bluuebunny/crossref_metadata_2025 huggingface dataset:
Fimbriation, capsulation, and iron-scavenging systems of Klebsiella strains associated with human urinary tract infection
Authors: Tarkkanen A M , Allen B L , Williams P H , Kauppi M , Haahtela K , Siitonen A , Orskov I , Orskov F , Clegg S , Korhonen T K
Published: January 2020

Thirty-two strains of Klebsiella pneumoniae and seven strains of Klebsiella oxytoca isolated from urinary tract infections in elderly adults were analyzed for capsular antigens, iron-scavenging systems, and fimbriation. All strains were capsulated. Twenty-seven different K antigens were identified among the strains, with no particular antigen dominating. All strains produced the iron-scavenging system enterochelin as analyzed by bioassay and DNA hybridization. In contrast, the aerobactin iron-sequestering system was not detected in any of the strains. All strains caused hemagglutination of tannin-treated human erythrocytes and reacted with an anti-type 3 fimbriae antiserum as well as in DNA hybridization with a type 3 fimbria-specific probe, indicating that the Klebsiella strains possessed this fimbrial type. Possession of type 1 fimbriae was analyzed by agglutination tests and by hybridization with DNA probes from two distinct Klebsiella type 1 fimbria gene clusters. Phenotypic expression of the type 1 fimbriae was found in 29 of 32 K. pneumoniae strains, whereas 30 strains reacted with either of the two type 1 fimbrial cluster DNA probes. In K. oxytoca, however, only three of seven strains expressed type 1 fimbriae and reacted with the DNA probes. The type 3 fimbriae were found to bind to a fraction of epithelial cells exfoliated in normal human urine, whereas the type 1 fimbriae bound strongly to urinary slime. No inhibitors of type 3 fimbrial binding were detected in human urine.
Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes
Authors: Yamaguchi R , Matsuo K , Yamazaki A , Takahashi M , Fukasawa Y , Wada M , Abe C
Published: January 2020

The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M. avium. Its gene was cloned by using a previously developed single-probe method and was sequenced. The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500. A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen. To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as beta-galactosidase fusion proteins, using pUR and pURS expression vectors. The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting B- and T-cell epitopes. A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined. The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs. This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity.
Antigenic analysis of the chlamydial 75-kilodalton protein
Authors: Zhong G , Brunham R C
Published: January 2020

Both B- and T-cell immunogenicity of a chlamydial 75-kDa protein was analyzed by using 131 partially overlapped decapeptide homologs of the 75-kDa protein from Chlamydia trachomatis serovar L2. Six rabbit antiserum specimens raised with serovars B, C, and L2 were used to assay the antibody reactivities of the decapeptides. Seventy-five of the 131 decapeptides were recognized by at least one antiserum specimen, and two peptides were found to be immunodominant and surface accessible on native organisms. The same set of decapeptides were cleaved from the pins and tested for their T-cell-stimulating activity in an in vitro proliferation assay. A single decapeptide was able to stimulate proliferation of chlamydial antigen-primed lymph node T cells from BALB/c mice.
Natural killer cells mediate protection induced by a Salmonella aroA mutant
Authors: Schafer R , Eisenstein T K
Published: January 2020

We have previously shown that an avirulent strain of Salmonella typhimurium, SL3235, blocked in aromatic synthesis, confers high levels of resistance to challenge with virulent Salmonella as early as 3 days postvaccination. In the present studies, it was found that immunization with SL3235 resulted in high levels of natural killer (NK) cell activity in the spleens and peritoneal cavities of C3H/HeJ mice, as measured by cytotoxicity against YAC-1 targets. NK cell activity was at its maximum 2 to 4 days after immunization and was ablated by in vivo or in vitro treatment with anti-asialo GM1. In vivo treatment with anti-asialo GM1 during the first week after immunization with SL3235 depleted NK cell activity and markedly increased mortality in mice challenged with a virulent Salmonella strain. These results are compatible with a role for NK cells as one important component in the resistance against virulent Salmonella infection induced by a live, attenuated vaccine.
Deformation factor: an extracellular protein synthesized by Bartonella bacilliformis that deforms erythrocyte membranes
Authors: Mernaugh G , Ihler G M
Published: January 2020

Bartonella bacilliformis, a hemotropic bacterium and the causative agent of the human disease bartonellosis, when incubated in a tryptone-based medium produces an extracellular factor, termed deformation factor (DF), which induces extensive indentations and trenches in trypsinized erythrocyte membranes. The factor is stable during storage at 4 degrees C. It can be inactivated by proteases or brief heating to 70 to 80 degrees C, can be precipitated by ammonium sulfate, is nondialyzable, and is retained by membranes with a 30,000-molecular-weight cutoff. These properties suggest that DF is probably a protein. Incubation of erythrocytes with phospholipase D renders them resistant to deformation by DF.
Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis
Authors: Barenkamp S J , Leininger E
Published: January 2020

A group of high-molecular-weight surface-exposed proteins of nontypeable Haemophilus influenzae are major targets of human serum antibody (S. J. Barenkamp and F. F. Bodor, Pediatr. Infect. Dis. J. 9:333-337, 1990). To further characterize these proteins, we cloned and sequenced genes encoding two related high-molecular-weight proteins from a prototype nontypeable Haemophilus strain. The gene encoding a 120-kDa Haemophilus protein consisted of a 4.4-kbp open reading frame, and the gene encoding a 125-kDa protein consisted of a 4.6-kbp open reading frame. The first 1,259 bp of the two genes were identical. Thereafter, the sequences began to diverge, but overall they were 80% identical, and the derived amino acid sequences showed 70% identity. A protein sequence homology search demonstrated similarity between the derived amino acid sequences of both cloned genes and the derived amino acid sequence of the gene encoding filamentous hemagglutinin, a surface protein produced by the gram-negative pathogen Bordetella pertussis. Antiserum raised against a recombinant protein encoded by the 4.6-kbp open reading frame recognized both the 120- and the 125-kDa proteins in the prototype strain as well as antigenically related high-molecular-weight proteins in 75% of a collection of 125 epidemiologically unrelated nontypeable H. influenzae strains. The antiserum directed against the recombinant protein also recognized purified filamentous hemagglutinin. A murine monoclonal antibody to filamentous hemagglutinin recognized both the 120-kDa and the 125-kDa protein in the prototype strain as well as proteins identical to those recognized by the recombinant-protein antiserum in 35% of the nontypeable H. influenzae strain collection. Thus, we have identified and partially characterized a group of highly immunogenic surface-exposed proteins of nontypeable H. influenzae which are related to the filamentous hemagglutinin of B. pertussis.
Protection of squirrel monkeys against virulent Plasmodium falciparum infections by use of attenuated parasites
Authors: Fandeur T , Gysin J , Mercereau-Puijalon O
Published: January 2020

We previously showed that the Uganda Palo Alto line of Plasmodium falciparum propagated in Saimiri monkeys and the line maintained in culture in human erythrocytes for many years in our laboratory are genetically unrelated (T. Fandeur, S. Bonnefoy, and O. Mercereau-Puijalon, Mol. Biochem. Parasitol. 47:167, 1991). When injected into a splenectomized Saimiri monkey, the in vitro-derived Palo Alto population procured a long-lasting, low-grade parasitemia that was spontaneously resolved by the animal. This line was propagated by serial blood transfers in two other monkeys without enhancement of the virulence of the parasites. The genetic characteristics of parasite samples corresponding to the different passages of the line in monkeys were stable for the several markers examined (pPF11.1, MSA1, and MSA2), although microheterogeneity was detected in telomeric and subtelomeric regions of chromosomes. Interestingly, in vitro-derived Palo Alto parasites induced a strong, potent immunity that enabled the monkeys to completely block subsequent challenge with two different heterologous lethal P. falciparum lines. These attenuated parasites are thus genetically stable in monkeys and represent an attractive model for assessing the feasibility of a live attenuated malaria vaccine.
Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components
Authors: Kesavalu L , Ebersole J L , Machen R L , Holt S C
Published: January 2020

Selected cell envelope components of Porphyromonas gingivalis were tested in a BALB/c mouse model in an attempt to elucidate further the outer membrane components of this putative oral pathogen that might be considered as virulence factors in host tissue destruction. Lipopolysaccharide (LPS), outer membrane, and outer membrane vesicles of P. gingivalis W50, ATCC 53977, and ATCC 33277 were selected to examine an immunological approach for interference with progressing tissue destruction. Mice were actively immunized with heat-killed (H-K) or Formalin-killed (F-K) whole cells or with the outer membrane fraction, LPS, or outer membrane vesicles of the invasive strain P. gingivalis W50. The induction of invasive spreading lesions with tissue destruction and lethality were compared among different immunization groups in normal, dexamethasone-treated (dexamethasone alters neutrophil function at the inflammatory site), and galactosamine-sensitized (galactosamine sensitization increases endotoxin sensitivity) mice after challenge infection with the homologous strain (W50) and heterologous strains (ATCC 53977 and ATCC 33277). Enzyme-linked immunosorbent assay analyses revealed significantly elevated immunoglobulin G and M antibody responses after immunization with H-K or F-K cells or the outer membrane fraction compared with those of nonimmunized mice. The killed whole-cell vaccines provided significantly greater protection against challenge infection in normal mice (decreased lesion size and death) than did either the outer membrane fraction or LPS immunization. The lesion development observed in dexamethasone-pretreated mice was significantly enhanced compared with that of normal mice after challenge with P. gingivalis. Immunization with P. gingivalis W50 provided less protection against heterologous challenge infection with P. gingivalis ATCC 53977; however, some species-specific antigens were recognized and induced protective immunity. Only viable P. gingivalis induced a spreading lesion in normal, dexamethasone-treated, or galactosamine-sensitized mice; F-K or H-K bacteria did not induce lesions. The F-K and outer membrane vesicle immunization offered greater protection from lesion induction than did the H-K immunogen after challenge infection simultaneous with galactosamine sensitization. The H-K cell challenge with galactosamine sensitization produced 100% mortality without lesion induction, suggesting that LPS or LPS-associated outer membrane molecules were functioning like endotoxin. Likewise, P. gingivalis W50 LPS (1 micrograms per animal) administered intravenously produced 80% mortality in galactosamine-sensitized mice. In contrast to the effects of immunization on lesion development, immunization with H-K or F-K cells or LPS provided no protection against intravenous challenge with LPS; 100% of the mice died from acute endotoxin toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)
Human tracheobronchial mucin: purification and binding to Pseudomonas aeruginosa
Authors: Reddy M S
Published: January 2020

Colonization of the respiratory tract with Pseudomonas aeruginosa is a serious problem in cystic fibrosis and seriously ill hospitalized patients. Human tracheobronchial mucin (HTBM), the major glycoprotein of human tracheobronchial secretions, is known to interact with this pathogen, which may then be cleared by mucociliary action. However, the mechanism of interaction is not known. To understand this process, pure HTBM was isolated from tracheobronchial secretions of a laryngectomee. Following initial fractionation on Sepharose CL-2B, the HTBM-containing fraction was subjected to reductive methylation and then gel filtration. Pure HTBM was employed in an overlay binding assay to identify the bacterial adhesin(s) and mucin receptors that participate in mucin-P. aeruginosa interactions. An approximately 16-kDa nonpilus protein component(s) of P. aeruginosa was found to be the adhesin(s) for HTBM. The mucin receptor for the 16-kDa component(s) was found in the peptide moiety. This study confirms that P. aeruginosa utilizes the nonpilus adhesin(s) to bind to HTBM. Identification of the specificity of the HTBM-P. aeruginosa interactions can lead to a better understanding of the predominance of P. aeruginosa colonization in individuals with cystic fibrosis.
Effects of Actinobacillus pleuropneumoniae hemolysin on porcine neutrophil function
Authors: Udeze F A , Kadis S
Published: January 2020

In an attempt to gain insight into the events that take place during Actinobacillus pleuropneumoniae infection, the present study was designed to ascertain the effects of bacterial toxicity on porcine neutrophil functions and viability. Incubation of phagocytes (2 x 10(6)) with opsonized A. pleuropneumoniae 4074 (2 x 10(7) CFU) resulted in phagocytic uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were not killed by the phagocytes. These effects were ascribed to hemolysin produced by the bacteria, because the presence of hemolysin-neutralizing antibody prevented overt cellular damage, significantly increased phagocytic uptake (P less than 0.001), and resulted in an approximately 10-fold decrease in the number of CFU of the ingested bacteria. Cytolytic doses of isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal activity of toxin-treated phagocytes for Escherichia coli, and decreased the ability of the phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and caused them to respond to phorbol myristic acetate with increased generation of superoxide anion. Because heated (100 degrees C, 5 min) hemolysin preparations did not produce similar effects, we contend that the observed effects were not due to contaminating endotoxin. The data presented herein indicate that A. pleuropneumoniae hemolysin is a potent antiphagocytic virulence factor by virtue of its leukocidal activity. Sublytic doses of the toxin may have important effects on the oxidative metabolism of phagocytic cells.
Molecular analysis of a Streptococcus mutans strain exhibiting polymorphism in the tandem gtfB and gtfC genes
Authors: Yamashita Y , Bowen W H , Kuramitsu H K
Published: January 2020

Streptococcus mutans UA101, which was previously demonstrated to be highly cariogenic in gnotobiotic rats, exhibited much lower water-insoluble glucan (IG) synthetic activity compared with that of S. mutans GS5 and was unable to express sucrose-dependent colonization of smooth surfaces in vitro. On the basis of Southern and Western blot (immunoblot) analyses, it was demonstrated that, unlike most S. mutans strains, strain UA101 contained a single copy of a gene coding for IG synthesis. The gene was isolated from a clone bank constructed with the plasmid pTH10 clone bank in Escherichia coli and had apparently evolved after homologous recombination of the gtfB and gtfC genes present on the chromosome of a recent ancestor of strain UA101. The enzyme expressed from the gene, gtfBC, was purified to near homogeneity by utilizing a single-step preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis system and was characterized. A derivative of strain UA101, UA101LBS, containing a chromosomal insertion of the GS5 gtfC gene was constructed after transformation. UA101LBS exhibited high IG synthetic activity and colonized smooth surfaces in vitro. By utilizing a conventional rat model system involving animals fed a high-sucrose diet, strain UA101 exhibited low levels of smooth surface caries activity relative to Streptococcus sobrinus 6715. By contrast, UA101LBS was as cariogenic as strain 6715. However, sulcal caries occurred equally well with all of the strains tested. These results are evaluated relative to the role of gtf gene products in cariogenicity.
Experimental Cryptosporidium parvum infections in immunosuppressed adult mice
Authors: Rasmussen K R , Healey M C
Published: January 2020

Five strains of adult mice were immunosuppressed with the synthetic glucocorticosteroid dexamethasone (DEX), administered either orally or intraperitoneally. The strains of mice used were C57BL/6N, DBA/2N, CBA, C3H/HeN, and BALB/cAnN. All mice were evaluated for susceptibility to Cryptosporidium parvum after intragastric inoculation with 10(6) oocysts per mouse. The DBA/2N, CBA, C3H/HeN, and BALB/cAnN mice given 0.25 micrograms of DEX per g per day orally (the dose and route previously used to infect rats with C. parvum) failed to develop chronic infections. However, the C57BL/6N mice sustained light infections during the entire 28-day experiment. The five strains of mice were also administered DEX intraperitoneally at concentrations ranging from 62.5 to 500 micrograms/day. Only the C57BL/6N mice given DEX at 125 micrograms/day developed chronic infections which persisted over 10 weeks, suggesting that the genetic background of the mouse plays a role in determining susceptibility to cryptosporidosis following immunosuppression with DEX. We believe that the C57BL/6N mouse model will prove to be superior to other animal models for evaluating potential anticryptosporidial agents, as well as for elucidating the immunological defects that allow C. parvum to establish chronic infections, because of cost effectiveness and ease in maintenance, breeding, and handling. We also evaluated the C3H/HeJ/beige mouse (lacks natural killer cell activity) and the C57BL/6N mouse maintained on a low-protein diet to induce immunosuppression. Neither of these mice exhibited heavy cryptosporidial infections.
Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis
Authors: Boerlin P , Peter O , Bretz A G , Postic D , Baranton G , Piffaretti J C
Published: January 2020

Fifty Borellia burgdorferi strains isolated from humans and ticks in Europe and the United States were analyzed by multilocus enzyme electrophoresis. Eleven genetic loci were characterized on the basis of the electrophoretic mobilities of their products. Ten loci were polymorphic. The average number of alleles per locus was 5.9, with a mean genetic diversity of 0.673 among electrophoretic types (ETs). The strains were grouped into 35 ETs constituting three main divisions (I, II, and III) separated at a genetic distance greater than 0.75. Divisions I, II, and III contained 13, 6, and 16 ETs, respectively. These findings, together with previous data from DNA hybridization and restriction enzyme analysis of rRNA genes, suggest that divisions I, II, and III may represent three distinct genomic species. All three divisions contained human clinical ETs. However, in division I, which includes the ET of the type strain of B. burgdorferi, the human pathogenic ETs constituted a single clone. The ETs of division I were from west-central Europe and the United States, whereas divisions II and III contained ETs from west-central and northern Europe but not from the United States. Finally, our data show that the genetic structure of B. burgdorferi populations is clonal.
Characterization of a neutralizing monoclonal antibody to Pasteurella haemolytica leukotoxin
Authors: Gerbig D G , Cameron M R , Struck D K , Moore R N
Published: January 2020

Six hybridoma clones producing monoclonal antibodies (MAbs) reactive with Pasteurella haemolytica A1 leukotoxin were derived from mice immunized with leukotoxin excised from sodium dodecyl sulfate-polyacrylamide gels. Of the six MAbs, only one, Ltx-2, neutralized leukotoxin in a BL-3 cell cytotoxicity assay. MAb Ltx-2 blocked association of A1 leukotoxin to BL-3 cells, as measured by flow cytometric analysis. The epitope recognized by Ltx-2 was localized to the carboxyl half of the native protein, between residues 450 and 939, by Western immunoblot analysis of CNBr fragments. Further analysis with leukotoxin deletion proteins indicated either that the Ltx-2-reactive epitope was localized in the carboxyl portion of the leukotoxin between amino acids 768 and 939 or that this region influences MAb recognition of the epitope. MAb Ltx-2 was tested for neutralizing activity against leukotoxin produced by P. haemolytica serotypes 1 through 12. The MAb neutralized leukotoxin produced by all of the A biotype isolates (serotypes 1, 5, 6, 7, 8, 9, and 12), with the exception of serotype A2, but did not neutralize any T biotype leukotoxin tested (T3, T4, or T10). The results indicate that MAb Ltx-2 neutralizes leukotoxin by interfering with target cell association and that the MAb-specific epitope is either not present or not critical for function in the leukotoxin produced by P. haemolytica serotypes A2, T3, T4, and T10.
Immunoglobulin G subclass response of localized juvenile periodontitis patients to Actinobacillus actinomycetemcomitans Y4 lipopolysaccharide
Authors: Wilson M E , Hamilton R G
Published: January 2020

Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass.
Vaccination of mice with a soluble protein fraction of Mycobacterium leprae provides consistent and long-term protection against M. leprae infection
Authors: Gelber R H , Murray L , Siu P , Tsang M
Published: January 2020

Groups of BALB/c mice were vaccinated intradermally with either Freund's incomplete adjuvant (FIA) alone, 10(7) heat-killed Mycobacterium leprae organisms in FIA, or a number of fractions of M. leprae containing soluble and/or cell wall components. At 1, 3, 6, 9, and 12 months later, vaccinated mice were challenged in the right hind footpad with 5,000 live M. leprae organisms, and vaccine protection was assessed 6 to 8 months later, at the peak of M. leprae multiplication in the negative control (FIA alone), by the two-sample rank-sum test. In these studies, a cell wall fraction rich in peptidoglycan was consistently ineffective. Both heat-killed M. leprae and a fraction containing cell wall and fixed proteins generally protected when the interval between vaccination and challenge was 1 or 3 months but not subsequently. On the other hand, soluble proteins of M. leprae alone or in combination (with cell wall fractions) consistently (14 of 14 instances) afforded highly significant protection (P less than or equal to 0.01) at all challenge intervals up to 1 year after vaccination. These results suggest that the soluble protein fraction of M. leprae offers promise for a vaccine against leprosy.
Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae
Authors: Briles D E , Forman C , Crain M
Published: January 2020

Previous studies have demonstrated that mouse antibodies to phosphocholine (PC) can protect mice against fatal infection caused by several, but not all, mouse-virulent laboratory strains of Streptococcus pneumoniae. Because the pneumococcal strains used in previous studies had been mouse passed and were propagated for many years outside of humans, it was not known whether antibody to PC would be able to protect mice against S. pneumoniae freshly isolated from humans. In the present study, we examined the ability of an immunoglobulin G (IgG) monoclonal antibody (MAb) to PC to protect against infections in mice caused by 14 pneumococcal strains of capsular types 3, 4, 6A, and 6B. Nine of these strains were selected as the most virulent strains for mice from a group of 69 fresh clinical isolates. Five were mouse-passed laboratory strains. Mouse IgG3 MAb to PC was able to exhibit protective effects (survival or increased time to death) against infection with virtually all of the strains injected intravenously and against infection with 70% of the strains injected intraperitoneally. The protective effects of antibody to PC appeared to be partially dependent on capsular type. MAb to PC was most effective against capsular type 3 strains and least effective against type 4 strains. With type 3 and type 4 strains, MAb to PC could frequently protect against larger numbers of CFU injected intravenously than intraperitoneally. For capsular type 6A and 6B strains the reverse was true.
Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E. coli hemolysin
Authors: Baldwin T J , Knutton S , Sellers L , Hernandez H A , Aitken A , Williams P H
Published: January 2020

A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region of E. coli hemolysin. Treatment of HEp-2 cells with enteroaggregative E. coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation.
A specific sequence of stimulation is required to induce synthesis of the antimicrobial molecule nitric oxide by mouse macrophages
Authors: Lorsbach R B , Russell S W
Published: January 2020

Nitric oxide production by macrophages required either simultaneous or sequential exposure to gamma interferon and lipopolysaccharide; exposure to lipopolysaccharide followed by exposure to gamma interferon gave little response. The apparently evanescent nature of the lipopolysaccharide signal, necessitating persistent stimulation, could be essential to down-regulating nitric oxide production after bacteria are cleared in vivo.
Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification
Authors: Cassels F J , Deal C D , Reid R H , Jarboe D L , Nauss J L , Carter J M , Boedeker E C
Published: January 2020

Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili. We are currently studying the importance of these defined epitope-containing regions as vaccine candidates.
Serum immunoglobulin G antibody to Porphyromonas gingivalis in rapidly progressive periodontitis: titer, avidity, and subclass distribution
Authors: Whitney C , Ant J , Moncla B , Johnson B , Page R C , Engel D
Published: January 2020

Porphyromonas gingivalis is a suspected pathogen in rapidly progressive periodontitis (RPP). We have determined the anti-P. gingivalis serum immunoglobulin G (IgG) isotype response and avidity and the subclass titer distributions for 30 RPP patients and 30 age-, sex-, and race-matched healthy subjects by using enzyme-linked immunosorbent assay technology. Patients and control subjects were classified as seropositive if their total IgG response to P. gingivalis was twofold or more than the median response in healthy subjects. The predominant antibody responses for both patients and healthy subjects were IgG2 and IgG3, with a subclass order of IgG2 greater than IgG3 greater than IgG1 greater than IgG4. The avidity of the IgG response was highest for the seropositive healthy subjects and was no different between seronegative and seropositive RPP patients. The subclass antibody responses did not depend on gender, and there were no correlations between titer, avidity, or subclass with disease severity in the RPP patients as measured by pocket depth or bone loss on dental X rays. The seronegative RPP patients exhibited antibody responses that were greater than the responses of seronegative healthy subjects for all four subclasses, while the seropositive RPP patients had higher IgG1 and IgG4 levels than seropositive healthy subjects. These findings are consistent with the hypothesis that both carbohydrate and protein antigens are important in the IgG response to P. gingivalis. The relative predominance of IgG2, a subclass which lacks strong complement fixation and opsonic properties, and the low avidity of patient anti-P. gingivalis IgG antibodies suggest that humoral responsiveness to infection with P. gingivalis may be ineffective in clearing this organism.
Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen
Authors: Kotloff K L , Herrington D A , Hale T L , Newland J W , Van De Verg L , Cogan J P , Snoy P J , Sadoff J C , Formal S B , Levine M M
Published: January 2020

A live, oral Shigella vaccine, constructed by transfer of the 140-MDa invasiveness plasmid from Shigella flexneri 5 and the chromosomal genes encoding the group- and type-specific O antigen of S. flexneri 2a to Escherichia coli K-12, was tested in humans. Designated EcSf2a-1, this vaccine produced adverse reactions (fever, diarrhea, or dysentery) in 4 (31%) of 13 subjects who ingested a single dose of 1.0 x 10(9) CFU, while at better-tolerated doses (5.0 x 10(6) to 5.0 x 10(7) CFU), it provided no significant protection against challenge with S. flexneri 2a. A further-attenuated aroD mutant derivative, EcSf2a-2, was then tested. Rhesus monkeys that received EcSf2a-2 in three oral doses of ca. 1.5 x 10(11) CFU experienced no increase in gastrointestinal symptoms compared with a control group that received an E. coli K-12 placebo. Compared with controls, the vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (60% efficacy; P = 0.001). In humans, EcSf2a-2 was well tolerated at inocula ranging from 5.0 x 10(6) to 2.1 x 10(9) CFU. However, after a single dose of 2.5 x 10(9) CFU, 4 (17%) of 23 subjects experienced adverse reactions, including fever (3 subjects) and diarrhea (209 ml) (1 subject), and after a single dose of 1.8 x 10(10) CFU, 2 of 4 subjects developed dysentery. Recipients of three doses of 1.2 to 2.5 x 10(9) CFU had significant rises in serum antibody to lipopolysaccharide (61%) and invasiveness plasmid antigens (44%) and in gut-derived immunoglobulin A antibody-secreting cells specific for lipopolysaccharide (100%) and invasiveness plasmid antigens (60%). Despite its immunogenicity, the vaccine conferred only 36% protection against illness (fever, diarrhea, or dysentery) induced by experimental challenge (P = 0.17). These findings illustrate the use of an epithelial cell-invasive E. coli strain as a carrier for Shigella antigens. Future studies must explore dosing regimens that might optimize the protective effects of the vaccine while eliminating adverse clinical reactions.
Staphylococcus aureus susceptibility to thrombin-induced platelet microbicidal protein is independent of platelet adherence and aggregation in vitro
Authors: Yeaman M R , Norman D C , Bayer A S
Published: January 2020

Bacterium-platelet interactions at the cardiac valve surface represent an important initial step in the induction of infective endocarditis (IE). This cell-cell interaction may play either a protagonistic role in the induction of IE via bacterial adherence to and aggregation of platelets or an antagonistic role via secretion of platelet-derived microbicidal molecules. We examined the spectrum and interrelationship of three aspects of the interaction of 20 clinical Staphylococcus aureus isolates with rabbit platelets in vitro: (i) S. aureus adherence to platelets; (ii) S. aureus-induced platelet aggregation; and (iii) S. aureus resistance to the action of thrombin-induced platelet microbicidal protein (PMP; low-molecular-weight cationic peptides contained in alpha granules). Among the 20 S. aureus isolates (11 bacteremia, 9 endocarditis), there was a heterogeneous distribution profile for each of the bacterium-platelet interaction parameters studied. For S. aureus-platelet adherence and S. aureus-induced platelet aggregation, 3 of 20 and 7 of 20 isolates tested were considered highly active for each respective parameter; 5 of 20 staphylococcal strains were deemed resistant to the bactericidal action of PMP. In addition, more endocarditis isolates (45%) were PMP resistant than strains from patients without endocarditis (19%). When analyzed concomitantly, there was a significant, positive correlation between S. aureus-platelet adherence and S. aureus-induced platelet aggregation among isolates (P = 0.003; r = 0.78). In contrast, there were no statistically significant relationships between either platelet adherence or aggregation and PMP resistance among these 20 S. aureus isolates. These data suggest that platelet adherence and aggregation are related abilities of S. aureus, while resistance to thrombin-induced PMP is an independent phenotypic characteristic and potential virulence factor.
Antibody-secreting cells in human peripheral blood after oral immunization with an inactivated enterotoxigenic Escherichia coli vaccine
Authors: Wennerås C , Svennerholm A M , Ahrén C , Czerkinsky C
Published: January 2020

Vaccine antigen-specific antibody-secreting cell (ASC) responses in peripheral blood of healthy adult volunteers were studied after oral immunization with a prototype enterotoxigenic Escherichia coli (ETEC) vaccine by means of the enzyme-linked immunospot technique. Three doses of vaccine consisting of formalin-killed ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) in combination with purified cholera toxin B subunit (CTB) were given 2 weeks apart. The ASC responses were detected 7 days after each immunization. Immunoglobulin A (IgA) was the predominant isotype produced by CFA/I- as well as CFA/II-specific ASCs. Moderate CFA/I- and CFA/II-specific IgM-secreting ASC (IgM-ASC) responses were also seen, whereas IgG-ASC responses to either of the CFAs were negligible. The ASC responses to CTB, on the other hand, comprised both IgA- and IgG-ASCs, with few if any specific IgM-ASCs. Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination. Maximal CFA-specific ASC responses were usually observed after a single dose or two doses of vaccine. A third dose of vaccine did not result in increased but rather resulted in decreased magnitudes of CFA-specific ASC responses. Furthermore, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB. These results suggest that two oral doses of ETEC vaccine induce a strong mucosal immune response, as reflected by the presence of large numbers of antigen-specific mucosal B cell immunoblasts in the blood.
Role of the bacterial cell wall in middle ear inflammation caused by Streptococcus pneumoniae
Authors: Carlsen B D , Kawana M , Kawana C , Tomasz A , Giebink G S
Published: January 2020

The pathogenesis of middle ear inflammation caused by Streptococcus pneumoniae was explored in the chinchilla model with different pneumococcal cell wall (CW) preparations, including isolated native CW, M1 muramidase CW (M1-CW) digest, amidase CW digest, and M1 peptidoglycan (M1-PG) digest. Inflammatory cell and lysozyme concentrations in middle ear fluid (MEF) were measured between 6 and 72 h after the middle ears were inoculated with one of the preparations or sterile saline. Middle ear histopathology was measured quantitatively at 72 h. Native CW, M1-CW digest, and amidase-CW digest caused significantly more inflammatory cell influx and lysozyme accumulation in MEF than saline did. M1-PG digest also caused more inflammatory cell influx and lysozyme accumulation in MEF than saline did but caused less inflammation than native CW or either CW digest. Epithelial metaplasia was significantly greater in ears inoculated with native CW than in ears inoculated with the CW or PG digest or with saline. Pneumococcal CW is, therefore, the principal factor that initiates middle ear inflammation in acute pneumococcal otitis media, and CW teichoication seems to be important in initiating this response.
Morphological changes of cultured endothelial cells after microinjection of toxins that act on the cytoskeleton
Authors: Müller H , von Eichel-Streiber C , Habermann E
Published: January 2020

Clostridium novyi alpha-toxin and C. difficile toxins A and B (all 200 to 300 kDa) and C. botulinum C2-I toxin (50 kDa) caused a delayed and persistent retraction and rounding of microinjected cells. Microinjected phalloidin acted fast and reversibly. Unlike C2-I toxin, phalloidin passed through the intercellular junctions. Specific antitoxin applied to the medium did not prevent the action of microinjected C. novyi or C. difficile toxin B. Microinjected antitoxin protected against the toxins applied with the medium or injected into the same cells.
Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes
Authors: Villalta F , Lima M F , Howard S A , Zhou L , Ruiz-Ruano A
Published: January 2020

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host.
Presence of parasite antigen on the surface of P388D1 cells infected with Ehrlichia risticii
Authors: Messick J B , Rikihisa Y
Published: January 2020

Indirect immunofluorescence staining of macrophages infected with Ehrlichia risticii by anti-E. risticii serum revealed a punctate staining pattern on the surface of the host cell. This pattern was distinguishable by fluorescence microscopy from E. risticii bound to the surface of the macrophage and from intracellular E. risticii. The surface localization of ehrlichial antigen on infected macrophages was confirmed by electron microscopy with immunoferritin labeling. As the intracellular ehrlichial burden increased, the amount of ehrlichial antigen on the host cell surface increased. Prokaryotic protein synthesis was necessary for the maintenance of ehrlichial antigen on the host cell surface, as demonstrated by disappearance of the surface antigen following treatment with oxytetracycline. However, host cell protein synthesis was not required, as demonstrated by the continued presence of ehrlichial antigen on the surface of host cells after cycloheximide treatment. Pronase treatment abolished the ehrlichial antigen present on the cell surface, indicating that this antigen is a protein. Anti-E. risticii serum or immunoglobulin G-mediated antibody-dependent cellular cytotoxicity of infected cells was demonstrated in a chromium release assay. These results imply that the parasite antigen on the host cell surface has a role in the pathogenesis of ehrlichiosis.
Comparison of susceptibility of inbred and outbred infant mice to Escherichia coli heat-stable enterotoxin STa
Authors: Bertin A
Published: January 2020

Comparison of the susceptibility of outbred OF1 and inbred BALB/c, C57BL/6, DBA/2, and CBA mice to heat-stable toxin (STa) of enterotoxigenic Escherichia coli was made at different levels of induced secretion. STa was able to elicit fluid accumulation into the intestine of each strain of mice; however, quantitatively different results were obtained. Results were as usual expressed by gut weight/remaining body weight ratios. Fluid accumulation weight and fluid accumulation weight/remaining body weight ratios were also estimated. Values obtained for BALB/c and OF1 mice were never significantly different, but values for OF1 mice were significantly higher than those for DBA and C57BL/6 mice at the highest concentrations of toxin (toxin dilutions of 1/2, 1/4, and 1/5). At the highest toxin concentration, gut weight/remaining body weight ratio in C57BL/6 mice was significantly lower than that for every other strain, but the fluid accumulation value obtained for DBA mice did not differ from that for C57BL/6 mice. Fluid accumulation values for DBA mice were also significantly lower at toxin dilutions of 1/5 and 1/8 than those for every other strain, and this was also the case when estimating the fluid accumulation weight/remaining body weight ratio at a dilution of 1/8. Although the intestine of each strain of mice was able to respond to STa by fluid accumulation, differences in susceptibility of the STa receptor could exist and make DBA mice more resistant to enterotoxigenic E. coli diarrhea.