bluuebunny/crossref_metadata_2025 huggingface dataset:
Egg Laying Is Delayed but Worm Fecundity Is Normal in SCID Mice Infected withSchistosoma japonicumandS. mansoniwith or without Recombinant Tumor Necrosis Factor Alpha Treatment
Authors: Cheever Allen W. , Poindexter Robert W. , Wynn Thomas A.
Published: December 2019

Mice with severe combined immunodeficiency (SCID mice) lack functional B and T cells. Egg laying bySchistosoma mansoniandS. japonicumwas delayed in SCID mice, but in a matter of weeks worm fecundity was equivalent to that in intact mice. SCID mice formed smaller hepatic granulomas and showed less fibrosis than did intact mice. The reduction in egg-associated pathology in SCID mice correlated with marked reductions in interleukin-4 (IL-4), IL-5, IL-13, and gamma interferon mRNA expression in the liver.S. mansoniinfections were frequently lethal for SCID mice infected for more than 9 weeks, whileS. japonicum-infected SCID mice died at the same rate as infected intact mice. We were unable to affect hepatic granuloma formation or egg laying by worms in SCID mice by administration of recombinant murine tumor necrosis factor alpha (TNF-α). In fact, SCID and BALB/c mice appeared to express nearly equivalent levels of TNF-α mRNA in their granulomatous tissues, suggesting that there is little or no deficit in TNF-α expression in infected SCID mice. The data indicate that TNF-α may be in large part derived from a non-T-cell source. Together, these findings provide little evidence that TNF-α alone can reconstitute early fecundity, granuloma formation, or hepatic fibrosis in schistosome-infected SCID mice.
Avidity as a Determinant of the Protective Efficacy of Human Antibodies to Pneumococcal Capsular Polysaccharides
Authors: Usinger William R. , Lucas Alexander H.
Published: December 2019

Antibodies reactive with capsular polysaccharides are considered the principal mediators of immunity against invasive diseases caused byStreptococcus pneumoniae. In this study, we tested the hypothesis that anti-pneumococcal capsular polysaccharide (PPS) antibody avidity can influence protective efficacy. We measured the avidities of individual adult postvaccination immunoglobulin G2 (IgG2) antibodies to PPS serotypes 6B and 23F and examined the relationship between avidity and opsonophagocytic and mouse-protective activities. The avidities of PPS 6B- and PPS 23F-specific IgG2 antibodies ranged from 6 to 31 nM−1and from 3 to 20 nM−1, respectively. We observed an inverse correlation between the magnitude of avidity and the amount of antibody required to protect mice against lethal bacteremia caused by serotype 6B pneumococci. Similarly, higher-avidity antibodies were more effective than lower-avidity antibodies in vitro in mediating complement-dependent opsonophagocytosis of both 6B and 23F pneumococci. These data suggest that in adults, PPS antibodies are sufficiently polymorphic to possess biologically significant variations in avidity. We conclude that avidity functions as an important determinant of anticapsular antibody protective efficacy against pneumococci.
Inverse Relationship between Severity of Experimental Pyelonephritis and Nitric Oxide Production in C3H/HeJ Mice
Authors: Nowicki Bogdan , Singhal Jyotsana , Fang Li , Nowicki Stella , Yallampalli Chandrasekhar
Published: December 2019

The contribution of nitric oxide to host resistance to experimental pyelonephritis is not well understood. We examined whether the inhibition of nitric oxide synthesis alters the sensitivity of lipopolysaccharide (LPS) responder (C3H/HeN) and nonresponder (C3H/HeJ) mice to experimentalEscherichia colipyelonephritis. C3H/HeJ and C3H/HeN mice were implanted subcutaneously with minipumps containing an inhibitor of nitric oxide,NG-nitro-l-arginine methyl ester (l-NAME), or a corresponding vehicle. Ascending urinary tract infection by bladder catheterization with two strains ofE. coli, an O75 strain bearing Dr fimbriae and an O75 strain bearing P fimbriae, was developed in tested animals. Twenty-four hours following bladder infection, the kidneys of C3H/HeN and C3H/HeJ mice were colonized at a similar rate. However, 5 weeks postinoculation, C3H/HeN mice cleared infection while C3H/HeJ mice showed persistent colonization. Twenty-four hours following infection, C3H/HeN mice treated withl-NAME showed no significant increase of renal tissue infection compared to the saline-treated control group. However,l-NAME-treated C3H/HeJ mice showed an approximately 100-fold increase inE. coliinfection rate compared to the saline-treated controls in the Dr+group but showed no change compared to those in the P+group. Dissemination of Dr+E. colibut not P+E. colito the liver and uterus was significantly enhanced withl-NAME treatment in C3H/HeJ mice only. Nitric oxide had no direct killing effect onE. coliin vitro. Nitrite production by various organs was found to be significantly lower in C3H/HeJ mice than in C3H/HeN mice. Alteration of nitric oxide and LPS responsiveness was significantly associated with the increased sensitivity of C3H/HeJ mice to experimental Dr+but not to P+E. colipyelonephritis. These findings are consistent with the hypothesis that nitric oxide synthase activity in concert with LPS responsiveness may participate in the antibacterial defense mechanisms of the C3H mouse urinary tract. This phenomenon is strain dependent and possibly related to the invasive properties ofE. coli.
Phylogenetic Analysis of Enteroaggregative and Diffusely Adherent
Escherichia coli
Authors: Czeczulin John R. , Whittam Thomas S. , Henderson Ian R. , Navarro-Garcia Fernando , Nataro James P.
Published: December 2019

The phylogenetics of the various pathotypes of diarrheagenic
Escherichia coli
are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative
E. coli
(EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent
E. coli
strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.
Interruption of Multiple Cellular Processes in HT-29 Epithelial Cells by
Pseudomonas aeruginosa
Exoenzyme S
Authors: Olson Joan C. , Fraylick Jennifer E. , McGuffie Eileen M. , Dolan Katherine M. , Yahr Timothy L. , Frank Dara W. , Vincent Timothy S.
Published: December 2019

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen
Pseudomonas aeruginosa
, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing
P. aeruginosa
388 and the non-ExoS-producing strain 388ΔS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388ΔS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388ΔS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the
P. aeruginosa
infectious process by rendering cells nonfunctional.
Structures inBacillus subtilisAre Recognized by CD14 in a Lipopolysaccharide Binding Protein-Dependent Reaction
Authors: Fan Xiaolong , Stelter Felix , Menzel Rene , Jack Robert , Spreitzer Ingo , Hartung Thomas , Schütt Christine
Published: December 2019

The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system. LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo. Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacteriumBacillus subtilis. Although CD14 does not interact with intactB. subtilisorganisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction. When serum-pretreatedB. subtilisorganisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed. The phagocytosis is strictly dependent both on LBP and on CD14. These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.
A
Neisseria gonorrhoeae
Immunoglobulin A1 Protease Mutant Is Infectious in the Human Challenge Model of Urethral Infection
Authors: Johannsen Diana B. , Johnston David M. , Koymen Hakan O. , Cohen Myron S. , Cannon Janne G.
Published: December 2019

Many mucosal pathogens, including
Neisseria gonorrhoeae
, produce proteases that cleave immunoglobulin A (IgA), the predominant immunoglobulin class produced at mucosal surfaces. While considerable circumstantial evidence suggests that IgA1 protease contributes to gonococcal virulence, there is no direct evidence that
N. gonorrhoeae
requires IgA1 protease activity to infect a human host. We constructed a
N. gonorrhoeae iga
mutant without introducing new antibiotic resistance markers into the final mutant strain and used human experimental infection to test the ability of the mutant to colonize the male urethra and to cause gonococcal urethritis. Four of the five male volunteers inoculated with the Iga
−
mutant became infected. In every respect—clinical signs and symptoms, incubation period between inoculation and infection, and the proportion of volunteers infected—the outcome of human experimental infection with FA1090
iga
was indistinguishable from that previously reported for a variant of parent strain FA1090 matching the mutant in expression of Opa proteins, lipooligosaccharide, and pilin. These results indicate that
N. gonorrhoeae
does not require IgA1 protease production to cause experimental urethritis in males.
Acute Clinical Disease in Cats following Infection with a Pathogenic Strain ofBartonella henselae(LSU16)
Authors: O’Reilly Kathy L. , Bauer Rudy W. , Freeland Rebecca L. , Foil Lane D. , Hughes Keith J. , Rohde Kristen R. , Roy Alma F. , Stout Rhett W. , Triche Patricia C.
Published: December 2019

Bartonella henselaeis the causative agent of human cat scratch disease as well as several serious sequelae of infections, including bacillary angiomatosis and bacillary peliosis. Conflicting reports describe the pathogenesis ofB. henselaein the cat. In this study, we characterized a strain ofB. henselaetermed LSU16. This strain was isolated on rabbit blood agar from a naturally infected 10-month-old female cat during a recurrent episode of bacteremia. The bacterial species was confirmed by PCR-restriction fragment length polymorphism analysis. Nine cats were infected intradermally with 5 × 107CFU of LSU16, and clinical signs, antibody responses, and bacteremia were monitored. All nine cats developed raised, erythematous areas at the site of inoculation within 72 h postinoculation; the swelling peaked at 14 days postinfection and was not palpable by 28 days postinfection. Fever developed in all nine cats between 6 and 16 days postinfection and lasted for 1 to 8 days. Between 6 and 16 days postinfection, all nine cats experienced lethargy which persisted 5 to 18 days. Seven of nine cats were bacteremic by day 7, and all nine cats had become bacteremic by 14 days postinfection. Bacteremia peaked at 14 to 28 days postinfection in all cats. In six of the nine infected cats, bacterial numbers reached nondetectable levels during the 7th week postinfection; however, a single animal maintained bacteremia to 18 weeks postinfection. All nine cats developed strong antibody responses toB. henselae, as determined by Western blot analysis and enzyme-linked immunosorbent assay. Subsequently, three naive cats were injected intradermally with blood from cats infected with LSU16 from a pure culture, and five naive cats were injected with feces from fleas which had been feeding on cats infected with a pure culture of LSU16. These cats developed signs similar to those described in the previous experiment and were euthanized at 5 weeks postinfection. We conclude thatB. henselaeLSU16 is a virulent strain ofB. henselaein cats and propose that the virulence ofB. henselaein cats is strain dependent.
Proinflammatory Activation of Neutrophils and Monocytes byHelicobacter pyloriin Patients with Different Clinical Presentations
Authors: Hansen Per Syrak , Go Mae F. , Varming Kim , Andersen Leif P. , Genta Robert M. , Graham David Y. , Nielsen Henrik
Published: December 2019

ChronicHelicobacter pyloriinfection is associated with mucosal inflammation. The aim of the present study was to assess human neutrophil and monocyte activation byH. pyloristrains obtained from patients with different clinical presentations. Bacterial sonicates from 12 strains were used to stimulate phagocyte upregulation of CD11b/CD18 adherence molecules assessed by fluorescence flow cytometry and oxidative burst responses assessed by chemiluminescence. A dose-dependent activation of CD11b/CD18 adherence molecules was observed with all strains on both neutrophils and monocytes. The activities were similar for strains from patients with duodenal ulceration and for strains from asymptomatic volunteers irrespective of histopathologic grades of the biopsy specimens from the antral mucosa. The neutrophil chemiluminescence response correlated with histopathologic severity. We conclude that upregulation of neutrophil and monocyte adherence molecules byH. pylorisonicates is not associated with clinical presentation of the infection.
Lymphotoxin InhibitsChlamydia pneumoniaeGrowth in HEp-2 Cells
Authors: Matsushima Hiroshi , Shirai Mutsunori , Ouchi Kazunobu , Yamashita Kenji , Kakutani Tetsu , Furukawa Susumu , Nakazawa Teruko
Published: December 2019

Cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-α) inhibit the intracellular replication ofChlamydia pneumoniaeorChlamydia trachomatis. In this study, we found that another cytokine, lymphotoxin (TNF-β), restricts the growth ofC. pneumoniaein HEp-2 cells. When lymphotoxin (10 U/ml) was added during incubation from 8 to 16 h postinoculation, inclusion body formation was severely reduced. In addition, we observed activation of nitric oxide production and the nuclear transition of NF-κB in HEp-2 cells in response to lymphotoxin. These results suggest that inhibition of chlamydial growth by lymphotoxin is mediated, at least in part, by nuclear transition of NF-κB, resulting in induction of nitric oxide synthase to produce nitric oxide, a potent bacteristatic agent. This is the first report on antichlamydial activity of lymphotoxin through induction of nitric oxide.
noxR3
, a Novel Gene from
Mycobacterium tuberculosis
, Protects
Salmonella typhimurium
from Nitrosative and Oxidative Stress
Authors: Ruan Jia , St. John Gregory , Ehrt Sabine , Riley Lee , Nathan Carl
Published: December 2019

Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) produced by activated macrophages participate in host defense against the facultative intracellular pathogens
Mycobacterium tuberculosis
and
Salmonella typhimurium
. To survive within macrophages, such pathogens may have evolved ROI and RNI resistance mechanisms. ROI resistance pathways have been intensively studied. Much less is known about the mechanisms of resistance to RNI. To identify possible RNI resistance genes in
M. tuberculosis
, a mycobacterial library was expressed in
S. typhimurium
and subjected to selection by exposure to the NO donor
S
-nitrosoglutathione (GSNO) in concentrations sufficient to kill the vast majority of nontransformed salmonellae. Among the rare surviving recombinants was a clone expressing
noxR3
, a novel and previously anonymous
M. tuberculosis
gene predicted to encode a small, basic protein. Expression of
noxR3
protected
S. typhimurium
not only from GSNO and acidified nitrite but also from H
2
O
2
.
noxR3
is the third gene cloned from
M. tuberculosis
that has been shown to protect heterologous cells from both RNI and ROI. This suggests diversity in the repertoire of mechanisms that help pathogens resist the oxidative and nitrosative defenses of the host.
Histoplasma capsulatum
Strain Variation in Both H Antigen Production and β-Glucosidase Activity and Overexpression of
HAG1
from a Telomeric Linear Plasmid
Authors: Fisher Kimber L. , Deepe George S. , Woods Jon P.
Published: December 2019

The H antigen of the dimorphic fungal pathogen
Histoplasma capsulatum
was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (
HAG1
) indicated sequence homology with genes for fungal β-glucosidases. To understand the biological role of this immunodominant antigen in
H. capsulatum
, enzymatic assays were performed to determine whether
H. capsulatum
contained a β-glucosidase enzyme activity and whether this activity was encoded by the
HAG1
gene. Substrate gels with
H. capsulatum
culture supernatants revealed β-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted β-glucosidase activities from three
H. capsulatum
restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the
H. capsulatum
classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an
HAG1
plasmid into
H. capsulatum
. Both a class II strain (G217B
ura5-23
) and a class III strain (G184AS
ura5-11
) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of β-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of
HAG1
. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a β-glucosidase.
Biased Immunoglobulin G1 Isotype Responses Induced in Cattle with DNA Expressingmsp1a ofAnaplasma marginale
Authors: Arulkanthan Appudurai , Brown Wendy C. , McGuire Travis C. , Knowles Donald P.
Published: December 2019

Immunization with the native major surface protein 1 (MSP1) (a heterodimer containing disulfide and noncovalently bonded polypeptides designated MSP1a and MSP1b) of the erythrocytic stage ofAnaplasma marginaleconferred protection against homologous challenge (G. H. Palmer, A. F. Barbet, W. C. Davis, and T. C. McGuire, Science 231:1299–1302, 1986). The MSP1a polypeptide possesses a conserved neutralization-sensitive epitope. In the present study, the immune response to DNA-mediated immunization usingmsp1a was studied. The plasmid pVCL/MSP1a, which encodes the completemsp1a gene ofA. marginaleunder the control of human cytomegalovirus immediate-early enhancer/promoter and intron A, was constructed. The immune responses elicited by immunization with pVCL/MSP1a into cardiotoxin-induced regenerating muscle were evaluated in mice and cattle. Antibody reactive with native MSP1a was detected in pooled sera of immunized BALB/c mice 3 weeks following primary immunization. Two calves seronegative forA. marginalewere immunized four times, at weeks 0, 3, 7, and 13, with pVCL/MSP1a. By 8 weeks, both calves responded to MSP1a with an antibody titer of 1:100, which peaked at 1:1,600 and 1:800 by 16 weeks after the initial immunization. Interestingly, immunoblotting with anti-immunoglobulin G1 (anti-IgG1) and anti-IgG2 specific monoclonal antibodies revealed a restricted IgG1 anti-MSP1a response in both animals. T-lymphocyte lines, established after the fourth immunization, proliferated specifically againstA. marginalehomogenate and purified MSP1 in a dose-dependent manner. These data provide a basis for an immunization strategy to direct bovine immune responses by using DNA vaccine vectors containing single or multiple genes encoding major surface proteins ofA. marginale.
Four Clones of
Borrelia burgdorferi
Sensu Stricto Cause Invasive Infection in Humans
Authors: Seinost Gerald , Dykhuizen Daniel E. , Dattwyler Raymond J. , Golde William T. , Dunn John J. , Wang Ing-Nang , Wormser Gary P. , Schriefer Martin E. , Luft Benjamin J.
Published: December 2019

Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring early in the course of infection. A major outer surface protein expressed by the spirochete early in infection is outer surface protein C (OspC). In
Borrelia burgdorferi
sensu stricto, OspC is highly variable. Based on sequence divergence, alleles of
ospC
can be divided into 21 major groups. To assess whether strain differences defined by
ospC
group are linked to invasiveness and pathogenicity, we compared the frequency distributions of major
ospC
groups from ticks, from the primary erythema migrans skin lesion, and from secondary sites, principally from blood and spinal fluid. The frequency distribution of
ospC
groups from ticks is significantly different from that from primary sites, which in turn is significantly different from that from secondary sites. The major groups A, B, I, and K had higher frequencies in the primary sites than in ticks and were the only groups found in secondary sites. We define three categories of major
ospC
groups: one that is common in ticks but very rarely if ever causes human disease, a second that causes only local infection at the tick bite site, and a third that causes systemic disease. The finding that all systemic
B. burgdorferi
sensu stricto infections are associated with four
ospC
groups has importance in the diagnosis, treatment, and prevention of Lyme disease.
Obligatory Role of Gamma Interferon for Host Survival in a Murine Model of Infection withBurkholderia pseudomallei
Authors: Santanirand P. , Harley V. S. , Dance D. A. B. , Drasar B. S. , Bancroft G. J.
Published: December 2019

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine model of either acute or chronic infection withB. pseudomalleiin Taylor Outbred (TO) mice which mimics many features of the human pathology. Intraperitoneal infection of TO mice at doses of >106CFU resulted in acute septic shock and death within 2 days. In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a 3- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested. Resistance to acute infection withB. pseudomalleiwas absolutely dependent upon the production of gamma interferon (IFN-γ) in vivo. Administration of neutralizing monoclonal antibody against IFN-γ lowered the 50% lethal dose from >5 × 105to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h. Neutralization of either tumor necrosis factor alpha or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo. Together, these results provide the first evidence of a host protective mechanism againstB. pseudomallei. The rapid production of IFN-γ within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic.
Human Monoclonal Immunoglobulin M Antibodies to Ganglioside GM1Show Diverse Cross-Reactivities with Lipopolysaccharides ofCampylobacter jejuniStrains Associated with Guillain-Barré Syndrome
Authors: Prendergast Martina M. , Willison Hugh J. , Moran Anthony P.
Published: December 2019

We examined the reactivity of a panel of anti-GM1immunoglobulin M monoclonal antibodies (MAbs) cloned from multifocal motor neuropathy patients with lipopolysaccharides (LPSs) ofCampylobacter jejunistrains, including serotype O:41 strains associated with Guillain-Barré syndrome. The MAbs reacted with ganglioside GM1to different degrees, and these differences in fine specificities for GM1were reflected in the different degrees of reactivity with each of theC. jejuniLPSs tested. Antibodies could also be discriminated by the varying patterns of inhibition by cholera toxin (a GM1ligand) in LPS binding studies. These results indicate that there is a substantial heterogeneity amongC. jejuniO:41 strains in their expression of GM1-like epitopes and among the fine specificities of different neuropathy-associated anti-GM1antibodies.
Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf-1/MEK1-MEK2/ERK1-ERK2 Pathway
Authors: van der Bruggen Tjomme , Nijenhuis Suzanne , van Raaij Estia , Verhoef Jan , Sweder van Asbeck B.
Published: December 2019

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) in response to endotoxin (lipopolysaccharide [LPS]). Several studies have identified signal transduction pathways that are activated by LPS, including activation of nuclear factor-κB (NF-κB) and activation of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38. In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-α production by primary human monocytes has been addressed with PD-098059, which specifically blocks activation of MAPK kinase (MEK) by Raf-1. TNF-α levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 μM). In addition, PD-098059 also reduced TNF-α mRNA expression when cells were stimulated for 1 h with LPS. On the other hand, LPS-induced interleukin-10 (IL-10) levels in the monocyte supernatant were only slightly inhibited by PD-098059. Ro 09-2210, a recently identified MEK inhibitor, completely abrogated TNF-α levels at nanomolar concentrations. IL-10 levels also were strongly reduced. To show the efficacy of PD-098059 and Ro 09-2210, ERK1 and -2 activation was monitored by Western blotting with an antiserum that recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2. Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 and 10 ng of LPS/ml)-dependent manner. Activation of ERK2 was blocked by PD-098059 (50 μM), whereas ERK1 seemed to be less affected. Ro 09-2210 completely prevented LPS-induced ERK1 and ERK2 activation. LPS-induced p38 activation also was prevented by Ro 09-2210. These data further support the view that the ERK signal transduction pathway is causally involved in the synthesis of TNF-α by human monocytes stimulated with LPS.
Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic
Aeromonas
sp. Serogroup O:34
Authors: Merino Susana , Aguilar Alicia , Nogueras Maria Mercedes , Regue Miguel , Swift Simon , Tomás Juan M.
Published: December 2019

Two different representative recombinant clones encoding
Aeromonas hydrophila
lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of
Aeromonas hydrophila
AH-3 (serogroup O34) introduced into
Escherichia coli
DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (
pla
) and C (
plc
) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of
A. hydrophila
AH-3 defective in either
pla
or
plc
genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic.
A. hydrophila
AH-3
plc
mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the
plc
single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic
Aeromonas
sp. serogroup O:34 infection process.
Activation of Phosphotyrosine Phosphatase Activity Attenuates Mitogen-Activated Protein Kinase Signaling and Inhibits c-FOS and Nitric Oxide Synthase Expression in Macrophages Infected withLeishmania donovani
Authors: Nandan Devki , Lo Raymond , Reiner Neil E.
Published: December 2019

Intracellular protozoan parasites of the genusLeishmaniaantagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact ofLeishmania donovanion mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PMA-induced phosphorylation of the MAP kinase substrate and transcription factor Elk-1. Simultaneously, leishmania infection markedly attenuated the induction of c-FOS and inducible nitric oxide synthase (iNOS) expression in response to PMA and gamma interferon (IFN-γ), respectively. These effects correlated with decreased phosphorylation of p44 and p42 MAP kinases on tyrosine residues. Consistent with the latter finding, lysates prepared from leishmania-infected cells contained an activity that dephosphorylated MAP kinase in vitro, suggesting the possibility of a phosphatase acting in vivo. Attenuation of both MAP kinase activity and c-FOS and iNOS expression was reversed by treatment of macrophages with sodium orthovanadate prior to infection. It was also found that the specific activity of the Src homology 2 domain containing tyrosine phosphatase (SHP-1) toward MAP kinase was markedly increased in leishmania-infected cells. These findings indicate that infection withL. donovaniattenuates MAP kinase signaling and c-FOS and iNOS expression in macrophages by activating cellular phosphotyrosine phosphatases. This may represent a novel mechanism of macrophage deactivation during intracellular infection.
Interleukin-1 and Tumor Necrosis Factor Activities Partially Account for Calvarial Bone Resorption Induced by Local Injection of Lipopolysaccharide
Authors: Chiang Cheng-Yang , Kyritsis George , Graves Dana T. , Amar Salomon
Published: December 2019

The present study was undertaken to test the hypothesis that tumor necrosis factor (TNF) and/or interleukin-1 (IL-1) activity mediates lipopolysaccharide (LPS)-induced bone resorption in vivo. To test this hypothesis,Escherichia coliLPS orPorphyromonas gingivalisLPS was injected into the subcutaneous tissues overlying mouse calvariae. Histological sections, prepared from the center of the lesion, were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the osteoclast number and the area of bone resorption. In time course experiments using normal mice, a peak of bone resorption occurred 5 days after endotoxin stimulation. In dose-response experiments, IL-1 receptor type 1 deletion (IL-1R−/−), TNF double-receptor p55/p75 deletion (TNF p55−/−/p75−/−), combined TNF p55 and IL-1 receptor type 1 deletion (TNF p55−/−/IL-1R−/−), and IL-1β-converting enzyme-deficient (ICE−/−) mice and the respective wild-type mice were injected with 500, 100, or 20 μg ofP. gingivalisLPS and sacrificed 5 days after LPS injection. At the highest dose (500 μg), significant decreases in osteoclast number occurred in mutant mice compared to wild-type mice: (i) a 64% reduction for the TNF p55−/−/IL-1R−/−mice, (ii) a 57% reduction for the IL-1R−/−mice, (iii) a 41% reduction for the TNF p55−/−/p75−/−mice, and (iv) a 38% reduction for the ICE−/−mice. At the two lower doses, bone resorption was apparent but no significant differences between mutant and wild-type animals were observed. The present data indicate that at higher doses, LPS-induced bone resorption is substantially mediated by IL-1 and TNF receptor signaling. Furthermore, IL-1 receptor signaling appears to be slightly more important than TNF receptor signaling. At lower LPS doses, other pathways leading to osteoclast activity that are independent of TNF and IL-1 are involved.
Staphylococcus schleiferi
subsp.
schleiferi
Expresses a Fibronectin-Binding Protein
Authors: Peacock S. J. , Lina G. , Etienne J. , Foster T. J.
Published: December 2019

Staphylococcus schleiferi
subsp.
schleiferi
is associated with a range of nosocomial infections, but the pathogenic mechanisms by which these occur are poorly understood. This study provides phenotypic and genotypic evidence for the expression of a cell wall-anchored fibronectin-binding protein by this species.
Intranasal Immunization with Heat-Inactivated
Streptococcus pneumoniae
Protects Mice against Systemic Pneumococcal Infection
Authors: Hvalbye Benedicte K. R. , Aaberge Ingeborg S. , Løvik Martinus , Haneberg Bjørn
Published: December 2019

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated
Streptococcus pneumoniae
type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.
Secretory Leukocyte Protease Inhibitor Interferes with Uptake of Lipopolysaccharide by Macrophages
Authors: Ding Aihao , Thieblemont Nathalie , Zhu Jing , Jin Fenyu , Zhang Jenny , Wright Samuel
Published: December 2019

Macrophages are among the most sensitive targets of bacterial endotoxin (LPS), responding to minute amounts of LPS by releasing a battery of inflammatory mediators. Transfection of macrophages with secretory leukocyte protease inhibitor (SLPI) renders these cells refractory to LPS stimulation. Here we show that uptake of LPS from soluble CD14 (sCD14)-LPS complexes by SLPI-overexpressing cells was only 50% of that seen in control cells. SLPI transfectants and mock transfectants did not differ in the surface expression of CD14 or CD18. We show, in addition, that recombinant human SLPI can bind to purified endotoxin in vitro. SLPI caused a decrease in the binding of LPS to sCD14 as assessed both by fluorescence quenching of labeled LPS and by nondenaturing polyacrylamide gel electrophoresis. These results suggest that the inhibitory effect of SLPI on macrophage responses to LPS may, in part, be due to its blockade of LPS transfer to soluble CD14 and its interference with uptake of LPS from LPS-sCD14 complexes by macrophages.
Functional Studies of a Fibrinogen Binding Protein from
Staphylococcus epidermidis
Authors: Pei Lei , Palma Marco , Nilsson Martin , Guss Bengt , Flock Jan-Ingmar
Published: December 2019

A gene encoding a fibrinogen binding protein from
Staphylococcus epidermidis
was previously cloned, and the nucleotide sequence was determined. A portion of the gene encompassing the fibrinogen binding domain has now been subcloned in an expression-fusion vector. The fusion protein can bind to fibrinogen in a capture enzyme-linked immunosorbent assay and can be purified by fibrinogen affinity chromatography. This protein can completely inhibit the adherence of
S. epidermidis
to immobilized fibrinogen, suggesting that the adherence of
S. epidermidis
to fibrinogen is mainly due to this protein. Antibodies against this fibrinogen binding protein were also found to efficiently block the adherence of
S. epidermidis
to immobilized fibrinogen. Despite homology with clumping factors A and B from
S. aureus
(cell surface-associated proteins binding to fibrinogen), binding involved the β chain of fibrinogen rather than the γ chain, as in clumping factor A.
The Autolysin-Encoding Gene (
lytA
) of
Streptococcus pneumoniae
Displays Restricted Allelic Variation despite Localized Recombination Events with Genes of Pneumococcal Bacteriophage Encoding Cell Wall Lytic Enzymes
Authors: Whatmore Adrian M. , Dowson Christopher G.
Published: December 2019

The
lytA
-encoded autolysin (
N
-acetylmuramoyl-
l
-alanine amidase) of
Streptococcus pneumoniae
is believed to play an important role in the pathogenesis of pneumococcal infection and has been identified as a putative vaccine target. Allelic diversity of
lytA
in an extensive collection of clinical isolates was assessed by restriction fragment length polymorphism and confirmatory sequencing studies. Genetic diversity within
lytA
is limited, especially compared to the high levels of diversity seen in other pneumococcal virulence factor genes, although small blocks generating mosaic structure were identified. Sequence comparisons with genes encoding cell wall lytic enzymes of pneumococcal bacteriophage suggest that localized recombination events have occurred between host
lytA
and these bacteriophage genes. These results confirm earlier suggestions that recombination between DNA encoding bacteriophage autolytic enzymes and chromosomally encoded
lytA
might be important in the evolution of
lytA
. The implications of these findings for understanding the evolution of
lytA
and the potential utility of LytA as a vaccine target are discussed.
Immunization of Mice with a TolA-Like Surface Protein ofTrypanosoma cruziGenerates CD4+T-Cell-Dependent Parasiticidal Activity
Authors: Quanquin Natalie M. , Galaviz Charles , Fouts David L. , Wrightsman Ruth A. , Manning Jerry E.
Published: December 2019

The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form ofTrypanosoma cruzihas been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins ofEscherichia coliandPseudomonas aeruginosa. Based on these similarities, we have designated this gene familytolT. Immunization of mice with recombinant TolT generates a population of CD4+T lymphocytes that recognizeT. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-γ), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-γ and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected withT. cruzialso produce significant levels of IFN-γ when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected withT. cruzi.
Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14
Authors: Caccavo Domenico , Afeltra Antonella , Pece Salvatore , Giuliani Giuseppe , Freudenberg Marina , Galanos Chris , Jirillo Emilio
Published: December 2019

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.
Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
Authors: Condon Michael R. , Viera Alejandro , D’Alessio Michael , Diamond Gill
Published: December 2019

Multicellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on α-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.
Cell Surface-Exposed Tetanus Toxin Fragment C Produced by RecombinantBacillus anthracisProtects against Tetanus Toxin
Authors: Mesnage Stéphane , Weber-Levy Martine , Haustant Michel , Mock Michèle , Fouet Agnès
Published: December 2019

Bacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinantB. anthracisstrain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.