bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Antiflagellar Human Monoclonal Antibody on Gut-Derived
Pseudomonas aeruginosa
Sepsis in Mice
Authors: Matsumoto Tetsuya , Tateda Kazuhiro , Miyazaki Shuichi , Furuya Nobuhiko , Ohno Akira , Ishii Yoshikazu , Hirakata Yoichi , Yamaguchi Keizo
Published: December 2019

We evaluated the effect of antiflagellar human monoclonal antibody on gut-derived
Pseudomonas aeruginosa
sepsis. Mice were given a suspension of
P. aeruginosa
SP10052 in their drinking water and were simultaneously treated with ampicillin (200 mg/kg of body weight) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce leukopenia and translocation of the
P. aeruginosa
that had colonized the gastrointestinal tract, thereby producing gut-derived generalized sepsis. In this model, intraperitoneal injection of 100 μg of antiflagellar human monoclonal antibody (SC-1225) per mouse for 5 consecutive days significantly (
P
< 0.01) increased the survival rate compared with that for mice treated with bovine serum albumin (BSA). Treatment with SC-1225 significantly reduced the average number of viable bacteria in portal blood, liver, and heart blood compared with the average number after treatment with BSA. Furthermore, the presence in serum of the inflammatory cytokines tumor necrosis factor alpha and interleukin 6 were evaluated as markers of severity of infection, and the results showed that the levels of these cytokines in mice treated with SC-1225 were significantly decreased in comparison with those in BSA-treated control mice. Although there was no significant difference in the number of bacteria that colonized the intestine, SC-1225 treatment significantly increased bacterial opsonophagocytosis by cultured peritoneal macrophages from mice with or without cyclophosphamide pretreatment. Our results indicate that antiflagellar human monoclonal antibody SC-1225 protects mice against gut-derived sepsis caused by
P. aeruginosa
and suggest that such an effect is due to its opsonophagocytic activity and the reduced motility of the translocated bacteria once the bacteria move from the intestine into the bloodstream.
Evaluation of Four Methods for Detection of Immunoglobulin M Antibodies to Dengue Virus
Authors: Branch Songee L. , Levett Paul N.
Published: December 2019

Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (mean time after onset, 11 days), and 36 patients in whom dengue was previously confirmed by serology (mean time after onset, 10 days). Thirty serum specimens from blood donors in a country where dengue is not endemic were used as negative controls. The methods evaluated were two IgM-capture enzyme-linked immunosorbent assays (ELISA) (MRL Diagnostics, Cypress, Calif., and PanBio, Queensland, Australia), a dot ELISA dipstick assay (Integrated Diagnostics, Baltimore, Md.), and a rapid immunochromatographic assay for dengue IgG and IgM (PanBio IC). IgG antibodies were also detected by an ELISA method (MRL Diagnostics). The sensitivities of the four assays were as follows: MRL Diagnostics IgM ELISA, 98.4%; PanBio IgM ELISA, 85.5%; Integrated Diagnostics IgM dot ELISA, 96.8%; and PanBio IC, 83.9%. The specificities of all tests were 100%. Evidence of secondary dengue was found in all patients with dengue hemorrhagic fever and in 83% of the remaining patients. The MRL Diagnostics IgM ELISA appears to be more sensitive than the PanBio IgM ELISA, and this may be significant when IgM titers are low, particularly in patients with secondary dengue infections. The dot ELISA dipstick assay is equally sensitive and may be more appropriate for use in laboratories with lower workloads.
Inhibition of Cytokine Gene Expression by Sodium Salicylate in a Macrophage Cell Line through an NF-κB-Independent Mechanism
Authors: Lemay Serge , Lebedeva Tatiana V. , Singh Ajay K.
Published: December 2019

Macrophage-derived cytokines and chemokines are involved at multiple steps of immune and inflammatory responses, and the transcriptional factor NF-κB appears to play a pivotal role in their coordinated upregulation. The anti-inflammatory agents salicylates have been proposed to act in part by inhibiting NF-κB. We have therefore studied the effects of sodium salicylate on lipopolysaccharide (LPS)-induced κB-binding activity and on cytokine and chemokine gene expression in the RAW264.7 murine macrophage cell line and compared them to those of an established NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). PDTC (100 μM) completely abrogated LPS-induced κB-binding activity and also profoundly inhibited the induction of interleukin 1α (IL-1α), IL-1β, IL-6, IL-10, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and MCP-1 and, to a lesser extent, leukemia inhibitory factor, RANTES, and IL-1Ra. In contrast, sodium salicylate (15 to 20 mM) had no effect on NF-κB activation but, nevertheless, suppressed several LPS-induced cytokine and chemokine genes to a degree similar to that obtained with PDTC. However, compared to PDTC, sodium salicylate caused significantly less inhibition of IL-1Ra and IL-10 gene expression after LPS stimulation. Neither LPS-induced MIP-1α, MIP-1β, nor MIP-2 was significantly affected by PDTC or sodium salicylate, demonstrating that NF-κB is dispensable for the transcriptional regulation of these genes by LPS. In summary, these results suggest that both NF-κB-dependent and NF-κB-independent pathways are necessary for the induction by LPS of a complex cytokine and chemokine response. In the RAW264.7 macrophage cell line, suprapharmacological concentrations of sodium salicylate exert a potent inhibitory effect on LPS-induced cytokine gene induction but appear to accomplish this by interfering with NF-κB-independent pathways of activation.
A Simple Saliva-Based Test for Detecting Antibodies to Human Immunodeficiency Virus
Authors: Schramm Willfried , Angulo Gustavo Barriga , Torres Patricia Castillo , Burgess-Cassler Anthony
Published: December 2019

This study was performed to determine the feasibility of using saliva as a diagnostic medium for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 under nonlaboratory conditions and to evaluate the performance characteristics of such a test. We developed for this purpose a self-contained kit (Saliva · Strip [ST]), which combines the collection and processing, as well as the analysis, of the specimen. The kit’s performance was evaluated in a blinded study. Saliva collection was facilitated with a specially designed device that contains a sample adequacy indicator, and immunochromatography test strips were used for the analysis. A total of 1,336 matched serum and saliva specimens (684 reactive and 652 nonreactive specimens) were tested. We tested sera using an enzyme immunoassay (EIA) and a rapid strip test. Sera reactive in one of the assays were also analyzed by Western blotting. Sensitivity and specificity were 99.4 and 99.4%, respectively, for ST, 100 and 99.1%, respectively, for EIA, and 99.7 and 100%, respectively, for the serum strip test. The saliva test performed well when HIV-2-positive sera or a low-titer performance panel (HIV-1) of serum or plasma specimens were diluted (1:2,000) in nonreactive saliva. Because the methodology we present here uses a noninvasively obtained medium, the methodology may be suitable for use in the field where laboratory support and personnel are limited, such as community outreach programs, doctors’ offices, surveillance studies, and community hospitals.
Long-Term Persistence of Immunoglobulin A (IgA) and IgM Antibodies against Human Cytomegalovirus in Solid-Organ Transplant Recipients
Authors: Eing Bodo R. , Baumeister Horst G. , Kuehn Joachim E. , May Guenter
Published: December 2019

The retrospective analysis of 494 solid-organ transplant recipients revealed that during the follow-up period (mean duration, 3.2 years) 184 (88%) of 209 anti-human cytomegalovirus (HCMV) immunoglobulin A (IgA)-positive patients remained IgA positive, as did 128 (74.85%) of 171 anti-HCMV IgM-positive patients. We conclude that anti-HCMV IgA and IgM testing for management of clinically relevant HCMV infections in solid-organ transplant recipients is dispensable.
Dot Blot Assay for Detection of Antidiacyltrehalose Antibodies in Tuberculous Patients
Authors: Vera-Cabrera Lucio , Rendon Adrian , Diaz-Rodriguez Manuel , Handzel Vera , Laszlo Adalbert
Published: December 2019

A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with other
Mycobacterium tuberculosis
immunogenic antigens.
Deficient Antibody-Dependent Cellular Cytotoxicity against Human Immunodeficiency Virus (HIV)-Expressing Target Cells in Perinatal HIV Infection
Authors: Ziegner Ulrike , Campbell Donald , Weinhold Kent , Frank Ian , Rutstein Richard , Starr Stuart E.
Published: December 2019

Peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus (HIV)-infected children, age-matched HIV-seronegative controls, and HIV-infected asymptomatic and symptomatic adults were compared for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity against target cells expressing HIV or herpes simplex virus (HSV) antigens. Target cells consisted of CD4 lymphocytes purified from PBMC of HIV-seronegative adults and incubated with the IIIB strain of HIV, HUT78 cells chronically infected with IIIB, and HSV-infected human fibroblasts. PBMC of asymptomatic HIV-infected adults were generally able to lyse CD4 cells expressing HIV antigens. Direct correlation was found between the magnitude of lysis and absolute CD4 cell counts in these individuals. In contrast to these results, PBMC from HIV-infected children were generally unable to lyse IIIB-expressing CD4 cells, regardless of the children’s clinical status, age, or absolute CD4 cell counts. Cells from HIV-seronegative adults and children did not directly lyse these target cells either but, in contrast to cells of HIV-seropositive children, were able to mediate cell lysis when serum from an HIV-seropositive adult was added. However, effector cells from these HIV-infected children were able to mediate both ADCC against HSV-infected fibroblasts and NK cell-mediated cytotoxicity against IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically HIV-infected children to mediate ADCC against HIV antigen-expressing CD4 cells may contribute to rapid progression to AIDS.
Evaluation of a Commercially Available Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay Kit for Diagnosing Acute Dengue Infections
Authors: Porter Kevin R. , Widjaja Susana , Darmawan Handinata , Lohita  , Hadiwijaya Sri Hartati , Maroef Chairin Nisa , Suharyono Wuryadi , Tan Ratna
Published: December 2019

Recently, commercially available kits for the detection of anti-dengue virus (anti-DEN) immunoglobulin M (IgM) antibodies have been developed. These standardized assays have greatly enhanced our ability to effectively diagnose DEN infections. We conducted an evaluation of a test kit manufactured by MRL Diagnostics Inc. that is designed to detect anti-DEN IgM antibodies. Eighty paired samples from DEN-infected individuals were tested by the MRL DEN Fever Virus IgM Capture enzyme-linked immunosorbent assay (ELISA), the PanBio Duo ELISA, the PanBio Rapid Immunochromatographic Test (PRIT), and the IgM-IgG antibody capture (MAC/GAC) ELISA. All infections were confirmed by either PCR-assisted detection of DEN transcripts or by DEN isolation in C6/36 cells. Seventeen paired samples from individuals with no evidence of acute DEN infection were used as negative controls. The PRIT had the best sensitivity (100%), whereas the MAC/GAC ELISA and the PanBio Duo assay had the highest levels of specificity. The MRL ELISA and the PanBio Duo assay were the top performers when taking into consideration both sensitivity and specificity. All assays were able to detect DEN-specific antibodies in samples from patients with either primary or secondary infections, regardless of the infecting DEN serotype.
Validation of a Gastrointestinal Explant System for Measurement of Mucosal Antibody Production
Authors: Losonsky Genevieve A. , Fantry George T. , Reymann Mardi , Lim Yu
Published: December 2019

A gastrointestinal explant culture system was developed and compared to the mononuclear cell extraction and enzyme-linked immunospot assay method for measurement of immunoglobulin A (IgA) and IgG antibody-secreting cells (ASCs) in gastric antral and duodenal biopsies of non-Helicobacter pylori-infected volunteers. IgA and IgG were detected in explant supernatants during 6 to 7 days of culture in all subjects. IgA containing secretory component was also detected throughout the culture period, although peak production occurred only in the first 3 days. During 7 days of culture, the cumulative geometric mean IgA levels produced were 2.2 and 8.02 μg/ml/10 mg of antral and duodenal biopsy tissues, respectively, while the cumulative geometric mean IgG levels were 1.54 and 2.92 μg/ml/10 mg of antral and duodenal biopsy tissues, respectively. Cycloheximide treatment resulted in a >90% reduction in both immunoglobulin classes after 6 days of treatment compared to levels in untreated controls. The detection of IgA and IgG ASCs extracted from biopsies on days 1 and 6 of culture confirmed that the antibody detected was derived from mucosal lamina propria. The IgA and IgG ASC responses were positively correlated with antibody concentrations detected in culture supernatants (r= 0.87 and 0.85, respectively). These results validate the potential usefulness of our gastrointestinal explant system for the evaluation of mucosal effector B-cell function.
Enhanced Secretory Leukocyte Protease Inhibitor in Human Immunodeficiency Virus Type 1-Infected Patients
Authors: Baqui A. A. M. A. , Meiller Timothy F. , Falkler William A.
Published: December 2019

Secretory leukocyte protease inhibitor (SLPI) has been found to possess activity against the human immunodeficiency virus type 1 (HIV-1) in vitro at physiological concentrations. A study was undertaken to evaluate SLPI levels in human saliva and plasma among HIV-positive (HIV
+
) patients with various HIV-1 viral loads in comparison to uninfected controls. Whole blood in EDTA and unstimulated saliva samples were collected from 37 HIV
+
patients, of whom 20 had a history of intravenous drug abuse (IVDA). Control samples were collected from 20 appropriate age- and sex-matched HIV-1-negative individuals. SLPI was estimated from both saliva and serum samples by an enzyme-linked immunosorbent assay. HIV viral load was determined using a quantitative reverse transcription-PCR. SLPI levels were increased 16.7% in plasma and 10.3% in saliva among HIV
+
patients in comparison to uninfected controls. SLPI levels were increased 5.9% in saliva and 3.9% in plasma among HIV
+
patients with a high viral load (>10,000 copies/ml) as compared to patients with a low viral load (<400 copies/ml). Only 23% of patients with a high viral load used combination therapy with protease inhibitor drugs, whereas 92.9% of HIV
+
patients with a low viral load used protease inhibitors. SLPI levels did not differ significantly among the IVDA patients, patients with different viral loads, or patients using protease inhibitor drugs. There was a statistically significant increase in SLPI levels in saliva among HIV patients in comparison to non-HIV-infected controls. An increase in SLPI levels among HIV
+
patients may be a natural consequence of HIV pathogenesis and an important factor in preventing oral transmission of HIV, but this increase may not be evident during plasma viremia in patients with a high viral load.
Analysis of the Humoral Immune Response to
Chlamydia pneumoniae
by Immunoblotting and Immunoprecipitation
Authors: Essig Andreas , Simnacher Ulrike , Susa Milorad , Marre Reinhard
Published: December 2019

Chlamydia pneumoniae
is a widely spread agent of respiratory tract infections in humans. A reliable serodiagnosis of the disease is hampered by the poor knowledge about immunodominant antigens in
C. pneumoniae
infections. We applied a novel strategy to identify immunogenic proteins of
C. pneumoniae
TW183 combining metabolic radiolabeling of de novo-synthesized chlamydial antigens with immunoprecipitation. By this technique
C. pneumoniae
antigens of approximately 160, 97 to 99, 60 to 62, 40, 27, and 15 kDa were detected in the vast majority of sera from patients with a current
C. pneumoniae
infection. By immunoblotting purified elementary bodies of
C. pneumoniae
TW183 with the same sera, only the 60- to 62-kDa antigen could be detected consistently. Sequential immunoprecipitation performed at different stages of the chlamydial developmental cycle revealed that the 60- to 62-kDa antigen is strongly upregulated after 24 to 48 h of host cell infection and is presented as a major immunogen in both
C. pneumoniae
-infected patients and mice. We conclude that, due to its high sensitivity and concurrent preservation of conformational epitopes, metabolic radiolabeling of chlamydial antigens combined with immunoprecipitation may be a useful method to reveal important immunogens in respiratory
C. pneumoniae
infection which might have been missed by immunoblot analysis.
Further Characterization of Human Salivary Anticandidal Activities in a Human Immunodeficiency Virus-Positive Cohort by Use of Microassays
Authors: Lin Alan L. , Shi Qinghong , Johnson Dorthea A. , Patterson Thomas F. , Rinaldi Michael G. , Yeh Chih-Ko
Published: December 2019

Salivary anticandidal activities play an important role in oral candidal infection. R. P. Santarpia et al. (Oral Microbiol. Immunol. 7:38–43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of
Candida albicans
blastoconidia and the formation of germ tubes by
C. albicans
. In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 μl). For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types. The susceptibility of several
Candida
isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of
Candida
isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole. Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (
P
< 0.05) and germ tube formation (
P
< 0.001). Stimulated whole-saliva flow rates were also significantly lower (
P
< 0.05) for HIV-infected patients. These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients. These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS.
Hemoglobin Toxicity in Experimental Bacterial Peritonitis Is Due to Production of Reactive Oxygen Species
Authors: Yoo Yeong-Min , Kim Ki-Mo , Kim Sung-Soo , Han Jeong-A , Lea Ho-Zoo , Kim Young-Myeong
Published: December 2019

Hemoglobin (Hb) is a toxic molecule responsible for the extreme lethality associated with experimental
Escherichia coli
peritonitis, but the mechanism has yet to be elucidated. Hb, but not globin, showed toxic effects in a live
E. coli
model but not in a model using killed
E. coli
. Methemoglobin, hematin, and the well-known Fenton reagents iron and iron-EDTA demonstrated the same lethal effect in
E. coli
peritonitis as Hb, while the addition of the Fenton inhibitors desferrioxamine (DF) and diethylenetriamine pentaacetate removed most of the cytotoxic activity of iron. Administration of a combined dose of superoxide dismutase and catalase minimized the action of Hb and iron-EDTA, suggesting that both O
2
⋅
−
and H
2
O
2
are involved in the toxic action of Hb in this rat model. The combination of the antioxidative enzymes and DF further suppressed iron-mediated lethality. An electron spin resonance technique with the spin-trapping reagent 5,5-dimethyl-1-pyroline-
N
-oxide (DMPO) showed O
2
⋅
−
generation in the peritoneal fluid of rats injected with
E. coli
alone or
E. coli
plus iron-DF, and
⋅
OH generation was detected in the peritoneal fluid of the rats injected with iron-EDTA. Hb did not show any spin adduct of oxygen radicals, suggesting that Hb produces non-spin-trapping radical ferryl ion, which decayed the spin adduct of DMPO. In the presence of Hb or iron-EDTA, O
2
−
-generating activity and viability of phagocytes decreased, whereas lipid peroxidation of peritoneal phagocytes increased. Generation of oxygen radicals and lipid peroxidation did not differ in the live and dead bacterial models. Bacterial numbers in the peritoneal cavity and blood were markedly increased in the live bacterial model with Hb and iron-EDTA. The Fenton inhibitor iron-DF prevented the loss of phagocyte function, lipid peroxidation, and bacterial proliferation. These results led us to conclude that the lethal toxicity of Hb in bacterial peritonitis is associated with a Fenton-type reaction, the products of which decrease phagocyte viability, through the induction of lipid peroxidation, allowing bacterial proliferation and resulting in mortality.
Role of theHaemophilus ducreyiTon System in Internalization of Heme from Hemoglobin
Authors: Elkins Christopher , Totten Pat A. , Olsen Bonnie , Thomas Christopher E.
Published: December 2019

By cloning intoEscherichia coliand construction of isogenic mutants ofHaemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. AnE. coli hemA tonBmutant expressingH. ducreyi hgbAgrew on low levels of hemoglobin as a source of heme only when an intactH. ducreyiTon system plasmid was present. In contrast, growth on heme by theE. coli hemA tonBmutant expressinghgbAwas observed only at high concentrations of heme, was TonB independent, and demonstrated thatH. ducreyiHgbA was not sufficient to function as a typical TonB-dependent heme receptor inE. coli. Allelic replacement of the wild-typeH. ducreyi exbB,exbD, andtonBloci with theexbB,exbD, andtonBdeletion resulted in anH. ducreyiisogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin inH. ducreyiis TonB independent. Additionally, theH. ducreyiTon system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes wasexbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of theseH. ducreyigenes, was most similar to those from other pasteurellae.
Regional Differences in the Cellular Immune Response to Experimental Cutaneous or Visceral Infection withLeishmania donovani
Authors: Melby Peter C. , Yang Yan-Zhu , Cheng Jun , Zhao Weiguo
Published: December 2019

Infection with the protozoanLeishmania donovanican cause serious visceral disease or subclinical infection in humans. To better understand the pathogenesis of this dichotomy, we have investigated the host cellular immune response to cutaneous or visceral infection in a murine model. Mice infected in the skin developed no detectable visceral parasitism, whereas intravenous inoculation resulted in hepatosplenomegaly and an increasing visceral parasite burden. Spleen cells from mice with locally controlled cutaneous infection showed strong parasite-specific proliferative and gamma interferon (IFN-γ) responses, but spleen cells from systemically infected mice were unresponsive to parasite antigens. The in situ expression of IFN-γ, interleukin-4 (IL-4), IL-10, IL-12, and inducible nitric oxide synthase (iNOS) mRNAs was determined in the spleen, draining lymph node (LN), and cutaneous site of inoculation. There was considerably greater expression of IFN-γ and IL-12 p40 mRNAs in the LN draining a locally controlled cutaneous infection than in the spleen following systemic infection. Similarly, there was a high level of IFN-γ production by LN cells following subcutaneous infection but no IFN-γ production by spleen cells following systemic infection. Splenic IL-4 expression was transiently increased early after systemic infection, but splenic IL-10 transcripts increased throughout the course of visceral infection. IL-4 and IL-10 mRNAs were also increased in the LN following cutaneous infection. iNOS mRNA was detected earlier in the LN draining a cutaneous site of infection compared to the spleen following systemic challenge. Thus, locally controlled cutaneous infection was associated with antigen-specific spleen cell responsiveness and markedly increased levels of IFN-γ, IL-12, and iNOS mRNA in the draining LN. Progressive splenic parasitism was associated with an early IL-4 response, markedly increased IL-10 but minimal IL-12 expression, and delayed expression of iNOS.
Role of Tumor Necrosis Factor Alpha in Gnotobiotic Mice Infected with an
Escherichia coli
O157:H7 Strain
Authors: Isogai Emiko , Isogai Hiroshi , Kimura Koichi , Hayashi Shunji , Kubota Toru , Fujii Nobuhiro , Takeshi Koichi
Published: December 2019

Gnotobiotic mice inoculated with an enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 strain developed a flaccid paresis, usually culminating in death. The bacteria colonized feces at 10
9
to 10
10
CFU per g (inoculum size: 2.0 × 10
9
CFU/mouse), and Shiga-like toxins (SLTs) were detected in the feces. A microscopic examination of colons showed mild inflammatory cell infiltration, thinning of the intestinal wall, or necrotic foci. Necrosis of tubular cells was noted in these symptomatic mice. Microhemorrhage, thrombosis, and edematous changes of the brain were also seen. Inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin 1α (IL-1α), and IL-6, were detected in the kidney after EHEC infection, but not in the serum. In the brain, only TNF-α was detected. When 2.0 × 10
2
CFU of EHEC O157:H7 was fed to germ-free mice, the number of bacteria began to rise rapidly on day 1 and was maintained at 10
8
to 10
9
CFU/g of feces. SLTs were detected in the feces of the mice. However, the mice showed no histological changes and no cytokine responses, similar to what was found for controls. Treatment with TNF-α modified the clinical neural signs, histopathological changes, and cytokine responses; mice treated with TNF-α developed severe neurotoxic symptoms and had higher frequencies of systemic symptoms and glomerular pathology. Strong cytokine responses were seen in the kidney and brain. Serum cytokines were also detected in this group. In contrast, a TNF-α inhibitor (protease inhibitor) inhibited these responses, especially in the brain. However, local synthesis of the cytokines was observed in the kidney. Thus, TNF-α and the other proinflammatory cytokines could be important in modifying the disease caused by EHEC.
Periplasmic Superoxide Dismutase in Meningococcal Pathogenicity
Authors: Wilks Kathryn E. , Dunn Kate L. R. , Farrant Jayne L. , Reddin Karen M. , Gorringe Andrew R. , Langford Paul R. , Kroll J. Simon
Published: December 2019

Meningococcal
sodC
encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A
sodC
mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The
sodC
mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of
Neisseria meningitidis
, most likely by reducing the effectiveness of toxic oxygen host defenses.
Borrelia burgdorferiInduces the Production and Release of Proinflammatory Cytokines in Canine Synovial Explant Cultures
Authors: Straubinger Reinhard K. , Straubinger Alix F. , Summers Brian A. , Erb Hollis N. , Härter Luc , Appel Max J. G.
Published: December 2019

Canine synovial membrane explants were exposed to high- or low-passageBorrelia burgdorferifor 3, 6, 12, and 24 h. Spirochetes received no treatment, were UV light irradiated for 16 h, or were sonicated prior to addition to synovial explant cultures. In explant tissues, mRNA levels for the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), IL-1β, and IL-8 were surveyed semiquantitatively by reverse transcription-PCR. Culture supernatants were examined for numbers of total and motile (i.e., viable) spirochetes, TNF-like and IL-1-like activities, polymorphonuclear neutrophil (PMN) chemotaxis-inducing activities, and IL-8. During exposure to synovial explant tissues, the total number of spirochetes in the supernatants decreased gradually by ∼30%, and the viability also declined. mRNAs for TNF-α, IL-1α, IL-1β, and IL-8 were up-regulated in synovial explant tissues within 3 h after infection with untreated or UV light-irradiatedB. burgdorferi, and mRNA levels corresponded to the results obtained with bioassays. During 24 h of coincubation, cultures challenged with untreated or UV light-irradiated spirochetes produced similar levels of TNF-like and IL-1-like activities. In contrast, explant tissues exposed to untreatedB. burgdorferigenerated significantly higher levels of chemotactic factors after 24 h of incubation than did explant tissues exposed to UV light-treated spirochetes. In identical samples, a specific signal for IL-8 was identified by Western blot analysis. High- and low-passage borreliae did not differ in their abilities to induce proinflammatory cytokines. No difference in cytokine induction between untreated and sonicated high-passage spirochetes was observed, suggesting that fractions of the organism can trigger the production and release of inflammatory mediators. The titration of spirochetes revealed a dose-independent cytokine response, where 103to 107B. burgdorferiorganisms induced similar TNF-like activities but only 107spirochetes induced measurable IL-1-like activities. The release of chemotactic factors was dose dependent and was initiated when tissues were infected with at least 105organisms. We conclude that intactB. burgdorferior fractions of the bacterium can induce the local up-regulation of TNF-α, IL-1α, and IL-1β in the synovium but that the interaction of viable spirochetes with synovial cells leads to the release of IL-8, which probably is a prime initiator of PMN migration during acute Lyme arthritis.
Early Cytokine and Chemokine Gene Expression duringPseudomonas aeruginosaCorneal Infection in Mice
Authors: Kernacki Karen A. , Goebel Dennis J. , Poosch Michael S. , Hazlett Linda D.
Published: December 2019

Using a multiprobe RNase protection assay, we examined cytokine and chemokine mRNAs that were expressed after corneal infection withPseudomonas aeruginosain mice. Cytokines that were upregulated included interleukin-1α (IL-1α) and -1β, IL-1 receptor antagonist, IL-6, IL-11, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, lymphotoxin β, transforming growth factor β1, and tumor necrosis factor alpha. Chemokine transcripts that were upregulated included Eotaxin; gamma-interferon-inducible protein 10; monocyte chemoattractant protein 1; macrophage inflammatory proteins 1α, 1β, and 2; and RANTES. Peak expression of these cytokines and chemokines was observed between 1 and 3 days after infection. These responses returned to or approached baseline preinfection levels by 7 days after ocular challenge. Identification of the various cytokines and chemokines upregulated during corneal infection provides important information relevant to unraveling the pathogenesis induced by this bacterium and provides hope that specific molecules can be targeted for therapy.
In Vivo Regulation of ReplicativeLegionella pneumophilaLung Infection by Endogenous Interleukin-12
Authors: Brieland J. K. , Remick D. G. , LeGendre M. L. , Engleberg N. C. , Fantone J. C.
Published: December 2019

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth ofLegionella pneumophilawas assessed by using a murine model of replicativeL. pneumophilalung infection. Intratracheal inoculation of A/J mice with virulent bacteria (106L. pneumophilacells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreatedL. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulateL. pneumophilagrowth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicativeL. pneumophilalung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, inL. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicativeL. pneumophilalung infection and suggest that regulation of intrapulmonary growth ofL. pneumophilaby endogenous IL-12 is mediated, at least in part, by TNF-α.
Antigenic Determinants of
Staphylococcus aureus
Type 5 and Type 8 Capsular Polysaccharide Vaccines
Authors: Fattom Ali I. , Sarwar Jawad , Basham Lisa , Ennifar Sofiane , Naso Robert
Published: December 2019

Bacterial capsular polysaccharides (CP) are carbohydrate polymers comprised of repeating saccharide units. Several of these CP have side chains attached to their backbone structures. The side chains may include O-acetyl, phosphate, sialic acid, and other moieties. Those moieties represent the immunodominant epitopes and the most functional ones. The clinically significant
Staphylococcus aureus
type 5 CP (CP 5) and type 8 CP (CP 8) are comprised of a trisaccharide repeat unit with one O-acetyl group attached to each repeat unit. The immunogenicity of these CP and the functionality of antibodies to the backbone and the O-acetyl moieties were investigated. Immunization with the native CP conjugates (CP with 75% O-acetylation) elicited a high proportion of antibodies directed against the O-acetyl moiety. Nonetheless, all of the vaccinees produced antibodies to the backbone moieties as well. Conjugate vaccines made of de-O-acetylated CP elicited backbone antibodies only. Antibodies to both backbone and O-acetyl groups were found to be opsonic against
S. aureus
strains which varied in their O-acetyl content. Absorption studies with O-acetylated and de-O-acetylated CP showed that (i) native CP conjugates generated antibodies to both backbone and O-acetyl groups and (ii) O-acetylated isolates were opsonized by both populations of antibodies while the non-O-acetylated strains were predominantly opsonized by the backbone antibodies. These results suggest that
S. aureus
CP conjugate vaccines elicit multiple populations of antibodies with diverse specificities. Moreover, the antibodies of different specificities (backbone or O-acetyl) are all functional and efficient against the variations in bacterial CP that may occur among clinically significant
S. aureus
pathogenic isolates.
Induction of Adrenomedullin mRNA and Protein by Lipopolysaccharide and Paclitaxel (Taxol) in Murine Macrophages
Authors: Zaks-Zilberman Meirav , Salkowski Cindy A. , Elsasser Theodore , Cuttitta Frank , Vogel Stefanie N.
Published: December 2019

Lipopolysaccharide (LPS), a potent inflammatory stimulus derived from the outer membrane of gram-negative bacteria, has been implicated in septic shock. Plasma levels of adrenomedullin (AM), a potent vasorelaxant, are increased in septic shock and possibly contribute to the characteristic hypotension. As macrophages play a central role in the host response to LPS, we studied AM production by LPS-stimulated macrophages. When peritoneal exudate macrophages from C3H/OuJ mice were treated with protein-free LPS (100 ng/ml) or the LPS mimetic paclitaxel (Taxol; 35 μM), an ∼10-fold increase in steady-state AM mRNA levels was observed, which peaked between 2 and 4 h. A three- to fourfold maximum increase in the levels of immunoreactive AM protein was detected after 6 to 8 h of stimulation. While LPS-hyporesponsive C3H/HeJ macrophages failed to respond to protein-free LPS with an increase in steady-state AM mRNA levels, increased levels were observed after stimulation of these cells with a protein-rich (butanol-extracted) LPS preparation. In addition, increased AM mRNA was observed following treatment of either C3H/OuJ or C3H/HeJ macrophages with solubleToxoplasma gondiitachyzoite antigen or the synthetic flavone analog 5,6-dimethylxanthenone-4-acetic acid. Gamma interferon also stimulated C3H/OuJ macrophages to express increased AM mRNA levels yet was inhibitory in the presence of LPS or paclitaxel. In vivo, mice challenged intraperitoneally with 25 μg of LPS exhibited increased AM mRNA levels in the lungs, liver, and spleen; the greatest increase (>50-fold) was observed in the liver and lungs. Thus, AM is produced, by murine macrophages, and furthermore, LPS induces AM mRNA in vivo in a number of tissues. These data support a possible role for AM in the pathophysiology of sepsis and septic shock.
The Number of Direct Repeats in
hagA
Is Variable among
Porphyromonas gingivalis
Strains
Authors: Kozarov Emil , Whitlock J. , Dong H. , Carrasco E. , Progulske-Fox A.
Published: December 2019

The coding sequence for the surface protein hemagglutinin A (HagA) of
Porphyromonas gingivalis
381 has previously been shown to contain four direct 1.35-kb repeats, designated
rep
HA
. This study was performed to determine if the number of
rep
HA
units in
hagA
is consistently 4 or if allelic polymorphism exists among strains and/or upon multiple passage of
P. gingivalis
. To this end, primers which were homologous to the regions directly 5′ and 3′ of the repeat domain in
hagA
were synthesized. PCR conditions which allowed amplification of the 8.4-kb repeat region between the primers in
P. gingivalis
381 were established. Genomic DNA templates from 13 other
P. gingivalis
strains and 9 fresh clinical isolates from patients were analyzed under the same conditions as used above. Analysis of these PCR products demonstrated that the strains tested had different numbers (two to four) of
rep
HA
units in the respective
hagA
genes. The PCR products of 8.4, 7.0, and 5.7 kb represent four, three, and two repeats, respectively. One strain from each group (381, four repeats; W83, three repeats; and AJW4, two repeats) was also tested to determine if the number of repeats remained invariant upon passaging onto solid medium. No variability in the number of repeats in
hagA
within a strain was detected after 18 passages.
P. gingivalis
381 was chosen for further testing in a mouse abscess model to determine if conditions of in vivo growth would select for deletions or duplications of the repeated sequences. Five days after infection, no change in the number of repeats was detected in cells recovered from either nonimmunized or preimmunized mice. This data indicates an interstrain variability of the number of repeat units and hence a size variability of the HagA protein of
P. gingivalis
, but unlike some surface antigens of other pathogenic species, the number of repeats remains relatively stable given the conditions of growth tested here.
Immunologic and Structural Relationships of the Minor Pilus Subunits among
Haemophilus influenzae
Isolates
Authors: McCrea Kirk W. , St. Sauver Jennifer L. , Marrs Carl F. , Clemans Daniel , Gilsdorf Janet R.
Published: December 2019

Two proteins, HifD and HifE, have been identified as structural components of
Haemophilus influenzae
pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b
H. influenzae
(Hib) and nontypeable
H. influenzae
(NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within
hifD
and
hifE
genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The
hifD
and
hifE
genes of all type b strains showed identical restriction patterns. Analysis of
hifD
and
hifE
genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.
Murine Dendritic Cells Pulsed In Vitro withToxoplasma gondiiAntigens Induce Protective Immunity In Vivo
Authors: Bourguin Isabelle , Moser Muriel , Buzoni-Gatel Dominique , Tielemans Françoise , Bout Daniel , Urbain Jacques , Leo Oberdan
Published: December 2019

The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case ofToxoplasma gondii, the available evidence indicates that CD4+protective cells belong to the Th1 subset. The aim of this study was to determine whetherT. gondiiantigens (inT. gondiisonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged withT. gondiicysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection withT. gondii76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum.T. gondiiantigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.
Borrelia burgdorferiand Interleukin-1 Promote the Transendothelial Migration of Monocytes In Vitro by Different Mechanisms
Authors: Burns Margaret J. , Furie Martha B.
Published: December 2019

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) orBorrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to eitherB. burgdorferior IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, orB. burgdorferi-stimulated HUVEC by 91% ± 3%, 65% ± 2%, or 25% ± 22%, respectively. Stimulation of HUVEC withB. burgdorferialso promoted a 6-fold ± 2-fold increase in the migration of human CD4+T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes acrossB. burgdorferi-treated HUVEC, migration of CD4+T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response toB. burgdorferiby approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium byB. burgdorferimay contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced byB. burgdorferiis significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates thatB. burgdorferiand IL-1 employ distinct mechanisms to activate endothelial cells.