bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of the
gbpA
Gene of
Streptococcus mutans
Alters Structural and Functional Aspects of Plaque Biofilm Which Are Compensated by Recombination of the
gtfB
and
gtfC
Genes
Authors: Hazlett Karsten R. O. , Mazurkiewicz Joseph E. , Banas Jeffrey A.
Published: December 2019

Inactivation of the
gbpA
gene of
Streptococcus mutans
increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which
gtfB
and
gtfC
have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180–2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters
S. mutans
plaque structure and that the presence of
gtfBC
recombinant organisms within a
gbpA
background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt
S. mutans
plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the
gbpA
mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the
gbpA
mutants used as inoculum were also
gtfBC
recombinants (
gbpA
/25%
gtfBC
), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin;
gbpA
plaque organisms were more susceptible than organisms in either the wt or
gbpA
/25%
gtfBC
plaques. These data allow the conclusion that GbpA contributes to
S. mutans
plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that
S. mutans
biofilm structure influences virulence. A potential model for this influence, which can account for the
gtfBC
recombination compensating
gbpA
inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.
Plasmin-Coated
Borrelia burgdorferi
Degrades Soluble and Insoluble Components of the Mammalian Extracellular Matrix
Authors: Coleman James L. , Roemer Elizabeth J. , Benach Jorge L.
Published: December 2019

Borrelia burgdorferi
, the spirochetal agent of Lyme disease, binds plasminogen in vitro. Exogenously provided urokinase-type plasminogen (PLG) activator (uPA) converts surface-bound PLG to enzymatically active plasmin. In this study, we investigated the capacity of a
B. burgdorferi
human isolate, once complexed with plasmin, to degrade purified extracellular matrix (ECM) components and an interstitial ECM. In a modified enzyme-linked immunosorbent assay using immobilized, soluble ECM components, plasmin-coated
B. burgdorferi
degraded fibronectin, laminin, and vitronectin but not collagen. Incubation of plasmin-coated organisms with biosynthetically radiolabeled native ECM resulted in breakdown of insoluble glycoprotein, other noncollagenous proteins, and collagen, as measured by release of solubilized radioactivity. Radioactive release did not occur with untreated spirochetes or spirochetes treated with uPA or PLG alone. Kinetic and inhibition studies suggested that the breakdown of collagen was indirect and due to prior disruption of supportive ECM proteins.
B. burgdorferi
is an invasive bacterial pathogen that may benefit by use of the host’s plasminogen activation system. The results of this study have identified mechanisms in which the spirochete can use this borrowed proteolytic activity to enhance invasiveness.
Trypanosoma cruziInfects Human Dendritic Cells and Prevents Their Maturation: Inhibition of Cytokines, HLA-DR, And Costimulatory Molecules
Authors: Van Overtvelt Laurence , Vanderheyde Nathalie , Verhasselt Valérie , Ismaili Jamila , De Vos Louis , Goldman Michel , Willems Fabienne , Vray Bernard
Published: December 2019

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas’ disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected byT. cruzitrypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected byT. cruziand can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, afterT. cruziinfection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected byT. cruziinfection. Indeed, secretion of cytokines such as IL-12, TNF-α, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced byT. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released byT. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used byT. cruzito escape the host immune responses in Chagas’ disease and thus might favor persistent infection.
Cell-Mediated Immune Responses in Four-Year-Old Children after Primary Immunization with Acellular Pertussis Vaccines
Authors: Ausiello Clara M. , Lande Roberto , Urbani Francesca , la Sala Andrea , Stefanelli Paola , Salmaso Stefania , Mastrantonio Paola , Cassone Antonio
Published: December 2019

Cell-mediated immune (CMI) responses to
Bordetella pertussis
antigens (pertussis toxin [PT], pertactin [PRN], and filamentous hemagglutinin [FHA]) were assessed in 48-month-old recipients of acellular pertussis [aP] vaccines (either from Chiron-Biocine [aP-CB] or from SmithKline Beecham [aP-SB]) and compared to CMI responses to the same antigens at 7 months of age, i.e., 1 month after completion of the primary immunization cycle. None of the children enrolled in this study received any booster of pertussis vaccines or was affected by pertussis during the whole follow-up period. Overall, around 75% of 4-year-old children showed a CMI-positive response to at least one
B. pertussis
antigen, independently of the type of aP vaccine received, and the proportion of CMI responders were at least equal at 48 and 7 months of age. However, longitudinal examination of individual responses showed that from 20 (against PT) to 37% (against FHA) of CMI responders after primary immunization became negative at 48 months of age. This loss was more than compensated for by conversion to positive CMI responses, ranging from 36% against FHA to 69% against PRN, in other children who were CMI negative at 7 months of age. In 60 to 80% of these CMI converters, a lack of decline or even marked elevation of antibody (Ab) titers against
B. pertussis
antigens also occurred between 20 and 48 months of age. In particular, the frequency of seropositivity to PRN and FHA (but not to PT) was roughly three times higher in CMI converters than in nonconverters. The acquisition of CMI response to
B. pertussis
antigens in 48-month-old children was not associated with a greater frequency of coughing episodes lasting ≥7 days and was characterized by a prevalent type 1 cytokine profile, with high gamma interferon and low or no production of interleukin-5, reminiscent of cytokine patterns following immunization with whole-cell pertussis vaccine or natural infection. Our data imply that vaccination-induced systemic CMI may wane by 4 years of age but may be acquired or naturally boosted by symptomless or minor clinical infection by
B. pertussis
. This might explain, at least in part, the persistence of protection against typical pertussis in aP vaccine recipients despite a substantial waning of both Ab and CMI responses induced by the primary immunization.
Identification of a Glycoprotein Produced by Enterotoxigenic
Escherichia coli
Authors: Lindenthal Christoph , Elsinghorst Eric A.
Published: December 2019

Enterotoxigenic
Escherichia coli
(ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (
tia
and
tib
) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of
E. coli
to adhere to and invade cultured human intestinal epithelial cells. The
tib
locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant
E. coli
expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with
tib
deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire
tib
locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering
E. coli
and with pertactin precursor from
Bordetella pertussis
. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the
tib
locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this
tib
locus open reading frame is proposed to be responsible for TibA modification. These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process.
Purification and Properties of Proline-Rich Antimicrobial Peptides from Sheep and Goat Leukocytes
Authors: Shamova Olga , Brogden Kim A. , Zhao Chengquan , Nguyen Tung , Kokryakov Vladimir N. , Lehrer Robert I.
Published: December 2019

We purified three proline-rich antimicrobial peptides from elastase-treated extracts of sheep and goat leukocytes and subjected two of them, OaBac5α and ChBac5, to detailed analysis. OaBac5α and ChBac5 were homologous to each other and to bovine Bac5. Both exhibited potent, broad-spectrum antimicrobial activity under low-concentration salt conditions. While the peptides remained active againstEscherichia coli,Pseudomonas aeruginosa,Bacillus subtilis, andListeria monocytogenesin 100 mM NaCl, they lost activity againstStaphylococcus aureusandCandida albicansunder these conditions. ChBac5 was shown to bind lipopolysaccharide, a property that could enhance its ability to kill gram-negative bacteria. Proline-rich Bac5 peptides are highly conserved in ruminants and may contribute significantly to their innate host defense mechanisms.
Expression of Fas and Fas Ligand on Mouse Renal Tubular Epithelial Cells in the Generalized Shwartzman Reaction and Its Relationship to Apoptosis
Authors: Koide Naoki , Narita Kayo , Kato Yutaka , Sugiyama Tsuyoshi , Chakravortty Dipshikha , Morikawa Akiko , Yoshida Tomoaki , Yokochi Takashi
Published: December 2019

Previously we reported that the consecutive injection of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) appeared to induce the injury of renal tubular epithelial cells via apoptosis. The aim of this study was to characterize the mechanism of renal tubular epithelial cell injury in GSR. The expression of Fas and Fas ligand was immunohistochemically detected on renal tubular epithelial cells from GSR-induced mice, although neither Fas nor Fas ligand was found in cells from untreated control mice or in cells from mice receiving a single injection of LPS. GSR-induced renal tubular epithelial cell injury was produced in neither Fas-negative MRL-lpr/lprmice nor Fas ligand-negative MRL-gld/gldmice. The administration of anti-gamma interferon antibody together with a preparative injection of LPS prevented the expression of Fas and Fas ligand and the apoptosis of renal tubular epithelial cells. A provocative injection of tumor necrosis factor alpha into LPS-sensitized mice augmented Fas and Fas ligand expression and the apoptosis of renal tubular epithelial cells. The administration of tumor necrosis factor alpha to interleukin-12-sensitized mice resulted in Fas and Fas ligand expression and the apoptosis. Sensitization with interleukin-12 together with anti-gamma interferon antibody did not cause the apoptosis of renal tubular epithelial cells. It was suggested that the Fas/Fas ligand system probably plays a critical role in the development of renal tubular epithelial cell injury through apoptotic cell death.
Comparative Analysis of
Legionella pneumophila
and
Legionella micdadei
Virulence Traits
Authors: Joshi Amrita D. , Swanson Michele S.
Published: December 2019

While the majority of Legionnaire’s disease has been attributed to
Legionella pneumophila
,
Legionella micdadei
can cause a similar infection in immunocompromised people. Consistent with its epidemiological profile, the growth of
L. micdadei
in cultured macrophages is less robust than that of
L. pneumophila
. To identify those features of the
Legionella
spp. which are correlated to efficient growth in macrophages, two approaches were taken. First, a phenotypic analysis compared four clinical isolates of
L. micdadei
to one well-characterized strain of
L. pneumophila
. Seven traits previously correlated with the virulence of
L. pneumophila
were evaluated: infection and replication in cultured macrophages, evasion of phagosome-lysosome fusion, contact-dependent cytotoxicity, sodium sensitivity, osmotic resistance, and conjugal DNA transfer. By nearly every measure,
L. micdadei
appeared less virulent than
L. pneumophila
. The surprising exception was
L. micdadei
31B, which evaded lysosomes and replicated in macrophages as efficiently as
L. pneumophila
, despite lacking both contact-dependent cytopathicity and regulated sodium sensitivity. Second, in an attempt to identify virulence factors genetically, an
L. pneumophila
genomic library was screened for clones which conferred robust intracellular growth on
L. micdadei
. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. Apparently,
L. pneumophila
and
L. micdadei
use distinct strategies to colonize alveolar macrophages, causing Legionnaire’s disease.
Myeloperoxidase Exerts Microbicidal Activity againstMycobacterium tuberculosis
Authors: Borelli Violetta , Banfi Elena , Perrotta Maria Giovanna , Zabucchi Giuliano
Published: December 2019

We investigated the antimycobacterial role of myeloperoxidase (MPO), one of the most abundant granule proteins in human neutrophils. Our data indicate that purified MPO, in the presence of hydrogen peroxide, exerts a consistent killing activity againstMycobacterium tuberculosisH37Rv and against a clinical isolate. The activity is time and dose dependent and requires the presence of chloride ions in the assay medium.
Infection of Primary Human Bronchial Epithelial Cells by
Haemophilus influenzae
: Macropinocytosis as a Mechanism of Airway Epithelial Cell Entry
Authors: Ketterer Margaret R. , Shao Jian Q. , Hornick Douglas B. , Buscher Ben , Bandi Venkata K. , Apicella Michael A.
Published: December 2019

Nontypeable
Haemophilus influenzae
is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable
H. influenzae
strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that
H. influenzae
cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable
H. influenzae
can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.
Differences in the Concentrations of Small, Anionic, Antimicrobial Peptides in Bronchoalveolar Lavage Fluid and in Respiratory Epithelia of Patients with and without Cystic Fibrosis
Authors: Brogden Kim A. , Ackermann Mark R. , McCray Paul B. , Huttner Kenneth M.
Published: December 2019

Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean ± standard deviation [SD], 0.78 ± 0.46 mM) than in 34 patients without CF (1.30 ± 0.66 mM) (
P
= 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF.
Streptococcal Pyrogenic Exotoxin F (SpeF) Causes Permeabilization of Lung Blood Vessels
Authors: Matsumoto Masakado , Ishikawa Naohisa , Saito Makoto , Shibayama Keigo , Horii Toshinobu , Sato Kumiko , Ohta Michio
Published: December 2019

Acute respiration distress syndrome (ARDS) is a typical complication in toxic shock-like syndrome (TSLS) caused by
Streptococcus pyogenes
. An isolated perfused rat lung model was used to identify the causative agent of ARDS in TSLS in this study. Some crude preparations separated from the culture supernatants of
S. pyogenes
isolates caused rapid increases in the weight of perfused lungs, indicating vascular permeabilization. Six samples from M type 1 and 3 isolates from TSLS and pharyngitis patients showed strong permeabilization activity, whereas preparations from isolates of other M types (although the number of isolates examined was limited) were negative. The active substance was purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using various columns, and the N-terminal amino acid sequence was determined. The resultant sequence of eight amino acids was identical to SpeF (mitogenic factor). Moreover, the vascular permeabilization activity of the purified band was abolished with anti-SpeF antiserum prepared by immunizing with the purified SpeF. This activity was also neutralized by the antiserum prepared by immunizing with a synthetic peptide derived from the published SpeF sequence. These results suggested that streptococcal SpeF is a major cause of permeabilization of lung blood vessels and sufficient for the pathogenesis of ARDS under the conditions of TSLS caused by
S. pyogenes
.
Group A Streptococcus Induces Apoptosis in Human Epithelial Cells
Authors: Tsai Pei-Jane , Lin Yee-Shin , Kuo Chih-Feng , Lei Huan-Yao , Wu Jiunn-Jong
Published: December 2019

Internalization of group A streptococcus (GAS) by epithelial cells may have a role in causing invasive diseases. The purpose of this study was to examine the fate of GAS-infected epithelial cells. GAS has the ability to invade A-549 and HEp-2 cells. Both A-549 and HEp-2 cells were killed by infection with GAS. Epithelial cell death mediated by GAS was at least in part through apoptosis, as shown by changes in cellular morphology, DNA fragmentation laddering, and propidium iodide staining for hypodiploid cells. A total of 20% of A-549 cells and 11 to 13% of HEp-2 cells underwent apoptosis after 20 h of GAS infection, whereas only 1 to 2% of these cells exhibited spontaneous apoptosis. We further examined whether streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease produced by GAS, was involved in the apoptosis of epithelial cells. ThespeBisogenic mutants had less ability to induce cell death than wild-type strains. When A-549 cells were cocultured with the mutant and SPE B for 2 h, the percentage of apoptotic cells did not increase although the number of intracellular bacteria increased to the level of wild-type strains. In addition, apoptosis was blocked by cytochalasin D treatment, which interfered with cytoskeleton function. The caspase inhibitors Z-VAD.FMK, Ac-YVAD.CMK, and Ac-DEVD.FMK inhibited GAS-induced apoptosis. These results demonstrate for the first time that GAS induces apoptosis of epithelial cells and internalization is required for apoptosis. The caspase pathway is involved in GAS-induced apoptosis, and the expression of SPE B in the cells enhances apoptosis.
Protective Role for Interleukin-5 during ChronicToxoplasma gondiiInfection
Authors: Zhang Yin , Denkers Eric Y.
Published: December 2019

To investigate the role of interleukin-5 (IL-5) duringToxoplasma gondiiinfection, IL-5 knockout (KO) mice and C57BL/6 control mice were infected intraperitoneally with ME49 cysts and the course of infection was monitored. The mortality rate during chronic infection was significantly greater in IL-5-deficient animals, and consistent with this finding, the KO mice harbored a greater number of brain cysts and tachyzoites than did their wild-type counterparts. Although the IL-5 KO animals did not succumb until late during infection, increased susceptibility, as measured by accelerated weight loss, was detectable during the acute stages of infection. The amounts of total immunoglobulin (Ig), IgM, and IgG2b were comparable in both strains, while the amount of IgG1 was much smaller in IL-5 KO mice. Spleen cell production of IL-12 in response toT. gondiiantigen was approximately threefold lower in the KO strain, and this decrease correlated with a selective loss of B lymphocytes during culture. A link between the presence of B cells and augmented IL-12 production was established by the finding that after removal of B cells with monoclonal antibody and complement, wild-type- and KO-derived cells produced equivalent levels of IL-12 in response toT. gondiiantigen. These results demonstrate a protective role of IL-5 againstT. gondiiinfection and suggest that IL-5 may play a role in the production of IL-12.
Intracellular Growth inAcanthamoeba castellaniiAffects Monocyte Entry Mechanisms and Enhances Virulence ofLegionella pneumophila
Authors: Cirillo Jeffrey D. , Cirillo Suat L. G. , Yan Ling , Bermudez Luiz E. , Falkow Stanley , Tompkins Lucy S.
Published: December 2019

SinceLegionella pneumophilais an intracellular pathogen, entry into and replication within host cells are thought to be critical to its ability to cause disease.L. pneumophilagrown in one of its environmental hosts,Acanthamoeba castellanii, is phenotypically different fromL. pneumophilagrown on standard laboratory medium (BCYE agar). Although amoeba-grownL. pneumophiladisplays enhanced entry into monocytes compared to BCYE-grown bacteria, the mechanisms of entry used and the effects on virulence have not been examined. To explore whether amoeba-grownL. pneumophiladiffers from BCYE-grownL. pneumophilain these characteristics, we examined entry into monocytes, replication in activated macrophages, and virulence in mice. Entry of amoeba-grownL. pneumophilainto monocytes occurred more frequently by coiling phagocytosis, was less affected by complement opsonization, and was less sensitive to microtubule and microfilament inhibitors than was entry of BCYE-grown bacteria. In addition, amoeba-grownL. pneumophiladisplays increased replication in monocytes and is more virulent in A/J, C57BL/6 Beige, and C57BL/6 mice. These data demonstrate for the first time that the intra-amoebal growth environment affects the entry mechanisms and virulence ofL. pneumophila.
Molecular and Idiotypic Analyses of the Antibody Response toCryptococcus neoformansGlucuronoxylomannan-Protein Conjugate Vaccine in Autoimmune and Nonautoimmune Mice
Authors: Nussbaum Gabriel , Anandasabapathy Sharmila , Mukherjee Jean , Fan Manxia , Casadevall Arturo , Scharff Matthew D.
Published: December 2019

The antibody response toCryptococcus neoformanscapsular glucuronoxylomannan (GXM) in BALB/c mice frequently expresses the 2H1 idiotype (Id) and is restricted in variable gene usage. This study examined the immunogenicity of GXM-protein conjugates, V (variable)-region usage, and 2H1 Id expression in seven mouse strains: BALB/c, C57BL/6, A/J, C3H, NZB, NZW, and (NZB × NZW)F1(NZB/W). All mouse strains responded to vaccination with GXM conjugated to tetanus toxoid (TT), the relative magnitude of the antibody response being BALB/c ∼ C3H > C57BL/6 ∼ NZB ∼ NZW ∼ NZB/W > A/J. Analysis of serum antibody responses to GXM with polyclonal and monoclonal antibodies to the 2H1 Id revealed significant inter- and intrastrain differences in idiotype expression. Thirteen monoclonal antibodies (MAbs) (two immunoglobulin M [IgM], three IgG3, one IgG1, three IgG2a, two IgG2b, and two IgA) to GXM were generated from one NZB/W mouse and one C3H/He mouse. The MAbs from the NZB/W mouse were all 2H1 Id positive (Id+) and structurally similar to those previously generated in BALB/c mice, including the usage of a VHfrom the 7183 family and Vκ5.1. Administration of both 2H1 Id+and Id−MAbs from NZB/W and C3H/H3 mice prolonged survival in a mouse model of cryptococcosis. Our results demonstrate (i) that V-region restriction as indicated by the 2H1 Id is a feature of both primary and secondary responses of several mouse strains; and (ii) that there is conservation of V-region usage and length of the third complementarity-determining region in antibodies from three mouse strains. The results suggest that V-region restriction is a result of antibody structural requirements necessary for binding an immunodominant antigen in GXM.
Analysis of Antigenic Structure and Human Immune Response to Outer Membrane Protein CD of
Moraxella catarrhalis
Authors: Murphy Timothy F. , Kirkham Charmaine , DeNardin Ernesto , Sethi Sanjay
Published: December 2019

Moraxella catarrhalis
is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Outer membrane protein CD (OMP CD) is a 45-kDa protein which is a potential vaccine antigen to prevent infections caused by
M. catarrhalis
. Eight monoclonal antibodies were used to study the antigenic structure of the OMP CD molecule by assaying recombinant peptides corresponding to the sequence of the protein. This approach identified two surface-exposed epitopes, including one near the amino terminus (amino acids 25 to 44) and one in the central region of the molecule (amino acids 261 to 331). Assays with serum and sputum supernatants of adults with COPD revealed variable levels of antibodies to OMP CD among individuals. To determine which portions of the OMP CD molecule were recognized by human antibodies, three human serum samples were studied with six recombinant peptides which span the sequence of OMP CD. All three sera contained immunoglobulin G antibodies which recognized exclusively the peptide corresponding to amino acids 203 to 260 by immunoblot assay. Adsorption experiments with whole bacteria established that some of the human antibodies are directed at surface-exposed epitopes on OMP CD. We conclude that OMP CD is a highly conserved molecule which contains at least two separate epitopes which are exposed on the bacterial surface. While individual adults with COPD show variability in the immune response to OMP CD, a specific region of the OMP CD molecule (amino acids 203 to 260) is important as a target of the human immune response.
Pseudomonas aeruginosaExoenzyme S Stimulates Murine Lymphocyte Proliferation In Vitro
Authors: Barclay Neil G. , Spurrell Jason C. L. , Bruno Tony F. , Storey Douglas G. , Woods Donald E. , Mody Christopher H.
Published: December 2019

The exuberant immunoinflammatory response that is associated withPseudomonas aeruginosainfection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients. Previous studies have established that an exoproduct ofP. aeruginosa(exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response. An animal model would facilitate studies of the pathophysiologic consequences of this activation. As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined. When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated. The optimum response occurred after 2 to 3 days in culture, at 4 × 105cells per well and 5.0 μg of exoenzyme S per ml. The response was not due to lipopolysaccharide, sinceRhodobacter sphaeroideslipid A antagonist did not block the response. Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1. There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S. Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S. Thus, murine lymphocytes, like human lymphocytes, respond toP. aeruginosaexoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis ofP. aeruginosainfections in CF patients.
Heat-KilledStreptococcus suisCapsular Type 2 Strains Stimulate Tumor Necrosis Factor Alpha and Interleukin-6 Production by Murine Macrophages
Authors: Segura Mariela , Stankova Jana , Gottschalk Marcelo
Published: December 2019

Streptococcus suiscapsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since mononuclear phagocytes have been suggested to play a central role in the pathogenesis of meningitis, the objective of the present study was to evaluate the capacity of whole killedS. suistype 2 organisms to induce the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by murine macrophages. Induction of cytokines was evaluated in the presence or absence of phorbol ester (phorbol 12-myristate 13-acetate [PMA]) costimulation. Results showed thatS. suistype 2 stimulated the production of both cytokines in a concentration- and time-dependent fashion. Although large doses of bacteria were required for maximal cytokine release, titers were similar to those obtained with the lipopolysaccharide (LPS) positive control. An increase in cytokine release was observed with bothS. suisand LPS with PMA costimulation. Experiments with cytochalasin-treated macrophages showed that the stimulation of cytokine production was phagocytosis independent. When macrophages were stimulated with an unencapsulated mutant, an increase in TNF production was observed, but the absence of the capsule had no effect on IL-6 production. In fact, whereas purified capsular polysaccharide ofS. suisfailed to induce cytokine release, purifiedS. suiscell wall induced both TNF and, to a lesser extent, IL-6. IL-6 secretion probably requires some distinct stimuli which differ from those of TNF. Finally, theS. suisputative virulence factors suilysin and extracellular protein EF showed no cytokine-stimulating activity. The ability ofS. suisto trigger macrophages to produce proinflammatory cytokines may have an important role in the initiation and development of meningitis caused by this microorganism.
Preliminary Characterization of a
Mycobacterium abscessus
Mutant in Human and Murine Models of Infection
Authors: Byrd Thomas F. , Lyons C. Rick
Published: December 2019

The ability to persist in the host after the establishment of infection is an important virulence determinant for mycobacteria.
Mycobacterium abscessus
is a rapidly growing mycobacterial species which causes a variety of clinical syndromes in humans. We have obtained a rough, wild-type human clinical isolate of
M. abscessus (M. abscessus-R)
and a smooth, attenuated mutant
(M. abscessus-S
) which spontaneously dissociated from the clinical isolate. We have found that
M. abscessus-R
is able to persist and multiply in a murine pulmonary infection model in contrast to
M. abscessus-S
, which is rapidly cleared. To understand the basis for this difference, we characterized the behavior of these variants in human tissue culture models of infection.
M. abscessus-R
is able to persist and multiply in human monocytes, while
M. abscessus-S
is deficient in this ability. Both of these variants are phagocytized by human monocytes.
M. abscessus-R
resides in a phagosome typical for pathogenic mycobacteria with a tightly adherent phagosomal membrane. In contrast,
M. abscessus-S
resides in a “loose” phagosome with the phagosomal membrane separated from the bacterial cell wall. Both
M. abscessus
variants also have distinctive growth patterns in a recently described fibroblast-mycobacterium microcolony assay, with
M. abscessus-R
exhibiting growth characteristics similar to those previously reported for virulent
M. tuberculosis
and
M. abscessus-S
exhibiting growth characteristics similar to those previously reported for avirulent
M. tuberculosis
. In both the monocyte infection assay and the murine pulmonary infection model, numerous infected mononuclear phagocyte aggregates develop at sites of
M. abscessus-R
infection, but are absent with
M. abscessus-S
infection. We conclude that a mutation has occurred in the
M. abscessus-S
variant which has altered the ability of this organism to persist and multiply in host cells and that this may be related to the phenotypic changes we have observed in our tissue culture models of infection.
Isolation of Recombinant Protective
Helicobacter pylori
Antigens
Authors: Hocking D. , Webb E. , Radcliff F. , Rothel L. , Taylor S. , Pinczower G. , Kapouleas C. , Braley H. , Lee A. , Doidge C.
Published: December 2019

A total of seven clones producing both new and previously described
Helicobacter pylori
proteins were isolated from a library of
H. pylori
genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with
Helicobacter felis
sonicate plus cholera toxin, a regimen which protects mice from
H. pylori
challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from
H. pylori
infection. Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens. The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into
Escherichia coli
expression vectors. The proteins produced from these expression systems were purified to allow testing for protective efficacy in an
H. pylori
mouse model. All five proteins were able to facilitate the clearance of a challenge with
H. pylori
, as judged by an assay of gastric urease activity and light microscopy on stomach sections. These results clearly indicate that the screening strategy has successfully identified candidate vaccine antigens.
Pulmonary Outcome in Cystic Fibrosis Is Influenced Primarily by Mucoid
Pseudomonas aeruginosa
Infection and Immune Status and Only Modestly by Genotype
Authors: Parad Richard B. , Gerard Craig J. , Zurakowski David , Nichols David P. , Pier Gerald B.
Published: December 2019

Whether allelic variants of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) independently contribute to pulmonary outcome in CF patients has not been resolved. We used both cross-sectional and mixed-model longitudinal analyses of data from CF patients that were at least 12 years old to determine the influence on pulmonary function (percent predicted forced expiratory volume [FEV
1
]) of the CFTR gene genotype, gender, mucoid
Pseudomonas aeruginosa
(MPA) infection status, presence of total opsonic antibody to MPA, and, separately, the opsonic antibody activity specific to the mucoid exopolysaccharide (MEP) surface antigen. Two different factors were independently associated with the lack of MPA infection: a high level of MEP-specific opsonic activity (MSOA), implicating an immunologically based mechanism of resistance to infection, and a lack of any type of opsonic antibody to MPA, indicative of no significant exposure or infection. This latter phenotype was found in a subset of CF patients who carried at least one uncommon CFTR gene allele suggestive of a genetic basis for resistance to infection in this group of older CF patients. For CF patients in whom both CFTR gene alleles were identified by screening for the 12 most common variants (75% of alleles), cross-sectional analysis showed that MPA infection was best correlated with lower percent predicted FEV
1
, while genotype (two versus one ΔF508 CFTR gene allele) and a low level of MSOA were associated with increased risk of infection. A mixed-model analysis of longitudinal spirometric measurements that considered multiple risk factors to derive regression equations was used to determine which clinical parameters had the greatest effect on the annual rate of decline in percent predicted FEV
1
. This analysis showed that the CFTR gene genotype only modestly modified the constant (
y
intercept) of the derived equations, while gender and MPA infection status had the largest effects on annual rates of decline in percent predicted FEV
1
. These results indicate that the CFTR genotype is usually not a primary determinant of pulmonary function in most CF patients, but gender and MPA infection status are. Infection status is potentially influenced by both immunologic (a high level of MSOA) and genetic factors, such as carriage of a CFTR gene allele that leads to a diagnosis of CF but still confers resistance to infection that is comparable to that of the wild-type CFTR gene.
Porin fromPseudomonas aeruginosaInduces Apoptosis in an Epithelial Cell Line Derived from Rat Seminal Vesicles
Authors: Buommino Elisabetta , Morelli Francesco , Metafora Salvatore , Rossano Fabio , Perfetto Brunella , Baroni Adone , Tufano Maria Antonietta
Published: December 2019

Micromolar concentrations of porin, purified from the outer membranes ofPseudomonas aeruginosa, induced in vitro the classic morphological and biochemical signs of apoptosis in an epithelial cell line (SVC1) derived from the rat seminal vesicle secretory epithelium. The programmed cell death (PCD) was p53 independent and associated with significant decrease ofbcl-2expression, a marked increase of c-myctranscriptional activity, and an absence of the mRNA coding for tissue transglutaminase. The Ca2+influx, caused by the porin treatment of SVC1 cells, appears to play an important role in the triggering of apoptosis in our biological model. The possibility that the porin property of inducing PCD plays a role in the infertility of individuals chronically infected by gram-negative bacteria is discussed.
Human β-Defensin-1 mRNA Is Transcribed in Tympanic Membrane and Adjacent Auditory Canal Epithelium
Authors: Bøe Roald , Silvola Juha , Yang Jinghui , Moens Ugo , McCray Paul B. , Stenfors Lars-Eric , Seljfelid Rolf
Published: December 2019

The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human β-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.
B- and T-Cell Immune Responses to Pneumococcal Conjugate Vaccines: Divergence between Carrier- and Polysaccharide-Specific Immunogenicity
Authors: McCool Tera L. , Harding Clifford V. , Greenspan Neil S. , Schreiber John R.
Published: December 2019

Conjugation of various serotypes of pneumococcal polysaccharide (PnPS) to carrier protein enhances the magnitude of the polysaccharide-specific antibody response, presumably by eliciting T-cell help. However, variability in PnPS serotype-specific immunogenicity has been observed. CBA/J mice immunized with either 6B or 19F PnPS conjugated to the protein carrier Cross Reactive Material197(CRM197) produce a strong anti-PnPS antibody response; however, when mice are immunized with 23F PnPS conjugated to CRM197, they fail to produce a significant anti-PnPS response. In order to determine whether this difference was related to alterations in antigen processing of the carrier protein and the subsequent T-cell responses, we studied proliferation of lymphocytes from CBA/J mice immunized with CRM197alone or conjugated to 6B, 19F, or 23F PnPS. T-cell proliferative responses to synthetic peptides demonstrated that lymph node cells elicited by the poorly immunogenic conjugate 23F-CRM197recognized many, but not all, of the epitopes recognized by lymph node cells elicited by 6B- and 19F-CRM197as well as additional epitopes. Despite marked differences in PnPS-specific immunogenicity, all mice made high titers of CRM197antibodies of the immunoglobulin G1isotype. Cells from mice immunized with any of the conjugates yielded vigorous T-cell responses to whole antigen. We conclude that the serotype of PnPS can alter the peptide specificities of T-cell responses, but even a poorly immunogenic PnPS conjugate can elicit a significant T-cell response. Thus, conjugation of PnPS to a carrier protein that elicits carrier-specific T- and B-cell responses does not necessarily enhance PnPS immunogenicity.
Observed Differences in Virulence-Associated Phenotypes between a Human Clinical Isolate and a Veterinary Isolate ofMycobacterium avium
Authors: Birkness Kristin A. , Swords W. Edward , Huang Pei-Hsiu , White Elizabeth H. , Dezzutti Charlene S. , Lal Renu B. , Quinn Frederick D.
Published: December 2019

Mycobacterium avium, the most common opportunistic pathogen in patients with AIDS, is frequently isolated from a variety of environmental sources, but rarely can these environmental isolates be epidemiologically linked with isolates known to cause human disease. Using a number of in vitro tissue culture assays, we found significant pathogenic differences between a serotype 4 human clinicalM. aviumisolate and a serotype 2 veterinary isolate. Cell association of the patient strain with a human intestinal cell line was 1.7 times that of the veterinary strain. Growth of this clinical strain in human peripheral blood mononuclear cell-derived macrophages increased from 12-fold higher than that of the veterinary isolate after 2 days to 200-fold higher after 4 days. By the conclusion of each experiment, lysis of all examined host cell types and accumulation of cell debris were observed in infections with the human isolate, but monolayers remained relatively intact in the presence of the animal isolate. The two strains also differed in the ability to stimulate human immunodeficiency virus replication in coinfected host cells, with p24 antigen levels after 6 days threefold higher in the cells coinfected with the clinical strain than in those infected with the veterinary strain. If the genetic differences responsible for the phenotypes observed in these assays can be identified and characterized, it may be possible to determine whichM. aviumstrains in the environment are potential human pathogens.
Differential Regulation of Enteric and Systemic Salmonellosis by
slyA
Authors: Watson Patricia R. , Paulin Sue M. , Bland A. Patricia , Libby Stephen J. , Jones Philip W. , Wallis Tim S.
Published: December 2019

Mutation of
slyA
, which reduces
Salmonella typhimurium
virulence in mice, caused only minor attenuation of
S. typhimurium
virulence in orally inoculated calves. This correlated with modest reductions in intestinal invasion and enteropathogenic responses in bovine ligated ileal loops.
slyA
appears to regulate virulence genes involved in systemic, but not enteric, salmonellosis.
Molecular Cloning of thePasteurella haemolytica pomAGene and Identification of Bovine Antibodies against PomA Surface Domains
Authors: Zeng Hui , Pandher Karamjeet , Murphy George L.
Published: December 2019

The gene (pomA) encoding PomA, an OmpA-like major outer membrane protein of the bovine respiratory pathogenPasteurella haemolytica, was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of PomA has significant identity with the sequences of other OmpA family proteins. Absorption of three different bovine immune sera with wholeP. haemolyticacells resulted in a reduction of bovine immunoglobulin G reactivity with recombinant PomA in Western immunoblots, suggesting the presence of antibodies against PomA surface domains.