bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Recombinant Flagellin Protein as a Tracer Antigen in a Fluorescence Polarization Assay for Diagnosis of Leptospirosis
Authors: Bughio Nasreen I. , Lin Min , Surujballi Om P.
Published: December 2019

The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of
Leptospira interrogans
serovar
pomona
in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against
L. interrogans
serovar
pomona
and
L. bergpetersenii
serovars
hardjo
and
sejroe
to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83.7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-
Leptospira
antibodies.
Modulation of T-Cell Responses to a Recall Antigen in Human T-Cell Leukemia Virus Type 1-Infected Individuals
Authors: Suzuki Muneou , Dezzutti Charlene S. , Okayama Akihiko , Tachibana Nobuyoshi , Tsubouchi Hirohito , Mueller Nancy , Lal Renu B.
Published: December 2019

To determine the mechanism of the purified protein derivative (PPD)-specific hyporesponsiveness inMycobacterium bovisBCG-vaccinated human T-cell leukemia virus type 1 (HTLV-1)-infected individuals, we examined cytokine production in response to PPD in the following four groups of individuals: (i) HTLV-negative, PPD nonresponders (n= 11; NN); (ii) HTLV-negative, PPD responders (n= 18; NP); (iii) HTLV-positive, PPD nonresponders (n= 15; PN); and (iv) HTLV-positive, PPD responders (n= 15; PP). In vitro stimulation with PPD resulted in both proliferative responses and gamma interferon (IFN-γ) production in NP and PP (P< 0.02), with minimal proliferation and IFN-γ production in the NN and PN groups. Further, PPD-specific interleukin 10 (IL-10) production was significantly reduced in the PN group (P< 0.01), while the other groups had comparable levels. Cytokine reconstitution experiments demonstrated that while addition of recombinant IL-12 (rIL-12) plus anti-IL-4 restored PPD-specific responses in the NN group, it had no effect in the PN group. However, addition of rIL-12 resulted in the increased production of IFN-γ in both nonresponder groups (NN and PN), suggesting that the lack of IFN-γ production was not responsible for the PPD anergy. We conclude that PPD-specific anergy in HTLV-1-infected individuals appears to be due in part to their inability to respond to rIL-12.
A New Method with General Diagnostic Utility for the Calculation of Immunoglobulin G Avidity
Authors: Korhonen Maria H. , Brunstein John , Haario Heikki , Katnikov Alexei , Rescaldani Roberto , Hedman Klaus
Published: December 2019

The reference method for immunoglobulin G (IgG) avidity determination includes reagent-consuming serum titration. Aiming at better IgG avidity diagnostics, we applied a logistic model for the reproduction of antibody titration curves. This method was tested with well-characterized serum panels for cytomegalovirus, Epstein-Barr virus, rubella virus, parvovirus B19, and
Toxoplasma gondii
. This approach for IgG avidity calculation is generally applicable and attains the diagnostic performance of the reference method while being less laborious and twice as cost-effective.
Variability of the Glycoprotein G Gene in Clinical Isolates of Herpes Simplex Virus Type 1
Authors: Rekabdar Elham , Tunbäck Petra , Liljeqvist Jan-Åke , Bergström Tomas
Published: December 2019

Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17
+
, F, and KOS 321. The reference strains Syn 17
+
and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K
122
to N, which is a gG-1–to–gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F
111
→V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F
111
→V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.
Antibiotics Modulate Vaccine-Induced Humoral Immune Response
Authors: Woo Patrick C. Y. , Tsoi Hoi-Wah , Wong Lei-Po , Leung Harry C. H. , Yuen Kwok-Yung
Published: December 2019

The effects of antibiotics on the antigen-specific humoral immune response are not known. Macrolides, tetracyclines, and beta-lactams are commonly prescribed antibiotics. The first two are known to have immunomodulatory activities. The effects of clarithromycin, doxycycline, and ampicillin on the primary and secondary antibody responses to tetanus toxoid, a pneumococcal polysaccharide vaccine, a hepatitis B virus surface antigen (HBsAg) vaccine, and live attenuatedSalmonella typhi(Ty21a) were investigated using a mouse model. For the mice receiving the tetanus toxoid, the immunoglobulin M (IgM) level of the clarithromycin group at day 7 was significantly lower than the corresponding antibody level of the normal saline (NS) group. For the mice receiving the pneumococcal polysaccharide vaccine, the total antibody and IgM levels of the clarithromycin group and the IgM level of the doxycycline group at day 7 were significantly lower than the corresponding antibody levels of the ampicillin and NS groups. For the mice receiving the HBsAg vaccine, the IgM level of the doxycycline group at day 7 was significantly lower than the corresponding antibody levels of the clarithromycin and NS groups, while the IgM level of the clarithromycin group at day 28 was significantly lower than the corresponding antibody levels of the doxycycline, ampicillin, and NS groups. For the mice receiving all three vaccines, there were no statistically significant differences between any of the antibody levels of the ampicillin group and the corresponding antibody levels of the NS group. For the mice receiving Ty21a, the total antibody levels of the ampicillin group at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Moreover, the IgM levels of the clarithromycin, doxycycline, and ampicillin groups at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Furthermore, the total antibody level of the ampicillin group at day 21 was significantly higher than the corresponding antibody level of the doxycycline group. For all four vaccines, there were no statistically significant differences among the serum levels of interleukin-10 and gamma interferon for the mice treated with the various antibiotics. We conclude that clarithromycin and doxycycline, but not ampicillin, suppress the antibody responses of mice to T-cell-dependent and T-cell-independent antigens, whereas all three antibiotics enhance the antibody response to live attenuated mucosal bacterial vaccines.
Development of a Recombinant Antigen for Antibody-Based Diagnosis of
Mycoplasma bovis
Infection in Cattle
Authors: Brank Marion , Le Grand Dominique , Poumarat François , Bezille Pierre , Rosengarten Renate , Citti Christine
Published: December 2019

Mycoplasma bovis
induces various clinical manifestations in cattle, such as mastitis, arthritis, and pneumonia. We have evaluated the immunoreactivity of three variable surface proteins (Vsps) of
M. bovis
, namely VspA, VspB, and VspC, with sera collected from herds with mycoplasmosis or from cattle experimentally infected with
M. bovis
. Western blot analysis revealed that the Vsps are the predominant antigens recognized by the host humoral response during
M. bovis
infection. The immunoreactivity of VspA, VspB, and VspC with host antibodies was independent of the clinical manifestations, the geographical origin of the
M. bovis
isolates, the mode of infection, and the animal’s history. Moreover, the results showed that Vsp-specific host antibodies can be detected about 10 days after experimental infection and for up to several months. The full-length or truncated versions of the VspA product were overexpressed in
Escherichia coli
as fusion proteins (FP-VspA). Recombinant products showed strong immunoreactivity with the Vsp-specific monoclonal antibodies 1A1 and 1E5, with the corresponding epitopes localized at the VspA N-terminal and C-terminal ends, respectively. Anti-
M. bovis
sera of cattle naturally or experimentally infected also strongly recognized the full-length FP-VspA. The seroreactivity of sera collected from cattle between 6 and 10 days after experimental infection was weaker with truncated versions of VspA lacking the 1E5 epitope than with the full-length VspA or the truncated versions lacking the 1A1 epitope. Overall, the results indicate that the Vsps, despite their inter- and intraclonal variability, may be applied as target antigens in serodiagnostic assays for epidemiological studies.
Decay of Passively Acquired Maternal Antibodies against Measles, Mumps, and Rubella Viruses
Authors: Nicoara Corina , Zäch Kristina , Trachsel Daniel , Germann Daniel , Matter Lukas
Published: December 2019

The decay of maternally derived antibodies to measles, mumps, and rubella viruses in Swiss infants was studied in order to determine the optimal time for vaccination. A total of 500 serum or plasma samples from infants up to 2 years of age were tested by enzyme-linked immunosorbent assay and fluorescent-antibody testing. The decline of antibody prevalence was slowest against the measles virus. By 9 to 12 months of age, only 5 of 58 (8.6%; 95% CI, 2.9 to 19.0) infants were antibody positive for the measles virus, and only 2 had levels above 200 mIU/ml. Mumps and rubella virus antibody seropositivity was lowest at 9 to 12 months of age with 3 of 58 (5.2%; 95% CI, 1.1 to 14.4) infants and at 12 to 15 months with 1 of 48 (2.1%; 95% CI, 0.1 to 11.1) infants, respectively. Concentrations of passively acquired antibodies decreased rapidly within the first 6 months of life. We observed no significant differences in antibody prevalence or concentration according to gender in any age group. In conclusion, MMR vaccination at 12 instead of 15 months of age could reduce the pool of susceptible subjects in infancy and support the efforts to eliminate these infections, particularly in combination with a second vaccine dose before school entry.
Role of p38 in the Priming of Human Neutrophils by Peritoneal Dialysis Effluent
Authors: Daniels Ian , Fletcher John , Haynes Andrew Paul
Published: December 2019

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight substance that is able to prime human neutrophils for the release of arachidonic acid and superoxide anion. Conventional priming agents, such as tumor necrosis factor alpha (TNF-α), are known to signal via mitogen-activated protein (MAP) kinases; at least one possible substrate for MAP kinases is cytosolic phospholipase A2(cPLA2). Phosphorylation of this enzyme results in arachidonic acid release, and this fatty acid is a potent primer and activator of the human neutrophil NADPH oxidase. Because of the striking similarities between the priming of neutrophils with agents such as TNF-α and PDE, we have investigated the signalling pathways evoked by PDE and explored the possibility that cPLA2is a target for activated MAP kinases. Our results show that PDE treatment of human neutrophils results in the phosphorylation of the p38 kinase rather than the p42 and p44 kinases. Phosphorylation of p38 is transient with maximal activity being observed 1 min after exposure to PDE. We were unable to demonstrate that activation of p38 resulted in phosphorylation of cPLA2; furthermore, translocation of this enzyme to a membrane-containing fraction was not enhanced in PDE-treated neutrophils. Taken together, these data suggest that, in a manner similar to that of TNF-α, PDE primes human neutrophils by the activation of the p38 kinase. However, unlike the cytokine, the activation of this protein does not result in phosphorylation or activation of cPLA2.
Can an Antibiotic (Macrolide) Prevent
Chlamydia pneumoniae
-Induced Atherosclerosis in a Rabbit Model?
Authors: Fong Ignatius W. , Chiu Brian , Viira Esther , Jang Dan , Fong Michael W. , Peeling Rosanna , Mahony James B.
Published: December 2019

There is increasing data implicating
Chlamydia pneumoniae
in the pathogenesis of atherosclerosis, and antibiotics may theoretically be useful to prevent secondary vascular complications. Three groups of New Zealand White specific-pathogen-free rabbits, fed cholesterol-free chow, were inoculated via the nasopharynx on three occasions, 2 weeks apart, with
C. pneumoniae
. Group I (
n
= 23) rabbits were untreated; group II (
n
= 24) rabbits were treated with azithromycin at 30 mg/kg of body weight daily for 3 days and then once every 6 days, starting 5 days after first inoculation and continuing until sacrifice (early treatment); and group III (
n
= 24) rabbits were treated with the same dose of azithromycin but initiated 2 weeks after the last inoculation. All animals were sacrificed at 10 to 11 weeks after initial inoculation and examined for signs of atherosclerosis of the aorta. Eight (34.8%) untreated rabbits developed early signs of atherosclerosis, whereas only one (4.2%) in the early-treatment group had such signs (
P
= 0.02). However, eight rabbits (33.3%) of the delayed-treatment group had atherosclerotic changes of the aorta and no significant reduction compared to untreated rabbits. Early treatment of
C. pneumoniae
-infected rabbits with azithromycin was highly effective (87%) in preventing atherosclerotic changes, but delayed treatment was ineffective. It is possible that longer or more aggressive antibiotic treatment may be needed to reverse preformed lesions or that antibiotics may not be of value once lesions have formed.
Sézary T-Cell Activating Factor Is aChlamydia pneumoniae-Associated Protein
Authors: Abrams J. Todd , Vonderheid Eric C. , Kolbe Sonya , Appelt Denah M. , Arking E. James , Balin Brian J.
Published: December 2019

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it’s role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated withChlamydia pneumoniaebacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein ofC. pneumoniaeor against the lipopolysaccharide ofChlamydiaespp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence ofC. pneumoniaeDNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of theC. pneumoniaeantigens and SAF appears to be associated with active disease in thatC. pneumoniaeantigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is aChlamydia-associated protein and that further investigation is warranted to determine whether SAF andC. pneumoniaeplay a role in the pathogenesis of CTCL.
Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization
Authors: Romano Joseph W. , Tetali Surya , Lee Eun Mi , Shurtliff Roxanne N. , Wang Xue Ping , Pahwa Savita , Kaplan Mark H. , Ginocchio Christine C.
Published: December 2019

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.
Relative Avidities of Human Immunoglobulin G Antibodies for Streptococcal Pyrogenic Exotoxins A and B
Authors: Mascini Ellen M. , Jansze Margriet , Verhoef Jan , van Dijk Hans
Published: December 2019

In this pilot study, we investigated the relative avidities for streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B of antibodies in sera from patients with fatal streptococcal toxic shock-like syndrome and from healthy individuals and in intravenous immunoglobulin (IVIG) preparations. We observed a great variation in the relative avidities of patient, control, and IVIG immunoglobulin G (IgG) (values estimated to be between 10
−7
and 10
−11
M), with mean values for patient IgG about 10-fold lower than those of control IgG.
Distinct Characteristics of Resistance toBorrelia burgdorferi-Induced Arthritis in C57BL/6N Mice
Authors: Ma Ying , Seiler Kathleen Petri , Eichwald Ernst J. , Weis John H. , Teuscher Cory , Weis Janis J.
Published: December 2019

Studies of mice infected withBorrelia burgdorferihave indicated that the severity of arthritis is influenced by the genetic composition of the host: the C3H mouse develops severe arthritis while BALB/c and C57BL/6 mice develop mild arthritis. In this study, the effects of increasing infectious dose on the severity of arthritis were determined in these three mouse strains. C3H/He mice developed severe arthritis at all infectious doses, with 100% infection requiring 200 spirochetes. In BALB/cAnN mice, arthritis severity was dependent on infectious dose; symptoms were mild with infection by 200B. burgdorferiand progressively more severe with increasing infectious dose. Infection of BALB/cAnN mice with 2 × 104B. burgdorferiresulted in arthritis with severity identical to that in C3H/He mice. Spirochete levels in rear ankle joints of C3H/HeJ and C3H/HeN mice were relatively high, as detected by PCR, and did not increase with infectious dose. Spirochete levels in joints from BALB/cAnN mice increased with increasing infectious dose to levels found in severely arthritic C3H/He mice. Thus, resistance to severe arthritis in BALB/cAnN mice was conditional: it could be overcome by high infectious dose and the arthritis became severe when high levels ofB. burgdorferiwere present in joints. A unique response to increasing infectious dose was seen in C57BL/6N mice, which displayed mild to moderate arthritis at all doses ofB. burgdorferitested, up to 2 × 105. At all infectious doses, the levels of spirochetes in ankle joints of C57BL/6N mice were high, equivalent to those found in the severely arthritic C3H/He mice. The arthritis observed in infected (C57BL/6N × C3H/HeN)F1mice was of severity intermediate between those of the two parental strains. The finding that resistance to severe arthritis in C57BL/6N mice could not be overcome by high infectious doses and was independent of spirochete levels in joints suggested that it was mediated by a distinct mechanism from that operating in BALB/cAnN mice.
Identification and Characterization of Protective T Cells in hsp65 DNA-Vaccinated andMycobacterium tuberculosis-Infected Mice
Authors: Bonato Vania L. D. , Lima Valeria M. F. , Tascon Ricardo E. , Lowrie Douglas B. , Silva Celio L.
Published: December 2019

Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulentMycobacterium tuberculosisH37Rv. During infection or after immunization, CD4+/CD8−and CD8+/CD4−hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44lo) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44hi) and produced gamma interferon (IFN-γ). In adoptive transfer of protection to naive mice, the total CD8+/CD4−cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4+/CD8−and CD8+/CD4−types and then into CD44hiand CD44lotypes, CD44locells were essentially unable to transfer protection, the most protective CD44hicells were CD8+/CD4−, and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44loIL-4-producing phenotype prevailed during infection, protection was associated with the CD8+/CD44hiIFN-γ-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.
A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species
Authors: Poulsen Knud , Reinholdt Jesper , Jespersgaard Christina , Boye Kit , Brown Thomas A. , Hauge Majbritt , Kilian Mogens
Published: December 2019

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains ofStreptococcus pneumoniae,Streptococcus oralis,Streptococcus mitis, andStreptococcus sanguiswas carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing ofigagenes fromS. oralisandS. mitisbiovar 1, sequencing of an additional sevenigagenes fromS. sanguisbiovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses ofigagenes of another 10 strains ofS. mitisbiovar 1 and 6 strains ofS. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity betweenS. oralisandS. mitisIgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those ofS. pneumoniaeandS. sanguis, respectively. Combined with the results of RFLP analyses using differentigagene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer ofigagene sequences among individual strains ofS. sanguisas well as amongS. mitisand the two speciesS. pneumoniaeandS. oralis. Whileigagenes ofS. sanguisandS. oraliswere highly homogeneous, the genes ofS. pneumoniaeandS. mitisshowed extensive polymorphism reflected in different degrees of antigenic diversity.
Mycoplasma pulmonis
Inhibits Electrogenic Ion Transport across Murine Tracheal Epithelial Cell Monolayers
Authors: Lambert Linda C. , Trummell Hoa Q. , Singh Ashvani , Cassell Gail H. , Bridges Robert J.
Published: December 2019

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by
Mycoplasma pulmonis
, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of
M. pulmonis
upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers.
M. pulmonis
-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na
+
absorption, cyclic AMP, and cholinergic-stimulated Cl
−
secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to
M. pulmonis
was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na
+
and Cl
−
, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 Ω/cm
2
; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in
M. pulmonis
-infected animals. Furthermore, the ability of
M. pulmonis
to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-
M. pulmonis
model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport.
MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells
Authors: Harboe Morten , Wiker Harald G. , Ulvund Gunni , Lund-Pedersen Bent , Andersen Åse Bengård , Hewinson R. Glyn , Nagai Sadamu
Published: December 2019

Culture fluids after growth ofMycobacterium bovisBCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosisandMycobacterium paratuberculosiswere tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.
Immunization of the Female Genital Tract with a DNA-Based Vaccine
Authors: Livingston Julie B. , Lu Shan , Robinson Harriet , Anderson Deborah J.
Published: December 2019

Vaccines are being sought for contraception and the prevention of sexually transmitted diseases. However, progress is slow in this area largely because of lack of information on induction of protective immune responses in genital tract mucosa. In this study, we investigated whether in vivo transfection with a model DNA-based antigen delivered by gene gun technology would induce an antibody response detectable in vaginal secretions. Female rats were immunized with plasmids encoding human growth hormone (HGH) under the control of a cytomegalovirus promoter (pCMV/HGH) via vaginal mucosa (V), Peyer’s patch (PP), and/or abdominal skin (S) routes. Localization of HGH in the target tissues demonstrated that all three sites can be transfected in vivo with pCMV/HGH. Vaginal tissues expressed roughly the same level of plasmid as skin. Antibodies to HGH were detectable in serum and vaginal secretions in rats immunized with pCMV/HGH. In the rats primed and boosted vaginally, vaginal immunoglobulin A (IgA) and IgG antibody titers to HGH were sustained for at least 14 weeks, whereas rats immunized via other routes and protocols (S/V, S/S, PP/PP, or PP/V) did not consistently sustain significant vaginal antibody titers beyond week 6. DNA-based immunizations administered by the gene gun may be an effective method of inducing local immunity in the female genital tract.
Characterization of the Immunostimulatory Properties ofLeishmania infantumHSP70 by Fusion to theEscherichia coliMaltose-Binding Protein in Normal andnu/nuBALB/c Mice
Authors: Rico Ana I. , Del Real Gustavo , Soto Manuel , Quijada Luis , Martinez-A. Carlos , Alonso Carlos , Requena Jose M.
Published: December 2019

Leishmania infantumHSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis. In this study, we usedL. infantumHSP70 fused toEscherichia colimaltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice. Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP,L. infantumHSP70, and MBP-HSP70, which consists of MBP fused to theL. infantumHSP70 amino terminus. Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70. Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response. Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response. Immunization of athymicnu/numice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response. Thus, we present evidence thatL. infantumHSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.
Extensive Immunoglobulin Binding of
Plasmodium falciparum
-Infected Erythrocytes in a Group of Children with Moderate Anemia
Authors: Scholander Carin , Carlson Johan , Kremsner Peter G. , Wahlgren Mats
Published: December 2019

Immunoglobulins (Ig) from healthy, nonimmune individuals bind to the surfaces of
Plasmodium falciparum
-infected erythrocytes (RBC). In order to investigate the presence of this parasite phenotype in wild isolates and its potential association with malarial anemia, we conducted a study of 207 anemic or nonanemic children with malaria in Gabon. Surface Ig binding to infected RBC was detected for 83% of the isolates. No difference in Ig binding between the groups was observed, but all isolates which exhibited extensive Ig binding were found in a group of moderately anemic children.
Prevalence and Distribution of the
hmw
and
hia
Genes and the HMW and Hia Adhesins among Genetically Diverse Strains of Nontypeable
Haemophilus influenzae
Authors: St. Geme Joseph W. , Kumar Vini V. , Cutter David , Barenkamp Stephen J.
Published: December 2019

Nontypeable
Haemophilus influenzae
is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. In previous work we identified high-molecular-weight adhesins referred to as HMW1 and HMW2, expressed by nontypeable strain 12, and determined that most strains of nontypeable
H. influenzae
express one or two antigenically related proteins. More recently, we determined that some strains lack HMW1- and HMW2-like proteins and instead express an adhesin called Hia. In the present study, we determined the prevalence and distribution of the
hmw
and
hia
genes in a collection of 59 nontypeable strains previously characterized in terms of genetic relatedness. Based on Southern analysis, 47 strains contained sequences homologous to the
hmw1
and
hmw2
genes and nine strains contained homologs to
hia
. No strain harbored both
hmw
and
hia
, and three strains harbored neither. Although the
hmw
and
hia
genes failed to define distinct genetic divisions, the
hmw
-deficient strains formed small clusters or lineages within the larger population structure. Additional analysis established that the IS
1016
insertion element was uniformly absent from strains containing
hmw
sequences but was present in two-thirds of the
hmw
-deficient strains. As IS
1016
is associated with the capsule locus (
cap
) in most encapsulated strains of
H. influenzae
, we speculate that
hmw
-deficient nontypeable strains evolved more recently from an encapsulated ancestor.
Molecular Characterization of an Outer Membrane Protein of
Actinobacillus actinomycetemcomitans
Belonging to the OmpA Family
Authors: White Peter A. , Nair Sean P. , Kim Mi-Jurng , Wilson Michael , Henderson Brian
Published: December 2019

The major outer membrane protein (OMP) of
Actinobacillus actinomycetemcomitans
is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of
A. actinomycetemcomitans
NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in
Escherichia coli
and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of
A. actinomycetemcomitans
Omp34, and its corresponding gene has been named
omp34.
Analysis of Inhibitory Epitopes in thePlasmodium falciparumRhoptry Protein RAP-1 Including Identification of a Second Inhibitory Epitope
Authors: Howard Randall F. , Jacobson Kym C. , Rickel Erika , Thurman Joyce
Published: December 2019

Immune responses toPlasmodium falciparumrhoptry-associated protein 1 (RAP-1), RAP-2, and RAP-3 appear to contribute to protection against infection by this human malarial parasite. This conclusion is suggested by results of monkey immunization trials and of cell culture studies showing antibody-dependent inhibition of erythrocyte invasion. In the present study, splenectomized owl monkeys were infected withP. falciparumin order to monitor anti-RAP-1 antibody production as antiparasite immunity developed. The monkeys responded to a primary infection with the production of antibodies to a fragment of RAP-1 containing amino acids 1 to 294 (RAP-11–294). After drug cure and reinfection, the monkeys had a prolonged prepatent period, indicating they had already developed partial immunity to the parasite. Sera from these animals showed major increases in anti-RAP-11–294antibodies. In contrast, only low levels of antibodies to inhibitory B-cell epitope 1 (iB-1), an inhibitory epitope in RAP-11–294with the sequence N200TLTPLEELYPT211, was observed after the initial parasite infection, and the anti-iB-1 antibodies were not readily boosted upon reinfection. These results suggest that iB-1 is an immunogenic but not immunodominant epitope and that anti-iB-1 antibodies do not substantially contribute to early stages of naturally acquired immunity in the owl monkey model. To identify additional epitopes bound by inhibitory antibodies, mouse monoclonal antibodies were produced with a recombinant fusion protein containing RAP-11–294. Monoclonal antibody 1D6 inhibited parasite invasion of erythrocytes in vitro. 1D6 did not bind peptide iB-1 but rather bound a second inhibitory epitope called iB-2. iB-2, like iB-1, is found near the amino terminus of p67, a RAP-1 processing product thought to be involved in merozoite invasion of erythrocytes. Since anti-iB-1 antibodies were not readily produced during parasite infection, it may be desirable to direct antibody responses to particular epitopes in RAP-1, such as iB-1 and iB-2.
Virulence of aSalmonella typhimuriumOmpD Mutant
Authors: Meyer Paul N. , Wilmes-Riesenberg Mary R. , Stathopoulos Christos , Curtiss Roy
Published: December 2019

AnompDmutation caused by a Tn10insertion was transduced intoSalmonella typhimuriumSL1344 and UK-1. The adherence and invasion capabilities of the resultantompDmutants were examined by tissue culture analysis. The virulence of theS. typhimurium ompDmutants was ascertained by a 50% lethal dose (LD50) study and by determining colonization ability with BALB/c mice. We found no statistically significant difference in adherence and invasion capacities between theS. typhimuriumwild type strains and their correspondingompDmutants. Furthermore, the LD50and colonization studies revealed that there is no statistically significant difference in virulence between theS. typhimuriumwild type strains and their correspondingompDmutants. These results differ from those reported previously (C. J. Dorman, S. Chatfield, C. F. Higgins, C. Hayward, and G. Dougan, Infect. Immun. 57:2136–2140, 1989).
Capsular Polysaccharide-Fimbrial Protein Conjugate Vaccine Protects againstPorphyromonas gingivalisInfection in SCID Mice Reconstituted with Human Peripheral Blood Lymphocytes
Authors: Choi Jeom-Il , Schifferle Robert E. , Yoshimura Fuminobu , Kim Byung-Woo
Published: December 2019

The effect of immunization with either aPorphyromonas gingivalisfimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate fromP. gingivaliscould potentially be developed as a vaccine against periodontal infection byP. gingivalis.
Microevolution of a Standard Strain ofCryptococcus neoformansResulting in Differences in Virulence and Other Phenotypes
Authors: Franzot Sarah P. , Mukherjee Jean , Cherniak Robert , Chen Lin-Chi , Hamdan Junia S. , Casadevall Arturo
Published: December 2019

Cryptococcus neoformansis a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence forC. neoformanslaboratory strains. A serendipitous observation in our laboratory that one isolate ofC. neoformansATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply thatC. neoformansis able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate thatC. neoformans(i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.
Streptococcus pyogenes
Serotype M1 Encodes Multiple Pathways for Entry into Human Epithelial Cells
Authors: Cue D. , Dombek P. E. , Lam H. , Cleary P. P.
Published: December 2019

The ability of a serotype M1 strain of
Streptococcus pyogenes
to efficiently invade A549 human lung epithelial cells was previously shown to be dependent on bacterial exposure to human or bovine serum proteins or synthetic peptides containing the sequence RGD. In this study, stimulation by invasion agonists was determined to be dependent on expression of the streptococcal cell surface protein, M1. Fetal bovine serum (FBS), fibronectin (Fn), the extracellular matrix protein laminin (Lm), and RGD-containing peptides were tested for their abilities to promote epithelial cell invasion and adherence by isogenic M1
+
and M1
−
strains of
S. pyogenes
. In the absence of an agonist, invasion and adherence were comparable for the two bacterial strains. FBS, Fn, and Lm stimulated invasion of the M1
+
strain as much as 70-fold but failed to significantly affect invasion by the M1
−
mutant. Adherence of the wild-type strain was stimulated by these same agonists. Epithelial cell adherence by the M1
−
strain, however, was unaffected by the presence of Fn or Lm. Several RGD-containing peptides were found to promote invasion independently of M1 expression. Binding of
125
I-Fn was reduced 88% by the M1
−
mutation and Fn was found to bind purified M1 protein, suggesting that Fn mediates invasion by direct binding to M1. To determine if host integrins might be involved in internalization of streptococci, several anti-integrin monoclonal antibodies (MAbs) were tested for their abilities to inhibit invasion. Antibody directed against integrin β1 inhibited FBS-, Fn-, and Lm-mediated invasion but did not abrogate RGD-peptide-stimulated invasion. MAb directed against the epithelial cell Fn receptor, integrin α5β1, inhibited Fn and FBS-mediated invasion but did not specifically inhibit Lm-mediated invasion. These results indicate that
S. pyogenes
has evolved multiple mechanisms for invasion of eukaryotic cells, at least two of which involve interactions between M1 protein, host integrins, and integrin ligands.
Surface-Associated Hsp60 Chaperonin of
Legionella pneumophila
Mediates Invasion in a HeLa Cell Model
Authors: Garduño Rafael A. , Garduño Elizabeth , Hoffman Paul S.
Published: December 2019

HeLa cells have been previously used to demonstrate that virulent strains of
Legionella pneumophila
(but not salt-tolerant avirulent strains) efficiently invade nonphagocytic cells. Hsp60, a member of the GroEL family of chaperonins, is displayed on the surface of virulent
L. pneumophila
(R. A. Garduño et al., J. Bacteriol. 180:505–513, 1988). Because Hsp60 is largely involved in protein-protein interactions, we investigated its role in adherence-invasion in the HeLa cell model. Hsp60-specific antibodies inhibited the adherence and invasiveness of two virulent
L. pneumophila
strains in a dose-dependent manner but had no effect on the association of their salt-tolerant avirulent derivatives with HeLa cells. A monospecific anti-OmpS (major outer membrane protein) serum inhibited the association of both virulent and avirulent strains of
L. pneumophila
to HeLa cells, suggesting that while both Hsp60 and OmpS may mediate bacterial association to HeLa cells, only virulent strains selectively displayed Hsp60 on their surfaces. Furthermore, the surface-associated Hsp60 of virulent bacterial cells was susceptible to the action of trypsin, which rendered the bacteria noninvasive. Additionally, pretreatment of HeLa cells with purified Hsp60 or precoating of the plastic surface where HeLa cells attached with Hsp60 reduced the adherence and invasiveness of the two virulent strains. Finally, recombinant Hsp60 covalently bound to latex beads promoted the early association of beads with HeLa cells by a factor of 20 over bovine serum albumin (BSA)-coated beads and competed with virulent strains for association with HeLa cells. Hsp60-coated beads were internalized in large numbers by HeLa cells and remained in tight endosomes that did not fuse with other vesicles, whereas internalized BSA-coated beads, for which endocytic trafficking is well established, resided in more loose or elongated endosomes. Mature intracellular forms of
L. pneumophila
, which were up to 100-fold more efficient than agar-grown bacteria at associating with HeLa cells, were enriched for Hsp60 on the bacterial surface, as determined by immunolocalization techniques. Collectively, these results establish a role for surface-exposed Hsp60 in invasion of HeLa cells by
L. pneumophila
.