bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates
Authors: McSweegan E , Walker R I
Published: January 2020

Campylobacter jejuni is able to colonize the human intestinal mucosa and cause disease. For this reason, it was important to investigate mechanisms by which C. jejuni adheres to epithelial cells and intestinal mucus gel. All strains of C. jejuni used were able to adhere to INT 407 epithelial cells and mucus, but high adherence to one substrate did not necessarily indicate comparable adherence to the other. The adherence of C. jejuni to cells was inhibited partially by treating the bacterial cells with proteases or glutaraldehyde or by adding a certain carbohydrate (fucose or mannose) to the medium. The flagellum of C. jejuni was identified as a potential adhesin by comparing adherence of flagellated and aflagellated variants. Shearing of the bacterial cells to remove the flagella reduced bacterial adhesion, whereas immobilization of the flagellum with KCN increased adhesion. Purified flagella showed specific, fucose-resistant binding to epithelial cells but not to intestinal mucus. The presence of a second, nonproteinaceous adhesin was suggested because no single treatment of the bacteria completely inhibited adhesion. Lipopolysaccharide (LPS) was identified as another C. jejuni adhesin. [3H]LPS specifically bound to epithelial cells, and this phenomenon was inhibited by periodate oxidation of the LPS or glutaraldehyde fixation of the epithelial cells. LPS, unlike flagella, was fucose sensitive and inhibited binding of whole bacterial cells to INT 407 cells. LPS was also able to bind to intestinal mucus gel. These data indicate that both flagella and LPS are important in adhesion to the mucosal surface.
Bactericidal action of eosinophils from normal human blood
Authors: Yazdanbakhsh M , Eckmann C M , Bot A A , Roos D
Published: January 2020

The ability of normal human eosinophils to ingest and kill Staphylococcus aureus and Escherichia coli was investigated and compared with the reactions shown by neutrophils from the same donors. The rate of phagocytosis of S. aureus by eosinophils was 50% of that shown by neutrophils. Unlike neutrophils, eosinophils were not able to kill ingested S. aureus at low bacterium/phagocyte ratios. The degree of S. aureus killing increased with increasing ratios, being equal to that of neutrophils when bacterium/phagocyte ratios of about 15 were used. This was probably due to a better triggering of the eosinophil oxidase system at high bacterium/phagocyte ratios. The early kinetics of the association of bacteria with eosinophils, the perforation of the bacterial envelope and the inactivation of bacterial proteins, was monitored in the ML-35 mutant strain of E. coli. The association of E. coli with eosinophils was 70% of that with neutrophils. Eosinophils had only 25% of the capacity of neutrophils to perforate the E. coli envelope. E. coli loses its colony-forming ability when the bacterial envelope has been perforated, indicating that eosinophils also kill E. coli more slowly than do neutrophils. This was confirmed with a plating assay for colony formation. The perforation of E. coli is independent of peroxidase-mediated reactions. Hence, the defective bactericidal action of eosinophils is probably not related to the differences between myeloperoxidase and eosinophil peroxidase. On the other hand, the inactivation of bacterial proteins is peroxidase dependent and was also seen to occur to a lesser extent in eosinophils compared with neutrophils. We conclude that eosinophils ingest E. coli but only slowly perforate (kill) these bacteria and barely inactivate the bacterial enzymes. In contrast, neutrophils quickly ingest and perforate (kill) E. coli and quickly inactivate the bacterial enzymes.
Nonopsonic phagocytosis of nonmucoid Pseudomonas aeruginosa by human neutrophils and monocyte-derived macrophages is correlated with bacterial piliation and hydrophobicity
Authors: Speert D P , Loh B A , Cabral D A , Salit I E
Published: January 2020

We have shown previously that some strains of Pseudomonas aeruginosa from patients with cystic fibrosis are phagocytized by human polymorphonuclear leukocytes in the absence of serum opsonins. The purpose of this study was to identify the bacterial features which render certain strains susceptible to nonopsonic phagocytosis. Three strains were phagocytized by human neutrophils and monocyte-derived macrophages, and two were not, as determined by luminol-enhanced chemiluminescence, visual inspection of stained smears, and bactericidal assay. Strains that were phagocytized formed pellicles when grown in static broth, but the phagocytosis-resistant strains did not. The phagocytosis-susceptible strains were more heavily piliated and more hydrophobic than the resistant strains. Bacteria exposed to heat (60 degrees C) or UV irradiation were depiliated, as assessed by electron microscopy, and rendered resistant to phagocytosis. When P. aeruginosa was grown on agar, it was piliated, hydrophobic, and susceptible to nonopsonic phagocytosis, but when grown to stationary phase in shaken broth, it was nonpiliated, less hydrophobic, and resistant to phagocytosis. It appears that nonopsonic phagocytosis of certain P. aeruginosa strains by human polymorphonuclear leukocytes and macrophages is facilitated by hydrophobic interactions which may be determined in part by pili.
Modulation of cellular immune responses in mice with disseminated histoplasmosis by recombinant interleukin-2
Authors: Deepe G S , Taylor C L , Harris J E , Bullock W E
Published: January 2020

Depression of the cellular immune responses in mice with disseminated histoplasmosis is associated with deficient production of interleukin-2 (IL-2) by splenocytes. Therefore, we examined whether a highly purified preparation of IL-2, recombinant human IL-2 (rIL-2), could modify the cellular immune responses in infected mice and whether this lymphokine could alter the severity of histoplasmosis in animals. Exogenous rIL-2, at concentrations of up to 1,000 U/ml, failed to augment the proliferative responses to concanavalin A by unfractionated splenocytes or splenic T cells from mice infected for 1 week. In addition, rIL-2 did not modulate the plaque-forming cell response to sheep erythrocytes by splenocytes from these same mice. However, at week 3, rIL-2 in concentrations ranging from 10 to 1,000 U/ml considerably augmented the proliferative response to concanavalin A and plaque-forming cell response to sheep erythrocytes by splenocytes from infected mice. Kinetics studies demonstrated that rIL-2 exerted maximal immunoregulatory activity when added on day 0 or 1 to cultures of splenocytes. In vivo administration of rIL-2, 200 to 20,000 U/day, for 10 days to normal and 3-week-infected mice did not alter the proliferative activity of splenocytes to concanavalin A; 200,000 U of rIL-2 per day actually depressed the proliferative responses of splenocytes from normal and infected mice. In vivo, rIL-2 did not modify delayed-type hypersensitivity responses to sheep erythrocytes or to histoplasmin by normal and infected mice. Moreover, treatment with rIL-2 in vivo did not reduce the number of Histoplasma CFU in spleens of mice. Thus, despite the immunoenhancing effect of rIL-2 in vitro, this lymphokine failed to exert similar effects in vivo.
Difference in structure between type b and nontypable Haemophilus influenzae populations
Authors: Porras O , Caugant D A , Gray B , Lagergård T , Levin B R , Svanborg-Edén C
Published: January 2020

The extent of chromosomal genetic variability and the genetic structure of Haemophilus influenzae populations was analyzed. A total of 119 isolates from humans in Göteborg, Sweden, and Birmingham, Ala., and 16 strains from a type culture collection were characterized for capsular type, biotype, outer membrane protein profile, and enzyme electrophoretic type (ET). The results of this study indicate that the bacteria identified as H. influenzae are a genetically extremely variable array of organisms. For the six enzymes studied, the estimated mean genetic diversity was 0.57 (approximately 20% higher than the corresponding estimate for Escherichia coli). Two lines of evidence indicate that despite its ability to recombine by transformation, H. influenzae maintains a largely clonal population structure. Although there is considerable potential for generating different genotypes, there were only 88 distinct ETs among the 135 strains, and isolates of the same ET and biotype were recovered at frequencies greater than would be anticipated at random. This evidence for a clonal population structure holds for uncapsulated as well as capsulated strains. However, these data also suggest that the stability of H. influenzae clones (clone persistence time) may be less than that of the nontransforming species E. coli. The ET data indicate that there is somewhat less variability among H. influenzae strains that express the same capsular antigens, biotype, and outer membrane proteins than among randomly chosen isolates. Nevertheless, there is substantial genetic variation among isolates within each of these classes and combinations thereof. There is also variation in these typing characteristics among strains of the same ET. These observations and those on genetic variability and population structures have implications for the characterization of H. influenzae isolates in clinical and epidemiological studies.
Lectinlike adhesins in the Bacteroides fragilis group
Authors: Rogemond V , Guinet R M
Published: January 2020

Lectinlike adhesins were identified in the Bacteroides fragilis group by using sugars immobilized on agarose beads either with whole bacteria by direct microscopic examination or with soluble extracts by immunoaffinoelectrophoresis. These two methods allowed the identification of two sugars reacting with whole bacteria and with the corresponding extracts: alpha-D-glucosamine and D-galactosamine. Among eight strains tested representing seven species, the two strains of B. fragilis were equally adhesive and showed the greatest adhesions. The lectinlike adhesin was purified by affinity chromatography on glucosamine-agarose or galactosamine-agarose and showed one band at 70,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This lectinlike adhesin may help to elucidate the roles of the B. fragilis group in the colonization of intestinal surfaces and in the predominance of B. fragilis in infections alone and in synergy with other bacteria.
In vitro assay for curative activity in blood of mice infected with Trypanosoma musculi
Authors: Wechsler D S , Kongshavn P A
Published: January 2020

An in vitro assay for curative antibody present in plasma of mice cured of Trypanosoma musculi is described. The assay involves the addition of plasma to a sample of infected blood, followed by hourly monitoring of the parasite count therein. Immune plasma effected a significant reduction in parasite number, whereas normal mouse plasma did not. Heat treatment of immune plasma for 30 min at 56 degrees C abolished its ability to effect any reduction. The assay is simple, rapid, and economical and correlates well with results of all in vivo studies performed to date.
Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system
Authors: Clark R A , Leidal K G , Taichman N S
Published: January 2020

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.
Inhibition of the interaction of Streptococcus sanguis with hexadecane droplets by 55- and 60-kilodalton hydrophobic proteins of human saliva
Authors: Babu J P , Beachey E H , Simpson W A
Published: January 2020

The effect of salivary secretions on the hydrophobicity of Streptococcus sanguis was investigated. Pretreatment of the bacteria with paraffin-stimulated whole saliva resulted in a 79% inhibition of adhesion to hexadecane droplets. Column chromatography on Sepharose 4B and sodium dodecyl sulfate gel electrophoretic analysis indicated that the inhibitory activity of saliva resided in a fraction containing material of approximately 60,000 molecular weight. The active components, which we have termed the hydrophobic components (HC), bind to octyl-Sepharose beads. Pretreatment of S. sanguis with HC resulted in a dose-dependent inhibition of the streptococcus-hexadecane interaction that reached a maximum of 85%. Furthermore, HC effectively blocked the ability of S. sanguis to adhere to hydroxyapatite beads coated with either whole saliva or HC. Sodium dodecyl sulfate-polyacrylamide gel analysis indicated that HC eluted from octyl-Sepharose consisted primarily of two proteins (60 kDa and 55 kilodaltons) which could be resolved by high-pressure liquid chromatography. Both of these proteins were able to inhibit the binding of S. sanguis to hexadecane in a dose-dependent manner; however, the 60-kilodalton molecule was slightly more effective in this assay. Amino acid analysis of these proteins showed that both proteins contained a high percentage of nonpolar amino acids. These findings suggest that certain components of saliva influence the interaction of S. sanguis with hydrophobic surfaces.
Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc
Authors: Krivan H C , Clark G F , Smith D F , Wilkins T D
Published: January 2020

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.
Functional and ultrastructural changes in alveolar macrophages from rabbits colonized with Bordetella bronchiseptica
Authors: Zeligs B J , Zeligs J D , Bellanti J A
Published: January 2020

Alveolar macrophages from rabbits colonized with Bordetella bronchiseptica in their respiratory tract exhibited significant decreases in cell adherence, phagocytic uptake, and bactericidal activity compared with macrophages from uncolonized animals. These dysfunctions were accompanied by ultrastructural changes, including a decrease in overall cell density, a vacuolation of the rough endoplasmic reticulum, and an increase in organelle-poor cell surface projections.
Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease
Authors: Heck L W , Morihara K , Abrahamson D R
Published: January 2020

Purified Pseudomonas aeruginosa elastase and alkaline protease rapidly cleaved soluble laminin, with each enzyme yielding different cleavage products. These cleavage fragments were separated from the intact laminin A and B polypeptide chains by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and detected by their characteristic Coomassie blue staining patterns. Pseudomonas elastase produced rapid and extensive degradation of both A and B chains, including the disulfide-rich regions. Apparently complete degradation to limit digests was obtained after 30 min with a substrate/enzyme ratio of 30:0.5. Under similar conditions, alkaline protease rapidly degraded the A chain while slowly degrading the B chain. In addition, immunoreactive laminin was released from authentic basement membranes after incubation with either enzyme as detected by an enzyme-linked immunoabsorption assay and by immunofluorescence. The results from these studies suggest a direct role for elastase and alkaline protease in both tissue invasion and hemorrhagic tissue necrosis in P. aeruginosa infections.
Interactions of gonococci with HeLa cells: attachment, detachment, replication, penetration, and the role of protein II
Authors: Bessen D , Gotschlich E C
Published: January 2020

Colony variants of Neisseria gonorrhoeae differ in their interactions with eucaryotic cells. When gonococci were cultivated with HeLa cell monolayers, the opacity phenotype (Op) became increasingly dominant in the subpopulation of organisms which adhered to the HeLa cells. Once bound, Op organisms displayed very low levels of detachment. Adherent Op gonococci exhibited generation times up to threefold greater than cultures containing gonococci in the absence of HeLa cells. In addition, the progeny of adherent Op organisms remained bound to the HeLa cell monolayer. Both piliated (P+) and transparent (Tr) colony types attached to HeLa cells, but their progeny were retained less efficiently. Gonococci bound to HeLa cells were subjected to the bactericidal action of fresh rat serum and approximately 0.5 to 2.5% survived, irrespective of their opacity or piliation phenotype. Incubation with gentamicin resulted in a 10- to 50-fold further reduction in viability. Pretreatment of HeLa cell monolayers with the microfilament-disrupting agent cytochalasin b diminished gonococcal survival in either serum or gentamicin by up to eightfold. In contrast, cytochalasin b treatment did not decrease survival of the commensal organism N. sicca. The data suggest that very few gonococci are completely interiorized and a small proportion of adherent gonococci are partially protected from the soluble-phase environment by HeLa cells.
Control of trypanodestructive antibody responses and parasitemia in mice infected with Trypanosoma (Duttonella) vivax
Authors: Mahan S M , Hendershot L , Black S J
Published: January 2020

After infection with a cloned population of Trypanosoma vivax, C57BL/6 mice controlled parasitemia during the exponential growth phase and survived, with intermittent parasitemia, for several weeks. In contrast, most mice of the C3H/He strain did not control the first wave of parasitemia and died within 9 to 13 days after infection. Control of parasitemia in C57BL/6 mice was mediated by the production of a variant surface glycoprotein-specific trypanodestructive antibody response which was accompanied by production of antibodies against antigens shared between procyclic and bloodstream T. vivax as well as antibodies against trinitrophenyl (TNP) and sheep erythrocytes. The infected C3H/He mice did not produce trypanodestructive antibodies or antibodies against procyclic antigens or TNP but did produce antibodies against sheep erythrocytes. Although infected C57BL/6 mice produced levels of serum immunoglobulin M four times higher than infected C3H/He mice, their parasite-induced B-cell DNA synthetic responses were similar, and both sets of mice developed similar numbers of spleen cells with cytoplasmic immunoglobulin M, a proportion of which could react with TNP. In vitro biosynthetic labeling studies accompanied by immunoglobulin precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the immunoglobulin-containing cells of infected C3H/He mice synthesized and secreted less immunoglobulin than similar cells from infected C57BL/6 mice. We concluded that some parasite-induced antibody-forming cells in C3H/He mice, perhaps including parasite-specific and certainly including TNP-specific cells, had an impaired capacity to make and release immunoglobulin. Within 24 h after Berenil-mediated elimination of T. vivax from infected C3H/He mice, a population of cyclophosphamide-sensitive spleen cells produced large amounts of parasite-specific and TNP-specific antibody. We concluded that the defect in terminal B-cell function leading to suppressed parasite-specific and TNP-specific antibody responses was induced either by living trypanosomes or short-lived factors from degenerating trypanosomes or by short-lived parasite-induced host responses.
Bactericidal-activities of human polymorphonuclear leukocyte proteins against Escherichia coli O111:B4 coated with C5 or C8
Authors: Tedesco F , Rottini G , Roncelli L , Basaglia M , Menegazzi R , Patriarca P
Published: January 2020

The postnuclear supernatant of disrupted polymorphonuclear leukocytes exhibited bactericidal activity on Escherichia coli O111:B4 coated with immunoglobulin M antibodies and C5 or C8 but not on C3- or C7-coated bacteria. To characterize this antimicrobial activity further, granules obtained from the postnuclear supernatant were extracted with sodium acetate (pH 4) and the soluble extract was subsequently fractionated through carboxymethyl cellulose and Sephacryl S-200. Over 90% of the activity present in the starting material was recovered in the soluble granule extract. Kinetic and dose-response analyses of the bacterial activity of the polymorphonuclear leukocyte extract on BAC1-5 and BAC1-8 revealed different susceptibilities to killing of these two bacterial intermediates; they also differed for their susceptibilities to killing at 37 degrees C and at room temperature. The suggestion raised by these data, that BAC1-5 and BAC1-8 could be killed by different bactericidal factors, was confirmed by the findings that separate fractions of the soluble granule extract obtained by carboxymethyl cellulose and Sephacryl S-200 chromatography exhibited specific activity on either BAC1-5 or BAC1-8, whereas other fractions were active on both intermediates.
Fate of Chlamydia trachomatis in human monocytes and monocyte-derived macrophages
Authors: Manor E , Sarov I
Published: January 2020

The fate of Chlamydia trachomatis (L2/434/Bu) in human peripheral blood monocytes and human monocyte-derived macrophages was studied by transmission electron microscopy (TEM) and by measuring the yield of infectious C. trachomatis in one-step growth experiments. Two main types of phagosome were seen by TEM in the cytoplasm of C. trachomatis-infected human monocytes (1 h postinfection [p.i.]): one in which the elementary body (EB) was tightly surrounded by the membrane of the phagosome and another in which the EB appeared in an enlarged phagosome. Later, 24 to 48 h p.i., each phagosome contained a single EB-like particle, an atypical reticulate body, or a damaged particle. One-step growth experiments showed that infection of human monocytes with C. trachomatis results in a decrease of infectious particles between 24 and 96 h p.i., whereas infection of the monocytes by C. psittaci (6BC strain) results in productive infection with, however, a 3.5-log lower yield than in control MA-104 cells. In contrast to the abortive replication of C. trachomatis in monocytes, monocyte-derived macrophages permitted replication as indicated by one-step growth experiments and TEM. in C. trachomatis-infected, monocyte-derived macrophages 72 h p.i., inclusions of two kinds were observed by TEM. One was very similar to the typical inclusions appearing in infected MA-104 (control) cells; the other was atypical, pleomorphic, often contained "channels," and held relatively few EB and reticulate bodies, some of which appeared damaged or abnormal. The significance of the responses to infection with C. trachomatis in monocytes compared with monocyte-derived macrophages and the role of these cells in sustaining chronic or latent infection and in dissemination of the infection to various parts of the body is discussed.
Toxicity and induction of resistance to Listeria monocytogenes infection by amphotericin B in inbred strains of mice
Authors: Brajtburg J , Elberg S , Kobayashi G S , Medoff G
Published: January 2020

Amphotericin B (AmB) treatment before infection with the bacterium Listeria monocytogenes prolonged survival of AKR mice but shortened survival of C57BL/6 mice compared with survival of untreated infected controls. C57BL/6 mice were also more sensitive to the acute toxic effects of AmB than AKR mice, as were (C57BL/6 X AKR)F1 hybrid mice. Spleen cells and erythrocytes (RBCs) from the C57BL/6 and the F1 hybrid mice were both more sensitive to the lytic and lethal effects of AmB than corresponding cells from AKR mice. Biochemical analysis indicated that catalase levels in RBCs from C57BL/6 and F1 hybrid mice were about 60% of those found in RBCs from AKR mice. The lysis by AmB of RBCs from all these strains of mice was inhibited by catalase or incubation in a low-oxygen environment. These findings suggest that (i) the low catalase levels in C57BL/6 and F1 hybrid mice may limit the protection of cells from the oxidant damage involved in AmB action, and (ii) the toxicity which occurs at low concentrations of AmB in the mouse strains with low intracellular catalase levels may interfere with or ablate the AmB-induced increases in mouse resistance to L. monocytogenes infection.
Localization of binding sites for purified Escherichia coli P fimbriae in the human kidney
Authors: Korhonen T K , Virkola R , Holthöfer H
Published: January 2020

Binding sites in the human kidney for purified P fimbriae of pyelonephritogenic Escherichia coli were determined. The purified KS71A (F7(1)) fimbriae bound only to epithelial elements of the kidney, i.e., to the apical aspect of proximal and distal tubular cells, as well as to the apical and cytoplasmic sites of collecting ducts. In addition, binding was seen at the vascular endothelium throughout the kidney and at the parietal epithelium of the glomeruli. The binding was specifically inhibited by the receptor analog of E. coli P fimbriae, globotriose. The binding sites identified suggested a possible pathogenetic mechanism for the invasion of P-fimbriated bacteria into the renal parenchyma, as well as for their subsequent spread into the circulatory system.
Immunogenic properties of Klebsiella pneumoniae type 2 capsular polysaccharide
Authors: Robert A , Jouin H , Fournier J M
Published: January 2020

The immunoprotective activity of Klebsiella pneumoniae K2 cell surface preparations and purified capsular polysaccharide was tested in mice. The 50% protective dose (PD50), expressed as capsular polysaccharide content, was 2 ng for cell surface preparations and 50 ng for purified capsular polysaccharide. Both preparations lost their immunoprotective activity after alkali treatment. Immune sera were raised in rabbits immunized with cell surface preparations. The precipitating and hemagglutinating capacity of these antisera was tested against either purified capsular polysaccharide or alkali-treated capsular polysaccharide. No difference was observed between the reactivity of the antisera against each antigen. The protective activity of these sera was tested on mice in passive transfer experiments, before and after absorption with either purified capsular polysaccharide or alkali-treated capsular polysaccharide. The sera lost their protective activity after absorption with purified capsular polysaccharide and after absorption with alkali-treated capsular polysaccharide. These experiments show that the difference in immunoprotective activity of cell surface preparations, purified capsular polysaccharide, and alkali-treated capsular polysaccharide is not due to a difference in their antigenic determinants. Cell surface preparations and purified capsular polysaccharide were fractionated by gel filtration on Sepharose 4B and by ultracentrifugation on cesium chloride density gradients. Three forms of capsular polysaccharide have been characterized. (i) A form of capsular polysaccharide with a very high protective activity (PD50 = 2 ng) that copurified with protein and lipopolysaccharide and was characterized by a low coefficient of distribution (Kd = 0.20) and a low density (1.5 to 1.6 g/cm3). (ii) A form of capsular polysaccharide with an intermediate protective activity (PD50 = 50 ng), contamined by less than 3% protein and 1% lipopolysaccharide, with a Kd of 0.35, and a density of 1.7 to 1.8 g/cm3. (iii) A nonimmunoprotective capsular polysaccharide obtained after alkali treatment of either cell surface preparations or purified capsular polysaccharide. The Kd of these fractions varied from 0.20 to 0.90 and their density from 1.7 to 1.8 g/cm3.
Effect of rat surfactant lipids on complement and Fc receptors of macrophages
Authors: Coonrod J D , Jarrells M C , Yoneda K
Published: January 2020

Murine resident alveolar macrophages have very low numbers of complement receptors detectable by rosetting with erythrocyte complexes. These macrophages are relatively inactive in tests of complement- or antibody-mediated bacterial phagocytosis in vitro. It is not known whether these characteristics are intrinsic or are environmentally modulated. We found previously that rat bronchoalveolar lavage fluid or isolated rat surfactant lipid causes a unique and marked reduction in the ability of peritoneal macrophages to form rosettes with immunoglobulin G or complement containing erythrocyte complexes in vitro. In this study the antirosetting activity of rat surfactant was found to be due to its neutral lipid component and, specifically, to the free fatty acid (FFA) fraction of the neutral lipids. Rat surfactant contained a higher level of FFA than has been reported for canine, guinea pig, or human surfactant. Studies with pure FFA showed that activity in blocking macrophage Fc and complement receptors correlated with increasing unsaturation and chain length. A mixture of eight commercially available FFAs, representing the most abundant FFA in rat surfactant and consisting mostly of saturated FFA, had much less effect on rat macrophage receptors than the naturally occurring FFA mixture. These findings suggest that long-chain-unsaturated FFAs in rat surfactant are the most important for the antireceptor activity. The antireceptor activity of rat surfactant or FFA on peritoneal macrophage receptors in vitro and known intrinsic properties of murine alveolar macrophages could not be precisely correlated, suggesting that impaired binding of immune complexes by murine alveolar macrophages and a high level of FFAs in rat surfactant may be independent rather than causally related phenomena.
Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype
Authors: Daskaleros P A , Payne S M
Published: January 2020

The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding.
Rabbit antibodies to the cell wall polysaccharide of Streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci
Authors: Szu S C , Schneerson R , Robbins J B
Published: January 2020

A conjugate, composed of the cell wall polysaccharide (C polysaccharide) of Streptococcus pneumoniae and bovine serum albumin (BSA), was prepared with the bifunctional agent N-succinimidyl-3-(2-pyridyldithio)-propionate. Analysis with monoclonal antibodies provided evidence that the phosphocholine (PC) moiety of the C polysaccharide was retained during the conjugation procedure. The C polysaccharide-BSA conjugate elicited antibodies to C polysaccharide in rabbits; no PC-specific antibodies were detected in globulins prepared from these hyperimmune sera obtained early and late after a second immunization. Rabbit hyperimmune sera were taken after multiple intravenous injections of the pneumococcus strain SRC-2, which has a capsulelike structure composed of the C polysaccharide. Globulin prepared from these antisera had both C polysaccharide- and PC-specific antibodies. Antibodies to C polysaccharide elicited by the C polysaccharide-BSA conjugate failed to protect mice against intraperitoneal challenge with a strain of type 3 or type 6A pneumococci. The anti-SRC-2 globulin conferred protection against both of these pneumococcal strains. Absorption of the SRC-2 globulin with C polysaccharide, however, failed to change its protective activity. These data provide evidence that antibodies to the C polysaccharide do not confer immunity against infection of mice with encapsulated pneumococci inoculated by the intraperitoneal route.
Strong mitogenic effect for murine B lymphocytes of an immunosuppressor substance released by Streptococcus intermedius
Authors: Arala-Chaves M P , Ribeiro A S , Santarém M M , Coutinho A
Published: January 2020

A noncytotoxic protein substance, produced by Streptococcus intermedius, with very potent immunosuppressive properties (F3'EP-Si) was tested for lymphocyte mitogenic activity. Although devoid of T-cell mitogenicity, F3'EP-Si stimulated proliferation and led to high numbers of plaque-forming cells in cultures of normal or T-cell-depleted, small or large splenic B cells from both lipopolysaccharide-responding and -nonresponding mice. The B-cell mitogenic activity of F3'EP-Si was quantitatively comparable to that of lipopolysaccharide, and the simultaneous exposure to both mitogens stimulated additive B-cell responses. Injection of F3'EP-Si into normal mice resulted in increased numbers of spleen cells, higher rates of mitotic activity, and very large numbers of plaque-forming cells, predominantly of the immunoglobulin G2a and -b isotypes. In preliminary experiments, the analysis of surface markers among the lymphocytes participating in the blastogenic response in vivo revealed a T-cell component in the response to F3'EP-Si. These observations are discussed in the context of the immunosuppressive activity of this and other microbial substances.
Experimental abortion and the systemic immune response to "Haemophilus somnus" in cattle
Authors: Widders P R , Paisley L G , Gogolewski R P , Evermann J F , Smith J W , Corbeil L B
Published: January 2020

"Haemophilus somnus" has been identified in the etiology of bovine abortion on the basis of the isolation of the organism from aborted fetal and placental tissues. To investigate the role of hematogenous dissemination of "H. somnus" in the pathogenesis of abortion and to monitor the humoral immune response to infection, 19 pregnant cows (gestation ages, 1.4 to 7 months) were challenged intravenously (11 cows) or intrabronchially (8 cows). Five cows challenged intravenously aborted, and one cow challenged intrabronchially resorbed her fetus. "H. somnus" was isolated in large numbers from aborted tissues, and placental lesions were similar to those reported in a field case of "H. somnus" abortion. Antibody titers in serum were measured by the microagglutination test (MAT) and by enzyme-linked immunosorbent assay (ELISA). A response to challenge was measured by MAT; it was also measured by ELISA within the immunoglobulin G1 (IgG1), IgG2, and IgM isotypes. On comparison of pre- and postchallenge antibody titers, the greatest and most persistent response was detected within the IgG2 isotype. Prechallenge antibody titers (measured by MAT and by IgG2 ELISA) were lower in animals that aborted than in normal calving animals, indicating that IgG2 antibody may have a role in limiting hematogenous dissemination of "H. somnus."
Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide
Authors: Brade L , Schramek S , Schade U , Brade H
Published: January 2020

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.
Plasmid RP1-mediated susceptibility of Acinetobacter calcoaceticus to rat polymorphonuclear leukocyte granule contents
Authors: Loeffelholz M J , Modrzakowski M C
Published: January 2020

The resistance plasmid RP1 was transferred by conjugation to a plasmidless strain of Acinetobacter calcoaceticus. Acquisition and expression of RP1 by A. calcoaceticus HO1-N was associated with an increase in sensitivity to the antimicrobial activity of extracted contents from rat polymorphonuclear leukocyte granules. Plasmid RP1-associated antibiotic resistance and sensitivity to granule contents were cured by exposure to acridine orange. Assays with granule extract fractions separated by fast protein liquid chromatography showed myeloperoxidase, protease, and lysozyme fractions to possess little or no antimicrobial activity against the A. calcoaceticus strains. A protein fraction designated peak D, containing two low-molecular-weight cationic peptides (M. J. Loeffelholz and M. C. Modrzakowski, Anal. Biochem., in press), did possess antimicrobial activity against both HO1-N and Ho1-N(RP1) strains, with the HO1-N(RP1) strain being significantly more sensitive.
Effect of T-lymphocyte suppression on the parasite burden in Leishmania major-infected, genetically susceptible BALB/c mice
Authors: Solbach W , Forberg K , Röllinghoff M
Published: January 2020

Treatment of Leishmania major-infected, genetically susceptible BALB/c mice with the T-lymphocyte-immunosuppressive drug cyclosporin A (CyA) resulted in a significantly reduced parasite burden in the local site of infection and in the draining lymph nodes. These data indicate that T cells are pivotal for the propagation of L. major in vivo.
Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology
Authors: Burns D L , Kenimer J G , Manclark C R
Published: January 2020

The mechanism by which pertussis toxin induces morphological changes in Chinese hamster ovary cells was studied to determine whether the resulting clustered growth pattern is due to toxin-catalyzed ADP-ribosylation of a cellular substrate. While pertussis toxin was extremely potent in inducing morphological changes in Chinese hamster ovary cells, preparations of isolated A subunit or B oligomer exhibited greatly reduced activity. The clustered growth response of these cells correlated with ADP-ribosylation of a 41-kilodalton cellular substrate for the toxin in that the toxin concentration and time of exposure to the toxin required for ADP-ribosylation were the same as those needed for alterations in cellular morphology. Moreover, pertussis toxin modified by either chemical or photolytic methods exhibited similar decreases in the ability to ADP-ribosylate the cellular substrate and alter cell morphology. These results suggest that clustering of Chinese hamster ovary cells is due to toxin-catalyzed ADP-ribosylation of a 41-kilodalton substrate. Therefore, alteration in Chinese hamster ovary cell morphology can be used as a measure of toxin activity. This assay should prove to be a useful tool in the development and evaluation of new pertussis vaccines.
Tear and serum antibody response to Chlamydia trachomatis antigens during acute chlamydial conjunctivitis in monkeys as determined by immunoblotting
Authors: Caldwell H D , Stewart S , Johnson S , Taylor H
Published: January 2020

In this study, we examined the temporal antibody response by immunoblotting analysis in tears and sera of three cynomolgus monkeys (Macaca fasicularis) with primary acute Chlamydia trachomatis serovar B conjunctivitis. The objective was to identify chlamydial antigens stimulating antibody during the host responses in the course of this self-limiting infection with the rationale that they may be protective antigens. The major outer membrane protein (MOMP), lipopolysaccharide (LPS), and polypeptides of 60 and 68 kilodaltons (kDa) were the predominant antigens recognized by immunoglobulin A (IgA) in monkey tears. Tear IgA antibody specific for the MOMP was first detected 14 days postinfection, whereas tear IgA reactive with LPS or the 68- and 60-kDa polypeptides was first detectable on day 21. Tear IgA antibodies specific for each of these antigens persisted in tears through day 56, 4 weeks after both peak clinical disease and recovery of the organism from the conjunctivae. In contrast, tear IgG antibodies peaked at approximately 28 days postinfection, the time of maximal inflammatory response. The IgG response in monkey sera was similar to that observed for tear antibodies, in that the MOMP, 60-, and 68-kDa polypeptides were the primary immunogens. The exception was that IgG antibody against these antigens was detected 1 week later than that observed for tear IgA antibodies. Of three monkeys that responded with tear IgA antibody against LPS, one did not have detectable serum IgG LPS antibody. The specificity of the tear IgA antibody response of monkeys was determined by immunoblotting nine other C. trachomatis serovars in addition to the homologous B serovar. The tear IgA response to the MOMP was predominantly B complex subspecies-specific (serovars B, Ba, D, and E), whereas the response to chlamydial LPS was found to be species-specific. The significance of these observations in relation to previous vaccine studies in nonhuman primates is discussed.
Differential growth characteristics and immunogenicity of tight and coasting temperature-sensitive mutants of Pseudomonas aeruginosa
Authors: Morris Hooke A , Sordelli D O , Cerquetti M C , Bellanti J A
Published: January 2020

Genetically attenuated vaccines capable of limited replication in the vaccinate may elicit stronger, longer-lasting immunity than that induced by component, killed whole-cell, or nonreplicating live vaccines. We have isolated and partially characterized temperature-sensitive Pseudomonas aeruginosa mutants of two different phenotypes: a tight mutant, which ceases all growth immediately after its transfer to 36 degrees C, and a coaster, which continues to replicate for five generations at 36 degrees C. The growth profiles of the two temperature-sensitive phenotypes were compared both in vitro and in vivo; maintenance of the coasting phenotype in vivo was confirmed. The immunogenicity of the two phenotypes was compared in two models. In model 1, ICR mice were immunized intraperitoneally (i.p.) with graded doses of either mutant and challenged 3 weeks later i.p. with lethal doses of the wild-type strain. In model 2, DBA/2J mice were immunized intranasally with either mutant and subsequently challenged with an aerosolized inoculum of the wild-type strain, and lung clearance was measured over 4 h. In both models, the coaster demonstrated slightly higher immunogenic potential and, in addition, induced significantly higher levels of immunotype-specific serum immunoglobulin G after i.p. immunization.