bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon
Authors: Heinzel F P , Rerko R M , Ling P , Hakimi J , Schoenhaut D S
Published: January 2020

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.
Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis
Authors: Fletcher H M , Schenkein H A , Macrina F L
Published: January 2020

Porphyromonas gingivalis has been implicated as a contributing etiological agent of adult periodontitis and generalized forms of early-onset periodontitis. Proteases of P. gingivalis may contribute to its pathogenicity by destroying connective tissue as well as inactivating key plasma proteins that might mediate protective host functions. In order to explore this problem, antiserum raised against membrane vesicles of P. gingivalis W83 was used to screen a genomic library of strain W83 constructed by using the lambda DASH vector system. A recombinant phage (lambda 34) expressing a P. gingivalis protease from the library was identified and characterized. Casein substrate zymography of lambda 34 lysates revealed a protease with an apparent molecular mass of 97 kDa. The gene encoding this protease was designated prtH. It was localized to a 3.7-kb HindIII-BamHI fragment and specified an enzyme which hydrolyzed the human C3 complement protein under defined conditions. The nucleotide sequence of this 3.7-kb fragment was determined, and one 2.9-kb open reading frame (992 amino acids) corresponding to a 110-kDa protein was detected, suggesting it might be a precursor of the 97-kDa active protease. prtH is not similar to any previously cloned protease gene from P. gingivalis.
Effects of a Porphyromonas gingivalis infection on inflammatory mediator response and pregnancy outcome in hamsters
Authors: Collins J G , Windley H W , Arnold R R , Offenbacher S
Published: January 2020

This study examines the effects of various localized, nondissemination challenges of Porphyromonas gingivalis on inflammatory mediator production and pregnancy outcome in the golden hamster. Live or heat-killed (HK) organisms were inoculated into a previously implanted subcutaneous tissue chamber on the 8th day of gestation to determine the effects on fetal weight, viability, and resorption. In one group of animals, HK organisms were inoculated prior to mating to determine the effects of previous exposure on day-8 gestational challenges. Chamber contents were assayed at 1 and 5 days after challenge for prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-alpha). All P. gingivalis challenges caused a significant increase in chamber PGE2 and TNF-alpha at P < 0.01 in the following order of potency: HK < Live < HK+Live. For example, following the HK+Live challenge, PGE2 levels increased from 4.7 pg/ml at baseline to 362 pg/ml at day 5 and TNF-alpha increased from 26.4 pg/ml to 724 pg/ml at day 5. The same order of potency of the various challenges was maintained with regard to the toxic effects of P. gingivalis on pregnancy outcome. For the HK+Live challenge, fetal weight was decreased 24%; embryolethality increased to 26.5% and the percent fetal resorption increased to 10.6% compared with control animal levels. There was a statistically significant association between increasing levels of both PGE2 and TNF-alpha and fetal growth retardation and embryolethality at P < 0.001. These data suggest that infections with gram-negative periodontal pathogens can elicit adverse pregnancy outcomes and that the levels of PGE2 and TNF-alpha produced as a result of challenge are associated with the severity of fetal effect.
Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells
Authors: Zhang Q , Young T F , Ross R F
Published: January 2020

Glycolipid receptors for Mycoplasma hyopneumoniae attachment were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, and monosialoganglioside GM3. No binding to sphingomyelin, cerebroside, lactosyl ceramide, ceramide trihexoside, monosialogangliosides GM1 and GM2, disialogangliosides (GD1a, GD1b, and GD3), trisialoganglioside (GT1b), cholesterol, cholesterol sulfate, palmitic acid, tripalmitin, or cholesteryl palmitate was detected. Total lipids extracted from cilia of the swine respiratory epithelium, the natural targets of M. hyopneumoniae infection, were also separated on TLC plates and overlaid with mycoplasmas. M. hyopneumoniae bound specifically to three ciliary glycolipids identified as La, Lb, and Lc. Binding to Lc was stronger than to La and Lb. All three lipids were believed to be sulfated glycolipids, as determined by laminin binding and staining with azure A. Lc was identified as a putative sulfatide because it has a mobility similar to that of authentic sulfatide and comigrated with sulfatide on TLC plates. Laminin bound to La, Lb, and Lc and produced dose-dependent inhibition of adherence of the mycoplasma to the three ciliary receptors. Binding of the mycoplasma to sulfatide, La, Lb, and Lc was partially inhibited by dextran sulfate, heparin, fucoidan, mucin, and chondroitin sulfate B. These substances blocked the adherence of M. hyopneumoniae to cilia and ciliated cells as shown in a previous study (Q. Zhang, T. F. Young, and R. F. Ross, Infect. Immun. 62:1616-1622, 1994). These results indicate that La, Lb, and Lc are the major native receptors for M. hyopneumoniae adherence to ciliated cells.
Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein conjugate vaccines
Authors: Szu S C , Taylor D N , Trofa A C , Clements J D , Shiloach J , Sadoff J C , Bryla D A , Robbins J B
Published: January 2020

To improve its immunogenicity for children and adults and to make it suitable for routine immunization of infants against typhoid fever, the capsular polysaccharide of Salmonella typhi (Vi) was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa. The conjugates elicited higher levels of antibodies (micrograms per milliliter of serum) in mice and in guinea pigs than did Vi and, unlike Vi alone, elicited booster antibody responses in both species. In adult volunteers, Vi-LT-B and Vi-rEPA, respectively, elicited higher levels of antibodies than Vi alone after the first injection (4.74 versus 1.77 and 4.91 versus 1.77; P < 0.005) and 26 weeks later (2.32 and 2.69 versus 0.54; P < 0.04); a second injection of the conjugates did not elicit a booster response of Vi antibodies. None of the 51 vaccinees had fever or significant local reactions. Vi-rEPA elicited slightly higher levels of Vi antibodies than did Vi-LT-B at all intervals after injection, but these differences were not significant. Each conjugate elicited antibodies to its carrier protein. The antibody responses elicited in adults by Vi bound to LT-B and rEPA are similar to those of other polysaccharide-protein conjugates. These conjugates promise to be an improved Vi vaccine. Studies of Vi conjugates with adults and infants in areas where typhoid is endemic are planned.
Cytotoxicity of Pseudomonas aeruginosa internal lectin PA-I to respiratory epithelial cells in primary culture
Authors: Bajolet-Laudinat O , Girod-de Bentzmann S , Tournier J M , Madoulet C , Plotkowski M C , Chippaux C , Puchelle E
Published: January 2020

Pseudomonas aeruginosa is the most important bacterial pathogen associated with chronic airway infection, especially in cystic fibrosis. We addressed the question of whether the galactophilic internal lectin of P. aeruginosa (PA-I) could represent a virulence factor for the respiratory epithelium. PA-I lectin was localized in all the bacteria of P. aeruginosa ATCC 33347 as determined by immunofluorescence staining. We investigated the dose-dependent effect of P. aeruginosa PA-I lectin on the growth, the ciliary beating frequency, and the morphology of human respiratory cells in primary culture of nasal polyps collected from non-cystic fibrosis patients. PA-I lectin significantly (P < 0.01) inhibited the growth of respiratory cells at a concentration of > or = 10 micrograms/ml. The percentage of active ciliated cell surface of the cultures decreased significantly (P < 0.05) at a PA-I lectin concentration of 50 micrograms/ml. Exposed to a low concentration of PA-I lectin (10 micrograms/ml), respiratory epithelial cells showed intracytoplasmic vacuoles when examined by light and transmission electron microscopy. At a higher concentration of PA-I lectin (100 micrograms/ml), major cell damage and severe epithelial shedding occurred. These results demonstrate that the P. aeruginosa internal PA-I lectin has a dose-dependent cytotoxic effect on respiratory epithelial cells in vitro. The P. aeruginosa PA-I lectin may represent a virulence factor by contributing to the respiratory epithelial damage during P. aeruginosa respiratory infections.
Naturally acquired human antibodies which recognize the first epidermal growth factor-like module in the Plasmodium falciparum merozoite surface protein 1 do not inhibit parasite growth in vitro
Authors: Chappel J A , Egan A F , Riley E M , Druilhe P , Holder A A
Published: January 2020

Merozoite surface protein 1, one of the major surface proteins of the invasive blood stage of the malaria parasite, is a prime candidate for the development of a vaccine against the human disease. Previously, monoclonal antibodies which both inhibited the growth of Plasmodium falciparum in vitro and bound to the first of two epidermal growth factor-like modules located near the carboxy terminus of the protein had been identified. In this study, we have used affinity chromatography on a recombinant fusion protein corresponding to the first epidermal growth factor-like module in P. falciparum merozoite surface protein 1 to prepare antibody induced by natural infection. The antibody was purified from the total immunoglobulin G fraction of adult West African donors, shown to passively confer immunity against falciparum malaria. Such affinity-purified antibodies were shown to recognize the native protein by a number of separate criteria and to block the binding of an inhibitory monoclonal antibody, but they failed to inhibit parasite invasion in an in vitro growth assay. These results indicate that antibody alone is not sufficient to interfere with erythrocyte invasion.
Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster
Authors: Collins J G , Smith M A , Arnold R R , Offenbacher S
Published: January 2020

This report describes the effects of two gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) preparations on hamster pregnancy outcome variables. Single intravenous challenges with Escherichia coli and Porphyromonas gingivalis LPS on day 8 of pregnancy produced dose-dependent effects on fetal weight malformation and fetal resorption with E. coli LPS having potent embryolethal effects. Premating maternal exposure to P. gingivalis produced embryolethal effects similar to those of E. coli. These data suggest that maternal exposure to P. gingivalis LPS prior to and during pregnancy can induce deleterious effects on the developing fetus.
Arginine aminopeptidase, an integral membrane protein of the Cryptosporidium parvum sporozoite
Authors: Okhuysen P C , DuPont H L , Sterling C R , Chappell C L
Published: January 2020

Cryptosporidium parvum oocysts were studied for the expression of aminopeptidase by using amino acids bound to the synthetic fluorescent substrate 7-amino-4-trifluoromethyl coumarin. After 1 h of incubation, intact oocysts showed no activity; however, homogenization and solubilization with Triton X-114 followed by phase separation yielded a 22-fold increase in aminopeptidase activity in the detergent fraction. With arginyl-6-amino-2-styrylquinoline as a substrate, aminopeptidase activity was observed in permeabilized oocysts and freshly excysted sporozoites but not on intact oocysts or empty oocyst membranes after excystation. These results suggest that C. parvum expresses an arginine aminopeptidase that is an integral protein of the sporozoite membrane.
Soluble peptidoglycan-induced monokine production can be blocked by anti-CD14 monoclonal antibodies and by lipid A partial structures
Authors: Weidemann B , Brade H , Rietschel E T , Dziarski R , Bazil V , Kusumoto S , Flad H D , Ulmer A J
Published: January 2020

We have investigated the interaction of soluble peptidoglycan (sPG), in comparison with lipopolysaccharide (LPS), with human mononuclear cells (MNC) by determining the capacity of sPG to induce interleukin-6 (IL-6) and IL-1 release. In addition, we investigated the modulation of their interaction by anti-CD14 monoclonal antibody and by partial structures of LPS. We found that sPG, like LPS, was able to induce IL-6 and IL-1 production by MNC. However, dose-response experiments revealed that at least 3,000 ng of sPG per ml was necessary for induction, whereas the optimal LPS concentration was 1 ng/ml. Anti-CD14 monoclonal antibody reduced sPG- and LPS-induced IL-6 and IL-1 production. Moreover, partial structures of LPS were able to reduce monokine production induced by sPG and LPS. We conclude that sPG constitutes, like LPS, an inflammatory cytokine inducer and that CD14 is involved in the activation of human monocytes not only by LPS but also by sPG.
Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei
Authors: Scheller L F , Wirtz R A , Azad A F
Published: January 2020

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.
Structural characteristics of polysaccharides that induce protection against intra-abdominal abscess formation
Authors: Tzianabos A O , Onderdonk A B , Zaleznik D F , Smith R S , Kasper D L
Published: January 2020

Bacteroides fragilis is the anaerobe most commonly isolated from clinical cases of intra-abdominal sepsis. In a rodent model of this disease process, intraperitoneal injection of the capsular polysaccharide complex (CPC) from B. fragilis provokes abscess formation, while subcutaneous administration of this complex confers protection against B. fragilis-induced intra-abdominal abscesses. The CPC consists of two discrete polysaccharides, polysaccharides A and B (PS A and PS B), each possessing oppositely charged structural groups critical to the ability of these carbohydrates to induce the formation of abscesses. Other bacterial polysaccharides that possess oppositely charged groups (such as the group antigen or capsular polysaccharide from Streptococcus pneumoniae type 1 strains) also exhibited potent abscess-inducing capabilities. We report here that positively and negatively charged groups on polysaccharides are also essential for inducing protection against abscess formation. Vaccination of rats with B. fragilis PS A, PS B, or the S. pneumoniae type 1 capsule protected against intra-abdominal abscesses subsequent to intraperitoneal challenge with each of these polysaccharides. Chemical conversion of the free amino or carboxyl groups on PS A to uncharged N-acetyl or hydroxymethyl groups, respectively, abrogated the ability of this polymer to confer protection against polysaccharide-mediated abscess formation. Adoptive transfer of splenic T cells from polysaccharide-vaccinated rats to naive animals demonstrated that T cells mediated this protective activity. T cells transferred from animals vaccinated with a polysaccharide repeating unit (Salmonella typhi Vi antigen) that normally contains one carboxyl group but was chemically converted to a polymer that possesses both free amino and carboxyl groups (accomplished by de-N-acetylating the Vi antigen) protected naive T-cell recipients against polysaccharide-induced abscesses. These results demonstrate that a distinct structural motif associated with the B. fragilis polysaccharides is necessary for induction of protective immunity against abscess formation associated with intra-abdominal sepsis. However, protection is not antigen specific in a traditional sense. Rather, the protective ability of these structurally dissimilar polysaccharides is conferred by, and perhaps specific for, a motif of oppositely charged groups.
Primate antibody response to immunotoxin: serological and computer-aided analysis of epitopes on a truncated form of Pseudomonas exotoxin
Authors: Roscoe D M , Jung S H , Benhar I , Pai L , Lee B K , Pastan I
Published: January 2020

NLysPE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) in which domain Ia (amino acids 1 to 252) and part of domain Ib (365 to 380) are deleted and an 11-amino-acid N-terminal peptide is added. LMB-1 is an immunotoxin in which the monoclonal antibody B3 is coupled to NLysPE38 near its N terminus. LMB-7 is a single-chain immunotoxin in which the Fv fragment of B3 is fused to PE38. To identify the antigenic regions of PE38, 12 polyclonal serum samples from monkeys immunized with the immunotoxins LMB-1 (six monkeys) and LMB-7 (six monkeys) were tested for their reactivity to a panel of 120 synthetic, overlapping peptides representing the amino acid sequence of NLysPE38. The antibody responses to peptides were similar among the 12 serum specimens, identifying several major immunodominant B-cell epitopes. Predominant reactivity was seen in six locations: amino acids 272 to 287, 341 to 359, 504 to 516, 540 to 564, and 573 to 591 and the C-terminal amino acids 591 to 613. The sera did not react with approximately 75% of the peptides. Furthermore, a computer-aided analysis was done to predict the immunologically relevant areas and revealed the same antigenic regions defined by serum reactivity to peptides. Competition enzyme-linked immunosorbent assays and neutralization assays were performed with domain II, III, or III plus Ib of PE38 and confirmed the immunodominance of domain III. To analyze the role of specific amino acids in antibody binding, individual amino acids of PE38 with large accessible surface areas were altered by site-directed mutagenesis. These results also show that the predicted areas of immunogenicity agree with the reactivity of the anti-PE38 antibodies to peptides and to the mutants of PE.
A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis
Authors: Behrens A , Heller M , Kirchhoff H , Yogev D , Rosengarten R
Published: January 2020

A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.
Virulence studies of Aspergillus nidulans mutants requiring lysine or p-aminobenzoic acid in invasive pulmonary aspergillosis
Authors: Tang C M , Smith J M , Arst H N , Holden D W
Published: January 2020

To identify steps in fungal intermediary metabolism required by Aspergillus spp. during invasive pulmonary aspergillosis, we have developed murine models involving Aspergillus nidulans as the inoculum. The advantages of using A. nidulans over Aspergillus fumigatus or Aspergillus flavus, which are the most common agents of clinical disease, are the well-understood genetics of A. nidulans and a large range of mutants of this species which are affected in a variety of metabolic pathways. Comparison of the virulence of A. nidulans strains carrying mutations which block the biosynthesis of lysine (lysA2) and p-aminobenzoic acid (pabaA1) shows that lysA2 strains have reduced virulence while pabaA1 strains are entirely nonpathogenic. The pathogenicity of pabaA1 strains can be restored by supplementing the drinking water of animals with p-aminobenzoic acid. The results indicate that the availability of lysine in the lung is limited, and p-aminobenzoic acid is probably not available at all. Thus, models of invasive pulmonary aspergillosis involving A. nidulans can be used to identify metabolic pathways that may be essential for the pathogenicity of A. fumigatus, the predominant pathogenic species, suggesting potential new targets for antifungal therapy.
Live Brucella spp. fail to induce tumor necrosis factor alpha excretion upon infection of U937-derived phagocytes
Authors: Caron E , Peyrard T , Köhler S , Cabane S , Liautard J P , Dornand J
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) plays a central role in activation of first-line defenses of a host against foreign organisms. To determine whether Brucella infection modulated TNF-alpha production, we measured the biological activity of this cytokine in supernatants of U937 cell-derived macrophages and of fresh human monocytes infected with Brucella spp. Neither the smooth nor rough Brucella strains used induced any measurable TNF-alpha excretion upon infection. On the contrary, as reported before for other gram-negative bacteria, phagocytosis of nonpathogenic Escherichia coli was followed by a rapid and transient induction of TNF-alpha release, suggesting an involvement of this cytokine in some autocrine process. As expected, the Brucella strains tested survived and/or multiplied within U937-derived macrophages, whereas E. coli was rapidly eliminated after phagocytosis. Immunoglobulin G opsonization of E. coli strains enhanced their intracellular killing and strongly potentiated TNF-alpha secretion. Immunoglobulin G opsonization of Brucella strains, in contrast, did not lead to TNF-alpha production, although their rate of intracellular multiplication was reduced. Killed brucellae, however, promoted a significant excretion of TNF-alpha from U937-derived macrophages into cell culture supernatants. We finally demonstrated that pretreatment of U937-derived macrophages with exogenous TNF-alpha significantly inhibited intracellular multiplication of Brucella spp. These results and experiments performed on fresh human monocytes or with isolated lipopolysaccharide (LPS) showed that (i) differences in TNF-alpha production observed during macrophage infection by Brucella spp. and E. coli were not due to differences in LPS structure but resulted from active inhibition of TNF-alpha production by a specific process linked to Brucella spp. and (ii) the capacity of Brucella spp. to use pathways avoiding TNF-alpha production during infection may be considered a major attribute of virulence.
Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis
Authors: Hodgson A L , Tachedjian M , Corner L A , Radford A J
Published: January 2020

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.
Pseudomonas aeruginosa selective adherence to and entry into human endothelial cells
Authors: Plotkowski M C , Saliba A M , Pereira S H , Cervante M P , Bajolet-Laudinat O
Published: January 2020

The pathogenesis of Pseudomonas aeruginosa disseminated infections depends on bacterial interaction with blood vessels. We have hypothesized that in order to traverse the endothelial barrier, bacteria would have to adhere to and damage endothelial cells. To test this hypothesis, we studied the adherence to human endothelial cells in primary culture of the piliated P. aeruginosa strain PAK and of two isogenic nonpiliated strains: PAK/p-, which carries a mutation in the pilin structural gene, and PAK-N1, a mutant defective in the regulatory rpoN gene. PAK adhered significantly more than did the pilus-lacking strains. P. aeruginosa was also taken up by endothelial cells, as determined by quantitative bacteriologic assays and by transmission electron microscopy. This internalization of P. aeruginosa seems to be a selective process, since the piliated strain was taken up significantly more than the nonpiliated bacteria and the avirulent Escherichia coli DH5 alpha, even following bacterial centrifugation onto the cell monolayers. A significant fraction of the internalized P. aeruginosa PAK was recovered in a viable form after 6 h of residence within endothelial cells. Progressive endothelial cell damage resulted from PAK intracellular harboring, as indicated by the release of lactate dehydrogenase. An increasing concentration of PAK cells was recovered from the extracellular medium with time, suggesting that ingested bacteria were released from endothelial cells and multiplied freely. We speculate that in vivo the ability of some P. aeruginosa strains to resist intracellular residence would afford protection from host defenses and antibiotics and that the release of viable bacteria into bloodstream may represent a central feature of the pathogenesis of bacteremia in compromised patients.
Humoral immune response to plasmid protein pgp3 in patients with Chlamydia trachomatis infection
Authors: Comanducci M , Manetti R , Bini L , Santucci A , Pallini V , Cevenini R , Sueur J M , Orfila J , Ratti G
Published: January 2020

We identified, by two-dimensional electrophoretic analysis and microsequencing, a protein of Chlamydia trachomatis elementary bodies which corresponds to the polypeptide (pgp3) encoded by open reading frame 3 (ORF3). Amino acid analysis showed that the first residue (Gly) found in the native protein is the one encoded by the second ORF3 codon, implying a typical bacterial removal of the first Met residue. Relatively large amounts of recombinant pgp3 (r-pgp3) in a stable, water-soluble form were obtained by overexpressing ORF3 in Escherichia coli and purifying the product from periplasmic extracts under nondenaturing conditions. These r-pgp3 preparations allowed specific detection of anti-pgp3 antibodies by enzyme-linked immunosorbent assay. Analysis of a group of 170 sera from healthy blood donors and from patients who were seropositive or -negative for C. trachomatis and Chlamydia pneumoniae showed that an immune response to pgp3 occurs in the majority (ca. 81%) of patients with sexually transmitted diseases who are seropositive for C. trachomatis and generally correlates with the response to cell surface antigens. No reaction between r-pgp3 and C. pneumoniae-positive sera was detected.
Ability of Staphylococcus aureus coagulase genotypes to resist neutrophil bactericidal activity and phagocytosis
Authors: Aarestrup F M , Scott N L , Sordillo L M
Published: January 2020

This study investigated the functional capabilities of neutrophils against different Staphylococcus aureus genotypes isolated from cows with mastitis. Six strains of S. aureus were chosen for use in the study, two with a common genotype, two with an intermediate genotype, and two with a rare genotype. The interaction between bacteria and neutrophils was measured by phagocytosis and bactericidal effect. The average percent killing of bacteria was lowest (40.0%) with strains belonging to the most common genotype, medium (50%) with strains belonging to the intermediate type, and highest (64.2%) with strains belonging to the rare type (P < or = 0.001). Statistically significant differences (P < or = 0.001) in the numbers of phagocytized bacteria were also found between the most prevalent type (6.27 bacteria per cell) and the other two types (intermediate type, 9.26/cell; rare type, 10.5/cell). These findings suggest that one of the reasons for the variation in prevalence of different genotypes of S. aureus in the mammary gland is due to the superior ability of some types to resist phagocytosis and/or killing by bovine neutrophils.
Differentiation of Pseudomonas aeruginosa pili based on sequence and B-cell epitope analyses
Authors: Castric P A , Deal C D
Published: January 2020

The nucleotide sequences of three previously undescribed Pseudomonas aeruginosa pilin structural genes are presented. Comparisons of deduced pilin primary structure and flanking DNA sequence allowed placement of these and six previously published sequences into one of two groups. Epitope mapping, using overlapping immobilized peptides representing the pilin primary structure, with antipilin monoclonal antibodies revealed several B-cell determinants grouped near the carboxyl terminus of P. aeruginosa 1244 pilin. One determinant was found to reside near the pilin constant region. These determinants were found associated with the pili of 31 of 95 P. aeruginosa clinical isolates.
M protein mediates streptococcal adhesion to HEp-2 cells
Authors: Wang J R , Stinson M W
Published: January 2020

Streptococcus pyogenes adheres to human epithelial cells in vitro and in vivo. To identify adhesins, cell wall components were extracted from S. pyogenes M6 with alkali or by treatment with mutanolysin and lysozyme. HEp-2 cells were incubated with extracts of S. pyogenes M6 and then analyzed by Western blot (immunoblot) assays, using antibodies to S. pyogenes. Only one streptococcal component (62 kDa) was bound to HEp-2 cells and was identified serologically as M6 protein. Experiments with pepsin-cleaved fragments of M protein indicated that the binding site was located at the N-terminal half of the molecule. M protein was bound selectively to two trypsin-sensitive surface components, 97 and 205 kDa, of HEp-2 cells on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. Tritium-labeled lipoteichoic acid bound to different HEp-2 cell components, 34 and 35 kDa, in a parallel experiment, indicating that lipoteichoic acid was not complexed with M protein and does not mediate M-protein binding. The four HEp-2 components were unrelated to fibronectin since they did not react with specific antibodies. An M-protein-deficient (M-) strain of streptococcus (JRS75), grown in chemically defined medium, showed 73% less adhesion activity to HEp-2 monolayers than an M+ strain (JRS4). Streptococcal adhesion was insensitive to competitive inhibition by selected monosaccharides. These results indicate that M protein binds directly to certain HEp-2 cell membrane components and mediates streptococcal adhesion.
Cellular and humoral immune responses to well-defined blood stage antigens (major merozoite surface antigen) of Plasmodium falciparum in adults from an Indian zone where malaria is endemic
Authors: Kabilan L , Sharma V P , Kaur P , Ghosh S K , Yadav R S , Chauhan V S
Published: January 2020

Conserved and variant regions of two blood stage vaccine candidate antigens of Plasmodium falciparum, merozoite surface antigen (MSA-1) and ring-infected erythrocyte surface antigen (Pf155/RESA), have been shown to be immunogenic. However, the relative immunogenicity of these immunogens in different populations has not been studied. The conserved N-terminal region of MSA-1 was investigated for its immunogenicity by studying cellular (T cell) and humoral (B cell) immune responses in P. falciparum-primed individuals, living in malaria-hyperendemic areas (Orissa State, India), where malaria presents an alarming situation. MSA-1-derived synthetic peptides contained sequences that activated T cells to proliferate and release gamma interferon in vitro. There was considerable variation in the responses to different peptides. However, the highest responses (51% [18 of 35] by proliferation and 34% [12 of 35] by gamma interferon release) were obtained with a synthetic hybrid peptide containing sequences from conserved N- and C-terminal repeat regions of MSA-1 and Pf155/RESA, respectively. Antibody reactivities in an enzyme immunoassay of plasma samples from these donors to different peptides used for T-cell activation were heterogeneous. In general, there was poor correlation between DNA synthesis and either gamma interferon release or antibody responses in individual donors, underlining the importance of examining several parameters of T-cell activation to assess the total T-cell responsiveness of a study population to a given antigen. However, the results from our studies suggest that synthetic constructs containing sequences from the N- and C-terminal regions of MSA-1 and Pf155/RESA representing different erythrocytic stages of the P. falciparum parasite are more immunogenic in humans living in malaria-hyperendemic areas of India who have been primed by natural infection.
Regulation of Clostridium perfringens alpha-toxin-activated phospholipase C in rabbit erythrocyte membranes
Authors: Sakurai J , Ochi S , Tanaka H
Published: January 2020

The rapid phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin was stimulated by AlF4- in rabbit erythrocyte membranes. GTP[gamma S] [guanosine 5'-O-(3-thiotriphosphate)] stimulated the rapid 1,2-diacylglycerol formation and inositol 1,4,5-trisphosphate release induced by the toxin. On the other hand, treatment of erythrocyte lysates with phorbol 12-myristate 13-acetate (PMA) resulted in inhibition of toxin-induced PA production, and long-term PMA or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) treatment of the lysates led to stimulation of PA formation. Furthermore, treatment of erythrocytes with the toxin caused an increase of protein kinase C activity in membrane fractions. The results suggest that toxin-induced PA formation is mediated by endogenous phospholipase C regulated through GTP-binding protein and protein kinase C in rabbit erythrocytes.
Lack of effect of Candida albicans mannan on development of protective immune responses in experimental murine candidiasis
Authors: Garner R E , Domer J E
Published: January 2020

Candida albicans mannoprotein (MAN) administered to mice before or during immunization with viable C. albicans downregulates MAN-specific delayed hypersensitivity. In the experiments reported here we determined the effect of MAN downregulation on protective immunity in minimally immunized mice, i.e., mice exposed to C. albicans either intradermally or intragastrically, and in maximally immunized mice, i.e., mice immunized by a combination of intradermal and intragastric exposure, in experimental systemic candidiasis. MAN suppression did not induce statistically significant alterations in the protective responses in experimental candidiasis, although 8 of 12 groups of mice treated with MAN had fewer CFU of C. albicans in their kidneys than their non-MAN-treated counterparts. The results emphasize the lack of correlation of delayed hypersensitivity with protection in candidiasis and suggest that MAN may contain epitopes involved in the protective response.
Immunization against Porphyromonas gingivalis inhibits progression of experimental periodontitis in nonhuman primates
Authors: Persson G R , Engel D , Whitney C , Darveau R , Weinberg A , Brunsvold M , Page R C
Published: January 2020

Periodontitis is a common infectious disease in which the attachment tissues of the teeth and their alveolar bone housing are destroyed, resulting in tooth loss. The gram-negative anaerobic microorganism Porphyromonas gingivalis has been closely linked to severe forms of the disease. We show for the first time that immunization of the primate Macaca fascicularis with killed P. gingivalis in Syntex Adjuvant Formulation-M inhibits progression of periodontal tissue destruction.
Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis
Authors: Filler S G , Ibe B O , Ibrahim A S , Ghannoum M A , Raj J U , Edwards J E
Published: January 2020

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.
Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A
Authors: West S E , Kaye S A , Hamood A N , Iglewski B H
Published: January 2020

In Pseudomonas aeruginosa, production of exotoxin A, an ADP-ribosyltransferase, is a complex and highly regulated process. Two positively acting regulatory genes, regA and regB, have been cloned and characterized. To identify additional exotoxin A regulatory genes, we have characterized four N-methyl-N'-nitro-N-nitrosoguanidine-generated mutants of P. aeruginosa PA103 which are deficient in exotoxin A production. These mutants (PA103-8, PA103-15, PA103-16, and PA103-19) do not accumulate intracellular exotoxin A and are not complemented by the cloned toxA or regAB genes. This observation indicates that the lesion(s) in the mutants is probably in an exotoxin A regulatory gene(s) and is not in the genes for secretion of exotoxin A or in the toxA or regAB genes. To assess the effect of the putative regulatory mutations on the toxA and regAB genes, we compared the activity of the toxA and regAB promoters in the mutant and parental strains using plasmids containing the genes for beta-galactosidase or chloramphenicol acetyltransferase under the control of either the toxA or the regAB promoter. The toxA promoter-beta-galactosidase fusion plasmid could not be maintained in PA103-8. beta-Galactosidase expression driven by the toxA promoter was absent in the mutant PA103-19 and occurred at a low level, which was not repressed by iron in mutants PA103-15 and PA103-16. The regAB genes are temporally controlled by two promoters, P1 and P2. In all four mutants, regAB P1 promoter activity was reduced; however, expression under the control of the regAB P2 promoter was normal. These observations suggest the existence of one or more regulatory genes which directly affect expression of both the toxA and the regAB P1 promoters.
Spatial arrangement of gene products of the P1 operon in the membrane of Mycoplasma pneumoniae
Authors: Layh-Schmitt G , Herrmann R
Published: January 2020

The spatial arrangement of three P1 operon-encoded proteins--attachment protein P1 (ORF5 gene product) and the ORF6-derived proteins of 40 and 90 kDa--in the membrane of Mycoplasma pneumoniae M129 was investigated by nearest-neighbor analysis. For these studies, the homobivalent, thiol-cleavable, and nonmembrane-permeating cross-linking reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used. The cross-linked proteins were isolated by immunoprecipitation with antibodies directed against fusion proteins of selected regions of the 40-kDa, the 90-kDa, or the P1 protein. The individual components of the immunoprecipitated protein complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and immunoblot analysis. This study showed that the P1 protein, the ORF6 gene product, and an unidentified 30-kDa protein were linked to each other in the intact bacterial membrane by the reagent DTSSP, indicating that these proteins are located at a maximal distance of 12 A (1 A = 0.1 nm) on the tip structure of M. pneumoniae.
Role of polymorphonuclear neutrophil leukocytes and their integrin CD11a (LFA-1) in the pathogenesis of severe murine malaria
Authors: Senaldi G , Vesin C , Chang R , Grau G E , Piguet P F
Published: January 2020

Infection of CBA mice with Plasmodium berghei ANKA results in severe malaria, which is characterized by mortality 6 to 10 days after infection and is associated with alterations of the brain microcirculation. These alterations consist of (i) intravascular sequestration of monocytes, (ii) an increase in vascular permeability as documented by Evans blue diffusion, and (iii) microhemorrhages. This syndrome may be due to an increase of production of tumor necrosis factor alpha which upregulates the endothelial expression of ICAM-1 and thus leads to adhesion of CD11a/CD18 (LFA-1)-bearing cells. During severe malaria, we found an important sequestration of the CD11a-bearing polymorphonuclear neutrophil leukocytes (PMN) in the lung but not in the brain. Treatment with a monoclonal antibody (MAb) against PMN, which induces profound neutropenia, prevented mortality and Evans blue diffusion in the brain and the lung, while it unexpectedly increased the occurrence of microhemorrhages. The anti-PMN MAb abolished PMN sequestration in the lung and also partially decreased monocyte sequestration in the brain and the lung. Treatment with an anti-CD11a MAb also prevented mortality, Evans blue diffusion, and PMN and monocyte sequestration. This study shows that PMN contribute to the mortality and the microvascular lesions resulting from severe malaria. This may be due to their CD11a-dependent sequestration in the lung and also to their indirect influence on vascular permeability and the sequestration of monocytes.