bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of endotoxin-induced acute lethality in Propionibacterium acnes-primed rabbits by an antibody to leukocyte integrin beta 2 with concomitant reduction of cytokine production
Authors: Ikeda N , Mukaida N , Kaneko S , Fujioka N , Su S , Nariuchi H , Unoura M , Harada K , Nakanuma Y , Kobayashi K
Published: January 2020

Acute lethality was induced in rabbits by the sequential injection of Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes induced the infiltration of inflammatory cells into the liver lobules during the early phase, and LPS in the late phase caused death in association with pathological changes mimicking hepatocellular necrosis or degeneration around infiltrated mononuclear cells and fibrin deposition in the liver, lung, and kidney, suggestive of a systemic Schwartzman-like reaction. These pathological changes were accompanied by the elevation of plasma tumor necrosis factor (TNF) and interleukin-8 (IL-8) levels. A neutralizing antibody to a leukocyte adhesion molecule, integrin beta 2 (CD18), administered at the time of LPS challenge, prevented reduced the elevation of plasma TNF and IL-8 levels. An anti-TNF alpha antibody but not an anti-IL-8 mediator in this model. These results indicate that CD18 is critically involved in vivo in activating leukocytes to produce cytokines in response to LPS.
Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin
Authors: Ritchings B W , Almira E C , Lory S , Ramphal R
Published: January 2020

This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.
An oligomer of the major outer membrane protein of Chlamydia psittaci is recognized by monoclonal antibodies which protect mice from abortion
Authors: de Sa C , Souriau A , Bernard F , Salinas J , Rodolakis A
Published: January 2020

Monoclonal antibodies (MAbs) were generated against an ovine abortive strain of Chlamydia psittaci. A plaque reduction assay was used to select 19 neutralizing antibodies which appeared to be heterogeneous in isotype, specificity, and recognized proteins. Different neutralizing MAbs were tested for their protective abilities against abortion in a pregnant-mouse model. All of the protective MAbs selected had the same isotype, were serotype 1 specific, and recognized a protein of about 110 kDa by immunoblotting. The recognized epitopes were resistant to sodium dodecyl sulfate and reducing agents, but all of them were heat sensitive. The protein was able to form disulfide-linked polymers. Immunological cross-reaction studies with rabbit sera showed a link between the 110-kDa protein and the major outer membrane protein (MOMP). The 110-kDa protein was purified by immunoaffinity and shown to be dissociated after heating into MOMP by silver staining and immunoblotting. These results show homogeneity among protective MAbs directed to heat-sensitive epitopes located on an oligomer of the MOMP of C. psittaci.
A pneumolysin-negative mutant of Streptococcus pneumoniae causes chronic bacteremia rather than acute sepsis in mice
Authors: Benton K A , Everson M P , Briles D E
Published: January 2020

Pneumolysin is a cytoplasmic virulence factor of Streptococcus pneumoniae that can interfere with phagocyte function in vitro. We have examined the effects of pneumolysin in vitro and in vivo and have found that it protects intravenously injected pneumococci against infection-induced host resistance. We employed a virulent capsular type 2 pneumococcal strain, D39, and its isogenic pneumolysin-negative mutant, PLN. Strain D39 exhibited exponential net growth in mice (doubling time, 1.4 h); 24 to 28 h after infection with 10(4) CFU, the numbers of pneumococci reached 10(9) to 10(10) CFU/ml and the mice died. Strain PLN yielded identical net growth in mice until reaching 10(6) to 10(7) CFU/ml at 12 to 18 h postinfection. At this time, the increase in the level of PLN CFU per milliliter ceased and remained constant for several days. PLN exhibited wild-type growth kinetics in mice when coinfected simultaneously with strain D39. This observation suggests that pneumolysin exerts its effects at a distance. By 12 to 18 h postinfection with PLN, mice exhibited the following evidence of an induced inflammatory response: (i) elevated plasma interleukin-6, (ii) a halt in the net growth of PLN, and (iii) control of the net growth of pneumolysin-producing D39 pneumococci upon subsequent challenge. Our data suggest that pneumolysin plays a critical role in sepsis during the first few hours after infection by enabling pneumococci to cause acute sepsis rather than a chronic bacteremia. However, once chronic bacteremia was established, it appeared that pneumolysin was no longer able to act as a virulence factor.
Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP1(19), the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum
Authors: Egan A F , Chappel J A , Burghaus P A , Morris J S , McBride J S , Holder A A , Kaslow D C , Riley E M
Published: January 2020

The major merozoite surface protein of Plasmodium falciparum (PfMSP1) is a candidate antigen for a malaria vaccine. A 19-kDa C-terminal processing product of PfMSP1 (PfMSP1(19)) is composed of two domains sharing a cysteine-rich motif with epidermal growth factor (EGF) and is the target of monoclonal antibodies which block erythrocyte invasion in vitro. We have evaluated human antibody responses to PfMSP1(19) by using recombinant proteins representing the EGF motifs encoded by the two main alleles of the MSP1 gene. We find that both EGF motifs are antigenic but that only 10 to 20% of malaria-exposed individuals have serum antibodies that recognized either of the motifs. When both EGF motifs were expressed together as a single protein, they were recognized by more than 40% of sera from malaria-exposed individuals. Major epitopes recognized by human antibodies are dependent upon the correct tertiary structure of the protein and are cross-reactive between the different allelic sequences of PfMSP1(19). This suggests that antibodies induced by vaccination with one or the other allelic forms of the protein could recognize all strains of P. falciparum. Immunoglobulin G (IgG) subclass-specific enzyme immunoassays indicate that PfMSP1(19) antibodies are predominantly of the IgG1 subclass.
Induction of serine and threonine protein phosphorylation by endotoxin-associated protein in murine resident peritoneal macrophages
Authors: Abu-Lawi K I , Sultzer B M
Published: January 2020

Endotoxin-associated protein (EP) from Salmonella typhi activated murine resident peritoneal macrophages to produce prostaglandin E2 (PGE2). Cells from both endotoxin nonresponder (C3H/HeJ) and the endotoxin responder (C3H/OuJ) mouse strains were activated by EP. This EP-induced prostaglandin E2 production was blocked by the protein kinase C (PKC) inhibitor H-7 as well as the tyrosine kinase inhibitor genistein, suggesting the involvement of both serine and threonine phosphorylation and tyrosine phosphorylation pathways in the activation of resident peritoneal macrophages by EP. Immunoblot analysis using antiphosphoserine and antiphosphothreonine antibodies showed that EP induced the serine and threonine phosphorylation of a 14-kDa protein (p14). This phosphorylation was not induced by phorbol myristic acid or by lipopolysaccharide endotoxin. Inhibitors of PKC, PKA, and PKG did not block the phosphorylation of p14. However, the tyrosine kinase inhibitor piceatannol blocked p14 serine and threonine phosphorylation, suggesting that this phosphorylation is dependent upon and preceded by a tyrosine phosphorylation step.
Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells
Authors: LeClaire R D , Kell W M , Sadik R A , Downs M B , Parker G W
Published: January 2020

The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level.
DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain
Authors: Rakonjac J V , Robbins J C , Fischetti V A
Published: January 2020

The serum opacity factor (SOF) is a group A streptococcal protein that induces opacity of mammalian serum. The serum opacity factor 22 gene (sof22) from an M type 22 strain was cloned from an EMBL4 library by screening for plaques exhibiting serum opacity activity. DNA sequencing yielded an open reading frame of 3,075 bp. Its deduced amino acid sequence predicts a protein of 1,025 residues with a molecular weight of 112,735, a size that approximates that of the SOF22 protein isolated from both the original streptococcal strain and Escherichia coli harboring the cloned sof22 gene. The molecule is composed of three domains: an N-terminal domain responsible for the opacity reaction (opacity domain), a repeat domain with fibronectin-binding (Fn-binding) activity, and a C-terminal cell attachment domain. The C-terminal end of SOF22 is characterized by a hexameric LPXTGX motif, an adjacent hydrophobic region, and a charged C terminus, which are the hallmarks of cell-bound surface proteins found on nearly all gram-positive bacteria. Immediately upstream of this cell anchor region, SOF22 contains four tandem repeat sequence blocks, flanked by prolinerich segments. The repeats share up to 50% identity with a repeated motif found in other group A streptococcal Fn-binding proteins and exhibit Fn-binding activity, as shown by subcloning experiments. According to deletion analysis, the opacity domain is confined to the region N terminal to the repeat segment. Thus, SOF22 is unique among the known Fn-binding proteins from gram-positive bacteria in containing an independent module with a defined function in its N-terminal portion. Southern blot analysis with a probe from this N-terminal region indicates that the opacity domain of SOF varies extensively among different SOF-producing M types.
Immunization of rats with the 260-kilodalton Entamoeba histolytica galactose-inhibitable lectin elicits an intestinal secretory immunoglobulin A response that has in vitro adherence-inhibitory activity
Authors: Kelsall B L , Ravdin J I
Published: January 2020

The 260-kDa galactose-inhibitable lectin of Entamoeba histolytica mediates binding of amebic trophozoites to purified colonic mucins and intestinal epithelial cells. Parenteral immunization of Lewis rats with immuno-affinity-purified lectin with Freund's adjuvant and then intra-Peyer's patch inoculation of lectin with cholera toxin B subunit as adjuvant elicited a significant anti-lectin secretory immunoglobulin A response in the bowel lumen. Purified intestinal secretory immunoglobulin A from three or four immunized animals studied possessed inhibitory activity (P < 0.02) in an in vitro assay of trophozoite galactose-specific adherence to Chinese hamster ovary cells.
Identity of Streptococcus mutans surface protein antigen III and wall-associated protein antigen A
Authors: Russell M W , Harrington D J , Russell R R
Published: January 2020

Preparations of Streptococcus mutans surface proteins AgIII and antigen A from different laboratories were compared with regard to amino acid composition, N-terminal amino acid sequence, electrophoretic mobility, and antigenic similarity. Despite previous observations of differences in physical properties, data indicate that these two preparations represent the same protein.
Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14
Authors: Loppnow H , Stelter F , Schönbeck U , Schlüter C , Ernst M , Schütt C , Flad H D
Published: January 2020

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.
Lipopolysaccharide-induced apoptosis in swine lymphocytes in vivo
Authors: Norimatsu M , Ono T , Aoki A , Ohishi K , Takahashi T , Watanabe G , Taya K , Sasamoto S , Tamura Y
Published: January 2020

The in vivo effects of bacterial lipopolysaccharide (LPS) on the immune systems of piglets were investigated. Intravenous injection of 0.5 mg of LPS per kilogram of body weight induced apoptosis, which was characterized by nuclear chromatin condensation and fragmentation and a ladder formation of nucleosomal DNA in lymphocytes both in the cortex of the thymus and in the germinal centers and paracortical areas of mesenteric lymph nodes at 24 h postinjection. The levels of endotoxin, tumor necrosis factor alpha, and cortisol in serum increased, generally according to the dose of LPS. These findings suggest that LPS can induce in vivo apoptosis of lymphocytes in piglets and support the notion that cytokine and endocrine responses may play an important role in LPS-induced apoptosis in the immune system.
Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract
Authors: Klimpel G R , Asuncion M , Haithcoat J , Niesel D W
Published: January 2020

Salmonella infection of the gastrointestinal tract (GT) results in fluid secretion and inflammation. In contrast, cholera toxin (CT) induces fluid secretion but no inflammation. Using a murine ligated intestinal loop model, we investigated cytokine production (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha) in the GT following exposure to these agents. Salmonella typhimurium induced a Th1-like cytokine profile in loops obtained from either nonimmune mice or Salmonella-immunized mice. CT induced only IL-6 and IL-10 production in ligated loops from nonimmune mice but induced a Th2-like cytokine profile in ligated loops obtained from CT-immunized mice. These results show that CT and S. typhimurium induce very different cytokine profiles in the GT.
Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis
Authors: Lee N G , Sunshine M G , Apicella M A
Published: January 2020

The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.
Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism
Authors: Xu Y , Barbieri J T
Published: January 2020

Pertussis toxin (PT)-catalyzed ADP-ribosylation of target proteins in intact Chinese hamster ovary (CHO) cells was evaluated with an in vitro ADP-ribosylation assay. In this assay, a postnuclear supernatant was prepared from CHO cells and used as a source of PT-sensitive target proteins for in vitro [32P[ADP-ribosylation. The postnuclear supernatant contained three proteins that were ADP-ribosylated in vitro, with apparent molecular masses of 50, 45, and 42 kDa. The 42- and 45-kDa proteins were membrane associated, while the 50-kDa protein was soluble. Following PT treatment of CHO cells, the 42- and 45-kDa proteins were not available for in vitro ADP-ribosylation, while the soluble 50-kDa protein remained available for in vitro ADP-ribosylation. The decrease in the availability of the 42- and 45-kDa proteins to in vitro ADP-ribosylation was proportional to the PT concentration and time of incubation with CHO cells. Western immunoblot analysis showed that extracts from PT-treated CHO cells and control CHO cells possessed equivalent amounts of two proteins that were recognized by anti-Gi protein antiserum. The two proteins recognized by anti-Gi protein antiserum from PT-treated cells migrated with higher apparent molecular weights than the two proteins from control cells. This was consistent with the in vivo ADP-ribosylation of the two proteins by PT.(ABSTRACT TRUNCATED AT 250 WORDS)
Neutrophil alkaline phosphatase activity increase in bacterial infections is not associated with a general increase in secretory vesicle membrane components
Authors: Karlsson A , Khalfan L , Dahlgren C , Stigbrand T , Follin P
Published: January 2020

The content of alkaline phosphatase (ALP) was determined in neutrophils isolated from patients with acute bacterial infections by a standard enzyme assay. Compared with control cells, patient cells exhibited about a fivefold increase in ALP activity. There was no difference between the ALP Km values of control and patient cells, which indicates that the elevated activity in patient cells was due to the presence of increased amounts of the enzyme. The ALP isozyme in both cell types was determined to be the tissue-unspecific ALP. The fact that much of the ALP activity was measurable only in the presence of detergent suggested that the enzyme was localized in the secretory vesicles, a putative reservoir of plasma membrane components. The amount and subcellular distribution of two other secretory vesicle membrane proteins, i.e., cytochrome b and complement receptor 3, were not altered; hence, we conclude that there was no general increase in amounts of secretory vesicle membrane constituents in the patient cells.
Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain
Authors: Bracha R , Nuchamowitz Y , Mirelman D
Published: January 2020

A monoclonal antibody (MAb), 318-28, that specifically reacts with a 30-kDa antigen present on membrane surfaces of all nonpathogenic (NP) Entamoeba histolytica strains tested and which did not react with pathogenic (P) strains was used for the isolation of the cDNA coding for this antigen from an expression library of an NP E. histolytica strain. The deduced amino acid composition was rich in lysine residues (14.5%), with some sequence similarity to a polyadenylate-binding protein. Southern and Northern (RNA) blot analyses, as well as amplifications of DNA segments by PCR, indicate that a very similar gene (identity of 96.5%) exists in P strains of E. histolytica. Unexpectedly, the NP-specific antigen was also identified by MAb 318-28 on the surfaces of a cloned, xenically cultivated and well-characterized P strain (BNI:0591) that was recently isolated from a human liver abscess. Binding of the MAb, both to the cell surfaces and to Western blots (immunoblots), was abolished, however, upon axenization of the BNI:0591 cultures. Oligonucleotide primers, designed to anneal only to specific DNA sequences of the NP 30-kDa protein gene copy, amplified a DNA segment from P strain BNI:0591 which was identical in sequence to that of the NP 30-kDa protein gene. Our findings indicate that a P strain of E. histolytica can possess and express, under certain growth conditions, an antigen that is usually detected only in NP strains.
Hepatocytes can serve as accessory cells in the response of immune T lymphocytes to heat-killed Listeria monocytogenes
Authors: Jiang X , Gregory S H , Wing E J
Published: January 2020

Previous findings in our laboratory indicated that the bulk of Listeria monocytogenes injected intravenously into mice and recovered in the liver is taken up and replicates within hepatocytes. Other investigators have shown that hepatocytes can display costimulatory adhesion molecules, express major histocompatibility complex class I and II molecules, and secrete a number of cytokines, including interleukin-1 (IL-1), IL-6, and IL-8. These data suggest that hepatocytes may serve as accessory cells in the immune response to L. monocytogenes. The accessory function and capacity of hepatocytes to present listerial antigens, however, have never been explored. We undertook a series of experiments to examine the response of Listeria-immune T lymphocytes to murine hepatocytes preincubated with heat-killed listeriae (HKL). Electron micrographs showing the organism within membrane-limiting vacuoles demonstrated the capacity of hepatocytes to internalize HKL. T cells cocultured with hepatocytes pulsed with HKL exhibited a 5- to 10-fold increase in [methyl-3H]thymidine incorporation relative to T cells cultured with either hepatocytes or HKL alone. Similarly, gamma interferon production by immune T cells was elevated significantly in cultures that contained both hepatocytes and HKL. The optimal response of T cells required lysosomal processing of HKL by hepatocytes and contact between the two cell populations. Furthermore, maximum T-cell proliferation and gamma interferon production were dependent upon the presence of CD4+ T lymphocytes and the expression of Ia antigens. Taken together, these findings demonstrate that hepatocytes pulsed with HKL can stimulate the antigen-specific response of immune T lymphocytes. These results suggest that hepatocytes can serve as accessory cells in host defenses to listerial infections of the liver.
Effect of experimental influenza A virus infection on isolation of Streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects
Authors: Wadowsky R M , Mietzner S M , Skoner D P , Doyle W J , Fireman P
Published: January 2020

Intranasal challenge with both influenza A virus and Streptococcus pneumoniae promotes otitis media with S. pneumoniae in chinchillas. We investigated whether influenza A virus infection promotes oropharyngeal colonization with S. pneumoniae and other middle ear pathogens by selectively inhibiting commensal bacteria. On study day 0, 12 allergic and 15 nonallergic adult subjects were intranasally inoculated with influenza A/Kawasaki (H1N1) virus. Every subject was infected with the virus as demonstrated by nasal shedding or seroconversion. Average upper respiratory symptom scores and nasal secretion weights from the entire subject group were elevated between days 2 and 6 (acute phase) and were not significantly different between allergic and nonallergic subjects. S. pneumoniae was not isolated from any subject prior to the virus challenge but was isolated in heavy density from 4 (15%) subjects on day 6 (P = 0.055). Staphylococcus aureus was isolated more frequently from the nonallergic subjects than from the allergic subjects on days 2 (80 versus 25%, respectively) 4, (67 versus 17%, respectively), and 6 (73 versus 25%, respectively) (P < 0.05). The isolation rates of other middle ear pathogens were not significantly different before virus challenge and during the acute and resolution phases (days 27 to 30) of the experimental infection for the entire subject group or either the allergic or nonallergic subgroup. Densities and isolation rates of commensal bacteria from the entire subject group were similar throughout the observational period. These results suggest that the virus infection promoted S. pneumoniae colonization of the oropharynx and that nonallergic persons may be more vulnerable to colonization with S. aureus than allergic persons. The altered colonization rates were not attributed to inhibition of commensal bacteria.
Endogenous gamma interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice
Authors: Nakane A , Okamoto M , Asano M , Kohanawa M , Minagawa T
Published: January 2020

The production and roles of endogenous gamma interferon (IFN-gamma), tumor necrosis factor (TNF), and interleukin-6 (IL-6) in both lethal and nonlethal infections of Staphylococcus aureus were investigated in mice. In the case of nonlethal infection, although no bacteria were detected in the bloodstreams, bacteria that colonized and proliferated persistently for 3 weeks were found in the kidneys. All mice given lethal injections died within 7 days, and large numbers of bacteria were detected in the bloodstreams, spleens, and kidneys. The first peaks of IFN-gamma, TNF, and IL-6 were observed in the bloodstreams and spleens of the mice with nonlethal and lethal infections within 24 h. Thereafter, in the nonlethal cases, IFN-gamma, TNF, and IL-6 peaked again in the spleens and kidneys during the period of maximum growth of bacteria in the kidneys, although only IL-6 was detected in the sera. In contrast, in the case of lethal infection, the titers of IFN-gamma and IL-6 in the sera and TNF in the kidneys peaked before death. Effects of in vivo administration of monoclonal antibodies (MAbs) against IFN-gamma and TNF on the fates of S. aureus-infected mice were studied. In the nonlethal infections, anti-TNF alpha (anti-TNF-alpha) MAb-treated mice, but not anti-IFN-gamma MAb-treated mice, died as a result of worsening infection, suggesting that endogenous TNF plays a protective role in host resistance to S. aureus infection. In the mice that received lethal doses, injection of anti-TNF-alpha MAb accelerated death. However, although injection of anti-IFN-gamma MAb inhibited host resistance of the infected mice early in infection, most of the animals survived the lethal infection by injection of anti-IFN-gamma MAb, suggesting that endogenous IFN-gamma plays a detrimental role in S. aureus infection. Thus, this study demonstrated that IFN-gamma and TNF play different roles in S. aureus infection.
The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain
Authors: Potempa J , Pike R , Travis J
Published: January 2020

Porphyromonas gingivalis contains high concentrations of numerous cysteine proteinases with trypsin-like activity which have been implicated as important virulence factors in adult-onset periodontitis. We have analyzed the subfractions of six P. gingivalis strains for the presence of arginine-X- and lysine-X-specific proteinases (Arg-gingipain [RGP] and Lys-gingipain [KGP]) previously purified from P. gingivalis H66. Western blot (immunoblot) analysis using antibodies produced against RGP and the N-terminal peptides of RGP or the catalytic subunit of KGP indicated that these enzymes are synthesized by the strains studied and exist as multiple molecular mass species. The major forms of RGP were identified as 110-, 95-, 70- to 90-, and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinins, with or without an added membrane anchorage peptide. The other forms are single-chain enzymes. While the 95- and 50-kDa RGP were found predominantly in culture medium, the 110- and 70- to 90-kDa forms associated with membranous fractions of the bacteria. The predominant form of KGP in all strains was a complex of the 60-kDa catalytic domain with hemagglutinins, and vesicle- and membrane-associated KGP was about 15 kDa larger than the 105-kDa enzyme present in culture media. These data explain the apparent complexity of P. gingivalis proteinases and indicate that in all strains tested there are two identical enzymes, one with arginine-X specificity and the other with lysine-X specificity, which, working in concert, are responsible for the trypsin-like activity associated with this bacterium.
Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide
Authors: Pérez-Pérez G I , Shepherd V L , Morrow J D , Blaser M J
Published: January 2020

The mechanism by which Helicobacter pylori, which has little or no invasive activity, induces gastric-tissue inflammation and injury has not been well characterized. We have previously demonstrated that water-extracted proteins of H. pylori are capable of activating human monocytes by a lipopolysaccharide (LPS)-independent mechanism. We have now compared activation of macrophages by purified LPS from H. pylori and from Escherichia coli. LPS was prepared by phenol-water extraction from H. pylori 88-23 and from E. coli O55. THP-1, a human promyelomonocytic cell line, and macrophages derived from rat bone marrow each were incubated with the LPS preparations, and cell culture supernatants were assayed for production of tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), and nitric oxide. THP-1 cells showed maximal activation by the LPS molecules after cell differentiation was induced by phorbol 12-myristate 13-acetate. Maximal TNF-alpha and PGE2 production occurred by 6 and 18 h, respectively, in both types of cells. In contrast, NO was produced by rat bone marrow-derived macrophages only and was maximal at 18 h. The minimum concentration of purified LPS required to induce TNF-alpha, PGE2, and NO responses in both types of cells was 2,000- to 30,000-fold higher for H. pylori than for E. coli. Purified LPS from three other H. pylori strains with different polysaccharide side chain lengths showed a similarly low level of activity, and polymyxin B treatment markedly reduced activity as well, suggesting that activation was a lipid A phenomenon. These results indicate the low biological activity of H. pylori LPS in mediating macrophage activation.
Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen
Authors: Gonzalez C T , Maheswaran S K , Murtaugh M P
Published: January 2020

An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium.
Protective immunity induced by Bacillus anthracis toxin-deficient strains
Authors: Pezard C , Weber M , Sirard J C , Berche P , Mock M
Published: January 2020

The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). Six mutant strains that are deficient in the production of one or two of these toxin components have been previously constructed and characterized (C. Pezard, E. Duflot, and M. Mock, J. Gen. Microbiol. 139:2459-2463, 1993). In this work, we examined the antibody response to the in vivo production of PA, LF, and EF in mice immunized with spores of strains producing these proteins. High titers of antibody to PA were observed after immunization with all strains producing PA, while titers of antibodies to EF and LF were weak in animals immunized with strains producing only EF or LF. In contrast, immunization with strains producing either PA and EF or PA and LF resulted in an increased antibody response to EF or LF, respectively. The differing levels of protection from a lethal anthrax challenge afforded to mice immunized with spores of the mutant strains not only confirm the role of PA as the major protective antigen in the humoral response but also indicate a significant contribution of LF and EF to immunoprotection. We observed, however, that PA-deficient strains were also able to provide some protection, thereby suggesting that immune mechanisms other than the humoral response may be involved in immunity to anthrax. Finally, a control strain lacking the toxin-encoding plasmid was unable to provide protection or elicit an antibody response against bacterial antigens, indicating a possible role for pXO1 in the survival of B. anthracis in a host.
Expression of surface hydrophobic proteins by Candida albicans in vivo
Authors: Glee P M , Sundstrom P , Hazen K C
Published: January 2020

Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed.
Endogenous interleukin-12 is involved in resistance to Brucella abortus infection
Authors: Zhan Y , Cheers C
Published: January 2020

Protective immunity against Brucella abortus is mediated by acquired cellular resistance, with gamma interferon (IFN-gamma)-producing T cells playing a key role. Interleukin-12 (IL-12) is a cytokine that has a profound effect on the induction of IFN-gamma-producing Th1 and NK cells. Here we report that depletion of endogenous IL-12 before infection of mice significantly exacerbated brucella infection. IL-12-depleted mice also had reduced splenomegaly resulting from infection and showed a decrease in percentage and absolute numbers of macrophages compared with those in control infected mice. Furthermore, spleen cells from IL-12-depleted mice had a reduced ability to produce nitrite, a product of activated macrophages. This could be the result of the low production of IFN-gamma by splenic T cells observed in the IL-12-depleted mice. The mechanism whereby IL-12 controls antibacterial resistance is discussed.
Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide
Authors: Blake M S , Blake C M , Apicella M A , Mandrell R E
Published: January 2020

Previous evidence from our laboratory suggested that the tight intercellular adhesions between the outer membranes of gonococci displaying the opacity colony phenotype occurred because Opa proteins expressed on one gonococcus adhered to the lipooligosaccharide (LOS) of the opposing bacterium (M.S. Blake, p. 51-66, in G. G. Jackson and H. Thomas, ed., The Pathogenesis of Bacterial Infections, 1985, and M. S. Blake and E. C. Gotschlich, p. 377-400, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, 1986). A noncompetitive inhibition assay used previously to determine the carbohydrate structures recognized by the major hepatic asialoglycoprotein receptor was modified to determine the gonococcal LOS structures that bind Opa proteins (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). The LOS carbohydrates used in these assays were LOS structures purified from pyocin LOS mutants of Neisseria gonorrhoeae 1291 described by K. C. Dudas and M. A. Apicella (Infect. Immun. 56:499-504, 1988) and further characterized by C. M. John et al. (J. Biol. Chem. 266:19303-19311, 1991). Purified gonococcal Opa proteins were incubated with each of the parent and mutant LOS, and the amount of binding of Opa proteins was measured by a direct enzyme-linked immunosorbent assay using the Opa-specific monoclonal antibody 4B12. The affinities of the Opa proteins for each of the LOS were determined indirectly by measuring the concentrations of Opa proteins that noncompetitively inhibited 50% of the binding of LOS-specific monoclonal antibodies. This concentration is inversely proportional to the affinity of the inhibitor (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). Our data suggest that the gonococcal Opa proteins tested had the highest affinity for the Gal beta 1-4GlcNAc residue present on the gonococcal lactoneoseries LOS. This affinity was comparable to that reported for the binding of the major hepatic asialoglycoprotein receptor to glycoconjugates containing terminal galactose and N-acetylgalactosamine (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). After sialylation of the lactoneoseries LOS, presumably on the terminal galactose residue, the interaction with the Opa proteins was ablated. Therefore, the gonococcal Opa-LOS and mammalian epithelial cell asialoglycoprotein receptor-carbohydrate interactions have quite similar specificities.
Immunogenicity of genetically engineered glutathione S-transferase fusion proteins containing a T-cell epitope from diphtheria toxin
Authors: Pillai S , Dermody K , Metcalf B
Published: January 2020

Glutathione S-transferase (GST) has been shown to induce a marginal antibody response in experimental animals as well as partial protection against a number of parasitic worms, including Schistosoma and Fasciola species. The objective of our study was to increase the immunogenicity of GST by adding heterologous T-cell epitopes at the carboxy terminus of the protein. We generated recombinant GST proteins by attaching one or three tandem repeats of a T-cell epitope of CRM197, a nontoxic variant of diphtheria toxin. This T-cell epitope encoding the region of amino acids 366 to 383 of CRM197, when contained in a GST fusion protein and/or after purification as a recombinant peptide, retained the ability to induce a CRM197-specific T-cell response. The fusion protein containing a single T-cell epitope induced a strong T-cell proliferative response to GST and also enhanced anti-GST antibody production in mice. The addition of three repeats of the epitope did not augment the responses when compared with the responses of GST itself. The results suggest that the addition of a single T-cell epitope to a larger protein like GST increases the immunogenicity of the protein.
Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis
Authors: Navikas V , Haglund M , Link J , He B , Lindqvist L , Fredrikson S , Link H
Published: January 2020

Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.
Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice
Authors: Stanley S L , Zhang T , Rubin D , Li E
Published: January 2020

Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.
CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia
Authors: Peterson P K , Gekker G , Hu S , Sheng W S , Anderson W R , Ulevitch R J , Tobias P S , Gustafson K V , Molitor T W , Chao C C
Published: January 2020

This study was carried out to determine the role of CD14 receptors in the uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Treatment of microglial cell cultures with antibodies to CD14 or with soluble CD14 significantly blocked infection by M. tuberculosis H37Rv, suggesting that CD14 receptors could facilitate entry of nonopsonized tubercle bacilli into macrophages within the brain.