bluuebunny/crossref_metadata_2025 huggingface dataset:
Early gamma interferon production by natural killer cells is important in defense against murine listeriosis
Authors: Dunn P L , North R J
Published: January 2020

A spot enzyme-linked immunosorbent assay was used to show that subcutaneous inoculation of a sublethal number of Listeria monocytogenes resulted in the early appearance of gamma interferon (IFN-gamma)-producing cells in the draining lymph nodes. In contrast, inoculation of UV-killed L. monocytogenes failed to cause the appearance of IFN-gamma-producing cells. The appearance of IFN-gamma-secreting cells in response to the living organisms peaked at 24 h of infection and then declined. The draining lymph node cells responsible for secreting IFN-gamma belonged to a cell population that was positive for the NK1.1, asialo-GM1, and Thy-1 markers but negative for the CD4 and CD8 T cell subset markers. Early elimination of natural killer (NK) cells by treatment with anti-NK cell antibodies resulted in severe exacerbation of infection, as did early neutralization of endogenous IFN-gamma by treatment with a rat anti-murine IFN-gamma monoclonal antibody. In contrast, depletion of CD4+ and CD8+ T cells failed to exacerbate infection. The results serve to show that the early production of IFN-gamma by NK cells, rather than by T cells, is an essential event in resistance to listeriosis.
Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces
Authors: Gibbons R J , Hay D I , Schlesinger D H
Published: January 2020

Cells of several strains of Streptococcus gordonii attached in much higher numbers to experimental pellicles formed from samples of submandibular or parotid saliva on hydroxyapatite (HA) beads than to buffer controls. The nature of the salivary components responsible were investigated by preparing experimental pellicles from chromatographic fractions of submandibular saliva obtained from Trisacryl GF 2000M columns. Adhesion of S. gordonii Blackburn was promoted by two groups of fractions. The adhesion-promoting activity in the first group of fractions was associated with the family of acidic proline-rich proteins (PRPs), while that of the second group is as yet unidentified. Experimental pellicles prepared by treating HA with 2 micrograms of pure 150-amino-acid-residue PRPs (PRP-1, PRP-2, and PIF-s) promoted adhesion of S. gordonii Blackburn cells to an extent comparable to that obtained with unfractionated saliva. However, pellicles prepared from a 106-residue PRP (PRP-3) were significantly less effective, and those prepared from the amino-terminal tryptic peptide (residues 1 to 30) of the PRP and the salivary phosphoprotein statherin were completely ineffective in promoting adhesion. Although adhesion of several strains of S. gordonii was promoted by adsorbed PRP-1, the adhesion of several strains of Streptococcus sanguis or Streptococcus oralis was either not affected or only weakly enhanced by this protein. S. gordonii cells bound avidly to PRPs adsorbed onto HA beads, but the streptococci did not appear to bind PRPs in solution, since concentrations of PRP as high as 200 micrograms/ml did not inhibit binding of bacterial cells to pellicles prepared from pure PRP. S. gordonii cells also attached well to PRP or a synthetic decapeptide representing residues 142 to 150 of the PRP when the peptide was linked to agarose beads. Studies with a series of synthetic decapeptides indicated that the minimal segment of PRP which promoted high levels of S. gordonii adhesion was the carboxy-terminal dipeptide Pro-Gln (residues 149 and 150).
Oral Immunization with L-Forms of
Vibrio cholerae
in Human Volunteers
Authors: Agarwal S. C. , Ganguly N. K.
Published: January 2020

Oral administration of heat-inactivated lysates of
Vibrio cholerae
Ogawa L-forms elicited significantly high coproantibody, serum indirect hemagglutinating antibody, and vibriocidal antibody responses in human volunteers, against both homologous and heterologous subtypes of
V. cholerae
and
V. El Tor.
Five oral biweekly doses produced an adequate antibody response which persisted for at least 6 weeks after immunization. No untoward side effects were seen.
Serological Studies on the Teichoic Acids of
Lactobacillus plantarum
Authors: Knox K. W. , Wicken A. J.
Published: January 2020

Both the wall ribitol teichoic acid and the membrane glycerol teichoic acid (lipoteichoic acid) from
Lactobacillus plantarum
NCIB 7220 have α-
d
-glucosyl substituents. Antisera to the ribitol teichoic acid were obtained by injecting whole or disintegrated organisms, the antigenicity of the wall teichoic acid apparently depending on its association with protein. It was necessary to inject disintegrated organisms or purified lipoteichoic acid to ensure the production of antibodies to the glycerol teichoic acid; these antibodies did not react with ribitol teichoic acid. The specificity of antibodies to the wall ribitol teichoic acid depends primarily on the α-
d
-glucosyl substituents, as the antibodies cross-react with α-
d
-glucosyl-substituted glycerol teichoic acids but not with an unsubstituted ribitol teichoic acid. The specificity of antibodies to the membrane glycerol teichoic acid may be directed against either the glucose or glycerol components, depending on the preparation injected.
Complement-Fixing Antigens in Lymphoid Cell Lines of American Origin
Authors: McCormick Kenneth J. , Mumford David M. , Stenback Wayne A. , Trentin John J.
Published: January 2020

Lymphoid cell lines from Americans with infectious mononucleosis (Choate, EH IV, and OP), and from a normal American (Cassio), were tested for presence of herpes-like virus by electron microscopy (EM) and immunofluorescence (IF), and for complement-fixing (CF) antigens, using both American and African sera. Whereas earlier tests of seven African (Burkitt) lymphoma cell lines showed an absolute correlation between presence of herpes-like virus and CF reactivity with either African or American sera, the same was not true of the American cell lines. Herpes-like particles were found in the Cassio line by both EM and IF, and in a few cells of the OP line by IF, but not by EM. The virus was not found in the Choate or EH IV lines by either EM or IF. African sera from either normal individuals or patients with Burkitt lymphoma contained CF antibodies to extracts of Cassio and OP cells. Normal American sera contained CF antibodies to these extracts as well as to extracts of Choate cells. The EH IV cell line did not produce CF antigens detectable with either African or American sera. The data indicate that the CF antigens of the herpes virus-negative Choate cell line were serologically distinct from those in Burkitt lines. However, it is possible that the antigens from both sources are related to the presence of the genome of the herpes-like virus.
Responses of Lymphocytic Choriomeningitis Carrier Mice to X-Ray-Induced Lymphoma
Authors: Sharon Nehama , Pollard Morris
Published: January 2020

The data presented in this paper define the interrelationship between two endogenous diseases in the same host. The Haas strain of germfree mice with congenitally acquired persistent lymphocytic choriomeningitis (LCM) virus infection developed the X-ray-induced lymphatic leukemia independent of LCM viremia. The immunoproliferative LCM-related lesions appeared after age 7 months, and the radiogenic lymphatic leukemia appeared earlier or during the same age period. Both diseases evolved with distinctive lesions at the appropriate time, with no evidence of interference nor of synergism between the two syndromes. The Haas strain of mice showed an unusual sensitivity to X rays which was associated with depletion of blood components and coincident death. The sensitivity of the mice to X rays might be a characteristic of the Haas mouse or the result of an LCM virus-induced alteration in hematopoiesis.
Heterogeneity of
Streptococcus mutans
Strains Based on Their Mannitol-1-Phosphate Dehydrogenases: Criterion for Rapid Classification
Authors: Brown Albert T. , Patterson Constance E.
Published: January 2020

Hexitol phosphate dehydrogenases were inducible in all strains of
Streptococcus mutans
studied, and these organisms could be placed into one of four groups according to the electrophoretic mobility of their mannitol-1-phosphate dehydrogenases.
Role of the Macrophage in Natural Resistance to Salmonellosis in Mice
Authors: Maier T. , Oels H. C.
Published: January 2020

In vitro, macrophages from normal strain BRVR mice killed
Salmonella
more quickly than did macrophages from normal strain BSVS mice.
Salmonella
injected intraperitoneally multiplied more quickly in BSVS mice than in BRVR mice. BRVR macrophages injected intraperitoneally into BSVS mice protected against
Salmonella
multiplication better than did BSVS macrophages. The populations of peritoneal cells that could be washed from the peritoneal cavity of normal BRVR and BSVS mice were morphologically and numerically identical. In vitro, BSVS macrophages were as efficient as BRVR macrophages in phagocytizing virulent
Salmonella
. These findings all support the concept that the greater natural resistance of BRVR mice to
Salmonella
infection may be explained by the greater ability of normal BRVR macrophages to kill ingested
Salmonella
.
Response of Cultured Mammalian Cells to Diphtheria Toxin V. Concurrent Resistance to Ribonucleic Acid Viruses in Diphtheria Toxin-Resistant KB Cell Strains
Authors: Moehring T. J. , Moehring J. M.
Published: January 2020

It was determined that KB-R cell strains, isolated from the KB cell line and resistant to diphtheria toxin, also resist infection by poliovirus, Mengo virus, vesicular stomatitis virus, and Newcastle disease virus. This resistance manifests itself by reduction in yields of progeny virus (a reduction of more than 2 logs in some cases), reduced production of viral-specific ribonucleic acid (RNA), and delayed cytopathic effect. In three KB-R strains tested, resistance was related to a step which falls between adsorption of virus and uncoating or release of viral messenger RNA. In two of these three strains, a second resistance mechanism was also active, causing a reduced production of viral-specific RNA. A relationship between the resistance to diphtheria toxin and the resistance to viral infection of the KB-R strains is considered. It has been postulated that the native diphtheria toxin molecule must be “activated” at the surface of a susceptible cell by a proteolytic process before it can enter the cell and inhibit protein synthesis. It is also known that the eclipse of some viruses occurs at or near the cell membrane and involves proteolytic activity. Resistance to viruses and toxin in the KB-R strains may result from the loss or modification of related labilizing or activating principles associated with the surface receptors for these agents.
Role of a Protease in Natural Activation of
Clostridium botulinum
Neurotoxin
Authors: Gupta Bibhuti R. Das , Sugiyama H.
Published: January 2020

All tested proteolytic
Clostridium botulinum
type A, B, and F strains and certain non-proteolytic B and F cultures produced a protease having trypsin-like substrate specificity; none of the tested type E (non-proteolytic) strains produced the enzyme. Progenitor toxin (toxic form whose specific toxicity is increased by treatment with trypsin) was found in culture fluid concentrates of all strains not producing the protease; it was also present in some concentrates that had the enzyme. Activation of highly purified type E progenitor toxin (molecular weight 150,000) by essentially pure protease from a proteolytic type B culture was always less than that obtained with trypsin. The product of the type E progenitor toxin-protease reaction increased in toxicity when further treated with trypsin. Results suggest that at least two bonds are cleaved by trypsin during activation of type E progenitor toxin to toxin (form manifesting maximal possible specific toxicity). Natural activation of progenitor toxin of proteolytic strains may also involve cleavage of more than one bond.
Properties of Purified Pneumococcal Hemolysin
Authors: Johnson Mary K.
Published: January 2020

With a purified preparation of pneumococcal hemolysin, a sigmoid relationship was found to exist between lysin concentration and hemolytic activity. Hemolysis was inhibited by a high ratio of erythrocytes to lysin, suggesting a multi-hit mechanism of action. Reaction rate decreased rapidly with time, possibly due to competition between ghosts and unlysed erythrocytes for fixation of lysin. The effects of
p
H and various agents on the processes of lysin adsorption and hemoglobin release were determined. The pneumococcal preparation did not possess nicotinamide adenine dinucleotide glycohydrolase activity.
Adjuvant Activity of
Mycobacterium leprae
Authors: Stewart-Tull D. E. S. , Davies M.
Published: January 2020

Mycobacterium leprae
organisms isolated from infected spleen and liver tissue by zonal centrifugation were shown to possess adjuvant activity. Histochemical examination of the footpad macrophage epithelioid granuloma showed that macrophages contained large amounts of hemosiderin after the injection of
M. leprae
and much smaller amounts after the injection of
M. tuberculosis.
Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae
Authors: Briles D E , Crain M J , Gray B M , Forman C , Yother J
Published: January 2020

The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice.
Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27)
Authors: Benz I , Schmidt M A
Published: January 2020

The adherence of diarrhea-associated Escherichia coli to the small-bowel mucosa is an important step in the pathogenesis of diarrheal diseases. In tissue culture systems, diarrhea-associated strains show three distinct patterns of adherence: localized adherence, diffuse adherence (DA), and the recently described aggregative adherence. To study the molecular basis of the DA phenotype, we investigated the diarrhea-associated DA strain 2787 (O126:H27), isolated from a case of infantile diarrhea. The DA phenotype is mediated by a 6.0-kb DNA fragment derived from a 100-kb plasmid harbored by the wild-type strain. This fragment codes for a 100-kDa protein which can be released from the bacterial cell into the supernatant by mild heat treatment. Recombinant DA+ strains as well as the isolated 100-kDa protein were used to engender specific antisera in rabbits. As demonstrated by Western blotting (immunoblotting), the antibodies engendered by the recombinant DA+ strain recognized a 100-kDa protein in the wild-type strain 2787 and in all recombinant strains showing DA. Immunogold electron microscopy localized the 100-kDa protein to the bacterial cell surface. Serologically related proteins of similar size were detected by Western blotting in other DA+ diarrhea-associated strains belonging to enteropathogenic E. coli serotypes. The 100-kDa protein denoted AIDA-I (adhesin involved in diffuse adherence) binds in a saturable fashion to HeLa cells. AIDA-I-specific immunoglobulin G antibodies--and, to an even greater extent, Fab fragments derived thereof--inhibited bacterial attachment to HeLa cells. This is direct evidence that the 100-kDa protein is the adhesin mediating the DA phenotype of these diarrhea-associated strains and is representative of a group of serologically related proteins in other DA+ strains.
Tumor necrosis factor and interleukin-6 in Candida albicans infection in normal and granulocytopenic mice
Authors: Steinshamn S , Waage A
Published: January 2020

We administered a neutralizing monoclonal antibody to tumor necrosis factor (TNF) during infection with Candida albicans in normal and granulocytopenic mice. Mice were rendered granulocytopenic (less than 0.1 x 10(9) granulocytes per liter) with cyclophosphamide. Growth of C. albicans from the kidneys was significantly increased in normal mice treated with the antibody to TNF, compared with that in control mice, after 36 h (3.6 x 10(4) +/- 1.2 x 10(4) CFU per kidney versus 9.1 x 10(3) +/- 6.2 x 10(3) CFU per kidney; P less than 0.05) and after 72 h (3.7 x 10(6) +/- 2.7 x 10(6) CFU per kidney versus 2.3 x 10(4) +/- 1.3 x 10(4) CFU per kidney; P less than 0.01). In granulocytopenic mice, the antibody to TNF had no effect on the growth of C. albicans from the kidneys. Furthermore, our study showed that the cytokines TNF and interleukin-6 (IL-6) were produced in a dose-dependent manner during C. albicans infection. TNF was detectable between 6 and 60 h, with peak levels at 24 h. Both TNF and IL-6 levels were significantly higher in cyclophosphamide-treated mice than in normal mice. Heat-inactivated C. albicans induced a TNF response different from that induced by viable C. albicans, with an early peak occurring at 3 to 4 h and declining to non-detectable levels after 15 to 24 h. Peak levels of TNF obtained with heat-inactivated C. albicans were lower than those obtained with viable C. albicans. Our study demonstrates that TNF and IL-6 are produced systemically during C. albicans infection and suggests that TNF is essential for granulocyte antifungal activity in vivo.
The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen
Authors: Sundstrom J B , Cherniak R
Published: January 2020

The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.
Rhodopseudomonas sphaeroides lipid A derivatives block in vitro induction of tumor necrosis factor and endotoxin tolerance by smooth lipopolysaccharide and monophosphoryl lipid A
Authors: Henricson B E , Perera P Y , Qureshi N , Takayama K , Vogel S N
Published: January 2020

Rhodopseudomonas (Rhodobacter) sphaeroides diphosphoryl lipid A is a relatively inert species of lipid A but has been shown to antagonize the effects of toxic lipopolysaccharide (LPS) both in vivo and in vitro. The antagonist and its monophosphoryl derivative were examined for the ability to block tumor necrosis factor synthesis and reverse tolerance induction in vitro in macrophage cultures stimulated with bioactive preparations of smooth LPS, rough LPS, diphosphoryl lipid A, and monophosphoryl lipid A. Inhibition of agonist activity and reversal of tolerance by these novel penta-acylated lipid A antagonists provides new insight into macrophage-LPS interactions.
Invasin expression in Yersinia pseudotuberculosis
Authors: Simonet M , Falkow S
Published: January 2020

A 3.2-kb region on the chromosome of Yersinia pseudotuberculosis, called inv, encodes invasin, a 103-kDa protein of the bacterial outer membrane. Invasin mediates bacterial entry into cultured animal cells. Six Y. pseudotuberculosis strains isolated from animal or human infections were analyzed for the presence of inv-related sequences with a radiolabeled inv clone, pRI203. We found that inv-specific sequences were present in all strains studied. Strains cured of virulence plasmid pYV were studied by Western immunoblot analysis with a monoclonal antibody directed against invasin. All but one strain produced invasin, but some strains produced more invasin than others. A strong correlation was found between the level of invasin production by these strains and their ability to enter into HEp-2 or CHO cells. The virulence of these strains was assessed in a murine model by measuring the number of bacteria in the spleen after intravenous challenge or in the mesenteric lymph nodes after intragastric challenge. The capacities of strains to invade cultured mammalian cells and to colonize the spleen were strongly correlative. In contrast, the ability of strains to translocate from the intestinal lumen to the mesenteric lymph nodes after intragastric inoculation did not correlate with their in vitro invasiveness.
Influence of serotype of group B streptococci on C3 degradation
Authors: Campbell J R , Baker C J , Edwards M S
Published: January 2020

Serotype III strains of group B streptococci (GBS) are isolated from the majority of young infants with bacteremia or meningitis. We hypothesized that serotype-associated differences in structure of the type-specific capsular polysaccharide or the presence of c protein would influence the extent to which C3 degradation occurs on GBS and that type-specific antibody would alter C3 deposition or degradation patterns. When clinical isolates of GBS representing serotypes Ia, Ib/c, II (with or without c protein) and III were employed with hypogammaglobulinemic serum as an opsonic source, a remarkable similarity was observed in patterns of C3 deposition and degradation for each of the four GBS serotypes and between strains with or without c protein. Both C3b and iC3b were detected by 5 min and throughout a 90-min opsonization interval. Less deposition occurred at 5 min on serotypes Ia and Ib/c than on types II and III GBS. Minimal degradation to C3d or smaller fragments was observed. Type-specific antibody facilitated C3b deposition on GBS and C3b degradation to iC3b early in opsonization. Possibly, accessibility of C3 fragments to neutrophil receptors, rather than the extent to which the surface permits C3 degradation, accounts for differential virulence among GBS serotypes.
Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation
Authors: Allison C , Coleman N , Jones P L , Hughes C
Published: January 2020

Proteus mirabilis causes serious kidney infections which can involve invasion of host urothelial cells. We present data showing that the ability to invade host urothelial cells is closely coupled to swarming, a form of cyclical multicellular behavior in which vegetative bacteria differentiate into hyperflagellated, filamentous swarm cells capable of coordinated and rapid population migration. Entry into the human urothelial cell line EJ/28 by P. mirabilis U6450 isolated at different stages throughout the swarming cycle was measured by the antibiotic protection assay method and confirmed by electron microscopy. Differentiated filaments entered urothelial cells within 30 min and were 15-fold more invasive (ca. 0.18% entry in 2 h) than an equivalent dry weight of vegetative cells isolated before differentiation, which attained only ca. 0.012% entry in the 2-h assay. The invasive ability of P. mirabilis was modulated in parallel with flagellin levels throughout two cycles of swarming. Septation and division of intracellular swarm cells produced between 50 and 300 vegetative bacteria per human cell, compared with 4 to 12 intracellular bacteria after incubation with vegetative cells. Transposon (Tn5) mutants of P. mirabilis with specific defects in motility and multicellular behavior were compared with the wild-type for the ability to invade. Mutants which lacked flagella (nonmotile nonswarming) were entirely noninvasive, and those which were motile but defective in swarm cell formation (motile nonswarming) were 25-fold less invasive than wild-type vegetative cells. Mutants with defects in the coordination of multicellular migration and the temporal control of consolidation (cyclical reversion of swarm cells to vegetative cells) were reduced ca. 3- to 12-fold in the ability to enter urothelial cells. In contrast, a nonhemolytic transposon mutant which swarmed normally retained over 80% of wild-type invasive ability. Swarm cells and early consolidation cells were at least 10-fold more cytolytic than vegetative cells as a result of their high-level production of hemolysin.
Electrophoretic karyotypes of clinical isolates of Coccidioides immitis
Authors: Pan S , Cole G T
Published: January 2020

Chromosomes of the fungal respiratory pathogen, Coccidioides immitis, were separated by contour-clamped homogeneous electric field gel electrophoresis. Twelve isolates were examined, the majority of which showed four chromosomes with a range of molecular size from 11.5 to 3.2 Mb. Three isolates (C634, C735, and L) revealed three chromosomal bands under the conditions employed for electrophoretic separation. However, in two of these isolates (C634 and C735), four chromosomes were visible on membrane transfers of pulsed-field gels after Southern hybridization between the chromosomal DNA and selected DNA probes. The probes included a conserved ribosomal gene and three previously described cDNAs isolated from C. immitis expression libraries. The L isolate was determined to have the same genome size as a typical four-chromosome isolate on the basis of microspectrophotometric comparison of fluorescence intensity of the ethidium bromide-stained nuclear DNA. The genome size of C. immitis determined by microspectrophotometry was approximately 28.2 +/- 2.6 Mb. The calculated genome size based on addition of the average molecular weights of chromosomal bands separated by contour-clamped homogeneous electric field gel electrophoresis was approximately equal to the estimate derived from the spectrophotometric analyses. This is the first report of the electrophoretic karyotype of C. immitis.
Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques
Authors: Casanova M , Lopez-Ribot J L , Martinez J P , Sentandreu R
Published: January 2020

Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with 14C-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (beta ME) and with beta-glucanases (Zymolyase) after treatment with beta ME. The beta ME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.
Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface
Authors: Rambukkana A , Das P K , Burggraaf J D , Yong S , Faber W R , Thole J E , Harboe M
Published: January 2020

Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.
Frequency of antigen-specific B cells during experimental ocular Chlamydia trachomatis infection
Authors: Pal S , Taylor H R , Huneke R B , Prendergast R A , Whittum-Hudson J A
Published: January 2020

Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cervical lymph nodes by an ELISPOT assay in a cynomolgus monkey model of trachoma. These local sites contained numbers of chlamydia-specific B cells that were higher than those in distant inguinal lymph nodes and peripheral blood. The numbers of chlamydia-specific immunoglobulin G-secreting B cells observed were 5 to 57 per 10(6) cells in conjunctiva and 24 to 996 per 10(6) cells in cervical lymph nodes during conjunctival infection or after challenge of immune monkeys with the chlamydial 57-kDa heat shock protein (hsp60). These studies demonstrate a large chlamydia-specific B-cell component in the conjunctiva during ocular chlamydial infection. These results are similar to our findings for chlamydia-specific T-cell responses.
Nutritional interactions between two suspected periodontopathogens, Treponema denticola and Porphyromonas gingivalis
Authors: Grenier D
Published: January 2020

A mutual symbiotic enhancement of growth of Porphyromonas gingivalis and Treponema denticola is described in this report. Brain heart infusion broth supplemented with vitamin K did not support the individual growth of P. gingivalis or T. denticola. However, when inoculated as a mixture, both bacterial species did grow significantly. The growth-stimulating factors produced by P. gingivalis and T. denticola were dialyzable and heat stable and were further identified as isobutyric acid and succinic acid, respectively. Since some forms of periodontal disease are associated with the presence, in affected sites, of high numbers of P. gingivalis and spirochetes, it is suggested that the bacterial interaction described in this report might be of utmost importance in the initiation and progression of the disease.
Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis
Authors: Sommer P , Gleyzal C , Guerret S , Etienne J , Grimaud J A
Published: January 2020

There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies.
Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae
Authors: Gilsdorf J R , Chang H Y , McCrea K W , Bakaletz L O
Published: January 2020

Thirty-eight clinical isolates of nontypeable Haemophilus influenzae were tested for the presence of hemagglutinating pili similar to those of H. influenzae type b (Hib) that mediate buccal epithelial cell adherence. Four endogenously hemagglutinating (HA+) strains were identified, and eight additional HA+ variants were obtained from HA- strains by erythrocyte enrichment. All 12 HA+ nontypeable H. influenzae isolates bound antisera directed against denatured pilins of Hib, but none bound antisera against assembled native pili of Hib. In erythrocyte- and buccal-cell-binding assays, HA+ nontypeable H. influenzae binding was reduced compared with HA+ Hib binding and was not significantly different from HA- nontypeable H. influenzae binding. Both HA- and HA+ nontypeable H. influenzae binding was increased over binding of HA- Hib. HA+ nontypeable H. influenzae strains agglutinated adult erythrocytes that possess the Anton antigen, which is thought to be the receptor for Hib pili, and did not agglutinate cord or Lu(a-b-) dominant erythrocytes, which lack the Anton antigen. Electron microscopy of HA- and HA+ variants of three nontypeable H. influenzae strains showed few or no surface appendages on the HA- organisms, but piluslike structures were seen on many organisms from two HA+ nontypeable H. influenzae strains and on a few organisms from one strain. Thus, nontypeable H. influenzae appears to possess structures that are immunologically similar to the pilins that make up the hemagglutinating pili of Hib. However, nontypeable H. influenzae appears to also possess mechanisms for erythrocyte and buccal cell adherence that are not directly correlated with the presence of a hemagglutinating pilus.
Non-O1 Vibrio cholerae intestinal pathology and invasion in the removable intestinal tie adult rabbit diarrhea model
Authors: Russell R G , Tall B D , Morris J G
Published: January 2020

A modified removable intestinal tie adult rabbit diarrhea (RITARD) model was used to investigate the intestinal pathology, intestinal bacterial colonization, intestinal fluid volume, and onset of diarrhea caused by non-O1 Vibrio cholerae. Three strains of non-O1 V. cholerae were studied. RITARD rabbits challenged with 10(3) CFU of strain NRT36S (a strain previously shown to cause diarrhea in volunteers) developed grade 3 diarrhea at 48 to 72 h. The mean counts of non-O1 V. cholerae isolated were 9.3 +/- 0.07 and 8.7 +/- 0.7 CFU/g from the small and large intestines, respectively. Histologic examination showed necrosis of the luminal epithelium in the colon and mild inflammatory cell infiltration in the adjacent lamina propria. The severity and extent of intestinal damage by strain NRT36S was dose dependent. Higher doses of strain NRT36S caused severe necrotizing colitis and enteritis, with bacteremia and mortality at less than 24 h in RITARD rabbits challenged with 10(9) CFU and at less than 48 h in RITARD rabbits challenged with 10(4) CFU. Electron and light microscopy demonstrated invasion of NRT36S into the luminal epithelial cells of the intestine. Challenge of RITARD rabbits with non-O1 V. cholerae A-5 and 2076-79 (strains which did not cause diarrhea in volunteers) did not cause diarrhea or intestinal pathology. Intestinal colonization was transient: at 72 h postchallenge, animals inoculated with strain A-5 were culture negative, while only low numbers of strain 2076-79 were detectable (approximately 0.4 to 0.8 CFU/g). Our data highlight the utility of the RITARD model, when combined with appropriate pathologic and bacteriologic studies, for obtaining insights into pathophysiologic mechanisms of enteric disease by non-O1 V. cholerae. In agreement with volunteer studies, non-O1 V. cholerae NRT36S is clearly pathogenic in this model; direct cell invasion may play a role in its ability to cause illness.
Virulence of non-type 1-fimbriated and nonfimbriated nonflagellated Salmonella typhimurium mutants in murine typhoid fever
Authors: Lockman H A , Curtiss R
Published: January 2020

The virulence of Salmonella typhimurium mutants that were unable to synthesize type 1 fimbriae was tested in a murine typhoid fever model. Nonfimbriated mutants (fim) exhibited a lower 50% lethal dose than a wild-type (fim+) strain and produced significantly higher mortality (fim, 55%; fim+, 37% [P less than 0.002]) in mice that were challenged orally. There was no difference in virulence when the wild-type and mutant strains were injected intraperitoneally into mice. The progress of a short-term lethal infection was monitored after oral inoculation of mice with a mixture containing equivalent numbers of fim+ wild-type and fim mutant bacteria. The results indicated that while both strains colonized the intestinal tract equally well and invaded internal organs, the S. typhimurium fim mutant proliferated in the blood of the mice faster than the fim+ strain. The results of the mixed oral challenge suggested that bacteremia caused by fim+ S. typhimurium was reduced or delayed by the sequestration of the fimbriated bacteria in the spleen, liver, and kidneys. Thus, type 1 fimbriae were not virulence factors for S. typhimurium in this model, and the fimbriae may be an impediment to the pathogen in this setting. An S. typhimurium double mutant lacking type 1 fimbriae and flagella (fla) also was tested in mice. The virulence of the fim fla mutant was greatly reduced compared with that of the wild-type strain (mortality from fim fla challenge, 11% [P less than 0.0005]). The significance of this latter result is discussed in relation to host adaptation by pathogenic salmonellae.
Hyaluronidase from infective Ancylostoma hookworm larvae and its possible function as a virulence factor in tissue invasion and in cutaneous larva migrans
Authors: Hotez P J , Narasimhan S , Haggerty J , Milstone L , Bhopale V , Schad G A , Richards F F
Published: January 2020

During skin penetration, infective hookworm larvae encounter hyaluronic acid as they migrate between epidermal keratinocytes and through the ground substance of the dermis. A hyaluronidase would facilitate passage through the epidermis and dermis during larval invasion. Zoonotic hookworm larvae of the genus Ancylostoma were shown to contain a hyaluronidase activity that migrated on modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) hyaluronic acid gels with an apparent Mr of 49,000. A second form with an Mr of 87,000 was also identified. The major etiologic agent of cutaneous larva migrans, A. braziliense, was shown to have the greatest enzyme activity, hydrolyzing up to 3.3 micrograms of hyaluronic acid per h per micrograms of total parasite protein at pH 6.0, whereas A. caninum and A. tubaeforme each had much less enzyme activity. The differences in enzyme activities between species correlated with differences in the intensities of the lytic zones at 49 and 87 kDa on SDS-PAGE hyaluronic acid gels. Hookworm hyaluronidase activity exhibited a broad pH optimum between 6.0 and 8.0 and did not hydrolyze chondroitin sulfate, two features that suggest that the hookworm enzyme is more like the invertebrate leech hyaluronidase than mammalian testicular or lysosomal hyaluronidase. Larvae of A. braziliense were shown to release hyaluronidase activity and degrade radiolabeled hyaluronic acid in vitro. Gold sodium thiomalate was identified as an enzyme inhibitor. The hyaluronidase is the second major virulence factor that we have identified from infective hookworm larvae.
Identification and characterization of a surface-associated protein (Ssp) of Staphylococcus saprophyticus
Authors: Gatermann S , Kreft B , Marre R , Wanner G
Published: January 2020

A 95-kDa protein was isolated from Staphylococcus saprophyticus 7108 grown on dialysis membranes placed on the surface of brain heart infusion agar. Strain CCM883 did not produce this protein. Ultrathin sections revealed the presence of very thin, tuftlike, 50- to 75-nm-long structures on the surface of strain 7108, whereas strain CCM883 was comparably smooth. The surface material could be removed by digestion with proteinase K, suggesting that the surface structures contain protein. High-resolution scanning electron microscopy showed a thick layer of surface material on strain 7108, whereas strain CCM883 appeared smooth. The 95-kDa protein was purified by Sephacryl S-300 chromatography, and an antiserum was raised in rabbits. This antiserum was used in immunogold labeling experiments, which showed that the protein is associated with the surface structures. Our experiments thus demonstrate the presence of a fibrillar protein on the surface of S. saprophyticus (Ssp for S. saprophyticus surface-associated protein).
Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo
Authors: Ohtsuka H , Horigome K , Higuchi A , Nomura N , Ochi H , Yokota S , Kohzuki T , Noguchi H
Published: January 2020

Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. IN vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin G1 [IgG1]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgG1), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but only weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgG1), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.
Serological relationship of tumor necrosis factor-inducing exoantigens of Plasmodium falciparum and Plasmodium vivax
Authors: Bate C A , Taverne J , Karunaweera N D , Mendis K N , Kwiatkowski D , Playfair J H
Published: January 2020

Exoantigens of Plasmodium vivax-parasitized erythrocytes stimulated macrophages to secrete tumor necrosis factor, and antisera raised against the exoantigens inhibited this secretion. The antisera also inhibited the activity of Plasmodium falciparum and Plasmodium yoelii exoantigens, and conversely, antisera against the latter cross-reacted with the exoantigens of P. vivax.