 patients withH. pylori-associated gastritis and 25 fromH. pylori-negative individuals were first analyzed for ENA-78 and interleukin-8 (IL-8) mRNA by semiquantitative reverse transcription (RT)-PCR. InH. pylori-positive patients, significantly elevated levels were found for both chemokines (P< 0.05). Only IL-8 mRNA levels differed significantly (P< 0.05) inH. pylori-infected individuals who had serum antibodies for cytotoxin-associated protein CagA versusH. pylori-infected CagA-negative persons. Quantification of ENA-78 transcript levels by competitive RT-PCR yielded a significant 45-fold upregulation for ENA-78 transcripts in biopsies ofH. pylori-positive versusH. pylori-negative patients (P< 0.05). In contrast to earlier findings with IL-8, the degree of ENA-78 mRNA upregulation was independent of the grade of activity of gastritis. Immunofluorescence studies on tissues of antral biopsies localized ENA-78 protein expression mainly to the gastric epithelium ofH. pylori-positive patients, while control tissues were negative. Upregulation of ENA-78 and IL-8 mRNA and protein expression was also observed in an in vitro system using a gastric adenocarcinoma cell line. Only viableH. pyloriyielded a strong ENA-78 and IL-8 induction, whileH. pyloriouter membrane proteins or water-soluble proteins had no significant effect. These data provide evidence for the importance of both IL-8 and ENA-78 in the development and perpetuation ofH. pylori-associated gastritis.
Population Dynamics of
Streptococcus mitis
in Its Natural Habitat
Authors: Hohwy Jesper , Reinholdt Jesper , Kilian Mogens
Published: July 2002

The purpose of this study was to examine the genetic structure of the typical commensal
Streptococcus mitis
biovar 1 in its natural habitat in the human oral cavity and pharynx and to investigate the role that selected microbial properties and host, spatial, and temporal factors play in determining the structure of the bacterial population. Consecutive samples were collected from buccal and pharyngeal mucosal surfaces of two infants, their four parents, and two elderly individuals over a period of approximately 1 year. A total of 751 isolates identified as
S. mitis
biovar 1 were typed by restriction endonuclease analysis (REA) and representative clones were typed by multilocus enzyme electrophoresis (MLEE). The genetic diversity of the
S. mitis
biovar 1 isolates collected from single infant hosts over a period of 9 to 10 months was found to be between 0.69 and 0.76, which is considerably higher than that previously observed for intestinal populations of
Escherichia coli
. The study provides evidence of the existence of both transient and persistent clones in adult individuals. In the two infants, however, none of 42 demonstrated clones were detected on more than a single occasion. Statistical calculations showed that the ability to persist was not distributed at random in the
S. mitis
biovar 1 population. However, neither immunoglobulin A1 protease activity nor the ability to bind α-amylase from saliva was a preferential characteristic of persistent genotypes. In contrast to current concepts of climax ecosystems, the species niche in the habitat appears to be maintained predominantly by a succession of clones rather than by stable strains. Several lines of evidence suggest that the major origin of “new” clones is the many other habitats in the respiratory tract that are occupied by this species.
Epidemic Population Structure of
Pseudomonas aeruginosa
: Evidence for a Clone That Is Pathogenic to the Eye and That Has a Distinct Combination of Virulence Factors
Authors: Lomholt Jeanet A. , Poulsen Knud , Kilian Mogens
Published: July 2002

The genetic structure of a population of
Pseudomonas aeruginosa
, isolated from patients with keratitis, endophthalmitis, and contact lens-associated red eye, contact lens storage cases, urine, ear, blood, lungs, wounds, feces, and the environment was determined by multilocus enzyme electrophoresis. The presence and characteristics of virulence factors were determined by restriction fragment length polymorphism analysis with DNA probes for
lasA
,
lasB
,
aprA
,
exoS
,
exoT
,
exoU
, and
ctx
and by zymography of staphylolysin, elastase, and alkaline protease. These analyses revealed an epidemic population structure of
P. aeruginosa
, characterized by frequent recombination in which a particular successful clone may increase, predominate for a time, and then disasappear as a result of recombination. Epidemic clones were found among isolates from patients with keratitis. They were characterized by high activity of a hitherto-unrecognized size variant of elastase, high alkaline protease activity, and possession of the
exoU
gene encoding the cytotoxic exoenzyme U. These virulence determinants are not exclusive traits in strains causing keratitis, as strains with other properties may cause keratitis in the presence of predisposing conditions. There were no uniform patterns of characteristics of isolates from other types of infection; however, all strains from urinary tract infections possessed the
exoS
gene, all strains from environment and feces and the major part of keratitis and wound isolates exhibited high elastase and alkaline protease activity, and all strains from feces showed high staphylolysin activity, indicating that these virulence factors may be important in the pathogenesis of these infectious diseases.
Reassessing the Role of
Staphylococcus aureus
Clumping Factor and Fibronectin-Binding Protein by Expression in
Lactococcus lactis
Authors: Que Yok-Ai , François Patrice , Haefliger Jacques-Antoine , Entenza José-Manuel , Vaudaux Pierre , Moreillon Philippe
Published: July 2002

Since
Staphylococcus aureus
expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem,
S. aureus
clumping factor A (
clfA
) and fibronectin-binding protein A (
fnbA
) genes were constitutively expressed in poorly pathogenic
Lactococcus lactis
using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both
clfA
and
fnbA
increased the adherence of lactococci to their specific ligands to a similar extent as the
S. aureus
gene donor. In vivo, the minimum inoculum size producing endocarditis in ≥80% of the rats (80% infective dose [ID
80
]) with the parent lactococcus was ≥10
7
CFU. In contrast,
clfA
-expressing and
fnbA
-expressing lactococci required only 10
5
CFU to infect the majority of the animals (
P
< 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as
S. aureus
and streptococci (ID
80
= 10
4
to 10
5
CFU) in this model. The results confirmed the role of
clfA
in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified
fnbA
as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the
clfA
and
fnbA
products should be blocked for the therapy to be effective.
Vibrio cholerae-Induced Cellular Responses of Polarized T84 Intestinal Epithelial Cells Are Dependent on Production of Cholera Toxin and the RTX Toxin
Authors: Fullner Karla Jean , Lencer Wayne I. , Mekalanos John J.
Published: July 2002

To study the utility of in vitro-polarized intestinal cell monolayers for modelingVibrio cholerae-host cell interactions, we added liveV. choleraebacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.
Cloning, Nucleotide Sequence, and Expression of the
Brucella melitensis sucB
Gene Coding for an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein
Authors: Zygmunt Michel S. , Dı́az Marı́a A. , Teixeira-Gomes Ana P. , Cloeckaert Axel
Published: July 2002

The
Brucella melitensis sucB
gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from
Brucella abortus
and
Escherichia coli
, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.
Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells
Authors: Gutsmann Thomas , Müller Mareike , Carroll Stephen F. , MacKenzie Roger C. , Wiese Andre , Seydel Ulrich
Published: July 2002

The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.
High Prevalence of Human Antibodies to Recombinant Duffy Binding-Like α Domains of the
Plasmodium falciparum
-Infected Erythrocyte Membrane Protein 1 in Semi-Immune Adults Compared to That in Nonimmune Children
Authors: Oguariri Raphael M. , Borrmann Steffen , Klinkert Mo-Quen , Kremsner Peter G. , Kun Jürgen F. J.
Published: July 2002

We used a panel of nine fusion proteins that contain different Duffy binding-like α (DBL-α) domains of
Plasmodium falciparum
-infected erythrocyte membrane protein 1 to assess the levels of antibody activity in serum samples obtained from semi-immune or nonimmune individuals from Lambaréné, Gabon. Recognition was measured in terms of either the prevalence or the magnitude of the response. A strong correlation between the immune status of the patients and reactivity with recombinant proteins was observed, which was interpreted as a reflection of the number of infections acquired over time. The antibody responses were predominantly directed toward variable epitopes of the DBL-α domain. Antibody responses could be reduced by preincubation of the sera with various fusion proteins. A portion of individuals who exhibited high-level responses to all fusion proteins also had antibodies which recognized conserved epitopes. The possibility that a synergizing effect of anti-DBL-α domain antibodies could support chemotherapy is discussed.
Antibodies to Loop 6 of the P2 Porin Protein of Nontypeable
Haemophilus influenzae
Are Bactericidal against Multiple Strains
Authors: Neary John M. , Yi Kyungcheol , Karalus Richard J. , Murphy Timothy F.
Published: July 2002

The P2 porin protein is the most abundant protein in the outer membrane of nontypeable
Haemophilus influenzae
(NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.