bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of Various Intestinal Bacteria in Converting Germfree Mice to the Normal State
Authors: Freter Rolf , Abrams Gerald D.
Published: January 2020

Earlier work had shown that a collection of anaerobic bacteria, in conjunction with facultative anaerobes, may be implanted into germfree mice, thereby rendering the animals “normal” with respect to a variety of parameters tested. The present experiments indicate that a different collection of anaerobic bacteria, isolated from the cecum of normal mice, was necessary to convert germfree mice to the “normal” state when the animals were fed a crude diet, rather than the refined food which had been used in the earlier work. The nature and level of short-chain fatty acids associated with various natural or synthetic “normal” floras in the ceca of mice did not always correlate with the
Escherichia coli
population present, indicating that fatty acids were not the sole agents inhibiting bacterial populations in the intestine. Experiments are reported which indicate that intestinal anaerobes may under certain circumstances be sufficient to control the populations of other intestinal bacteria such as
E. coli
. In other instances, such as control of
Shigella
populations in the mouse intestine, intestinal anaerobes appeared to act synergistically with an
E. coli
strain, in spite of the fact that the population of the latter was itself suppressed by the anaerobes.
Murine Typhus Toxin: Studies on Identification of the Neutralizing Factor Present in Normal Human Serum
Authors: Bell E. John , Munoz John J. , Peacock Marius , Cole Robert L.
Published: January 2020

Studies were made on the isolation and identification of the
Rickettsia typhi
toxin-neutralizing factor (TNF) previously demonstrated in normal human serum. By means of various methods of separating serum proteins, such as filtration on Sephadex G-200, dextran precipitation, hydroxyapatite chromatography, and ultracentrifugation, TNF was found to be closely associated with purified serum β-lipoprotein, although no serological relationship with this protein was demonstrated. Lipase as well as trypsin digestion of purified preparations of β-lipoprotein destroyed the TN activity. No evidence was obtained for an association of TNF with the immunoglobulins or with any serum protein other than β-lipoprotein. Further studies revealed that (i) serum specimens with TN titers of 1:1024 and others with titers of 1:8 or less contained the same concentration of β-lipoprotein; (ii) purified preparations of β-lipoprotein isolated from TNF positive and negative sera, and which had the same protein concentration, differed as much as 250-fold in TN titer; and (iii) the TN activity of a serum could be removed by absorption with antiserum to β-lipoprotein from a positive donor, but not with antiserum to β-lipoprotein from a negative donor.
Identification of Different Measles Virus-Specific Antibodies in the Serum and Cerebrospinal Fluid from Patients with Subacute Sclerosing Panencephalitis and Multiple Sclerosis
Authors: Salmi Aimo A. , Norrby Erling , Panelius Martin
Published: January 2020

Matched serum and cerebrospinal fluid (CSF) samples from eight cases of subacute sclerosing panencephalitis (SSPE) and 15 cases of multiple sclerosis (MS) were characterized in neutralization, hemolysis-inhibition (HLI), hemagglutination-inhibition (HI) with Tween 80—ether-treated antigen, complement-fixation (CF), and immunodiffusion tests. CF tests were carried out with crude virus material, purified nucleocapsids, and small particle hemagglutinin as antigens. A certain diversity in the relative content of antibodies against different virus products in various sera was found. There was a high degree of correlation between titers of neutralizing and HLI antibodies, but a less strict correlation between titers of HLI and HI antibodies. Serum samples from two cases of MS and one case of SSPE contained high titers of HLI and neutralizing antibodies in the presence of only low titers of HI antibodies demonstrable with Tween 80—ether-treated antigen. The major fraction of antibodies detected in CF and immunodiffusion tests reacted with nucleocapsids. There was a tendency of nucleocapsid CF antibody titers, as compared to neutralization and HLI antibody titers, to be higher in samples from patients with SSPE than from cases of MS. No significant differences were found between antibody titers recorded in neutralization, HLI, and HI tests carried out with two different measles virus strains, Edmonston and a strain (LEC) derived from a case of SSPE. Comparison of antibodies against measles virus products and, as a reference, against a group-specific vertex capsomer antigen of adenovirus in matched serum and CSF samples revealed a production of measles virus-specific antibodies within the central nervous system of all cases of SSPE and 8 out of 15 cases of MS.
Cells Producing Low-Molecular-Weight Antibody to
Escherichia coli
Lipopolysaccharide
Authors: Jackson Anne L. , Walters Curla S.
Published: January 2020

CD-1 mice immunized with sheep red blood cells (SRBC) or
Escherichia coli
lipopolysaccharide (LPS), developed splenic plaque-forming cells (PFC) producing low-molecular-weight antibody; these cells were detected by means of purified rabbit antisera to mouse γ
1
, γ
2a
, and γ
2b
immunoglobulins. In contrast to SRBC, the primary γ
1
response to LPS was absent and γ
2a
and γ
2b
PFC were detected irregularly. Both immunogens elicited a secondary cellular response in all three subclasses without a corresponding increase in “direct” or γ
M
PFC. An increase in serum bactericidal antibody following a second injection of LPS was not parallelled by an increase in splenic γ
M
PFC; it might therefore involve the synthesis of γ
2
complement-fixing antibody.
Effects of Various Suspending Media on Plaque Formation by Rickettsiae in Tissue Culture
Authors: Wike David A. , Ormsbee Richard A. , Tallent George , Peacock Marius G.
Published: January 2020

Effects of some media used for suspending rickettsiae during purification, for metabolic studies, and in titrations of infectious rickettsiae were examined with respect to the plaque-forming ability of
Rickettsia rickettsi
and
R. typhi
in primary chicken embryo tissue cultures and the infectivity of
R. typhi
in mice. Brain heart infusion broth (BHI) was found superior to all other media tested in preventing both a significant decrease in plaque-forming units (PFU) and a delay in plaque formation. Skim milk, egg yolk, and some metabolic media were effective in maintaining PFU at 0 C, but did not prevent a significant delay in plaque formation. However, infectivity of
R. typhi
for tissue culture and mice was markedly decreased when suspended in metabolic media at 26 C. Addition of BHI to the routine tissue culture overlay reversed the deleterious effects of sucrose-phosphate solutions. The effects of Mg
2+
, Mn
2+
, K
+
, Na
+
, sucrose, and glutamate were also examined. No significant differences were observed between
R. rickettsi
and
R. typhi
in their responses to different media. The results of this study suggest the necessity for a reappraisal of previous studies of metabolism and infectivity of rickettsiae in these media.
Pseudomonas aeruginosa:
Immune Status in Patients with Cystic Fibrosis
Authors: Doggett R. G. , Harrison G. M.
Published: January 2020

In order to have a better understanding of the clinical significance of
Pseudomonas aeruginosa
, circulating and secretory antibodies were measured. Of 100 patients diagnosed as having cystic fibrosis (CF) and an atypical mucoid
P. aeruginosa
cultured from their sputum, each possessed serum precipitins. These immunoprecipitates, however, were not detected in the sera of 40 CF patients, some of whom were chronically ill with pulmonary colonization by typically rough-smooth strains of
P. aeruginosa.
The sera of 46 CF patients and 27 CF patient parents not colonized by
P. aeruginosa
were negative for the precipitins. The sera from 15 of 45 chronically ill patients not having CF, however, but harboring
P. aeruginosa
, also possessed serum precipitins. The sera from 85 subjects not having CF and not clinically infected with
P. aeruginosa
were negative for precipitins. Serum hemagglutination titers as high as 1:4096 were measured in older CF patients having advanced pulmonary disease and who were infected with mucoid
P. aeruginosa.
Salivary titers ranged from 1:8 to 1:64. Increased levels of both circulating and secretory antibodies of the immunoglobulin A and G classes were demonstrated in patients with CF. Once a patient with CF becomes colonized with
P. aeruginosa
a process of conversion from the rough and smooth forms to the mucoid form is almost inevitable. Although the mucoid form predominates in the sputum, intermediates of the various colony types are often present. Serum precipitins were demonstrable only after the appearance of mucoid strains in the sputum of patients with CF. Although antibiotics tend to reduce the number of mucoid microorganisms, they are rarely, if ever, eradicated from these patients' lungs. Recurrent episodes of servere pulmonary infection and the evidence of increasing antibody formation to mucoid strains indicates the invasiveness of these particular strains.
Action of a Phospholipase C Preparation on the First Component of Complement of Guinea Pig and Human Serum: Lack of Correlation with Enzyme Activity
Authors: Loos G. Michael , Borsos Tibor
Published: January 2020

Interaction of a phospholipase C (from
Clostridium welchii
) preparation with the first component of human and guinea pig complement in serum is due to contaminants devoid of phospholipase C activity.
Ecology of Human Oral Lactobacilli
Authors: van Houte Johannes , Gibbons Ronald J. , Pulkkinen Andrea J.
Published: January 2020

Lactobacilli were found in saliva, on teeth, and on the dorsum of the tongue, the vestibular mucosa, and the hard palate in humans. Their proportions in saliva, expressed as percentage of the anaerobically cultivable flora, were 10- to 100-fold higher than those on the tooth surface, but were comparable to those on the epithelial surfaces. The adherence of
Lactobacillus casei
and
L. fermenti
to oral surfaces was compared with that of streptomycin-labeled
Streptococcus sanguis
and
S. salivarius
by using in vitro-cultivated cells. The affinity of both
Lactobacillus
species for the tooth surface was very low compared with that of
S. sanguis
but was somewhat higher than that of
S. salivarius.
The lactobacilli and both
Streptococcus
species adhered to a similar extent to the dorsum of the tongue, whereas the affinity of the lactobacilli and
S. salivarius
for the vestibular mucosa was about one-half of that of
S. sanguis.
The results suggest that the affinity of lactobacilli for oral surfaces significantly influences their proportional distribution in the mouth. The relatively low affinity of lactobacilli for the tooth surface suggests that their recognized association with carious lesions and mechanical appliances under certain conditions may be due primarily to mechanical retention rather than unique growth conditions.
In Vitro Interactions Between Virus and Enhancer
Authors: Righthand Fay
Published: January 2020

Cell-free supernatant fluids of permissive host cells contained an enhancer for the
m
+ variant of echovirus 6. The enhancer activity remained in supernatant fractions after centrifugation at 110,000 ×
g
for 4 hr. Under in vitro conditions, a complex between enhancer and virus formed rapidly. The complex was not dissociated by dilution and was sedimented by centrifugation. The effect of enhancer on the distribution of virus in CsCl density gradients was determined. In the absence of enhancer, virus was recovered in one peak at 1.33 g/ml. Incubation of enhancer with virus before centrifugation resulted in the formation of two peaks of infectivity at 1.33 g/ml and 1.24 g/ml. The heavier virus peak represented 43% of the recovered infectivity and could be enhanced. The light virus peak contained 26% of the infectivity and could not be enhanced. The density of enhancer was 1.20 g/ml and approximated the density of the light virus population.
Plaque-Forming Cells in Rabbits Immunized with BCG Bacilli
Authors: Okuyama Harue , Morikawa Kazuo
Published: January 2020

Plaque-forming cells (PFC) in the regional lymph node, its efferent lymph, and the spleen of rabbits immunized with BCG were enumerated by localized hemolysis in gel, using sheep red blood cells coated with tuberculin protein. In the primary response, the maximal level of PFC production was reached at 5 days in the regional lymph node, and at 7 days in the spleen, whereas efferent lymphatic cells showed only a small number of PFC. Sharp peaks of PFC levels at 3 days were seen in the regional lymph node, spleen, and the efferent lymphatic cells in the secondary response. Blast cell outflow from the regional lymph node was maximal at 3 days in the secondary response as well as in the primary response. The relation between blast cell outflow and PFC production was discussed.
Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection
Authors: Herrera M A , Rosero F , Herrera S , Caspers P , Rotmann D , Sinigaglia F , Certa U
Published: January 2020

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.
Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model
Authors: Sawyer R T , Garner R E , Hudson J A
Published: January 2020

The isolated perfused mouse liver model was used to study the effect of Arg-Gly-Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD-treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells.
Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread
Authors: Vazquez-Boland J A , Kocks C , Dramsi S , Ohayon H , Geoffroy C , Mengaud J , Cossart P
Published: January 2020

The lecithinase gene of the intracellular pathogen Listeria monocytogenes, plcB, was identified in a 5,648-bp DNA fragment which expressed lecithinase activity when cloned into Escherichia coli. This fragment is located immediately downstream of the previously identified gene mpl (prtA). It contains five open reading frames, named actA, plcB, and ORFX, -Y, and -Z, which, together with mpl, form an operon, since a 5.7-kb-long transcript originates from a promoter located upstream of mpl (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991). A second promoter was detected in front of actA which encodes a putative membrane protein containing a region of internal repeats. plcB encodes the lecithinase, a predicted 289-amino-acid protein homologous to the phosphatidylcholine-specific phospholipases C of Bacillus cereus and Clostridium perfringens (alpha-toxin). plcB mutants produce only small plaques on fibroblast monolayers, and an electron microscopic analysis of infected macrophages suggests that lecithinase is involved in the lysis of the two-membrane vacuoles that surround the bacteria after cell-to-cell spread. On the opposite DNA strand, downstream of the operon, three more open reading frames, ldh, ORFA, and ORFB, were found. The deduced amino acid sequence of the first one is homologous to lactate dehydrogenases. Low-stringency Southern hybridization experiments suggest that these three open reading frames lie outside of the L. monocytogenes virulence region: mpl and actA were specific for L. monocytogenes, sequences hybridizing to plcB were detected in L. ivanovii and L. seeligeri, and sequences hybridizing to ORFX, -Y, and -Z were found in L. innocua. In contrast to this, sequences hybridizing to ldh or ORFB were detected in all Listeria species (including the nonpathogenic ones).
R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections
Authors: Darfeuille-Michaud A , Jallat C , Aubel D , Sirot D , Rich C , Sirot J , Joly B
Published: January 2020

Klebsiella pneumoniae strains involved in hospital outbreaks of nosocomial infections, such as suppurative lesions, bacteremia, and septicemia, were resistant to multiple antibiotics including broad-spectrum cephalosporins. Epidemiologic investigations revealed that the reservoir for these K. pneumoniae strains was the gastrointestinal tracts of the patients. The study of the adherence ability of the strains reported here showed that these bacteria adhered to the microvilli of the Caco-2 cell line. This adhesion was mediated by a nonfimbrial protein with a molecular mass of 29,000 Da designated CF29K. Pretreatment of bacteria with antibodies raised against CF29K or Caco-2 cells with purified CF29K prevented the adhesion of K. pneumoniae strains to Caco-2 cells. CF29K immunologically cross-reacted with the CS31A surface protein of Escherichia coli strains involved in septicemia in calves. Genes encoding CF29K were located on a high-molecular-weight conjugative R plasmid, which transferred to E. coli K-12. Transconjugants expressed a large amount of CF29K protein and adhered to the brush border of Caco-2 cells. These findings show that K. pneumoniae strains were able to colonize the human intestinal tract through a plasmid-encoded 29,000-Da surface protein. Hybridization experiments indicated that the gene encoding resistance to broad-spectrum cephalosporins by the production of CAZ-1 enzyme and the gene encoding the adhesive property to intestinal cells were both located on a 20- to 22-kb EcoRI restriction DNA fragment. Genes encoding aerobactin and the ferric aerobactin receptor were also found on this R plasmid.
Free versus liposome-encapsulated muramyl tripeptide phosphatidylethanolamide in treatment of experimental Klebsiella pneumoniae infection
Authors: Melissen P M , van Vianen W , Rijsbergen Y , Bakker-Woudenberg I A
Published: January 2020

The effect of free and liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) on resistance to Klebsiella pneumoniae infection in mice was investigated. It was shown that administration of MTPPE, at 24 h before bacterial inoculation, led to a dose-dependent antibacterial resistance in terms of increased clearance of bacteria from the blood and bacterial killing in various organs. The lowest effective dose of MTPPE was 50 micrograms per mouse. Administration of liposome-encapsulated MTPPE was also effective at a dose of 25 micrograms per mouse. The time of administration of both free and liposome-encapsulated MTPPE, with respect to the appearance of bacteria in the blood, was very important and indicated that repeated administration is necessary to obtain protection for a prolonged period. In view of the toxicity of MTPPE, it was an important observation that repeated administration of MTPPE in the liposome-encapsulated form also produced antibacterial resistance. Administration of free and liposome-encapsulated MTPPE resulted in increased numbers of granulocytes, monocytes, and lymphocytes in the blood of uninfected mice and prevented leukopenia in infected mice.
Modification in penicillin-binding proteins during in vivo development of genetic competence of Haemophilus influenzae is associated with a rapid change in the physiological state of cells
Authors: Dargis M , Gourde P , Beauchamp D , Foiry B , Jacques M , Malouin F
Published: January 2020

By using whole-cell labeling assay with 125I-penicillin V, we observed a reduction in the binding of the radiolabeled beta-lactam to four or five penicillin-binding proteins (PBPs) in Haemophilus influenzae cells cultivated under specific conditions. PBPs 3A, 3B, 4, and 6 were altered after the growth of bacteria in diffusion chambers implanted in the peritoneal cavity of rats. PBP 2 was also modified when cells were cultivated in human cerebrospinal fluids. Because this observation may have important consequences on the efficacy of beta-lactams during antibiotic therapy, we characterized the physiological state of bacteria cultivated in animals in the hope of explaining how such important changes in cell properties develop in vivo. Since the development of natural genetic competence occurs at the stationary phase of growth in H. influenzae, we used a DNA transformation assay to evaluate the physiological state of bacteria grown in diffusion chambers implanted in rats. Chromosomal DNA isolated from an antibiotic-resistant donor strain was mixed with bacteria in diffusion chambers. At different times during a 5-h incubation period, recipient bacteria were collected from the chambers, CFU were determined by plate counting, and antibiotic-resistant transformants were isolated on selective plates. Genetic competence rapidly developed in cells grown in rats, and the frequency of transformation by test DNA was elevated. Electron microscopy revealed an irregular cell shape and blebs at the surface of bacteria cultivated in animals and in cerebrospinal fluids. In an attempt to induce a similar physiological state in vitro, we supplemented broth cultures with cyclic AMP or synchronized cultures by a nutritional upshift. No changes in PBPs were observed with supplemental cyclic AMP or during a single cell cycle. Finally, a reduction in the affinity of PBPs for 125I-penicillin V identical to that observed in bacteria grown in rats was observed in cells isolated from the stationary phase of growth in vitro. These results clearly indicate that H. influenzae cells grown in animals undergo a rapid change to a physiological state similar to that found in late-stationary-phase cultures in vitro. This observation indicates that the rational design of future and improved antibiotic therapy of H. influenzae infections should consider cell properties of slow-growing or latent bacteria.
Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice
Authors: Hill M R , McCallum R E
Published: January 2020

The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level. In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice. Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg). Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml. A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF. C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity. Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators. Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively. These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved. The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.
Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells
Authors: Prévost M C , Lesourd M , Arpin M , Vernel F , Mounier J , Hellio R , Sansonetti P J
Published: January 2020

Shigella flexneri causes bacillary dysentery, an invasive disease of colonic epithelial cells in humans. The capacity of bacteria, once they have entered into a cell and escaped the phagocytic vacuole, to spread intracellularly and directly to adjacent cells without further extracellular passage is a key factor in invasion of the epithelial layer. Movement of intracellular bacteria is dependent upon the polymerization of actin; concentration of the formed filaments to one end of the bacterium is associated with initiation of movement. This movement may lead to the formation of a protrusion at the cell surface through which the bacterium passes to an adjacent cell. Development of these protrusions in infected HeLa cells is described, with emphasis on two critical observations. First, initiation of movement is coupled with bacterial division since elongation of the bacterial body is associated with relocalization of the previously uniformly distributed layer of actin to one pole of the bacterium. Second, the actin-bundling protein plastin appears to bundle the actin filaments just posterior to the bacterium, producing an ongoing contraction of the cylindrical actin tail that may be associated with forward movement of the bacterium within the protrusion.
Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents
Authors: Sibold C , Wang J , Henrichsen J , Hakenbeck R
Published: January 2020

Sixty-six strains of Streptococcus pneumoniae isolated in different parts of the world, 46 resistant and 22 susceptible to penicillin, were subdivided by multilocus enzyme electrophoresis into 28 distinct electrophoretic types (ETs). The ETs to which penicillin-susceptible strains were assigned differed from those containing resistant isolates of the same serotype. Five common clones could be recognized among the penicillin-resistant bacteria by combining the ETs, the antigenic properties of penicillin-binding proteins PBP 1a and 2b, and the tetracycline and chloramphenicol resistance profiles. Two clones were found in Finland and were associated with capsular serotypes 6B and 23F, respectively. Two clones were from Spain (type 6B and 9V, respectively). The fifth clone was isolated in South Africa and in Spain and contained both serotype 23F isolates and one type 19F strain. The other resistant strains were represented by rare isolates distributed among 12 other ETs, confirming that resistance to penicillin has evolved by multiple branches. Because capsular type was mixed in several ETs, the results also demonstrate that it may vary among very closely related pneumococci.
Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile
Authors: Borriello S P , Wren B W , Hyde S , Seddon S V , Sibbons P , Krishna M M , Tabaqchali S , Manek S , Price A B
Published: January 2020

A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.
Analysis of adhesion and cytotoxicity of Tritrichomonas foetus to mammalian cells by use of monoclonal antibodies
Authors: Burgess D E , McDonald C M
Published: January 2020

The relationship of Tritrichomonas foetus adhesion to mammalian cells and cytotoxicity to these targets was investigated. High-adherence and low-adherence lines of T. foetus, derived by repeated adhesion to HeLa cells, showed high and low cytotoxicity, respectively, to HeLa cells. When parasites were separated from targets by membranes (0.4-microns pore size), no cytotoxicity was detectable. Monoclonal antibodies elicited against T. foetus that lowered adhesion also lowered parasite-mediated cytotoxicity. Flow cytometry experiments revealed that the levels of an adhesion- and cytotoxicity-blocking antibody bound to the surface of high-adherence clones of T. foetus were higher than those in low-adherence clones. Western blots of parasite extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with an anti-T. foetus antibody. A molecule with a molecular weight of approximately 190,000 composed of subunits with molecular weights of approximately 140,000 to 150,000 and approximately 65,000 was identified. Immunoprecipitation experiments with metabolically labeled T. foetus and the same antibody confirmed that similar subunits were synthesized by the parasite. These results indicate that adhesion of T. foetus to mammalian cells is an important step in cytotoxic damage of these targets and that a surface adhesin on the parasite is involved in the adhesion mechanism.
Effect of exogenous sialylation of the lipooligosaccharide of Neisseria gonorrhoeae on opsonophagocytosis
Authors: Kim J J , Zhou D , Mandrell R E , Griffiss J M
Published: January 2020

Serum-sensitive Neisseria gonorrhoeae strains become serum resistant when grown in the presence of a sialic acid precursor, cytidine monophospho-N-acetylneuraminic acid. We examined the abilities of human neutrophils to phagocytose sialylated and nonsialylated gonococci and observed a decrease in the complement-dependent phagocytosis of sialylated gonococci compared with that of nonsialylated gonococci (50.7 versus 25.9% survival at 30 min). This decrease in opsonophagocytosis after sialylation may contribute to the pathogenicity of gonococcal infections.
Mycobacterium leprae produces extracellular homologs of the antigen 85 complex
Authors: Pessolani M C , Brennan P J
Published: January 2020

The antigen 85 complex is a set of at least three closely related secreted proteins (85A, 85B, and 85C) of 30 to 32 kDa produced by Mycobacterium tuberculosis and other mycobacteria. Their prominence in Mycobacterium leprae, the one obligate intracellular pathogen of the genus, had been assumed on the basis of immunological evidence and proof of the existence of the gene encoding the 85B protein of the complex. We have now observed the production of this family of proteins by M. leprae through analysis of various fractions by Western blotting (immunoblotting) with monospecific rabbit antisera raised against the individual Mycobacterium bovis BCG 85A, 85B, and 85C proteins. A predominant cross-reactive band with an apparent molecular mass of 30 kDa was detected in extracts of nondisrupted whole M. leprae and in soluble fractions prepared from the tissues of M. leprae-infected armadillos. Further studies of the subcellular distribution of this protein within the bacterium confirmed that it is secreted by the organism, an observation that explains past difficulties in detecting the antigen 85 complex in M. leprae. Confirmation that the M. leprae product is a member of the antigen 85 complex was obtained by comparison of peptide fingerprints with those from the BCG product. The pattern of reactivity of the M. leprae antigen 85 complex with anti-M. bovis BCG 85B serum, as well as two-dimensional electrophoresis, established that the 85B component was the predominant member of the complex in M. leprae. The fibronectin-binding capacity of the M. leprae and BCG 85 complexes was reinvestigated by new approaches and is questioned. Nevertheless, the results obtained with the native proteins reinforce previous reports, derived primarily from the use of homologous proteins, that the antigen 85 complex is one of the dominant protein immunogens of the leprosy bacillus.
Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases
Authors: Sorsa T , Ingman T , Suomalainen K , Haapasalo M , Konttinen Y T , Lindy O , Saari H , Uitto V J
Published: January 2020

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.
Interaction of the fish pathogen Aeromonas salmonicida with rainbow trout macrophages
Authors: Garduño R A , Kay W W
Published: January 2020

A procedure was developed to culture rainbow trout macrophages (M phi) on supported glass coverslips. Using this method and a variety of well-characterized Aeromonas salmonicida strains with normal or altered cell surfaces, we investigated the role of this unusual bacterial surface in the bacterium-M phi interaction. An intact crystalline protein array, the A-layer, mediated adherence of A. salmonicida cells to M phi even in the absence of opsonins. In contrast, unopsonized cells of an A-layer-negative (A-) mutant with a smooth lipopolysaccharide (LPS) layer were unable to interact with M phi. However, this ability was recovered when the A-layer was reconstituted onto the smooth LPS surface of these A- LPS+ cells. Two A. salmonicida mutants possessing the A-layer in different disorganized states had a reduced ability to interact with M phi. A+ cells grown under calcium limitation produced A-layers locked into an alternative conformation which mediated the highest levels of M phi association in the absence of opsonins or any other surface coating. Coating A+ cells with hemin greatly increased their levels of M phi association, and bacterial cells grown on trout blood agar plates also had a dramatic increase in their ability to interact with M phi. Only A+ A. salmonicida cells were highly cytotoxic to trout M phi, especially after being coated with hemin, presumably due to a more focused targeting of the bacterial cell onto the M phi surface and/or into the intracellular regions of the M phi.
Protection of mice against Salmonella typhimurium with an O-specific polysaccharide-protein conjugate vaccine
Authors: Watson D C , Robbins J B , Szu S C
Published: January 2020

Serious infections with salmonellae remain a threat in many human populations. Despite extensive study of salmonella infections in animals and clinical experience with killed cellular vaccines, there are no vaccines against serotypes other than Salmonella typhi licensed for human use. Serum antibodies to the O-specific polysaccharide (O-SP) of salmonellae protect mice against invasive infection. In order to render it immunogenic, we have conjugated the O-SP of Salmonella typhimurium to carrier proteins by various schemes. O-SP conjugated to tetanus toxoid (O-SP-TT) elicited antibodies in outbred mice after three subcutaneous injections without adjuvant. The O-SP alone elicited no detectable antibody. The antibody response to O-SP-TT was boosted by successive doses and consisted of immunoglobulin G (IgG) and IgM. Most mice only produced antibodies specific for the abequose (O:4 factor) region of the O-SP. Occasional animals also produced antibodies to the core oligosaccharide. Immunized mice were protected against intraperitoneal challenge with S. typhimurium, demonstrating a 160-fold increase in the 50% lethal dose. Passive immunization with conjugate-induced IgM or IgG also protected against challenge. These results indicate that an O-SP-TT conjugate, when given by a route and formulation acceptable for human use, protects mice against challenge with S. typhimurium.
High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide
Authors: Gazzano-Santoro H , Parent J B , Grinna L , Horwitz A , Parsons T , Theofan G , Elsbach P , Weiss J , Conlon P J
Published: January 2020

Bactericidal/permeability-increasing protein (BPI) is a 55-kDa cationic protein (nBPI55) elaborated by polymorphonuclear neutrophils (PMN). BPI has potent bactericidal activity against a wide variety of gram-negative organisms and neutralizes endotoxin activities. An N-terminal fragment of nBPI55 exhibits the bactericidal and antiendotoxin properties of the holoprotein. To further characterize the biological activities of the N-terminal fragment, a recombinant protein (rBPI23) corresponding to the first 199 amino acids of human BPI was produced and purified. rBPI23 had antibacterial activity equivalent to that of nBPI55 against Escherichia coli J5. Furthermore, both rBPI23 and nBPI55 bound identically to a broad range of R- and S-form lipopolysaccharides (LPS) and to natural and synthetic lipid A. Binding of radiolabeled nBPI55 to LPS was inhibited in an identical fashion by either nBPI55 or rBPI23. The binding of both proteins to immobilized E. coli J5 lipid A was inhibited in a comparable fashion by long- or short-chain LPS or lipid A. The binding of both rBPI23 and nBPI55 was specific, saturable, and of high affinity, with an apparent Kd of approximately 2 to 5 nM for all ligands tested. These results demonstrate that BPI recognizes the highly conserved lipid A region of bacterial LPS via residues contained within the amino-terminal portion of the BPI molecule.
A murine model of chronic mucosal colonization by Pseudomonas aeruginosa
Authors: Pier G B , Meluleni G , Neuger E
Published: January 2020

Chronic mucosal colonization by Pseudomonas aeruginosa is an integral part of the pathologic process associated with disease due to infection with this organism. We have adapted the streptomycin-treated murine model of chronic mucosal colonization by enteric pathogens to study colonization by P. aeruginosa. Mice first received 1 mg of streptomycin per ml of drinking water for 2 to 5 days and then ingested 10(7) CFU of P. aeruginosa per ml of drinking water for a minimum of 5 days. The result of this regimen was chronic mucosal colonization with P. aeruginosa for up to 10 weeks, which was determined by fecal cultures and confirmed by culture of the intestines after killing of the experimental animals. Bacterial counts were highest in the cecum and colon, with some evidence for extraintestinal bacterial translocation as well. Use of P. aeruginosa mutants deficient in the production of colonization factors such as pili and those dependent on the rpoN gene product resulted in a lower level of chronic colonization. Immune responses to type-specific lipopolysaccharide, pili, and flagellar antigens were measured, and increases in both serum and intestinal antibodies were usually elicited when a strain elaborated a given antigen. This model represents an easy method of routinely achieving chronic mucosal colonization by P. aeruginosa and should prove useful for the study of both bacterial virulence factors and host responses associated with this infectious process.
Gamma interferon modifies CD4+ subset expression in murine candidiasis
Authors: Romani L , Cenci E , Mencacci A , Spaccapelo R , Grohmann U , Puccetti P , Bistoni F
Published: January 2020

A single injection of monoclonal antibody to gamma interferon administered in conjunction with a live Candida albicans yeast cell vaccine resulted in the detection of nonprotective Th2 rather than protective Th1 responses and altered the early expression of interleukin 4 and gamma interferon mRNA in CD4+ cells.
Effect of Pasteurella multocida toxin on bone resorption in vitro
Authors: Felix R , Fleisch H , Frandsen P L
Published: January 2020

Pasteurella multocida toxin (PMT), which is the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs, was found to stimulate bone resorption in vitro. This stimulation was observed both in cultures of murine calvaria by measuring the release of calcium and of the lysosomal enzyme beta-glucuronidase and in murine long bone cultures by measuring the release of calcium. Both systems showed the same dose response curve, with the maximal effect at a concentration of 5 ng/ml. The effect on calvaria was studied in more detail. PMT increased bone resorption 24 h after its addition and always had to be present to express an effect. Calcitonin was able to inhibit this increase of resorption completely, and inhibitors of prostaglandin synthesis suppressed it partially. Although the data show an effect of PMT on bone tissue, the results do not exclude an action on cells in the nasal cavity, which could indirectly stimulate bone resorption.
Modulation of endotoxin-induced monokine release in human monocytes by lipid A partial structures that inhibit binding of 125I-lipopolysaccharide
Authors: Ulmer A J , Feist W , Heine H , Kirikae T , Kirikae F , Kusumoto S , Kusama T , Brade H , Schade U , Rietschel E T
Published: January 2020

We have previously shown that the synthetic tetraacyl precursor Ia (compound 406, LA-14-PP, or lipid IVa) was not able to induce the production of tumor necrosis factor, interleukin-1, and interleukin-6 in human monocytes but strongly antagonized lipopolysaccharide (LPS)-induced formation of these monokines. This inhibition was detectable at the level of mRNA production. To achieve a better understanding of molecular basis of this inhibition, we investigated whether lipid A precursor Ia (LA-14-PP), Escherichia coli-type lipid A (LA-15-PP), Chromobacterium violaceum-type lipid A (LA-22-PP), and synthetic lipid A partial structures and analogs (LA-23-PP, LA-24-PP, and PE-4) were able to influence the binding of 125I-LPS to human monocytes and compared this inhibitory activity with the agonistic and antagonistic action in the induction of monokines in human monocytes. 125I-LPS (20 ng per well) was added to human monocytes in the presence or absence of unlabeled rough Re mutant-derived LPS (Re-LPS) or lipid A compounds, and specific LPS binding was determined after 7 h. This binding was found to be dependent on CD14 as shown by the use of an anti-CD14 monoclonal antibody. Compound LA-14-PP was found to inhibit the binding of 125I-LPS to the cells in a similar dose-response to that of unlabeled LPS. This shows that the inhibitory capacity on LPS binding does not correlate with the monokine-inducing capacity because Re-LPS is active in inducing tumor necrosis factor, interleukin-1, and interleukin-6, while LA-14-PP is not. The strong capacity of LA-14-PP to inhibit 125I-LPS binding, however, correlates with the strong inhibitory capacity of this compound on LPS-induced monokine production. Compounds LA-15-PP, LA-23-PP, and LA-24-PP were active in the inhibition of 125I-LPS binding but were 5- to 10-fold weaker than Re-LPS and LA-14-PP. Of all lipid A structures tested, compound LA-22-PP expressed the weakest inhibitory capacity on LPS binding. These compounds showed again that the activity of binding inhibition does not correlate with the monokine-inducing capacity. We assume that the inhibitory effects of lipid A partial structures on LPS-induced monokine production have their origin in a competitive inhibition between LPS and the lipid A partial structures for the same binding site on the cell membrane.