bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between levels of Clostridium difficile toxin A and toxin B and cecal lesions in gnotobiotic mice
Authors: Vernet A , Corthier G , Dubos-Ramaré F , Parodi A L
Published: January 2020

Various Clostridium difficile strains were studied with respect to their pathogenicity in monoassociated mice in relation to levels of toxin A and toxin B in vivo and in vitro. Two strains which were the most potent toxin producers in vitro induced mortality (100%); mice monoassociated with these strains were found to have high levels of both toxins in their ceca and an intense cecal epithelial ulceration together with a severe inflammatory process. No mortality was observed with the other strains. Strains which were moderately toxinogenic in vitro induced inflammation of the cecum but no ulceration, and no toxin A was found. Inflammation intensity was not related to toxin B levels. After 3 weeks, ceca returned to normal in spite of a chronic cytotoxin production. When compared with in vitro results, which showed a good correlation between the levels of the two toxins, toxin A amounts in vivo were found to be lowered relative to toxin B levels. The lack of detectable toxin A levels in animals infected with all but the two most highly toxinogenic strains prevented death. This work points out the importance of investigation of toxin A for the understanding of C. difficile pathogenicity.
Inhibition of endotoxin-induced activation of human monocytes by human lipoproteins
Authors: Flegel W A , Wölpl A , Männel D N , Northoff H
Published: January 2020

Toxicity of lipopolysaccharide (LPS) (endotoxin) is, to a large extent, mediated by the activation of monocytes/macrophages and subsequent release of monokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). It is known that LPS binds readily to serum lipoproteins and that LPS-lipoprotein complexes are less toxic than unbound LPS. Here we present data analyzing the impact of the LPS-serum interaction at the cellular level. By measuring IL-1 TNF-alpha, and IL-6, the interaction of different LPSs or lipid A with human serum could be shown to prevent the activation of human monocytes. The amounts of LPS inactivated by normal human serum did not exceed 10 ng/ml. The LPS-inactivating capacity of serum was shown to be a function of the lipoproteins. Other serum components, such as naturally occurring anti-LPS immunoglobulin G, complement, or nutritive lipids, had no significant influence in our system. Our experiments suggest that serum lipoproteins control endotoxin-induced monocyte activation and monokine release.
Deficiency of interleukin-2 production upon addition of soluble egg antigen to cultures of isolated hepatic granulomas or hepatic granuloma cells from mice infected with Schistosoma japonicum
Authors: Stavitsky A B , Harold W W
Published: January 2020

Schistosoma japonicum-infected C57BL/6 mice show similar dynamics of hepatic granulomatous inflammation and delayed hypersensitivity elicited by soluble egg antigens (SEA) which reach peak levels at 9 weeks of infection and then spontaneously regress. In an attempt to link the level of interleukin 2 (IL-2) production to the spontaneous regression of hepatic granulomatous inflammation, the study determined the dynamics of IL-2 production by SEA-challenged isolated hepatic granulomas (HG) and cells isolated enzymatically from the HG. The production of IL-2 by SEA-stimulated HG or HG cells reached its peak when these preparations from 9-week-infected mice were stimulated and fell thereafter. Some possible mechanisms that might explain the IL-2 deficiency were examined. This deficiency is not due to the in vitro binding of IL-2 by the HG cells of infected mice and is, therefore, due rather to underproduction of IL-2. The deficiency was also not explained by reduced numbers of antigen-presenting cells (macrophages or B cells) or of L3T4+ T lymphocytes. In vitro SEA-induced IL-2 production by HG cells from acutely infected mice was suppressed consistently by Lyt-2+ T cells from the spleens and in the majority of our experiments by Lyt-2+ T cells from the HG of mice infected for 10 weeks. These findings are consistent with the main features of our working hypothesis, but it remains to be proven that in vivo deficiency of lymphokine(s) such as IL-2 is responsible for the spontaneous decrease in granulomatous inflammation and that this lymphokine deficiency is a result of suppression.
Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri
Authors: Leimeister-Wächter M , Chakraborty T
Published: January 2020

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.
Adherence targets of Vibrio parahaemolyticus in human small intestines
Authors: Yamamoto T , Yokota T
Published: January 2020

Formalin-fixed human small intestinal mucosa with mucus coating, villi, and lymphoid follicle epithelium at the mucosal surface was used to test the adherence sites of clinically isolated (Kanagawa phenomenon-positive) strains of Vibrio parahaemolyticus. V. parahaemolyticus strains grown on CFA agar (supplemented with 3% NaCl) for ca. 3 h at 37 degrees C possessed various levels of cell-associated hemagglutinins (HAs) which were detected with human or guinea pig erythrocytes. The observed adherence abilities of V. parahaemolyticus strains to human small intestinal mucosa correlated roughly with the HA levels of the strains. Under the test conditions, ileal lymphoid follicle epithelium (especially M cells) provided the best adherence target for V. parahaemolyticus. Adherence to villus absorptive cells or to mucus coating was observed at lower levels. In addition, all 3-h-grown V. parahaemolyticus strains tested produced high levels of HAs as detected with rabbit erythrocytes. The strains were all strikingly motile. In contrast, V. parahaemolyticus strains grown on CFA agar (supplemented with 3% NaCl) for ca. 20 h at 37 degrees C had much lower levels of HAs, adherence abilities, and motility. In contrast to the above observations, piliation of V. parahaemolyticus was more extensive at ca. 20 h of incubation at 37 degrees C than at ca. 3 h of incubation at 37 degrees C. The remarkable ability of V. parahaemolyticus to adhere to lymphoid follicle epithelium was also confirmed by using rabbit small intestinal mucosa.
A major antigen on the outer envelope of a human oral spirochete, Treponema denticola
Authors: Umemoto T , Namikawa I , Suido H , Asai S
Published: January 2020

Human oral spirochetes are prominent inhabitants of subgingival plaque in patients with periodontal disease. By immunoelectron microscopy using protein A-gold complexes and either polyclonal mouse antiserum against the 53-kDa antigen or 53-kDa-antigen-specific monoclonal antibody, a major polypeptide antigen, with a molecular weight of 53,000 (molecular size, 53 kilodaltons [kDa]), of a human oral spirochete, Treponema denticola ATCC 33520, was found to localize on the surface of the outer envelope.
Immunological characteristics of the glycolipid antigen of Leptospira interrogans serovar lai
Authors: Masuzawa T , Nakamura R , Shimizu T , Iwamoto Y , Morita T , Yanagihara Y
Published: January 2020

The protective antigen (PAg), a glycolipid substance, was extracted from Leptospira interrogans serovar lai strain 017 with a chloroform-methanol-water (1:2:0.8 [vol/vol/vol]) solution and partially purified by silica gel column chromatography. The PAg was not detected by Coomassie brilliant blue staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis but was observed as a smearlike band, which corresponded to a 24- to 30-kilodalton standard protein, by silver staining. The outer envelope (OE) fraction showed the same band, suggesting that the PAg was one of the chemical components of the OE. The immunogenicity and protective activity of the PAg were compared with those of the OE. The PAg as well as the OE and whole cells was able to induce agglutinating antibody against L. interrogans. Furthermore, the immune sera exhibited opsonic activity against L. interrogans, as observed by measurement of chemical luminescence derived from reactive oxygen. The PAg exhibited protective activity in hamsters challenged with lethal doses of L. interrogans. Therefore, the antigen may be useful as a component vaccine against leptospiral infection.
Pharmacological and biochemical studies of cytotoxicity of Clostridium novyi type A alpha-toxin
Authors: Bette P , Frevert J , Mauler F , Suttorp N , Habermann E
Published: January 2020

The actions of apparently homogeneous alpha-toxin from Clostridium novyi type A were studied in order to develop an in vitro system which closely mimics its in vivo effects and to search for the mode of poisoning. Time to death (by intravenous injection of mice) was inversely related to dose, with a detection limit of about 200 ng/kg of body weight at 100 h. Injections of 2.5 ng or more into the rat paw led to a slowly (maximum after about 30 h) developing, dose-dependent edema which was useful as a quantitative in vivo assay based on volumetry. Vascular leakage was due to gap formation between endothelial cells. Similarly, endothelial cells cultured from pig pulmonary artery lost their "cobblestone" arrangement after a dose-dependent lag period of some hours after poisoning. The morphological changes were accompanied by depression of uptake or incorporation of [3H]uridine. A quantitative in vitro assay was established on the inhibition of [3H]uridine incorporation. As in animals, the action of alpha-toxin started with a few nanograms per milliliter and proceeded slowly for at least 1 day but became resistant to antitoxin within 2 h of exposure. The toxin action is not limited to endothelial cells, since chicken embryonic cells, a mouse fibroblast line (L-929), and a rat phaeochromocytoma line (PC-12) behaved similarly. Alpha-toxin was found to differ from other bacterial toxins investigated whose modes of action are already known.
Plasmid-associated adherence of Shigella flexneri in a HeLa cell model
Authors: Pál T , Hale T L
Published: January 2020

The initial interaction of Shigella flexneri with HeLa cells was studied at 4 degrees C, a temperature that inhibits parasite-directed endocytosis. It was found that invasive strains were 10-fold more adherent to HeLa cells than were isogenic, noninvasive strains which had lost a 140-megadalton plasmid. Adherent strains were also more hydrophobic than were nonadherent strains.
In vivo antigenic variation of Campylobacter flagellin
Authors: Logan S M , Guerry P , Rollins D M , Burr D H , Trust T J
Published: January 2020

Campylobacter coli VC167 cells producing either antigenic phase 1 (P1) or phase 2 (P2) flagellins (as determined by characteristic protein and DNA patterns) were used to infect rabbits by the removable intestinal tie-adult rabbit diarrhea (RITARD) procedure. Rabbits infected with P2 cells shed predominantly P2 cells throughout the infection; in rabbits infected with P1 cells, a transition of fecal isolates from P1 to P2 was observed.
Nonionic block polymer surfactants modulate the humoral immune response against Streptococcus pneumoniae-derived hexasaccharide-protein conjugates
Authors: Zigterman G J , Schotanus K , Ernste E B , Van Dam G J , Jansze M , Snippe H , Willers J M
Published: January 2020

Nonionic block polymer surfactants (NBPs) were tested for the capacity to stimulate the antibody response against hexasaccharide (HS), derived from Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS), which was conjugated to proteins. The immune response was evaluated in the (CBA/N x BALB/c)F1 progeny, in which female mice are phenotypically normal whereas male mice carry an X-chromosome-linked immunodeficiency. NBPs L101, L121, 1101, and 1501 were able to increase anti-HS immunoglobulin M (IgM) and IgG levels in both normal and X-chromosome-linked immunodeficient mice (with up to 74-fold stimulation of antibody titers). Distribution of S3PS-specific antibodies over the various IgG isotypes was restricted after immunization with either HS-bovine serum albumin or HS-keyhole limpet hemocyanin (HS-KLH). Addition of NBPs (in particular 1501) resulted in a more diverse immune response with either antigen as judged by isotype distribution. Isoelectric focusing of individual sera and subsequent detection of S3PS-binding antibodies in these sera by immunochemical staining revealed a restricted number of different spectrotypes in the course of the immune response. Upon immunization of mice with HS-KLH, spectra of secreted antibodies were slightly more complex and more densely stained than after immunization with HS-bovine serum albumin. Furthermore, NBPs 1101 and 1501 appeared to be able to stimulate the secretion of antibodies, which were secreted only in small amounts without the use of NBPs. Different explanations for increased spectrotype diversity after immunization with KLH as the carrier and after administration of NBPs as the adjuvant are discussed.
Determination of the epidemiology and transmission of nontypable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints
Authors: Loos B G , Bernstein J M , Dryja D M , Murphy T F , Dickinson D P
Published: January 2020

It is assumed that the causative bacteria in children suffering from otitis media reach the middle ear via the eustachian tube. The purpose of this investigation was to use endonuclease restriction of bacterial chromosomal DNA to compare isolates of nontypable (NT) Haemophilus influenzae obtained from the nasopharynx and from middle ear (ME) effusions of patients with otitis media. Strains of NT H. influenzae were isolated from the nasopharynx (NP) and affected ME from a group of 13 unrelated children with otitis media with effusion (OME). For 12 of these children, identical strains were isolated from the NP and ME in a first episode of OME. Each of these 12 sets differed from the other 11. Six of these children suffered from a second episode of OME with NT H. influenzae. Five of these children with recurrence again had identical NP and ME strains. These results suggest that at the time of an episode of OME, there is one predominant strain of NT H. influenzae that colonizes both the NP and ME. The strains of NT H. influenzae isolated from all six of the second episodes were different from strains from the first episode, indicating turnover of the predominant strain in the NT H. influenzae population between episodes. When we investigated three siblings with concurrent episodes of OME, we found that they shared several similar strains of NT H. influenzae, thereby demonstrating that within a family, transmission of NT H. influenzae from child to child is possible. These results from DNA fingerprinting were essentially identical when compared with results from outer membrane protein subtyping performed on the same set of strains. The analysis of endonuclease restriction patterns of total genomic DNA provides a sensitive measure of genetic dissimilarity between strains and represents an easily applicable method for epidemiological and transmission studies of bacterial infections associated with NT H. influenzae.
Characterization of salivary alpha-amylase binding to Streptococcus sanguis
Authors: Scannapieco F A , Bergey E J , Reddy M S , Levine M J
Published: January 2020

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.
Molecular diversity of neurotoxins from Clostridium botulinum type D strains
Authors: Moriishi K , Syuto B , Kubo S , Oguma K
Published: January 2020

The molecular properties of Clostridium botulinum type D South African (D-SA) were compared with those of neurotoxins from type D strain 1873 (D-1873) and type C strains Stockholm and 6813. D-SA toxin, purified 610-fold from the culture supernatant in an overall yield of 30%, consisted of an intact peptide chain with a molecular weight of 140,000. Limited proteolysis of the toxin by trypsin formed a dichain structure consisting of a light chain (Mr, 50,000) and a heavy chain (Mr, 90,000) linked by a disulfide bond(s) and enhanced the lethal activity about fourfold. Antibodies against the D-SA toxin light chain reacted with D-1873 toxin but not with C1 toxins. On the other hand, antibodies against the heavy chain of D-SA toxin cross-reacted with type C strain Stockholm, D-1873, and type C strain 6813 toxins in that order. Amino-terminal sequences of heavy and light chains of D-SA and D-1873 toxins were similar but not identical. These results indicate that within the type D strains, neurotoxins differ in molecular structure and antigenicity.
Transformation of the dermatophyte Trichophyton mentagrophytes to hygromycin B resistance
Authors: Gonzalez R , Ferrer S , Buesa J , Ramon D
Published: January 2020

A transformation system for the ringworm-producing dermatophyte Trichophyton mentagrophytes has been developed. The system employs the plasmid pHIS, which contains a bacterial hygromycin B phosphotransferase gene linked to Cochliobolus heterostrophus regulatory sequences (B. G. Turgeon, R. C. Garber, and O. C. Yoder, Mol. Cell. Biol. 7:3297-3305, 1987). This plasmid confers hygromycin B resistance to T. mentagrophytes. The DNA was stably integrated into the fungal genome, and the number and sites of integrations varied among transformants. Transformant clones were capable of infecting guinea pigs. This system opens the way for the molecular genetic analysis of the interaction of T. mentagrophytes with epithelial animal tissues.
Isolation of tube precipitin antibody-reactive fractions of Coccidioides immitis
Authors: Kruse D , Cole G T
Published: January 2020

Patients presenting with primary coccidioidal infection have been shown by earlier investigators to produce immunoglobulin M (IgM) precipitin antibodies to lysates of mycelial and spherule phases of Coccidioides immitis. This humoral response has been detected by tube precipitin (TP) and immunodiffusion (ID)-TP assays of patient sera, which are valuable aids in early diagnosis of coccidioidomycosis. Several reports of antigenic fractions which show reactivity with patient TP antibody have been published. However, confusion persists with respect to the nature of the specific serologically reactive macromolecule(s). In this study we isolated two TP antibody-reactive antigens (TP-Ags) from an alkali-soluble, water-soluble fraction of the inner conidial wall and a culture filtrate plus toluene lysate of the mycelial phase of C. immitis. The crude antigens were first separated by concanavalin A (ConA) chromatography. The TP-Ags were identified in ID-TP assays as 120- and 110-kilodalton (kDa) fractions which were electroeluted from reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of the ConA-bound conidial wall extract and ConA-bound culture filtrate plus lysate preparation, respectively. Following electroelution, the 120-kDa fraction was subjected to gel filtration chromatography which yielded a major 240-kDa and minor 120-kDa component. The apparent dimer may be a product of disulfide bond formation resulting from reassociation of the reduced, monomeric components (120 kDa). The latter was suggested by the presence of cysteine in the isolated fraction. The electroeluted 110-kDa fraction was subjected to ion-exchange chromatography. The DEAE-isolated, TP antibody-reactive fraction was identified as antigen 2 in the coccidioidin-anti-coccidioidin reference system. Homogeneity of the TP-Ags was demonstrated in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the respective chromatographically isolated fractions. The two purified TP-Ags showed reactivity in the TP and ID-TP assays and were capable of binding patient IgM but comparatively little IgG antibody, as determined by an enzyme-linked immunosorbent assay. It appears that the diagnostic TP reaction between sera from patients with coccidioidomycosis and the ID reference antigens examined in this study is a composite of IgM binding to both a 120-kDa and a 110-kDa antigen.
Effect of Treponema hyodysenteriae infection on mucosal mast cells and T cells in the murine cecum
Authors: Nibbelink S K , Wannemuehler M J
Published: January 2020

The pathogenic mechanisms responsible for the development of lesions in swine and mice after infection with Treponema hyodysenteriae have not been fully characterized. The release of inflammatory mediators from mast cells has been postulated to play a role in lesion development during swine dysentery. Therefore, C3H/HeN mice were infected with T. hyodysenteriae, and mucosal mast cell (MMC) numbers were examined in cecal sections. An initial increase in MMC numbers from 13 to 22 per 50 crypt villus units was observed, but at 20 days postinfection the numbers significantly decreased (P less than 0.05) to 5 MMC per 50 crypt villus units. Immunohistochemical analysis performed on cecal sections failed to show a significant change in lamina proprial T-lymphocyte subsets. Numbers of T. hyodysenteriae CFU recovered from the cecum were stable throughout the experimental time period. Mast cell-deficient W/Wv mice and their mast cell-sufficient littermates were also infected to determine whether MMCs were necessary for the occurrence of T. hyodysenteriae-induced lesions. W/Wv mice were as susceptible to infection as their normal littermates and developed similar macroscopic and microscopic lesions. These results indicate that changes in MMC numbers can be detected after an infection with T. hyodysenteriae; however, on the basis of observations of infected W/Wv mice, mast cells are not required for lesion development in the murine model.
Role of endogenous gamma interferon in host response to infection with blood-stage Plasmodium chabaudi AS
Authors: Stevenson M M , Tam M F , Belosevic M , van der Meide P H , Podoba J E
Published: January 2020

The role of gamma interferon (IFN-gamma), a pluripotent lymphokine capable of activating macrophages, in acquired immunity to blood-stage malaria was investigated. C57BL-derived, lipopolysaccharide-resistant C57BL/10ScN mice, which were found to be resistant to intraperitoneal (i.p.) infection with 10(6) Plasmodium chabaudi AS parasitized erythrocytes, were treated with monoclonal anti-IFN-gamma antibody (MAb). Two MAbs were used: R4-6A2, a rat anti-mouse, neutralizing immunoglobulin G1, which was prepared against natural murine IFN-gamma, and DB-1, a murine anti-rat immunoglobulin G1 prepared against recombinant rat IFN-gamma, which can neutralize the murine molecule as well as the rat molecule. C57BL/10ScNH mice were injected i.p. with 200 micrograms of R4-6A2 1 day before infection and every 3 days through day 21. Control mice were treated with normal rat serum. In separate experiments, DB-1 (1.0 mg per week for 4 weeks) was administered i.p. to C57BL/10ScNH mice beginning on the day of infection; control mice were untreated. Control and MAb-treated mice were infected i.p. with 10(6) P. chabaudi AS parasitized erythrocytes, and the course and outcome of infection were determined. Control mice exhibited a course of infection that was characterized by a peak parasitemia between 30 and 40% parasitized erythrocytes and elimination of the parasite by 4 weeks. MAb-treated mice exhibited a significantly greater parasitemia 1 to 2 days before the peak parasitemia as well as a significantly greater peak parasitemia but also completely cleared the infection by 4 weeks. Thus, these results suggest that treatment with anti-IFN-gamma MAb impairs but does not completely abrogate host resistance to P. chabaudi AS. We also examined the kinetics of IFN-gamma production by spleen cells cultured in vitro with malaria antigen or concanavalin A. Spleen cells were recovered from individual C57BL/6 mice at various times after i.p. infection with 10(6) P. chabaudi AS parasitized erythrocytes. The amount of IFN-gamma produced was quantitated by enzyme-linked immunosorbent assay. In each case, the peak of IFN-gamma production occurred just before the peak parasitemia, followed by a decrease to little or no IFN-gamma production through 42 days postinfection. There was thus a parallel between the kinetics of production of IFN-gamma in vitro by spleen cells from infected animals and the requirement in vivo for the endogenous molecule just before and at the time of peak parasitemia. In conclusion, these results suggest that IFN-gamma-dependent and -independent mechanisms contribute to host resistance to P. chabaudi AS.
Pathophysiologic glucocorticoid elevations promote bacterial translocation after thermal injury
Authors: Jones W G , Minei J P , Richardson R P , Fahey T J , Calvano S E , Antonacci A C , Shires G T , Shires G T
Published: January 2020

Thermal injury results in transient elevations of plasma glucocorticoids and promotes translocation of bacteria from the gut to the mesenteric lymph nodes (MLN) in rats. Translocated organisms are quickly cleared following uncomplicated thermal injury. However, subsequent burn wound infection, in temporal association with sustained pathophysiologic elevations of plasma corticosterone, results in the continued presence of enteric bacteria in the MLN. To study the role of sustained pathophysiologic steroid elevations in the mediation of this prolonged bacterial translocation, Wistar rats were randomly placed in groups receiving one of the following: (i) a 30% total body surface area scald injury with placement of a subcutaneous corticosterone pellet, (ii) a 30% total body surface area scald and a sham pellet implantation, (iii) a sham burn and a corticosterone pellet implantation, or (iv) a sham burn and a sham pellet implantation. The animals were sacrificed on days 1 and 4 after injury, and cultures of the MLN, as well as the liver and spleen, were taken. Implantation of corticosterone pellets resulted in sustained elevations of plasma corticosterone compared with controls not receiving corticosterone pellets, similar to results seen in association with injury and infection. These pathophysiologic elevations were associated with the prolonged presence of organisms in the MLN (90% of burned rats with implanted corticosterone pellets versus 25% of rats with uncomplicated burns on postburn day 4; P less than 0.01), but only in the presence of burn injury. Pathophysiologic glucocorticoid elevations did not lead to progression of translocation to the viscera or blood. Thus, the pathophysiologic glucocorticoid response contributes to the translocation of enteric bacteria and their prolonged presence in the MLN after systemic injury.
Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis
Authors: Takashima T , Ueta C , Tsuyuguchi I , Kishimoto S
Published: January 2020

We studied the production of tumor necrosis factor alpha (TNF-alpha) by peripheral blood monocytes taken from patients with pulmonary tuberculosis and from healthy controls. It was found that the monocytes from patients with newly diagnosed tuberculosis released significantly greater amounts of TNF-alpha in vitro in response to lipopolysaccharide than did those from healthy controls (P less than 0.05). However, the monocytes from patients with chronic refractory tuberculosis released significantly lower amounts of TNF-alpha than did those from patients with newly diagnosed tuberculosis (P less than 0.005). Even when the cells were primed for 24 h with 500 U of recombinant interferon gamma per ml, the same pattern of results was observed. The depressed TNF-alpha production by the monocytes from patients with chronic refractory tuberculosis was also shown in response to Mycobacterium bovis BCG. This depressed TNF-alpha production did not recover, even when cultured for 1 to 7 days in the sera of healthy individuals. The sera from patients with chronic refractory tuberculosis did not have any suppressive effect on the lipopolysaccharide-induced TNF-alpha production. Thus, it was demonstrated that the levels of TNF-alpha produced by monocytes were related to the disease states of pulmonary tuberculosis and that the depressed TNF-alpha production by monocytes in patients with chronic refractory tuberculosis might not be acquired.
Pathogenesis of Cryptococcus neoformans in congenitally immunodeficient beige athymic mice
Authors: Salkowski C A , Balish E
Published: January 2020

Mortality after intravenous challenge with 10(4) Cryptococcus neoformans demonstrated that doubly immunodeficient beige athymic (bg/bg nu/nu) mice were more susceptible to systemic cryptococcosis than either bg/bg or nu/nu mice. Infected bg/bg nu/nu mice also had a shortened lifespan compared with their bg/bg nu/+ littermates. Beige athymic (bg/bg nu/nu) but not bg/bg nu/+mice developed cryptococcal lesions in the skin, demonstrating that C. neoformans is dermatotropic in a T-cell-deficient host. Higher numbers of C. neoformans were isolated from the lungs and spleen of infected bg/bg nu/nu than bg/bg nu/+ mice as early as day 3 after challenge, indicating that in lymphoid-rich organs, T cells can alter the course of systemic cryptococcosis early in the infection. Despite extensive abscess formation in the brains of bg/bg nu/+ mice, dissemination and growth rate of C. neoformans in the brain was similar in both genotypes. The primary histopathological feature in tissues from bg/bg nu/nu mice infected with C. neoformans consisted of foci of encapsulated yeast cells with minimal to no inflammatory response. In contrast to bg/bg nu/nu mice, bg/bg nu/+ mice mounted a vigorous inflammatory response to C. neoformans that progressed from acute to chronic inflammation. Beige athymic mice are a new animal model that will be useful in clarifying the innate and acquired immune factors important in resistance to cryptococcosis.
Diarrhea induced by Treponema hyodysenteriae: a young chick cecal model for swine dysentery
Authors: Sueyoshi M , Adachi Y
Published: January 2020

The experimental infection of 25 young chicks with Treponema hyodysenteriae was carried out. Treponemes were isolated from 21 of 25 chicks on day 21 after inoculation. The ceca of chicks infected with treponemes were atrophied. The lumen was filled with a white watery fluid instead of digested feed. In some infected chicks, a cecal core was observed with the fluid in the cecum. The cecal core was grayish, hard, and rod shaped. It consisted of eroded cells and debris of treponemes and resembled the pseudomembrane. Bloody mucus was also observed in one chick. The thickness of the mucosae in 17 of 25 chicks were markedly increased. The histological changes were classified into two types. In the case of regressive changes of epithelial cells which mean severe erosion, the laminae propriae were exposed. Hemorrhage, edema, and heterophil infiltration in the laminae propriae were also confirmed. Numerous treponemes were observed within the edematous area under the remaining epithelia and also invaded the epithelial cells and laminae propriae. In the other case, progressive changes, that is, hyperplasia of mucosal epithelial cells and elongation of the crypt, were observed. The epithelia consisted mainly of cuboidal basophilic cells, mitotic cells, and goblet cells. The mitotic cells increased in number and were also observed near the superficial luminal surface of the ceca. Mucous goblet cells were also considerably increased in number. The erosion of superficial luminal epithelial cells was not so severe, but edema in laminae propriae was frequently observed. Electron-microscopic observation demonstrated that the basophilic epithelial cells were polyribosome rich, mitochondria poor, and lipid droplet poor. Furthermore, tonofibril-like structures under the terminal web in cytoplasms were lost, and numerous membrane-bound vesicles at the terminal web with free ribosomes were observed. In places, a number of vesicles were observed between microvilli, and some vesicles were released from the apical cytoplasm of epithelial cells. Microvilli also became scarce and irregular. The junctional complexes between cuboidal cells became unclear, the interdigitations between cuboidal cells became loose, and the intercellular spaces were widened. In conclusion, the appearance of numerous membrane-bound vesicles at the terminal web and the dilated intercellular space indicates increased secretion of water and electrolytes, and the abnormal proliferation of such immature epithelial cells indicates impaired absorption. On the basis of our findings, we can say that diarrhea induced by T. hyodysenteriae can be developed by synergism between impaired absorption and increased secretion by intestinal mucosae with hyperplastic immature epithelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice
Authors: Goodwin M S , Weiss A A
Published: January 2020

Proliferation of Bordetella pertussis in the lungs of infant mice challenged by the intranasal route was examined. The bacteria rapidly proliferated in the lungs of mice challenged with a sublethal dose of a wild-type strain (BP338) or a filamentous hemagglutinin mutant (BPM409) from 500 at day 0 to 10(7) at day 15. The infection cleared in about 40 days. Pertussis toxin-deficient mutant BP357 gave a similar profile; however, the number of bacteria recovered was slightly reduced, suggesting that pertussis toxin is not essential for bacterial growth in the lungs. In contrast, adenylate cyclase toxin mutant BP348 was rapidly cleared from the lungs, with no viable bacteria remaining 10 days postchallenge, suggesting that the adenylate cyclase toxin is a colonization factor required for the bacteria to initiate infection.
Intracellular hemolysin-producing Listeria monocytogenes strains inhibit macrophage-mediated antigen processing
Authors: Cluff C W , Garcia M , Ziegler H K
Published: January 2020

We found that virulent hemolysin-producing (Hly+) Listeria monocytogenes strains inhibit antigen processing and presentation when added to macrophages in vitro. A virulent Hly- bacteria caused little or no inhibition. Live Hly+ bacteria inhibited presentation of both heat-killed L. monocytogenes and ovalbumin. Several observations indicate that hemolysin produced by intracellular bacteria was responsible for the inhibition. First, inhibition was observed even when extracellular bacteria were removed after a brief 10-min bacterial uptake period. Second, inhibition was not prevented by the addition of cholesterol, a substance which inactivates soluble hemolysin. Third, only very high concentrations of soluble hemolysin were inhibitory. Under conditions which inhibit antigen presentation (10(5) per well), macrophages retained normal levels of Ia, maintained normal morphology, and were not permeable when assayed by chromium release. The uptake and catabolism of 35S-labeled live bacteria by macrophages were similar for both Hyl+ and Hly- bacteria. Only a small decrease in uptake and catabolism of surface-iodinated heat-killed L. monocytogenes by macrophages pretreated with inhibitory numbers of live Hly+ bacteria was observed. Additionally, macrophages pretreated with live Hly+ bacteria and fixed 1.5 h later were able to effectively present an ovalbumin peptide (amino acids 323 to 339) to the T-cell hybridoma DO11.10. Hemolysin-producing bacteria inhibited the presentation of antigens that need processing better than they did of antigens that do not require a processing event. Thus, we have demonstrated inhibition of an intracellular antigen processing pathway by hemolysin-producing L. monocytogenes, which may contribute to the virulence of this pathogen.
Purification and characterization of a novel hemagglutinin from Vibrio cholerae
Authors: Banerjee K K , Ghosh A N , Dutta-Roy K , Pal S C , Ghose A C
Published: January 2020

A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates' of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other O1 and non-O1 V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae O1 and non-O1 strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.
Rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins
Authors: Skerlavaj B , Romeo D , Gennaro R
Published: January 2020

Bactenecins are a class of arginine-rich antibacterial peptides of bovine neutrophil granules. Two bactenecins with approximate molecular weights of 5,000 and 7,000 designated Bac5 and Bac7, respectively, exert in vitro a potent bactericidal activity toward several gram-negative bacteria (R. Gennaro, B. Skerlavaj, and D. Romeo, Infect. Immun. 57:3142-3146, 1989). We have now found that this activity shows an inverse relationship to the ionic strength of the medium and is inhibited by divalent cations and greatly potentiated by lactoferrin. Under conditions supporting marked bactericidal activity, the two peptides cause a rapid increase in the permeability of both the outer and inner membranes of Escherichia coli, as shown by unmasking of periplasmic beta-lactamase and of cytoplasmic beta-galactosidase. In addition, the two bactenecins inhibit the respiration of E. coli and Klebsiella pneumoniae but not of Bac5- and Bac7-resistant Staphylococcus aureus. Furthermore, they induce a drop in ATP content in E. coli, K. pneumoniae, and Salmonella typhimurium and a marked decrease in the rates of transport and incorporation of [3H]leucine and [3H]uridine into E. coli protein and RNA, respectively. In general, all these effects become evident within 1 to 2 min and reach their maximal expression within about 5 min. Overall, these data strongly suggest that the decrease in bacterial viability is causally related to the increase in membrane permeability and the subsequent fall in respiration-linked proton motive force, with the attendant loss of cellular metabolites and macromolecular biosynthesis ability.
Polymorphic expression of defensins in neutrophils from outbred rats
Authors: Eisenhauer P , Harwig S S , Szklarek D , Ganz T , Lehrer R I
Published: January 2020

We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7. Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans. The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4. This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others. These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents.
Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay
Authors: Cloeckaert A , de Wergifosse P , Dubray G , Limet J N
Published: January 2020

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.
Comparison of receptors for 987P pili of enterotoxigenic Escherichia coli in the small intestines of neonatal and older pig
Authors: Dean E A
Published: January 2020

Enterotoxigenic Escherichia coli isolates that express 987P pili colonize the small intestine and cause diarrhea in neonatal (less than 6-day-old) but not in older (greater than 3-week-old) pigs. However, 987P+ E. coli isolates adhere in vitro to small-intestinal epithelial cells from pigs of both ages. This indicates that older pigs as well as neonatal pigs contain receptors for 987P pili and that resistance in older pigs is not due to a lack of intestinal receptors for 987P pili. In this study, we demonstrated that 3-week-old gnotobiotic pigs, like neonatal pigs, were colonized and developed diarrhea when challenged with 987P+ E. coli. We compared 987P receptors in small-intestinal epithelial cell brush borders and in intestinal washes (luminal contents) from less than 1-day-old, 3-week-old gnotobiotic, and 3- to 4-week-old weaned pigs. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters, and 987P binding was demonstrated by immunoassay using purified 987P pili. Multiple 987P-binding components ranging from 33 to 40 kDa were found in brush borders from both 987P-susceptible (neonatal and gnotobiotic) and 987P-resistant (older) pigs: 987P binding to these receptors, which we called 987R, did not correlate with 987P susceptibility. A less than 17-kDa 987P receptor, 987M, was found in the mucus fraction of intestinal washes from 987P-resistant older pigs. Only trace amounts of 987M were detected in 987P-susceptible neonatal and gnotobiotic pigs. 987M comigrated with the 987P receptor previously isolated from adult rabbits. Receptors for 987P in the mucus of older pigs may inhibit 987P-mediated intestinal colonization by preventing the attachment of 987P+ enterotoxigenic E. coli to intestinal epithelial receptors for 987P.
Complete nucleotide sequence of the structural gene for alkaline proteinase from Pseudomonas aeruginosa IFO 3455
Authors: Okuda K , Morihara K , Atsumi Y , Takeuchi H , Kawamoto S , Kawasaki H , Suzuki K , Fukushima J
Published: January 2020

The DNA-encoding alkaline proteinase (AP) of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, the gene-incorporated bacteria expressed high levels of both AP activity and AP antigens. The amino acid sequence deduced from the nucleotide sequence revealed that the mature AP consists of 467 amino acids with a relative molecular weight of 49,507. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified AP reported previously. The amino acid sequence analysis revealed that both the N-terminal side sequence of the purified AP and several internal lysyl peptide fragments were identical to the deduced amino acid sequences. The percent homology of amino acid sequences between AP and Serratia protease was about 55%. The zinc ligands and an active site of the AP were predicted by comparing the structure of the enzyme with of Serratia protease, thermolysin, Bacillus subtilis neutral protease, and Pseudomonas elastase.