bluuebunny/crossref_metadata_2025 huggingface dataset:
Tuberculin-Purified Protein Derivative-, MPT-64-, and ESAT-6-Stimulated Gamma Interferon Responses in Medical Students before and afterMycobacterium bovisBCG Vaccination and in Patients with Tuberculosis
Authors: Johnson P. D. R. , Stuart R. L. , Grayson M. L. , Olden D. , Clancy A. , Ravn P. , Andersen P. , Britton W. J. , Rothel J. S.
Published: December 2019

QuantiFERON-TB(QIFN) (CSL Limited) is a whole-blood assay for the recognition of infection withMycobacterium tuberculosis. QIFN measures gamma interferon (IFN-γ) production when purified protein derivatives (PPDs) of mycobacteria are incubated with venous blood samples. The specificity of QIFN in medical students before and after BCG immunization was assessed, and sensitivity in patients with tuberculosis was assessed. Antigens were PPD derived fromM. tuberculosisand twoM. tuberculosis-specific proteins, ESAT-6 and MPT-64. Of 60 medical students, all of whom had 0-mm tuberculin skin tests (TSTs) at study entry, 58 (97%) were initially classified as negative forM. tuberculosisinfection by PPD QIFN. Five months after BCG immunization, 7 of 54 students (13%) had a TST result of ≥10 mm and 11 of 54 students (20%) tested positive by PPD QIFN. ESAT-6- and MPT-64-stimulated IFN-γ responses in the medical students were negative prior to and after BCG immunization. For patients with active tuberculosis, 12 of 19 (63%) were positive by PPD QIFN, 11 of 19 (58%) were positive by ESAT-6 QIFN, and 0 of 12 were positive by MPT-64 QIFN. In conclusion, PPD QIFN was negative in 97% of a low-risk population who had not received BCG and who had negative TSTs. The specificities of both the TST and PPD QIFN were reduced following BCG immunization. PPD QIFN and ESAT-6 QIFN were of similar and moderate sensitivity in patients with active tuberculosis, but ESAT-6 QIFN is likely to be more specific because it is not influenced by past BCG exposure.
Specificity and Prevalence of Natural Bovine Antimannan Antibodies
Authors: Srinivasan Abhay , Ni Yawei , Tizard Ian
Published: December 2019

Immune responses to the carbohydrate components of microorganisms, mediated both by antibodies and by lectins, are an important part of host defense. In the present experiments, the specificity and presence of natural bovine antibodies against mannan, a common fungal antigen, were examined by enzyme-linked immunosorbent assay (ELISA), usingSaccharomyces cerevisiaemannan as an antigen. The results showed that all serum samples from animals of three age groups (newborn, calf, and adult) tested contained antimannan antibodies, and the titer of these antibodies increased significantly in adults. However, titers among individual adult cattle differed widely. Inhibition assays showed that yeast mannan was the strongest inhibitor.d-Mannose exhibited only a minor inhibitory effect at high concentrations. This suggests that most of these antibodies recognize an oligosaccharide-based epitope(s) different from those recognized by lectins. Cattle possess three serum C-type lectins (collectins) capable of recognizing mannan in a calcium-dependent manner. Addition of EDTA to the reaction did not reduce antibody binding, suggesting that the binding of these antibodies to mannan was not affected by the presence of collectin. The antibodies purified from either calf or adult serum by mannan-Sepharose affinity chromatography consisted of mainly immunoglobulin G (IgG) and a smaller amount of IgM. IgG1 was shown to be the dominant antimannan IgG isotype by isotype-specific ELISA. Together, these results demonstrate the production of natural antimannan antibodies in cattle in an age-dependent manner. These antibodies might be involved in defending the host against mannan-containing pathogens as a specific line of defense in conjunction with the innate response by lectins.
Comparison and Characterization of Immunoglobulin G Subclasses among Primate Species
Authors: Shearer Michael H. , Dark Robyn D. , Chodosh James , Kennedy Ronald C.
Published: December 2019

Little information is available on the immunoglobulin G (IgG) subclasses expressed in the sera of nonhuman primate species. To address this issue, we compared the IgG subclasses found in humans (IgG1, IgG2, IgG3, and IgG4) to those of nonhuman primates, such as baboons and macaques. Cross-reactive antihuman IgG subtype-specific reagents were identified and used to analyze purified IgG from sera by solid-phase enzyme-linked immunosorbent assay. Protein A-purified human IgG obtained from sera was composed of IgG1, IgG2, IgG3, and IgG4, whereas baboon and macaque IgG was composed of IgG1, IgG2, and IgG4. Protein G-purified human IgG was composed of IgG1, IgG2, IgG3, and IgG4, whereas baboon and macaque IgG was composed of IgG1, IgG2, and IgG4. To test the possibility that baboon and macaque IgG3 is actually present, but is outcompeted for binding to proteins A and G by the other more abundant IgG subclasses, we repurified the IgG from sera that did not bind either protein A or protein G. We found a baboon IgG3 population in the sera that did not bind protein A, but bound protein G. No IgG3 subtype was detectable in macaque sera. These data suggest that baboon sera, like human sera, contain four IgG subtypes, whereas macaque sera exhibit only three of the human subclass analogs. In addition, the IgG subtype-specific reagents were shown to be useful in determining the IgG subclass distribution following vaccination of baboons with hepatitis B surface antigen.
Binding of
Candida albicans
to Immobilized Amino Acids and Bovine Serum Albumin
Authors: Hawser Stephen P. , Islam Khalid
Published: December 2019

In this study, we examined the binding of
Candida albicans
synchronized yeast-phase cells to plastic, immobilized amino acids and bovine serum albumin (BSA) and quantified the binding by using an XTT tetrazolium salt assay and absorbance determination. Our results show that
C. albicans
binds efficiently and specifically to several nonpolar aliphatic amino acids and positively charged amino acids and to BSA immobilized on tissue culture plastic but not to polar uncharged, negatively charged, or aromatic amino acids. Adhesion of yeasts to immobilized amino acids was not affected by preincubation of cells with BSA, whereas binding to immobilized BSA was affected by preincubation of yeasts with alanine, proline, and leucine but not by arginine or lysine. The ability to distinguish the chirality of these amino acids was also examined by using both the
d
and
l
amino acid configurations, and the results show that
C. albicans
yeasts recognize only the
l
configuration of these amino acids. The observations that
C. albicans
specifically binds to certain amino acids indicate that these amino acids may prove useful tools for studying the binding interactions of
C. albicans
yeasts with host proteins such as components of the extracellular matrix.
Interleukin-15 Augments Superoxide Production and Microbicidal Activity of Human Monocytes againstCandida albicans
Authors: Vázquez Nancy , Walsh Thomas J. , Friedman Daphne , Chanock Stephen J. , Lyman Caron A.
Published: December 2019

Interleukin-15 (IL-15) is a newly described cytokine that shares biological activities with IL-2. We report here results demonstrating the ability of IL-15 to enhance superoxide production and antifungal activity of human monocytes. After 18 and 48 h of treatment with IL-15, human elutriated monocytes manifested enhanced superoxide production in response to either phorbol myristate acetate or opsonizedCandida albicansblastoconidia. Similar results were obtained when monocytes were treated with IL-2, but to a lesser extent. Combination studies with IL-15 and IL-2 showed no additive or synergistic effects. Following incubation of monocytes with IL-15 for 18 h, there was no significant increase in mRNA transcripts for components of the NADPH oxidase complex, p40-phox, p47-phox, and gp91-phox, suggesting a posttranscriptional modulation of enhanced superoxide production. Antibodies against the γ chain of the IL-2 receptor and, to a lesser extent, against the β chain partially abrogated the IL-15-mediated enhanced superoxide production. Additionally, human monocytes showed enhanced killing activity againstC. albicansafter 18 h of incubation with IL-15 or IL-2, but this treatment did not enhance the ability of these cells to phagocytose the organism. In addition, the enhanced fungicidal activity seen after 18 h of treatment was no longer detectable after 48 h of cytokine treatment. Culture supernatants from the IL-15-treated monocytes were assayed for the presence of other proinflammatory cytokines. IL-15 treatment did not induce the release of detectable levels of tumor necrosis factor alpha, IL-1β, or IL-12. Our results indicate that IL-15 upregulates the microbicidal activity of human monocytes againstC. albicans.
Aggregation and Binding Substances Enhance Pathogenicity in Rabbit Models ofEnterococcus faecalisEndocarditis
Authors: Schlievert Patrick M. , Gahr Pamala J. , Assimacopoulos Aris P. , Dinges Martin M. , Stoehr Jennifer A. , Harmala John W. , Hirt Helmut , Dunny Gary M.
Published: December 2019

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models ofEnterococcus faecaliscardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 108CFU and observed for 2 days. No clinical signs of illness developed in animals given AS−EBS−organisms, and all survived. All rabbits given AS−EBS+organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+EBS−organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+EBS+organisms developed signs of illness and died. None of the rabbits receiving AS+EBS+organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+EBS+organisms. In contrast, cell extracts from AS−EBS−organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-α) and TNF-β, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 × 109CFU of test organisms. None of the animals given AS−EBS−organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS−EBS+or AS+EBS−organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+EBS+organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS−EBS−or AS+EBS+organisms developed vegetations, but animals given AS+EBS+organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.
Modulation of Murine Lyme Borreliosis by Interruption of the B7/CD28 T-Cell Costimulatory Pathway
Authors: Shanafelt Marie-Claude , Kang Insoo , Barthold Stephen W. , Bockenstedt Linda K.
Published: December 2019

Recent studies have implicated cytokines associated with Th2 cells in the genetic resistance to murine Lyme borreliosis. Because the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th-cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to the Th2 cytokine profile and development of arthritis in BALB/c mice infected withBorrelia burgdorferi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin 4 (IL-4) and upregulation of gamma interferon (IFN-γ) responses byB. burgdorferi-specific T cells and by reduction ofB. burgdorferi-specific immunoglobulin G. Despite the shift toward a Th1 cytokine pattern, which others have associated with disease susceptibility, the severity of arthritis was unchanged. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies or CTLA-4Ig enhanced IFN-γ production over that seen with CD86 blockade alone, yet augmentation of this Th1-associated cytokine did not enhance disease. These results demonstrate that IL-4 production by T cells inB. burgdorferi-infected BALB/c mice is dependent upon CD86/CD28 interaction and that this cytokine does not contribute significantly to host resistance to the development of arthritis. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-γ-producing T cells inB. burgdorferiinfection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. These studies may provide insight into the role of the B7/CD28 pathway in other infectious and autoimmune diseases in which deviation of Th cell immune responses occurs and antigen is persistently present.
Complementation Analysis of the
Dichelobacter nodosus fimN
,
fimO
, and
fimP
Genes in
Pseudomonas aeruginosa
and Transcriptional Analysis of the
fimNOP
Gene Region
Authors: Johnston Joanne L. , Billington Stephen J. , Haring Volker , Rood Julian I.
Published: December 2019

The causative agent of ovine footrot, the gram-negative anaerobe
Dichelobacter nodosus
, produces polar type IV fimbriae, which are the major protective antigens. The
D. nodosus
genes
fimN
,
fimO
, and
fimP
are homologs of the
Pseudomonas aeruginosa
fimbrial assembly genes,
pilB
,
pilC
, and
pilD
, respectively. Both the
pilD
and
fimP
genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the
D. nodosus
genes were introduced into
P. aeruginosa
strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that the
fimP
gene complemented a
pilD
mutant of
P. aeruginosa
for both fimbrial assembly and protein secretion. However, the
fimN
and
fimO
genes did not complement
pilB
or
pilC
mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the
P. aeruginosa
fimbrial assembly system. The transcriptional organization and regulation of the
fimNOP
gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two ς
70
-type promoters located upstream of ORFM, an open reading frame proximal to
fimN
. Transcription of the
D. nodosus
fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.
Intracellular
Staphylococcus aureus
Escapes the Endosome and Induces Apoptosis in Epithelial Cells
Authors: Bayles Kenneth W. , Wesson Carla A. , Liou Linda E. , Fox Lawrence K. , Bohach Gregory A. , Trumble W. R.
Published: December 2019

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a
Staphylococcus aureus
mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections.
S. aureus
cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of
S. aureus
, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization
S. aureus
escapes the endosome and induces apoptosis in nonprofessional phagocytes.
The
Pseudomonas aeruginosa
Quorum-Sensing Signal Molecule
N
-(3-Oxododecanoyl)-
l
-Homoserine Lactone Has Immunomodulatory Activity
Authors: Telford Gary , Wheeler D. , Williams Paul , Tomkins P. T. , Appleby P. , Sewell Herbert , Stewart Gordon S. A. B. , Bycroft Barrie W. , Pritchard David I.
Published: December 2019

Diverse gram-negative bacterial cells communicate with each other by using diffusible
N
-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population “quorate,” and the appropriate target gene is activated. In pathogenic bacteria, such as
Pseudomonas aeruginosa
, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs
N
-(3-oxododecanoyl)-
l
-homoserine lactone (OdDHL) and
N
-(3-oxohexanoyl)-
l
-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 × 10
−5
M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 × 10
−5
M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1–Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of
P. aeruginosa
infections by functioning as a virulence determinant per se.
Phase Variation in
Helicobacter pylori
Lipopolysaccharide
Authors: Appelmelk B. J. , Shiberu B. , Trinks C. , Tapsi N. , Zheng P. Y. , Verboom T. , Maaskant J. , Hokke C. H. , Schiphorst W. E. C. M. , Blanchard D. , Simoons-Smit I. M. , van den Eijnden D. H. , Vandenbroucke-Grauls C. M. J. E.
Published: December 2019

Helicobacter pylori
NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le
x
) in a polymeric form. Le
x
is β-
d
-galactose-(1-4)-[α-
l
-fucose-(1-3)]-β-
d
-acetylglucosamine. Schematically the LPS structure is (Le
x
)

n

-core-lipid A. In this report, we show that Le
x
expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of α1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le
x
minus fucose; LPS: (lactosamine)

n

-core-lipid A. The switch of Le
x
to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le
y
; LPS: (Le
y
)-core-lipid A. A third group of variants arose by acquisition of α1,2-linked fucose which hence expressed Le
x
plus Le
y
; LPS: (Le
y
)(Le
x
)

n

-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le
x
)

n

-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and
N
-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of
H. pylori
, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.
A Second Two-Component Regulatory System of
Bordetella bronchiseptica
Required for Bacterial Resistance to Oxidative Stress, Production of Acid Phosphatase, and In Vivo Persistence
Authors: Jungnitz Heidrun , West Nicholas P. , Walker Mark J. , Chhatwal Gursharan S. , Guzmán Carlos A.
Published: December 2019

Random minitransposon mutagenesis was used to identify genes involved in the survival of
Bordetella bronchiseptica
within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (
ris
) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in
Bordetella
spp., the
bvg
locus. A
risA′-′lacZ
translational fusion was constructed and integrated into the chromosome of
B. bronchiseptica
. Determination of β-galactosidase activity under different environmental conditions suggested that
ris
is regulated independently of
bvg
and is optimally expressed at 37°C, in the absence of Mg
2+
, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all
Bordetella
spp., is required for the expression of acid phosphatase by
B. bronchiseptica
. Although catalase and superoxide dismutase production were unaffected, the
ris
mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of
bvg
-positive and
bvg
-negative
ris
mutants with the intact
ris
operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the
ris
mutant is cleared by the host much earlier than the wild-type strain, suggesting that
ris
-regulated products play a significant role in natural infections. The identification of a second two-component system in
B. bronchiseptica
highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.
Cytotoxic T-Lymphocyte-Mediated Lysis ofToxoplasma gondii-Infected Target Cells Does Not Lead to Death of Intracellular Parasites
Authors: Yamashita Keizo , Yui Katsuyuki , Ueda Masakatsu , Yano Akihiko
Published: December 2019

CD8+T cells play a crucial role in the control of infection with intracellular microbes. The mechanisms underlying the CD8+T-cell-mediated clearance of the intracellular pathogenToxoplasma gondiiare, however, not completely understood. The effect of CD8+cytotoxic T-lymphocyte (CTL)-mediated lysis of host cells on the viability of intracellularT. gondiiwas investigated. Quantitative competitive PCR of the gene encodingT. gondiimajor surface antigen (SAG-1) was combined with treatment of the parasites with DNase, which removed the DNA template of nonviable parasites. The induction by CD8+CTLs of apoptosis in cells infected withT. gondiidid not result in the reduction of live parasites, indicating that intracellularT. gondiiremains alive after lysis of host cells by CTLs.
Identification of Two
Shigella flexneri
Chromosomal Loci Involved in Intercellular Spreading
Authors: Hong Mei , Gleason Yuki , Wyckoff Elizabeth E. , Payne Shelley M.
Published: December 2019

The ability of
Shigella flexneri
to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two
S. flexneri
chromosomal loci that are required for these processes were identified by screening a pool of Tn
phoA
insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of Tn
phoA
insertion were within two different regions that are almost identical to
Escherichia coli
K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the
E. coli
chromosome, upstream of
murZ
, while the other is at 28 min, downstream of
tonB
. The mutant with the insertion at 70 min was named
vpsC
because it showed an altered pattern of virulence protein secretion. The
vpsC
mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the
S. flexneri vpsA
, -
B
, and -
C
genes complemented all of the phenotypes of the
vpsC
mutant. A mutation in
vpsA
resulted in the same phenotype as the
vpsC
mutation, suggesting that these two genes are part of a virulence operon in
S. flexneri
. The mutant with the insertion at 28 min was interrupted in the same open reading frame as
S. flexneri ispA
. This
ispA
mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.
hgpB
, a Gene Encoding a Second
Haemophilus influenzae
Hemoglobin- and Hemoglobin-Haptoglobin-Binding Protein
Authors: Ren Zhen , Jin Hongfan , Morton Daniel J. , Stull Terrence L.
Published: December 2019

Haemophilus influenzae
requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in
H. influenzae
HI689. Mutation of
hgpA
did not affect binding or utilization of either heme source. The
hgpA
mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by
H. influenzae
. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to
hgpA
, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced
Escherichia coli
harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of
H. influenzae
at the newly identified locus, designated
hgpB
, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An
hgpA hgpB
double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of
H. influenzae
has been identified and characterized, and the presence of an additional protein of similar function has been revealed.
Comparison of the PspA Sequence fromStreptococcus pneumoniaeEF5668 to the Previously Identified PspA Sequence from Strain Rx1 and Ability of PspA from EF5668 To Elicit Protection against Pneumococci of Different Capsular Types
Authors: McDaniel Larry S. , McDaniel D. Olga , Hollingshead Susan K. , Briles David E.
Published: December 2019

PspA (pneumococcal surface protein A) is a serologically varied virulence factor ofStreptococcus pneumoniae. In mice, PspA has been shown to elicit an antibody response that protects against fatal challenge with encapsulatedS. pneumoniae, and the protection-eliciting residues have been mapped to the α-helical N-terminal half of the protein. To date, a published DNA sequence forpspAis available only forS. pneumoniaeRx1, a laboratory strain. PspA/EF5668 (EF5668 indicates the strain of origin of the PspA) is serologically distinct from PspA/Rx1. Sequencing of the gene encoding PspA/EF5668 revealed 71% identity with that of PspA/Rx1. The greatest amount of divergence between the two proteins was seen in their α-helical portions, which are surface exposed and probably under selective pressure to diversify serologically. In spite of the diversity within the α-helical regions of PspAs, we have observed that recombinant PspA (rPspA)/EF5668, like rPspA/Rx1, can elicit cross-protection against pneumococci of different capsular and PspA serological types.
Specificity of Bactericidal Antibody Response to Serogroup B Meningococcal Strains in Brazilian Children after Immunization with an Outer Membrane Vaccine
Authors: Milagres Lucimar G. , Gorla Maria Cecília A. , Sacchi Claudio T. , Rodrigues Mauricio M.
Published: December 2019

Pre- and postvaccination serum samples from 77 children aged 2 to 6 years, who received the Cuban BC vaccine (B:4:P1.15), were analyzed for bactericidal antibodies against a local B:4:P1.15 strain (N44/89). Sera from 16 individuals with bactericidal antibodies against the B:4:P1.15 strain were tested against 23 Brazilian isolates. These include B:4 strains of distinct serosubtypes: P1.15, P1.7,1, P1.3, P1.9, P1.nt, and a B:8,19,23:P1.16 strain. A Cuban B:4:P1.15 strain (Cu385/83) was also included in the study. The specificities of bactericidal antibodies were analyzed by using mutant strains lacking a class 1 protein (PorA protein) or a class 5 protein or both. The results indicated that PorA and class 5 proteins are the main targets recognized by the bactericidal antibodies of vaccinees. Nonetheless, a complex pattern of recognition by bactericidal antibodies was found, and vaccinees were grouped according to antibody specificity. Antibodies from some individuals recognized PorA of serosubtype P1.15. However, antibodies from these individuals could not kill all P1.15 strains tested. Antibodies from a second group recognized both PorA and class 5 proteins, and antibodies from a third group recognized an as yet unidentified target antigen. The results demonstrate the importance of determining the fine epitope specificity of bactericidal antibodies to improve the existing vaccines against B meningococci.
Age-Related Buildup of Humoral Immunity against Epitopes for Rosette Formation and Agglutination in African Areas of Malaria Endemicity
Authors: Barragan Antonio , Kremsner Peter G. , Weiss Walter , Wahlgren Mats , Carlson Johan
Published: December 2019

In this report, we show an age-related buildup of agglutinating activity as well as serum activity against rosette formation in children living in areas of Kenya and Gabon where malaria is endemic. Sera from Kenyans in general exhibited a stronger and wider immune response toward the epitopes, probably reflecting a difference in transmission patterns between the two areas. Thus, our results indicate that repeated malaria attacks in areas of endemicity, and consequently exposure to different isolate-specific antigens, will elicit an antibody-mediated response eventually enabling recognition of the majority of rosetting and agglutinating antigens. The correlation between antirosetting and agglutinating capacity was poor in individual cases, indicating that the rosetting epitopes are only a minor part of the highly diverse surface-exposed antigens (mainly PfEMP1) on the surface of parasitized erythrocytes toward which antibodies may react. These data together with our previous findings that the protection against cerebral malaria correlates with presence of antirosetting antibodies shed new light on our understanding of the gradual acquisition of immunity toward severe complications of malarial infection which children reared in areas of endemicity attain.
Toxoplasma GondiiBradyzoites Form Spontaneously during Sporozoite-Initiated Development
Authors: Jerome M. E. , Radke J. R. , Bohne W. , Roos D. S. , White M. W.
Published: December 2019

Tachyzoites (VEG strain) that emerge from host cells infected withToxoplasma gondiisporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following ∼20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker,BAG1. Within 24 h of the growth shift, 2% of the population expressedBAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. SpontaneousBAG1expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to expressBAG1earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >104parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressedBAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.
Mannan-Specific Immunoglobulin G Antibodies in Normal Human Serum Accelerate Binding of C3 toCandida albicansvia the Alternative Complement Pathway
Authors: Zhang Mason X. , Kozel Thomas R.
Published: December 2019

Candida albicansactivates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allowsC. albicansto initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca2+with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding toC. albicansincubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab′)2fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system andC. albicans.
Characterization of the Hemorrhagic Reaction Caused by
Vibrio vulnificus
Metalloprotease, a Member of the Thermolysin Family
Authors: Miyoshi Shin-ichi , Nakazawa Hiromi , Kawata Koji , Tomochika Ken-ichi , Tobe Kazuo , Shinoda Sumio
Published: December 2019

Vibrio vulnificus
is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (
V. vulnificus
protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.
Localization of a T-Cell Epitope of Superantigen Toxic Shock Syndrome Toxin 1 to Residues 125 to 158
Authors: Hu Wei-Gang , Zhu Xi-Hua , Wu Yu-Zhang , Jia Zheng-Cai
Published: December 2019

Toxic shock syndrome toxin 1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. So far, little is known about T-cell epitopes on superantigens. In this study, we developed an improved method for localizing T-cell epitopes on superantigens that involved synthetic peptides plus costimulation by CD28 or phorbol myristate acetate. Using this method, we localized a T-cell epitope to a 34-residue region, TSST-1 (residues 125 to 158), which possessed only two of four TSST-1-targeted β-chain variable element (Vβ) specificities of T-cell receptors in humans and mice, human Vβ2 and murine Vβ15.
Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens
Authors: Mason Kevin M. , Dryden Tricia D. , Bigley Nancy J. , Fink Pamela S.
Published: December 2019

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured withStreptococcus mutansproduced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.
Novel Selection for Isoniazid (INH) Resistance Genes Supports a Role for NAD
+
-Binding Proteins in Mycobacterial INH Resistance
Authors: Chen Ping , Bishai William R.
Published: December 2019

The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the
Escherichia coli oxyR
mutant, which is relatively sensitive to INH, with a
Mycobacterium tuberculosis
plasmid library and selected for INH-resistant clones. Three genes were identified and called
ceo
for their ability to complement the
Escherichia coli oxyR
mutant.
ceoA
was the previously identified
M. tuberculosis glf
gene, which encodes a 399-amino-acid NAD
+
- and flavin adenine dinucleotide-requiring enzyme responsible for catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The proteins encoded by the
ceoBC
pair were homologous with one another and with the N terminus of the potassium uptake regulatory protein TrkA. Each of the three Ceo proteins contains a motif common to NAD
+
binding pockets. Overexpression of the
M. tuberculosis glf
gene by placing it under the control of the
hsp60
promoter on a multicopy plasmid in
Mycobacterium bovis
BCG produced a strain for which the INH MIC was increased 50% compared to that for the control strains, while similar overexpression of the
ceoBC
pair had no effect on INH susceptibility in BCG. Mycobacterial extracts containing the overexpressed Glf protein did not bind radiolabeled INH directly, suggesting a more complex mechanism than the binding of unmodified INH. Our results support the hypothesis that upregulated mycobacterial proteins such as Glf may contribute to INH resistance in
M. tuberculosis
by binding a modified form of INH or by sequestering a factor such as NAD
+
required for INH activity.
Immunological Characterization of Asp f 2, a Major Allergen fromAspergillus fumigatusAssociated with Allergic Bronchopulmonary Aspergillosis
Authors: Banerjee Banani , Greenberger Paul A. , Fink Jordan N. , Kurup Viswanath P.
Published: December 2019

The 37-kDa recombinant protein Asp f 2, encoding an allergen ofAspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 fromAspergillus nidulansand fibrinogen binding protein fromCandida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereasA. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen ofA. fumigatusexhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis ofA. fumigatus-associated allergic diseases.
In Situ Detection of Apoptosis at Sites of Chronic Bacterially Induced Inflammation in Human Gingiva
Authors: Tonetti Maurizio S. , Cortellini Davide , Lang Niklaus P.
Published: December 2019

Apoptosis is a key phenomenon in the regulation of the life span of terminally differentiated leukocytes. Human gingiva represents an established model to study immune responses to bacterial infection. In this investigation, we used the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) technique to evaluate presence and topographic location of apoptosis-associated DNA damage in human gingival biopsies along with the expression of the p53 and Bcl-2 apoptosis-regulating proteins. Qualitative data analysis showed high densities of cells expressing DNA damage and p53 both within the epithelial attachment to the tooth and in the perivascular infiltrate (infiltrated connective tissue [ICT]) immediately underlying the site of chronic bacterial aggression. Topographic consistency between DNA damage- and p53-positive cells was consistently observed. Quantitative analysis of the ICT showed mean densities of DNA damage- and p53-positive cells of 345 ± 278 and 403 ± 182 cells/mm2, respectively. Numerical consistency was confirmed by multivariate regression analysis: densities of DNA damage-positive cells were significantly predicted by densities of p53-positive cells (P= 0.001,r2= 0.84). In the ICT, cells displaying biotinylated DNA nicks were 3.8% ± 2.7% of total cellularity, while p53- and Bcl-2-positive cells represented 4.4% ± 1.7% and 15.4% ± 6.7% of total cells, respectively. It is suggested that p53 expression associated with DNA damage is a prevalent phenomenon in chronically inflamed human gingiva, and that apoptosis may be a relevant process for the maintenance of local immune homeostasis at sites of chronic bacterial challenge in vivo.
The Major Outer Membrane Protein ofChlamydia psittaciFunctions as a Porin-Like Ion Channel
Authors: Wyllie Susan , Ashley Richard H. , Longbottom David , Herring Alan J.
Published: December 2019

The major outer membrane protein (MOMP) ofChlamydiaspecies shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified fromChlamydia psittacihas a predominantly β-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the “porin channel” hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK∼ 2) and permeable to ATP. They may therefore be a route by whichChlamydiacan take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.