bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Acute-Phase Parasite Load on Pathology, Parasitism, and Activation of the Immune System at the Late Chronic Phase of Chagas’ Disease
Authors: Marinho Claudio R. F. , D’Império Lima Maria Regina , Grisotto Marcos G. , Alvarez José M. 
Published: December 2019

ABSTRACTTo obtain low and high parasite loads in the acute phase of Chagas’ disease, A/J mice were infected with 103or 105Trypanosoma cruzitrypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8+and B220−CD5−cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220+), CD4+, and CD8+large lymphocytes; (iii) a shift of CD4+cells towards a CD45RBLowphenotype; (iv) increased frequencies of both CD45RBLowand CD45RBHighlarge CD4+cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase ofT. cruziinfection influences the activation of the immune system and development of Chagas’ disease pathology at the late chronic phase of the disease.
Identification of Fur, Aconitase, and Other Proteins Expressed by
Mycobacterium tuberculosis
under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry
Authors: Wong Diane K. , Lee Bai-Yu , Horwitz Marcus A. , Gibson Bradford W. 
Published: December 2019

ABSTRACT

Iron plays a critical role in the pathophysiology of
Mycobacterium tuberculosis
. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in
M. tuberculosis
under conditions of high and low iron concentration. Proteins in cellular extracts from
M. tuberculosis
Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.
Generation of Multinucleated Giant Cells In Vitro by Culture of Human Monocytes withMycobacterium bovisBCG in Combination with Cytokine-Containing Supernatants
Authors: Gasser Annette , Möst Johannes 
Published: December 2019

ABSTRACTMultinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself. Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products. The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro. Stimulation of monocytes withMycobacterium bovisbacillus Calmette-Guérin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27%. In contrast, stimulation with one component alone induced only low fusion rates of up to 10%. Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria. BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation. Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria. MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the β chain (CD18) of β2-integrins. These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.
Cathelicidin Gene Expression in Porcine Tissues: Roles in Ontogeny and Tissue Specificity
Authors: Wu Hua , Zhang Guolong , Ross Christopher R. , Blecha Frank 
Published: December 2019

ABSTRACTCathelicidins constitute a family of mammalian antimicrobial peptides that are synthesized in the bone marrow as prepropeptides, stored in neutrophil granules as propeptides, and released as active, mature peptides upon neutrophil degranulation. We investigated the developmental expression of two porcine cathelicidins, PR-39 and protegrin. Both cathelicidins were expressed constitutively in the bone marrow of all pigs at all of the ages tested. Peripheral blood neutrophils from young pigs expressed PR-39 and protegrin mRNA, which were not detectable at 42 days of age. At earlier ages, expression of PR-39 mRNA was detected in the kidney and liver and several lymphoid organs, including the thymus, spleen, and mesenteric lymph nodes, but disappeared at 4 weeks of age. These data provide the first evidence of cathelicidin gene expression in peripheral leukocytes and may indicate a role for these antimicrobial peptides in the development of host defense mechanisms.
Adhesin Expression in Matched Nasopharyngeal and Middle Ear Isolates of Nontypeable
Haemophilus influenzae
from Children with Acute Otitis Media
Authors: Krasan Graham P. , Cutter David , Block Stan L. , St. Geme Joseph W. 
Published: December 2019

ABSTRACT

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable
Haemophilus influenzae
. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable
H. influenzae
from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five
hif
genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable
H. influenzae
associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.
Localization of the Intracellular Activity Domain ofPasteurella multocidaToxin to the N Terminus
Authors: Wilson Brenda A. , Ponferrada Virgilio G. , Vallance Jefferson E. , Ho Mengfei 
Published: December 2019

ABSTRACTWe have shown thatPasteurella multocidatoxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 inXenopusoocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response inXenopusoocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using theXenopusoocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain ofP. multocidathe entiretoxAgene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein inEscherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments inE. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in theXenopusoocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.
An Opsonizing Monoclonal Antibody That Recognizes a Noncapsular Epitope Expressed on
Cryptococcus neoformans
Authors: Merkel Glenn J. , Scofield Barbara A. 
Published: December 2019

ABSTRACT

A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on
C. neoformans
was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all
C. neoformans
serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.
RecombinantStaphylococcusStrains as Live Vectors for the Induction of Neutralizing Anti-Diphtheria Toxin Antisera
Authors: Fromen-Romano Cécile , Drevet Pascal , Robert Alain , Ménez André , Léonetti Michel 
Published: December 2019

ABSTRACTWe have investigated whether the nonpathogenic gram-positive bacteriaStaphylococcus xylosusandS. carnosuscan display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE′mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins onS. xylosusandS. carnosuscell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface ofS. xylosuscells whereas it is efficiently exposed onS. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally withS. carnosusdisplaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, onlyS. carnosuscan display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.
Complement-Dependent Accumulation and Degradation of Platelets in the Lung and Liver Induced by Injection of Lipopolysaccharides
Authors: Shibazaki Masahiko , Kawabata Yoshihiro , Yokochi Takashi , Nishida Akira , Takada Haruhiko , Endo Yasuo 
Published: December 2019

ABSTRACTWe found unique behaviors among platelets within a few minutes of the intravenous injection of lipopolysaccharide (LPS) into mice. Platelets accumulated primarily in the liver at lower doses of LPS, but at higher doses they accumulated largely in the lungs. When the platelets accumulated in these organs were degraded, there was a rapid anaphylactoid shock. The platelet response depended on the strain of mouse and on the source of LPS. Of various LPSs tested, the LPS from the smooth type ofKlebsiellaO3 (KO3-S LPS) was the most potent at inducing the platelet response and shock. K-76 monocarboxylic acid, an inhibitor of complement C5, effectively prevented theKO3-S LPS-induced degradation (but not accumulation) of platelets and the ensuing rapid shock in BALB/c mice. Moreover, in DBA/2 mice (which are deficient in complement C5), platelets accumulated in the lungs and liver in response toKO3-S LPS but soon returned to the circulation without degradation, and there was no rapid shock. The LPS from the rough type ofKO3 induced an accumulation of platelets in the liver and lungs but not a degradation of platelets. On the basis of these results and those reported by other investigators, we propose that in the platelet response to LPS, the lectin pathway to form C3 convertase from C4 and C2 is involved in the rapid accumulation of platelets in the liver and lungs and that the pathway from C5 to C9 is involved in the destruction of platelets and the consequent anaphylactoid shock.
Characterization of HumanMycobacterium bovisBacille Calmette-Guérin-Reactive CD8+T Cells
Authors: Smith Steven M. , Malin Adam S. , Pauline T.  , Lukey  , Atkinson Sara E. , Content Jean , Huygen Kris , Dockrell Hazel M. 
Published: December 2019

ABSTRACTGamma interferon (IFN-γ)-secreting CD4+T cells have long been established as an essential component of the protective immune response againstMycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+T cells. These cells are capable of acting as both IFN-γ secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence ofMycobacterium bovisBCG-reactive CD8+T cells in healthy BCG-vaccinated donors and that these CD8+T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with liveM. bovisBCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-γ and tumor necrosis factor alpha (TNF-α). These CD8+T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs againstM. bovisBCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+T cells, while whole-M. bovisBCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+T cells may play a critical role in the human immune response to tuberculosis infection.
Common and Specific Characteristics of the High-Pathogenicity Island of
Yersinia enterocolitica
Authors: Rakin A. , Noelting C. , Schubert S. , Heesemann J. 
Published: December 2019

ABSTRACT

Yersinia pestis
,
Y. pseudotuberculosis
O:1, and
Y. enterocolitica
biogroup 1B strains carry a high-pathogenicity island (HPI), which mediates biosynthesis and uptake of the siderophore yersiniabactin and a mouse-lethal phenotype. The HPI of
Y. pestis
and
Y. pseudotuberculosis
(Yps HPI) are highly conserved in sequence and organization, while the HPI of
Y. enterocolitica
(Yen HPI) differs significantly. The 43,393-bp Yen HPI sequence of
Y. enterocolitica
WA-C, serotype O:8, was completed and compared to that of the Yps HPI of
Y. pseudotuberculosis
PB1, serotype O:1A. A common GC-rich region (G+C content, 57.5 mol%) of 30.5 kb is conserved between yersinia strains. This region carries genes for yersiniabactin biosynthesis, regulation, and uptake and thus can be considered the functional “core” of the HPI. In contrast, the second part of the HPI is AT rich and completely different in two evolutionary lineages of the HPI, being 12.8 kb in the Yen HPI and 5.6 kb in the Yps HPI. The variable part acquired one IS
100
element in the Yps HPI and accumulated four insertion elements, IS
1328
, IS
1329
, IS
1400
, and IS
1222
, in the Yen HPI. The insertion of a 125-bp ERIC sequence modifies the structure of the promoter of the
ybtA
yersiniabactin regulator in the Yen HPI. In contrast to the precise excision of the Yps HPI in
Y. pseudotuberculosis
, the Yen HPI suffers imprecise deletions. The Yen HPI is stably integrated in one of the three
asn
tRNA copies in
Y. enterocolitica
biogroup 1B (serotypes O:8, O:13, O:20, and O:21), probably due to inactivation of the putative integrase. The 17-bp duplications of the 3′ end of the
asnT
RNA are present in both
Yersinia
spp. The HPI attachment site is unoccupied in nonpathogenic
Y. enterocolitica
NF-O, biogroup 1A, serotype O:5. The HPI of
Yersinia
is a composite and widely spread genomic element with a highly conserved yersiniabactin functional “core” and a divergently evolved variable part.
Cryptosporidium parvumApical Complex Glycoprotein CSL Contains a Sporozoite Ligand for Intestinal Epithelial Cells
Authors: Langer Rebecca C. , Riggs Michael W. 
Published: December 2019

ABSTRACTCryptosporidiosis, caused by the apicomplexan parasiteCryptosporidium parvum, has become a well-recognized diarrheal disease of humans and other mammals throughout the world. No approved parasite-specific drugs, vaccines, or immunotherapies for control of the disease are currently available, although passive immunization withC. parvum-specific antibodies has some efficacy in immunocompromised and neonatal hosts. We previously reported that CSL, an ∼1,300-kDa conserved apical glycoprotein ofC. parvumsporozoites and merozoites, is the antigenic species mechanistically bound by neutralizing monoclonal antibody 3E2 which elicits the circumsporozoite precipitate (CSP)-like reaction and passively protects againstC. parvuminfection in vivo. These findings indicated that CSL has a functional role in sporozoite infectivity. Here we report that CSL has properties consistent with being a sporozoite ligand for intestinal epithelial cells. For these studies, native CSL was isolated from whole sporozoites by isoelectric focusing (IEF) following observations that the ∼1,300-kDa region containing CSL as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was comprised of approximately 15 molecular species (pI 3 to 10) when examined by two-dimensional (2-D) electrophoresis and silver staining. A subset of six ∼1,300-kDa species (pI 4.0 to 6.5) was specifically recognized by 3E2 in 2-D Western immunoblots of IEF-isolated CSL. Isolated native CSL bound specifically and with high affinity to permissive human intestinal epithelial Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner. Further, CSL specifically bound to the surface of live Caco-2 cells inhibited sporozoite attachment and invasion. In addition, sporozoites having released CSL after incubation with 3E2 and occurrence of the CSP-like reaction did not attach to and invade Caco-2 cells. These findings indicate that CSL contains a sporozoite ligand which facilitates attachment to and invasion of Caco-2 cells and, further, that ligand function may be disrupted by CSL-reactive monoclonal antibody. We conclude that CSL is a rational target for passive or active immunization against cryptosporidiosis.