bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Interleukin-8-Induced Degranulation of Polymorphonuclear Neutrophils from Human Immunodeficiency Virus Type 1-Infected Individuals
Authors: Meddows-Taylor Stephen , Martin Desmond J. , Tiemessen Caroline T.
Published: December 2019

Degranulation of peripheral blood polymorphonuclear leukocytes (PMNLs) was monitored in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals with or without pulmonary tuberculosis (HIV/TB and HIV groups, respectively) by measuring the release of β-glucuronidase induced by interleukin-8 (IL-8). This was increased in a dose-dependent manner in the control groups consisting of healthy blood donors and patients with pulmonary tuberculosis. In contrast, PMNLs from the HIV and HIV/TB groups responded reciprocally in the same assay; that is, higher IL-8 input concentrations resulted in the release of less enzyme than lower IL-8 input concentrations. The degranulation response of PMNLs from HIV-1-infected individuals was similarly altered for another agonist,N-formyl-methionyl-leucyl-phenylalanine, suggesting that impairment of the nonoxidative armature of PMNL was a more generalized phenomenon. However, impaired IL-8-induced degranulation was found to be associated with the reduced expression of both IL-8 receptors, A and B, on whole-blood PMNLs from HIV-1-infected patients compared with that on whole-blood PMNLs from healthy persons. The density of IL-8RA, in particular, was most reduced on the surfaces of PMNLs from those patients with the poorest degranulation in response to IL-8. Inefficient agonist-induced degranulation may contribute to the increased susceptibility of HIV-1-infected persons to secondary microbial infections, this being further exacerbated in HIV/TB patients who, in addition, display defects in phagocytosis and oxidative burst.
Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific forEntamoeba histolytica
Authors: Tachibana Hiroshi , Cheng Xun-Jia , Watanabe Katsuomi , Takekoshi Masataka , Maeda Fumiko , Aotsuka Satoshi , Kaneda Yoshimasa , Takeuchi Tsutomu , Ihara Seiji
Published: December 2019

Genes coding for human antibody Fab fragments specific forEntamoeba histolyticawere cloned and expressed inEscherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced intoEscherichia coli. Three thousand colonies were screened for the production of antibodies toE. histolyticaHM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from fiveEscherichia coliclones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of theEscherichia colilysates to nine reference strains ofE. histolyticawere examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted withEntamoeba disparreference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific forE. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishingE. histolyticafromE. disparand for use in the serodiagnosis of amebiasis.
Molecular Cloning of the Gene for a Conserved Major Immunoreactive 28-Kilodalton Protein ofEhrlichia canis: a Potential Serodiagnostic Antigen
Authors: McBride Jere W. , Yu Xue-jie , Walker David H.
Published: December 2019

A gene encoding a 28-kDa protein ofEhrlichia caniswas cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes ofE. canis,Cowdria ruminantium, andEhrlichia chaffeensiswas performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). TheE. canis p28gene has significant nucleic acid and amino acid sequence homologies with theE. chaffeensisouter membrane protein-1 (omp-1) gene family, with theCowdria ruminantium map-1gene, and with otherE. canis28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologousp28gene copies in theE. canisgenome, confirming thatp28is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed thatE. canisP28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs withE. chaffeensisP28. Thep28genes from seven differentE. canisisolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.
A New Cell Enzyme-Linked Immunosorbent Assay Demonstrates Gamma Interferon Suppression by Beta Interferon in Multiple Sclerosis
Authors: Bakhiet Moiz , Özenci Volkan , Withagen Carin , Mustafa Maha , Fredrikson Sten , Link Hans
Published: December 2019

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system of unknown etiology. Immune mechanisms involving the proinflammatory cytokine gamma interferon (IFN-γ) are believed to play an important role in the pathogenesis of MS. IFN-β-1b has been introduced as a treatment for MS and was found to reduce the number and severity of clinical exacerbations. To examine the influence of IFN-β-1b on myelin basic protein (MBP)-specific and phytohemagglutinin-induced IFN-γ production, we developed a cell-released capturing enzyme-linked immunosorbent assay (CRC-ELISA), which rapidly measures spontaneous and antigen- or mitogen-induced cellular IFN-γ production. CRC-ELISA documented a significant MBP-specific T-cell response in the blood of untreated MS patients, as assessed by IFN-γ production. This response was suppressed in MS patients treated with IFN-β-1b. The present work confirms in vivo the in vitro suppressive effects of IFN-β-1b on IFN-γ production in MS. Moreover, it provides a powerful new technique for detection of cytokines.
Estimation of Growth Rates of
Escherichia coli
BJ4 in Streptomycin-Treated and Previously Germfree Mice by In Situ rRNA Hybridization
Authors: Rang Camilla U. , Licht Tine Rask , Midtvedt Tore , Conway Patricia L. , Chao Lin , Krogfelt Karen A. , Cohen Paul S. , Molin Søren
Published: December 2019

The growth physiology of
Escherichia coli
during colonization of the intestinal tract was studied with four animal models: the streptomycin-treated mouse carrying a reduced microflora, the monoassociated mouse with no other microflora than the introduced strain, the conventionalized streptomycin-treated mouse, and the conventionalized monoassociated mouse harboring a full microflora. A 23S rRNA fluorescent oligonucleotide probe was used for hybridization to whole
E. coli
cells fixed directly after being taken from the animals, and the respective growth rates of
E. coli
BJ4 in the four animal models were estimated by correlating the cellular concentrations of ribosomes with the growth rate of the strain. The growth rates thus estimated from the ribosomal content of
E. coli
BJ4 in vivo did not differ in the streptomycin-treated and the monoassociated mice. After conventionalization there was a slight decrease of the bacterial growth rates in both animal models.
Effect of Early Antibiotic Treatment on the Antibody Response to Cytoplasmic Proteins of
Brucella melitensis
in Mice
Authors: Bowden Raul A. , Racaro Graciela C. , Baldi Pablo C.
Published: December 2019

To test whether antibiotic therapy hampers the antibody response to
Brucella
antigens, 30 BALB/c mice were infected with
Brucella melitensis
H38 and randomized for treatment with doxycycline administered intraperitoneally for 42 days starting at 7 or 28 days postinfection (p.i.) (groups DOX7 and DOX28, respectively) or for no treatment (control group). Antibodies to smooth lipopolysaccharide (LPS) reached peak levels (mean optical density [OD] = 2.618) between days 56 and 70 p.i. in the control group, and similar peak levels (mean OD = 2.486) were observed in the DOX28 group, but significantly lower peak levels (mean OD = 0.821) were observed at 28 days p.i. in the DOX7 group. The antibody response against cytoplasmic proteins depleted of LPS (CPs) reached maximal levels (mean OD = 2.402) between days 56 and 70 p.i. in the control group, but no response was detected in the DOX7 group. Anti-CP antibodies were detected in only three animals from the DOX28 group, at levels significantly lower than those in the control group (mean maximal OD = 0.791). The pattern of antibody response to an 18-kDa cytoplasmic protein of
Brucella
spp. was similar to that against the CP antigen. This study shows that early antibiotic treatment affects the antibody response of mice to cytoplasmic proteins of
Brucella
and, to a lesser extent, to LPS.
CD4-Positive and CD8-Positive Cytotoxic T Lymphocytes Contribute to Human Papillomavirus Type 16 E6 and E7 Responses
Authors: Nakagawa Mayumi , Stites Daniel P. , Palefsky Joel M. , Kneass Zachary , Moscicki Anna-Barbara
Published: December 2019

Cytotoxic T-lymphocyte (CTL) responses to E6 and E7 were previously shown to be more commonly detectable in human papillomavirus type 16 (HPV-16)-positive women without squamous intraepithelial neoplasia (SIL) than in HPV-16-positive women with SIL (M. Nakagawa, D. P. Stites, S. Farhat, J. R. Sisler, B. Moss, F. Kong, A. B. Moscicki, and J. M. Palefsky, J. Infect. Dis. 175:927–931, 1997). The objective of this study was to characterize the phenotype(s) of the effector cell population responsible for HPV-16 E6- and E7-specific cytotoxic responses. Peripheral blood mononuclear cells were stimulated with HPV-16 E6 or E7 fusion protein. Cells from an autologous B-lymphoblastoid cell line, infected with vaccinia virus expressing E6 or E7, served as target cells. The effector cells were characterized by using natural-killer-cell removal, antibody blocking, and T-cell subset separation. Our results suggest that both CD4 and CD8 T lymphocytes contribute to HPV-16 E6- and E7-specific CTL responses although their relative contributions vary from individual to individual. On the other hand, natural killer cells in the effector cell population contribute to background activities but not to HPV-specific responses in this assay system.
Similar Humoral and Cellular Immunological Reactivities to Human Herpesvirus 6 in Patients with Multiple Sclerosis and Controls
Authors: Enbom Malin , Wang Fu-Zhang , Fredrikson Sten , Martin Claes , Dahl Helena , Linde Annika
Published: December 2019

Several studies have suggested an association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS). We have previously studied intrathecal production of antibody to lymphotropic herpesviruses in MS patients and the presence of human herpesvirus 1 to 7 DNAs in cerebrospinal fluid (CSF). In the present study anti-HHV-6 immunoglobulin M (IgM) in serum and anti-HHV-6 IgG subclasses in serum and CSF were examined and the lymphoproliferative response to HHV-6 was analyzed. The PCR examination was refined by purifying DNA from CSF and retesting the samples for HHV-6 DNA. There were no statistically significant differences between the groups concerning IgM positivity, distribution of IgG subclasses, or lymphoproliferative response to HHV-6. The purification of DNA increased the number of PCR-positive samples from 0 of 71 to 4 of 68. The study does not give additional support to the possibility that HHV-6 is a common cause of MS, but a role for the virus in a subset of patients cannot be excluded.
Use of a Monoclonal Antibody against anEscherichia coliO26 Surface Protein for Detection of Enteropathogenic and Enterohemorrhagic Strains
Authors: Kerr Paul , Ball Hywel , China Bernard , Mainil Jacques , Finlay David , Pollock David , Wilson Ian , Mackie Dermot
Published: December 2019

A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain ofEscherichia coli(EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26eae- and VT-negative strain, or O157eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence ofeaeand the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which wereeaepositive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors includedeae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained sixeae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at −70°C, after which none were found to contain eithereaeor VT factors. The association of the antigen detected by the MAb with significant enteropathogenicE. coliand EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.
A Novel Enzyme-Linked Immunosorbent Assay Using the RecombinantActinobacillus pleuropneumoniaeApxII Antigen for Diagnosis of Pleuropneumonia in Pig Herds
Authors: Leiner Gero , Franz Burkart , Strutzberg Katrin , Gerlach Gerald-F.
Published: December 2019

For the surveillance of pig herds infected with porcine pleuropneumonia, an enzyme-linked immunosorbent assay (ELISA) using the recombinantActinobacillus pleuropneumoniaeApxII protein as species- but not serotype-specific antigen was developed. Using this ELISA, 243 of 400 animals from 22A. pleuropneumoniae-infected herds were classified as seropositive.
A New Sensitive Serological Assay for Detection of Lentivirus Infections in Small Ruminants
Authors: Saman Eric , Van Eynde Geertrui , Lujan Luis , Extramiana Belén , Harkiss Gordon , Tolari Francesco , Gonzàlez Lorenzo , Amorena Beatriz , Watt Neil , Badiola Juan
Published: December 2019

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in
Escherichia coli
and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99.8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (
n
= 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.
Inducible and Constitutive In Vitro Neutrophil Chemokine Expression by Mammary Epithelial and Myoepithelial Cells
Authors: Barber Michele R. , Pantschenko Alexander G. , Hinckley Lynn S. , Yang T. J.
Published: December 2019

Previously, our laboratory showed that bovine and caprine mammary secretions are chemotactic and that chemoattractants found in these secretions are qualitatively different according to infection status and/or lactation stage. However, the cellular source of the chemoattractants has not been defined. In this study we used a modified Boyden chamber assay to examine the ability of previously established caprine mammary epithelial cell (CMEC) and myoepithelial cell (CMMyoEC) lines to produce chemoattractants for neutrophils. We found that CMEC culture supernatants, but not those of CMMyoEC cultures, induced in vitro neutrophil chemotaxis. Further characterization showed that chemotactic activity was produced when the cells underwent contact-induced differentiation. Neutrophil migration was chemotactic, not chemokinetic, and was augmented when the epithelial and myoepithelial cells were cocultured. Additionally, chemotactic activity was inducible byStaphylococcus aureusplus alpha-toxin,Escherichia coli, and interleukin-1β (IL-1β) in CMEC cultures. However, CMMyoEC cultures could not be induced to produce chemotactic activity. Anti-IL-8 antibody was able to block some constitutively produced chemotactic activity and chemotactic activity induced by IL-1β andS. aureusplus alpha-toxin. These results indicate that epithelial cells may play a major role in producing chemoattractants, specifically IL-8, in the mammary gland.
Regeneration and Tolerance Factor: a Correlate of Human Immunodeficiency Virus-Associated T-Cell Activation
Authors: Givens Tara S. , DuChateau Brian K. , Boomer Jonathan S. , Westerman Maxwell P. , Gilman-Sachs Alice , Beaman Kenneth D.
Published: December 2019

Human immunodeficiency virus (HIV) infection causes extensive phenotypic alterations in lymphocytes. Cellular markers that are normally absent or expressed at low levels on quiescent cells are upregulated throughout the disease course. The transmembrane form of regeneration and tolerance factor (RTF) is expressed at negligible levels on resting T cells but is quickly upregulated following in vitro stimulation and activation. Recently, we reported that expression of RTF was significantly higher in cells from HIV-seropositive (HIV+) individuals than in cells from HIV-seronegative (HIV−) individuals. Because T cells from HIV+individuals express markers reflecting chronic activation, we hypothesized that these in vivo-activated cells would coexpress RTF. Flow cytometry was used to assess RTF expression on activated (CD38+and HLA-DR+) CD4+and CD8+T cells. HIV+individuals had higher percentages of RTF+CD38+(P< 0.0001) or RTF+HLA-DR+(P= 0.0001) CD4+T cells than HIV−individuals. In HIV+individuals, increased percentages of CD4+T cells that were RTF+, RTF+CD38+, and RTF+HLA-DR+correlated inversely with the absolute number and percentage of CD4+T cells and correlated positively with plasma β2-microglobulin concentrations. HIV+individuals had higher percentages of CD8+T cells that were RTF+CD38+(P= 0.0001) or RTF+HLA-DR+(P= 0.0010). In HIV+individuals, increased percentages of CD8+T cells that were RTF+HLA-DR+correlated inversely with the percentage of CD4+T cells, and high percentages of CD8+T cells that were RTF+CD38+correlated positively with plasma β2-microglobulin levels. These findings strongly suggest that increased RTF expression is a correlate of HIV-associated immune system activation.
Detection of Borreliacidal Antibodies in Lyme Borreliosis Patient Sera Containing Antimicrobial Agents
Authors: Jobe Dean A. , Rawal Nenoo , Schell Ronald F. , Callister Steven M.
Published: December 2019

The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis. However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies. In this study, we developed a
Bacillus subtilis
agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum. We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies. High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies. These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.
Declining Immunoglobulin A Production in Prostates of Men with AIDS
Authors: Carson Henry J. , Lueck Nathan E.
Published: December 2019

We studied immunoglobulin A production (IgA) in prostates of men with AIDS. Prostate sections from AIDS patients and human immunodeficiency virus-negative men were stained for IgA with immunoperoxidase. Prostate sections from nondiseased men were positive for IgA, while prostate sections from AIDS patients were essentially negative for IgA. Diminishing secretory IgA production may represent a characteristic of AIDS.
bca
, Beta Gene, and Gene Product Divergency in Reference and Prototype Strains of
Streptococcus agalactiae
Authors: Maeland Johan A. , Bevanger Lars , Iversen Grethe , Lyng Randi Valsoe
Published: December 2019

Reference and prototype strains of
Streptococcus agalactiae
(GBS) were originally selected on the basis of phenotypic traits which, however, do not always mirror genotypic traits. A total of 14 reference and prototype GBS strains were examined by PCR designed to detect the
bca
and beta genes, encoding the c proteins c
α
and c
β
, respectively. The cognate proteins were detected by whole-cell-based fluorescent antibody testing and Western blotting. The PCR for beta gene detection and the antibody-based c
β
protein detection showed concordant results with all of the isolates, whereas 7 of 14 strains which did not express c
α
protein at detectable levels contained
bca
gene elements, consistent with
bca
gene and gene product divergency in these strains. The results emphasize the importance of genetic characterization of reference and prototype strains of GBS which, in the past, have been selected on the basis of phenotypic traits.
Listeria monocytogenesVirulence Factors That Stimulate Endothelial Cells
Authors: Drevets Douglas A.
Published: December 2019

Listeria monocytogenesinfection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-typeL. monocytogenesorL. monocytogenesmutants; then surface expression of E-selectin and neutrophil adhesion were measured. The results showed that ΔhlyandprfAmutants were the most crippled, requiring 100-fold more mutant bacteria than wild-type bacteria for analogous stimulation. By comparison,L. monocytogenesmutants with deletions ofactA,inlA,inlB,inlAB,plcA, andplcBresembled their parent strains, and a ΔplcA ΔplcBmutant displayed decreased intracellular growth rate but only a minor decrease in stimulation of E-selectin or neutrophil adhesion. Other experiments showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but internalization and increased surface E-selectin and intercellular adhesion molecule-1 expression were profoundly inhibited. However, cytochalasin D had no effect on the HUVEC response to stimulation with lipopolysaccharide or tumor necrosis factor alpha. These data suggest that listeriolysin O production by infectingL. monocytogenescontributes to increased expression of surface E-selectin and intercellular adhesion molecule-1, but neither it nor intracellular replication are directly responsible for this event. Nonetheless it is possible that listeriolysin O potentiates the effect(s) of an other molecule(s) that directly triggers this response. Additionally, cellular invasion byL. monocytogenesappears to be critical for initiating the HUVEC response, potentially by providing a signal which results in upregulation of the necessary bacterial genes.
Bacterial Cytolysin Perturbs Round Window Membrane Permeability Barrier In Vivo: Possible Cause of Sensorineural Hearing Loss in Acute Otitis Media
Authors: Engel Frank , Blatz Rosemarie , Schliebs Reinhard , Palmer Michael , Bhakdi Sucharit
Published: December 2019

The passage of radioiodinated streptolysin-O (SLO) and albumin through the round window membrane (RWM) was studied in vivo. When applied to the middle ear, SLO became quantitatively entrapped in this compartment and no passage to the cochlea occurred. However, flux of radioiodinated albumin through the toxin-damaged RWM was observed. We propose that the passage of noxious macromolecules, such as proteases, from a purulent middle-ear effusion may be facilitated by pore-forming toxins, resulting in cochlear damage and sensorineural hearing loss.
Immunization of
Aotus
Monkeys with Recombinant
Plasmodium falciparum
Hybrid Proteins Does Not Reproducibly Result in Protection from Malaria Infection
Authors: Kocken Clemens H. M. , Hundt Erika , Knapp Bernhard , Brazel Dieter , Enders Burkhard , Narum David L. , Wubben Jaqueline A. M. , Thomas Alan W.
Published: December 2019

Plasmodium falciparum
antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in
Aotus
monkeys, but solid protection from a
P. falciparum
blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.
Effect of Prior Experimental Human Enteropathogenic
Escherichia coli
Infection on Illness following Homologous and Heterologous Rechallenge
Authors: Donnenberg Michael S. , Tacket Carol O. , Losonsky Genevieve , Frankel Gad , Nataro James P. , Dougan Gordon , Levine Myron M.
Published: December 2019

Two studies of adult volunteers were performed to determine whether prior enteropathogenic
Escherichia coli
(EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic
eae
deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.
Saccharomyces cerevisiae-Secreted Fusion Proteins Pfs25 and Pfs28 Elicit PotentPlasmodium falciparumTransmission-Blocking Antibodies in Mice
Authors: Gozar Mary Margaret G. , Price Virginia L. , Kaslow David C.
Published: December 2019

Transmission-blocking vaccines based on sexual-stage surface antigens ofPlasmodium falciparummay assist in the control of this lethal form of human malaria. Two vaccine candidates, Pfs25 and Pfs28, were produced as single recombinant fusion proteins. The 39-kDa chimeric proteins, having a C-terminal His6tag, were secreted bySaccharomyces cerevisiae, using the prepro-α-factor leader sequence. Pfs25-28 fusion proteins were significantly more potent than either Pfs25 or Pfs28 alone in eliciting antibodies in mice that blocked oocyst development inAnopheles freebornimosquitoes: complete inhibition of oocyst development in the mosquito midgut was achieved with fewer vaccinations, at a lower dose, and for a longer duration than with either Pfs25 or Pfs28 alone. Increased antigen-specific immunoglobulin G titers and highly significant lymphoproliferative stimulation by Pfs28-containing antigens suggest the presence of an immunodominant helper T-cell epitope in the Pfs28 portion of the fusion proteins. This epitope may be responsible for the enhanced humoral response to both Pfs25 and Pfs28 antigens. Protein production of the fusion protein was improved 12-fold by converting Pfs28 codons to yeast-preferred codons (TBV28), using a modifiedADH2promoter and incorporating a (Glu-Ala)2repeat after the Kex2 cleavage site.
Role of
Fusobacterium nucleatum
and Coaggregation in Anaerobe Survival in Planktonic and Biofilm Oral Microbial Communities during Aeration
Authors: Bradshaw David J. , Marsh Philip D. , Watson G. Keith , Allison Clive
Published: December 2019

Coaggregation is a well-characterized phenomenon by which specific pairs of oral bacteria interact physically. The aim of this study was to examine the patterns of coaggregation between obligately anaerobic and oxygen-tolerant species that coexist in a model oral microbial community. Obligate anaerobes other than
Fusobacterium nucleatum
coaggregated only poorly with oxygen-tolerant species. In contrast,
F. nucleatum
was able to coaggregate not only with both oxygen-tolerant and other obligately anaerobic species but also with otherwise-noncoaggregating obligate anaerobe–oxygen-tolerant species pairs. The effects of the presence or absence of
F. nucleatum
on anaerobe survival in both the biofilm and planktonic phases of a complex community of oral bacteria grown in an aerated (gas phase, 200 ml of 5% CO
2
in air · min
−1
) chemostat system were then investigated. In the presence of
F. nucleatum
, anaerobes persisted in high numbers (>10
7
· ml
−1
in the planktonic phase and >10
7
· cm
−2
in 4-day biofilms). In an equivalent culture in the absence of
F. nucleatum
, the numbers of black-pigmented anaerobes (
Porphyromonas gingivalis
and
Prevotella nigrescens
) were significantly reduced (
P
≤ 0.001) in both the planktonic phase and in 4-day biofilms, while the numbers of facultatively anaerobic bacteria increased in these communities. Coaggregation-mediated interactions between
F. nucleatum
and other species facilitated the survival of obligate anaerobes in aerated environments.
An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis
Authors: Lyadova Irina , Yeremeev Vladimir , Majorov Konstantin , Nikonenko Boris , Khaidukov Sergei , Kondratieva Tatiana , Kobets Natalia , Apt Alexander
Published: December 2019

I/St mice, previously characterized as susceptible toMycobacterium tuberculosisH37Rv, were given 103or 105CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44−CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+CD45RB−/lowcells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+CD8−T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+IL-4−) clone and one Th0-like (IFN-γ+IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.
Organ-Dependent Variation of Capsule Thickness inCryptococcus neoformansduring Experimental Murine Infection
Authors: Rivera Johanna , Feldmesser Marta , Cammer Michael , Casadevall Arturo
Published: December 2019

In studies of murine infection, the capsule thickness ofCryptococcus neoformansvaried depending on the organ. The relative order of thickness was as follows: lung > brain > in vitro isolates. The differences in capsule thickness suggest that there are organ-related differences in the expression of genes responsible for capsule thickness.
Helicobacter pylori
Containing Only Cytoplasmic Urease Is Susceptible to Acid
Authors: Krishnamurthy Partha , Parlow Mary , Zitzer Jason B. , Vakil Nimish B. , Mobley Harry L. T. , Levy Marilyn , Phadnis Suhas H. , Dunn Bruce E.
Published: December 2019

Helicobacter pylori
, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of
H. pylori
both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of
H. pylori
to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of
H. pylori
cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured
H. pylori
with flurofamide (1 μM), a potent, but poorly diffusible urease inhibitor.
H. pylori
with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast,
H. pylori
with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly,
Escherichia coli
SE5000 expressing
H. pylori
urease and the Ni
2+
transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type
H. pylori
, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of
H. pylori
in acid; the activity of surface-localized urease is essential for resistance of
H. pylori
to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of
H. pylori
, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria (“altruistic autolysis”), is essential for survival of
H. pylori
in an acid environment. The ability of
H. pylori
to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.
Helicobacter hepaticusTriggers Colitis in Specific-Pathogen-Free Interleukin-10 (IL-10)-Deficient Mice through an IL-12- and Gamma Interferon-Dependent Mechanism
Authors: Kullberg Marika C. , Ward Jerrold M. , Gorelick Peter L. , Caspar Patricia , Hieny Sara , Cheever Allen , Jankovic Dragana , Sher Alan
Published: December 2019

Mice rendered deficient in interleukin-10 (IL-10) by gene targeting (IL-10−/−mice) develop chronic enterocolitis resembling human inflammatory bowel disease (IBD) when maintained in conventional animal facilities. However, they display a minimal and delayed intestinal inflammatory response when reared under specific-pathogen-free (SPF) conditions, suggesting the involvement of a microbial component in pathogenesis. We show here that experimental infection with a single bacterial agent,Helicobacter hepaticus, induces chronic colitis in SPF-reared IL-10−/−mice and that the disease is accompanied by a type 1 cytokine response (gamma interferon [IFN-γ], tumor necrosis factor alpha, and nitric oxide) detected by restimulation of spleen and mesenteric lymph node cells with a solubleH. hepaticusantigen (Ag) preparation. In contrast, wild-type (WT) animals infected with the same bacteria did not develop disease and produced IL-10 as the dominant cytokine in response toHelicobacterAg. StrongH. hepaticus-reactive antibody responses as measured by Ag-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, IgG3, and IgA were observed in both WT and IL-10−/−mice. In vivo neutralization of IFN-γ or IL-12 resulted in a significant reduction of intestinal inflammation inH. hepaticus-infected IL-10−/−mice, suggesting an important role for these cytokines in the development of colitis in the model. Taken together, these microbial reconstitution experiments formally establish that a defined bacterial agent can serve as the immunological target in the development of large bowel inflammation in IL-10−/−mice and argue that in nonimmunocompromised hosts IL-10 stimulated in response to intestinal flora is important in preventing IBD.
Minimum Chemical Requirements for Adhesin Activity of the Acid-Stable Part of
Candida albicans
Cell Wall Phosphomannoprotein Complex
Authors: Kanbe Toshio , Cutler Jim E.
Published: December 2019

This study was conducted to define adhesive characteristics of the acid-stable moiety of the
Candida albicans
phosphomannoprotein complex (PMPC) on adherence of this fungus to marginal zone macrophages of the mouse spleen. Complete digestion of the acid-stable moiety (Fr.IIS) of the
C. albicans
PMPC with an α-mannosidase or hydrolysis with 0.6 N sulfuric acid destroyed adhesin activity, as determined by the inability of the soluble digests to inhibit yeast cell adherence to the splenic marginal zone. Fr.IIS adhesin activity was decreased following digestion with an α-1,2-specific mannosidase. Oligomannosyls consisting of one to six mannose units, which were isolated from the acid-stable part of the PMPC, did not inhibit yeast cell binding and thus do not function alone as adhesin sites in the PMPC. To gain more insight into the minimum requirements for adhesin activity, PMPCs were isolated from a
Saccharomyces cerevisiae
wild-type strain and from mutant strains
mnn
1,
mnn
2, and
mnn
4; the PMPCs were designated scwt/Fr.II, scmn1/Fr.II, scmn2/Fr.II, and scmn4/Fr.II, respectively.
S. cerevisiae
scmn2/Fr.II lacks oligomannosyl side chain branches from the outer core mannan, and scmn2/Fr.II was the only PMPC without adhesin activity.
S. cerevisiae
scwt/Fr.II, scmn1/Fr.II, and scmn4/Fr.II showed adhesin activities less than that of
C. albicans
Fr.II. These three
S. cerevisiae
PMPCs are generally similar to Fr.IIS, except that the
S. cerevisiae
structure has fewer and shorter side chains. Immunofluorescence microscopy show that the acid-stable part of the PMPC is displayed homogeneously on the
C. albicans
yeast cell surface, which would be expected for a surface adhesin. Our results indicate that both the mannan core and the oligomannosyl side chains are responsible for the adhesin activity of the acid-stable part of the PMPC.
Role of NK Cells in Early Host Response to Chlamydial Genital Infection
Authors: Tseng Chien-Te Kent , Rank Roger G.
Published: December 2019

The cell-mediated immune response has been documented to be the major protective immune mechanism in mice infected genitally with the agent of mouse pneumonitis (MoPn), a biovar ofChlamydia trachomatis. Moreover, there is strong evidence to indicate that gamma interferon (IFN-γ) is a major effector mechanism of the cell-mediated immune response. Previous studies from this laboratory have also reported that the dominant cell population in the genital tract is the CD4 Th1 population. When experiments were performed by the enzyme-linked immunospot assay, high numbers of cells producing IFN-γ were found in the genital tract, concomitant with resolution of the infection; however, in addition, an increase in IFN-γ-producing cells which were CD4−was seen early in the infection. Since natural killer (NK) cells produce IFN-γ and have been found to participate in the early responses in other infections, we hypothesized that NK cells are responsible for early IFN-γ production in the murine chlamydial model. NK cells were quantified by the standard YAC-1 cytotoxicity assay and were found to appear in the genital tract as early as 12 h after intravaginal infection with MoPn. The cells were confirmed to be NK cells by abrogation of YAC-1 cell cytotoxicity by treatment in vitro and in vivo with anti-asialo-GM1. The early IFN-γ response could also be depleted by treatment with anti-asialo-GM1, indicating that NK cells were responsible for the production of this cytokine. Of interest was our observation that depletion of NK cells also exacerbated the course of infection in the mice and elicited a Th2 response, as indicated by a marked increase in immunoglobulin G1 antibody. Thus, these data demonstrate that NK cells are not only responsible for the production of IFN-γ early in the course of chlamydial genital tract infection but are also, via IFN-γ, a significant factor in the development of the Th1 CD4 response and in the control of the infection.
Complementary Recognition of Alternative Pathway Activators by Decay-Accelerating Factor and Factor H
Authors: Kraus Damian , Medof M. Edward , Mold Carolyn
Published: December 2019

The alternative complement pathway (ACP) functions as a surveillance mechanism by which microorganisms are opsonized with C3b in the absence of specific antibodies. The effectiveness of the ACP relies on its ability to distinguish self from non-self. This recognition function is mediated by C3 regulatory proteins including serum factor H, membrane cofactor protein (MCP), and membrane decay-accelerating factor (DAF). H activity against bound C3b can be increased by host components such as sialic acid and decreased by microbial polysaccharides. DAF and MCP may also recognize cell surface changes such as the presence of viral glycoproteins, since some virus-infected and tumor cells activate the ACP. In the present study, liposomes containing wild-type and mutantSalmonella minnesotalipopolysaccharide (LPS) were tested for ACP activation in serum. LPS-containing liposomes with bound C3b were then tested for their susceptibility to C3 convertase regulation by H and membrane DAF and for the sensitivity of their bound C3b to the cofactor activity of H. The results indicate that while the shortest mutant, Re595 LPS, did not induce ACP activation, R7 LPS containing an additional disaccharide did. This activation was poorly regulated by DAF but was inhibited by H. The regulatory activity of H for liposome-bound C3b, however, decreased when LPS of greater polysaccharide size was present in the membrane. In contrast the ACP activation induced by the phospholipid phosphatidylethanolamine was effectively inhibited by DAF but only poorly inhibited by H.
The Spirochete
Borrelia crocidurae
Causes Erythrocyte Rosetting during Relapsing Fever
Authors: Burman Nils , Shamaei-Tousi Alireza , Bergström Sven
Published: December 2019

Several species of the genus
Borrelia
exhibit antigenic variation of variable major proteins on their surface during relapsing fever. We have investigated the African relapsing fever species
Borrelia crocidurae
during infections in mice and compared it with the thoroughly studied North American species
Borrelia hermsii
. A major difference between the two species is that
B. crocidurae
can bind and become completely covered with erythrocytes. In addition,
B. crocidurae
causes a prolonged spirochetemia which coincides with a delayed appearance of antiborrelial antibodies. We show that the antibody response against an unrelated antigen is not delayed and that antibiotic treatment, which dissociates rosettes and inhibits the spirochetes, also leads to an early antibody response. Taken together, the erythrocyte aggregation and prolonged spirochetemia hint at a new mode of immune evasion where erythrocyte-covered spirochetes may avoid contact with the phagocytic cells and B cells of the immune system, thereby delaying the onset of a specific immune response.
Expression and Purification of the Recombinant Lethal Factor of
Bacillus anthracis
Authors: Gupta Pankaj , Batra Smriti , Chopra Arun P. , Singh Yogendra , Bhatnagar Rakesh
Published: December 2019

The structural gene for the 90-kDa lethal factor (LF) isolated from
Bacillus anthracis
was expressed as a fusion protein with six histidine residues in
Escherichia coli
. Expression of LF in
E. coli
under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.
Siderophore Production by Cystic Fibrosis Isolates of
Burkholderia cepacia
Authors: Darling Patricia , Chan Maria , Cox Andrew D. , Sokol Pamela A.
Published: December 2019

Sixty-one
Burkholderia cepacia
isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of
B. cepacia
correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.