bluuebunny/crossref_metadata_2025 huggingface dataset:
Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate
Authors: Collins F M , Lamb J R , Young D B
Published: January 2020

Mycobacterium tuberculosis culture filtrate (MTCF) protein antigens were isolated from mid-logarithmic-phase cultures grown in liquid medium and examined by high-pressure liquid chromatography and Western blot (immunoblot) analysis. A major protein band with a molecular mass of about 68 kilodaltons (kDa) and several fainter bands in the 38- and 24-kDa range were observed. The MTCF protein produced a significant delayed footpad hypersensitivity response in Mycobacterium bovis BCG-vaccinated C57BL/6 mice, comparable to that observed with standard purified protein derivative (PPD). The same proteins induced a blastogenic response in tuberculin-sensitive human peripheral blood monocytes and in T-cell clones developed from these cells. The proliferative responses to the MTCF antigens were equivalent to those observed following stimulation with PPD or M. tuberculosis sonic extracts. However, the MTCF sensitins were not recognized by five monoclonal antibodies directed against killed M. tuberculosis antigens in an enzyme immunoassay, although some response was seen with a monoclonal antibody (ML34) directed against M. leprae antigens. The ability of the MTCF to stimulate T-cell responses both in vivo and in vitro while not being recognized by antibodies directed against dead mycobacterial antigens suggests that they may be of interest as potential protective immunogens.
Characterization of an in vitro-stimulated, Cryptococcus neoformans-specific second-order suppressor T cell and its precursor
Authors: Fidel P L , Murphy J W
Published: January 2020

Using a cryptococcal culture filtrate antigen (CneF) in a murine model, we have demonstrated previously that a cascade of Cryptococcus neoformans-specific suppressor T cells and soluble factors function in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response. In addition, we have successfully hybridized the C. neoformans-specific, first-order T-suppressor (Ts1) cell and have established that the culture supernatant (hTsF1) from this hybridoma induces second-order T-suppressor (Ts2) cells in vivo. Here we report the in vitro induction of expression-phase suppressor cells. The suppressor cells were induced by culturing nylon wool-nonadherent splenic cells from naive mice with hTsF1 in the absence of CneF. Nylon wool-nonadherent splenic cells similarly cultured with supernatants from the BW5147 thymoma cells, the fusion partners of the hybridoma, did not significantly suppress the cryptococcal DTH response. The suppressor cells were designated Ts2 cells based on their similarities in function, specificity, and phenotype, i.e. L3T4-, Lyt-2+, and I-J+, to the in vivo-induced Ts2 cells. By employing the in vitro culture technique, we demonstrated that the precursors of the functional Ts2 cells were L3T4- Lyt-1-2+ I-J- cells. The induction of Ts2 cells was not associated with [3H]thymidine incorporation; therefore, we concluded that hTsF1 induces the Lyt-2+ I-J- cells to differentiate into Lyt-2+ I-J+ functional Ts2 cells without a significant amount of proliferation. From the results of this study, a better understanding of the processes involved in the regulation of the DTH response to CneF was achieved. The in vitro culture technique will allow for further detailed studies of the interactions between the various cell populations and the Ts1 cell-derived soluble factor during the induction of Ts2 cells.
Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis
Authors: Damiani G , Biano A , Beltrame A , Vismara D , Mezzopreti M F , Colizzi V , Young D B , Bloom B R
Published: January 2020

Three monoclonal antibodies (H60.15, H61.3, and H105.10) directed to protein antigens of Mycobacterium tuberculosis were obtained and characterized. H60.15 recognizes a protein with a molecular mass of 28 kilodaltons (kDa) with broad cross-reactivity on a panel of 12 species and strains of mycobacteria. H61.3 reacts with a 35-kDa protein present in M. tuberculosis, Mycobacterium bovis BCG, and M. africanum. On the basis of the antigen molecular masses and competition experiments with other monoclonal antibodies, H60.15 and H61.3 seem to be the first described monoclonal antibodies to these M. tuberculosis proteins. H105.10 binds to the cross-reactive 65-kDa protein present in mycobacteria. Epitope mapping of H105.10 was performed by using the M. leprae DNA sublibrary available in bacteriophage lambda gt11 for this antigen and revealed that its epitope resides in the region from amino acids 20 to 54. The 28-, 35-, and 65-kDa antigens isolated by immunoblotting and presented on nitrocellulose to pleural effusion T cells from tuberculosis patients induced a proliferative response, indicating the presence of T-cell epitopes. These observations indicate that two protein antigens should be added to the list of antigens detectable in M. tuberculosis by monoclonal antibodies. The common feature of such proteins, the elicitation of an immune response of limited or broad cross-reactivity for mycobacteria, encourages the search for their role in the pathogenesis of mycobacterioses.
Induction of inflammation by Escherichia coli on the mucosal level: requirement for adherence and endotoxin
Authors: Linder H , Engberg I , Baltzer I M , Jann K , Svanborg-Edén C
Published: January 2020

Bacterial infection of the mouse urinary tract is followed by the recruitment of leukocytes to the mucosal surface. This study examined the bacterial components involved in the induction of this response. Escherichia coli of serotype O75:K5:H- expressing adhesins specific for the Gal alpha 1-4Gal beta- (Gal, galactose) and mannose-containing receptors were instilled into the urinary bladder of lipopolysaccharide responder (C3H/HcN) and lipopolysaccharide nonresponder (C3H/HeJ) mice. The inflammation was quantitated as the number of leukocytes excreted into the urine at various times after infection. The response was first shown to depend on the Lps genotype of the mouse. The leukocyte excretion that occurred within 24 h after infection of C3H/HeN mice was absent in C3H/HeJ mice. The components triggering the response were present on both live and Formalin-killed bacterial cells, and the response was mimicked by intravesical inoculation of isolated lipid A. Pretreatment of bacteria with soluble receptor oligosaccharides resulted in inhibition of attachment in vitro and of the inflammation in vivo. A direct synergy between adhesins specific for Gal alpha 1-4Gal beta receptors and lipid A was demonstrated. Mixtures of these components induced a leukocyte response higher than the sum of the responses to each component alone. These results suggest that the inflammation induced by gram-negative bacteria in the urinary tract can be triggered at the level of the epithelial cells by endotoxin presented by an attaching bacterial cell and that intact function at the Lps locus of the host is required for this to occur.
Induction of tolerance to lipopolysaccharide (LPS)-D-galactosamine lethality by pretreatment with LPS is mediated by macrophages
Authors: Freudenberg M A , Galanos C
Published: January 2020

In mice treated with D-galactosamine, lipopolysaccharide (LPS) exhibits enhanced toxicity (C. Galanos, M. A. Freudenberg, and W. Reutter, Proc. Natl. Acad. Sci. USA 76:5939-5943, 1979). Pretreatment of mice with LPS before D-galactosamine rendered them tolerant to the enhanced lethal effect of LPS. Tolerance was established at 1 h after LPS injection and, depending on the dose of LPS used for pretreatment, lasted for up to 48 h. With C3H/HeJ mice with acquired sensitivity to LPS (M. A. Freudenberg, D. Keppler, and C. Galanos, Infect. Immun. 51:891-895, 1986), i.e., mice that had been administered C3H/HeN macrophages, pretreatment with LPS induced tolerance only if the C3H/HeN macrophages were already present at the time of pretreatment. This indicates that, like lethality, induction of tolerance by LPS is a macrophage-mediated phenomenon. Direct interaction of LPS with macrophages is the first step in the initiation of tolerance or toxicity. C3H/HeN macrophages (2 X 10(7], incubated with minute amounts of LPS (0.5 to 0.02 microgram) in vitro and transferred subsequently to C3H/HeJ mice, induced lethality when administered together with or after D-galactosamine and tolerance when injected before D-galactosamine. Macrophages activated in vitro lost their tolerance- and lethality-inducing properties upon further incubation in LPS-free culture medium for 18 h. Such macrophages could be successfully restimulated by a new addition of LPS.
Plasmodium chabaudi antigen Pch105, Plasmodium falciparum antigen Pf155, and erythrocyte band 3 share cross-reactive epitopes
Authors: Holmquist G , Udomsangpetch R , Berzins K , Wigzell H , Perlmann P
Published: January 2020

By immunoblotting with a number of monoclonal antibodies raised in human and murine malaria systems, we have been able to establish the presence of cross-reactive epitopes on the Plasmodium falciparum vaccine candidate antigen Pf155/RESA and its proposed Plasmodium chabaudi analog Pch105. These findings were confirmed when the same antibodies were tested in an immunofluorescence assay. By using short synthetic peptides corresponding to repeated sequences in the C terminus of the Pf155 and enzyme-linked immunosorbent assays, the cross-reacting epitope was found to be localized to this repeat segment. Furthermore, a monoclonal antibody to murine erythrocyte band 3 which also cross-reacted with human band 3 bound to both Pch105 and Pf155 as well as to the synthetic peptides, suggesting that these proteins share a related epitope. The cross-reactions reflect the existence of sequence homologies of band 3 with these plasmodial proteins. This molecular similarity may be used by the parasite to disturb the rigidity of the erythrocyte membrane, thereby facilitating its entrance into the cell.
Possible role of a proteinase in endosporulation of Coccidioides immitis
Authors: Yuan L , Cole G T , Sun S H
Published: January 2020

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antiserum in rabbits against the purified proteinase for use in immunoelectron microscopy. We showed that immunolabel was localized in the cell wall of both the saprobic and parasitic phases but was most concentrated in the wall of the segmentation apparatus of spherules just prior to endospore differentiation. The total wall fractions of the mycelial phase, as well as those of presegmented and endosporulating spherules, were isolated from in vitro grown cells and then treated with a proteinase inhibitor (phenylmethylsulfonyl fluoride [PMSF]) which irreversibly binds to the residual proteolytic enzyme in the wall isolates. Each fraction was dialyzed, lyophilized, and separately incubated with the active, purified 36,000-Mr proteinase. The reaction mixtures were examined spectrophotometrically (A280) for decomposition of the substrates. Only the PMSF-treated wall isolated from endosporulating spherules was significantly digested. Active, 36,000-Mr proteinase was isolated from intact and viable, endosporulating spherules by brief extraction of the cells with 1% octyl-beta-D-thioglucoside, a nonionic detergent. The serine proteinase may be partly responsible for autolysis of the segmentation apparatus of mature spherules, a morphogenetic process which is pivotal for release of endospores and subsequent proliferation of the pathogen.
Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma marginale
Authors: Oberle S M , Palmer G H , Barbet A F , McGuire T C
Published: January 2020

A major surface protein complex from the Florida isolate of Anaplasma marginale has been previously shown to induce protection in immunized cattle and has been proposed as the basis of a subunit vaccine against anaplasmosis. This complex in the Florida isolate is composed of two noncovalently associated polypeptides with molecular masses of 105 and 100 kilodaltons (kDa). The analogous protein complex from four geographically different isolates of A. marginale was immunoprecipitated and compared with the protein complex of the Florida isolate. The polypeptides of the complex varied in apparent molecular mass among the isolates. By using antibodies recognizing epitopes on each polypeptide of the Florida isolate, the antigenic identity of the polypeptides in the analogous complexes was determined. The polypeptides recognized by the neutralizing monoclonal antibody 22B1, which recognizes a 105-kDa polypeptide in the Florida isolate, ranged from 70 to 100 kDa in the other isolates. Those polypeptides recognized by rabbit antiserum R911, which recognizes a 100-kDa polypeptide in the Florida isolate, ranged from 97 to 100 kDa. The surface-exposed peptides in the complexes were compared by limited enzymatic digestion to assess structural homology among isolates. Despite the marked variations in molecular weight, there were conserved peptides between the 22B1-reactive polypeptides and between the R911-reactive peptides. Determination of the role of the conserved peptides in inducing immunity will be critical in the application of these polypeptides as the basis of a subunit vaccine for bovine anaplasmosis.
Deficiency of interleukin-2 activity upon addition of soluble egg antigen to cultures of spleen 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 (HGI) and delayed hypersensitivity (DH) elicited by soluble egg antigens (SEA) which reach peak levels at 9 weeks of infection and then spontaneously regress. The in vitro SEA-induced proliferation of spleen cells (SC) from infected animals attained its high point and then declined when SC from 5-week-infected mice were used. The present study determined the dynamics of interleukin-2 (IL-2) production by SEA-challenged SC from infected mice in an attempt to link the level of IL-2 production to the spontaneous regression of the aforementioned T-cell-mediated immune responses. The production of IL-2 by SEA-stimulated SC reached its peak when cells from 7-week-infected mice were challenged at least 2 weeks after the peak of the proliferative response, but declined at about the same time as the HGI and DH responses. Therefore, the decline in IL-2 activity cannot alone explain the diminished proliferative response but could account for the reduction in HGI and DH in vivo. 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 SC of infected mice and is, therefore, likely to be due to underproduction of IL-2. Nor is the deficiency explained by reduced numbers of antigen-presenting cells (macrophages and B cells) or of L3T4+ T lymphocytes or by suppression of IL-2 production by macrophages or macrophage products such as prostaglandins. However, suppression of IL-2 production was observed consistently upon coculture of SC from acutely infected mice with SC from mice infected for 10 weeks. The cells which suppress appear to be Lyt2+ T cells. The data are consistent with the hypothesis that suppressor T cells inhibit the production of IL-2 and perhaps of other cytokines or lymphokines and that this suppression explains the spontaneous down-regulation of HGI which occurs during schistosomiasis japonica.
Histologic, immunofluorescence, and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii
Authors: Khavkin T , Tabibzadeh S S
Published: January 2020

A histologic, immunofluorescence, and electron microscopic study of the intracellular parasitism of Coxiella burnetii (the Q fever agent) in mouse lungs after intranasal challenge was undertaken. It was shown that this microorganism invades type I and, rarely, type II pneumocytes as well as pulmonary fibroblasts and histiocytes. The infectious process can be described as a focal intra-alveolar inflammation with the macrophages prevailing in the exudate. It is self-limited, with a complete resolution. The inflammation is associated with atelectases and with increased secretory activity by type II pneumocytes. Alveolar macrophages and granulocytes degrade C. burnetii. This degradation is followed by damage to and eventual disintegration of some macrophages and by damage to some bacterium-free pneumocytes and vascular endothelial cells in the vicinity of macrophages degrading organisms. The cell damage might be caused by lipopolysaccharide released from degraded organisms. The infectious process is also associated with the influx of T cells in the pneumonic foci, T-cell attachment to the macrophages degrading organisms, and fusion of some macrophages. These are considered a morphologic expression of cell-mediated immunity involved in the infectious process.
Clonal diversity of Neisseria meningitidis from a population of asymptomatic carriers
Authors: Caugant D A , Kristiansen B E , Frøholm L O , Bøvre K , Selander R K
Published: January 2020

Genetic diversity and relationships among 109 isolates of Neisseria meningitidis obtained from throat cultures of healthy individuals in Norway in 1984 were assessed by analyzing electrophoretically demonstrable allelic variation at 15 enzyme-encoding chromosomal genes. Seventy-eight distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. The mean genetic diversity per locus among the 78 ETs (0.538) was equivalent to that among 19 ETs represented by 66 isolates collected from patients with meningococcal disease in Norway in the first 5 months of 1984. The clonal composition of the collection of carrier strains was, however, quite different from that of strains from patients. The two groups of clones, the ET-5 complex and the ET-37 complex, that were responsible for 91% of the cases of systemic disease in Norway in 1984 were identified in only 7 and 9%, respectively, of the throat cultures from healthy individuals, and their frequencies in the human population sampled were only 0.7% for clones of the ET-5 complex and 0.9% for those of the ET-37 complex. The complex of clones that was most frequently represented by isolates from carriers (19%) has never been recovered from patients with meningococcal disease in Norway or elsewhere, which suggests that these clones have a low virulence potential. Children attending the same day care center or school seldom harbored the same clone in their throats.
Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates
Authors: Andersen A A , Van Deusen R A
Published: January 2020

Monoclonal antibodies (MAbs) were produced to four Chlamydia psittaci isolates: NJ1 and TT3 from turkeys, VS1 from a parakeet, and B577 from an ovine abortion. MAbs were tested for reactivity with each isolate by the indirect immunofluorescent antibody technique and for neutralization by an inclusion reduction neutralization technique in tissue culture. Two genus-specific and 14 serovar-specific MAbs were produced. Genus-specific MAbs reacted with all avian and mammalian isolates; however, each failed to neutralize its homologous chlamydial isolate. Turkey isolates NJ1 and TT3 were antigenically similar; serovar-specific MAbs produced to each reacted equally with both isolates yet showed little or no reaction with other serovars. Serovar-specific MAbs to the parakeet and abortion isolates were distinct; no cross-reactions were seen with other serovars. None of the serovar-specific MAbs reacted with an ovine arthritis isolate. Of the 14 serovar-specific MAbs, 13 partially neutralized homologous strains with or without the addition of complement. Because of the high specificity, the serovar-specific MAbs used with the immunofluorescence technique provided a rapid and precise method to identify three serovars of C. psittaci.
Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain
Authors: Popoff M R , Rubin E J , Gill D M , Boquet P
Published: January 2020

By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin.
Induction of lyme arthritis in LSH hamsters
Authors: Schmitz J L , Schell R F , Hejka A , England D M , Konick L
Published: January 2020

In studies of experimental Lyme disease, a major obstacle has been the unavailability of a suitable animal model. We found that irradiated LSH/Ss Lak hamsters developed arthritis after injection of Borrelia burgdorferi in the hind paws. When nonirradiated hamsters were injected in the hind paws with B. burgdorferi, acute transient synovitis was present. A diffuse neutrophilic infiltrate involved the synovia and periarticular structures. The inflammation was associated with edema, hyperemia, and granulation tissue. Numerous spirochetes were seen in the synovial and subsynovial tissues. The histopathologic changes were enhanced in irradiated hamsters. The onset and duration of the induced swelling were dependent on the dose of radiation and the inoculum of spirochetes. Inoculation of irradiated hamsters with Formalin-killed spirochetes or medium in which B. burgdorferi had grown for 7 days failed to induce swelling. This animal model should prove useful for studies of the immune response to B. burgdorferi and the pathogenesis of Lyme arthritis.
Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey
Authors: Clark W B , Magnusson I , Abee C , Collins B , Beem J E , McArthur W P
Published: January 2020

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.
Endothelial cell contraction increases Candida adherence to exposed extracellular matrix
Authors: Klotz S A , Maca R D
Published: January 2020

Bovine vascular endothelial cells treated with EDTA, urea, or thrombin underwent a marked, reversible contraction resulting in exposure of the subendothelial extracellular matrix (ECM). Candida yeasts adhered more to contracted monolayers than to confluent monolayers (P less than 0.01) by preferentially adhering to the ECM. Two strains of Candida albicans and one strain of Candida tropicalis bound avidly to exposed ECM, but Pseudomonas aeruginosa did not. However, treatment of endothelium with forskolin, which induces cell shape changes without exposure of the ECM, did not cause an increase in adherence.
Inhibition of Chlamydia trachomatis growth by recombinant tumor necrosis factor
Authors: Shemer-Avni Y , Wallach D , Sarov I
Published: January 2020

Purified human recombinant tumor necrosis factor (rTNF) alpha inhibited the growth of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cell cultures. The inhibition of C. trachomatis yield could be achieved even when the rTNF alpha (200 ng/ml) was added up to 12 h after infection. The effect of rTNF alpha on chlamydial infection was synergistic with that of gamma interferon.
Role of endogenous gamma interferon in host defense against Chlamydia trachomatis infections
Authors: Zhong G M , Peterson E M , Czarniecki C W , Schreiber R D , de la Maza L M
Published: January 2020

BALB/c mice (6 to 8 weeks old) infected with Chlamydia trachomatis serovar L1 were sacrificed, and the yield of Chlamydia inclusion-forming units from the liver and lungs was measured in HeLa 229 cells. The yield of inclusion-forming units reached a peak at 3 days postinfection and then progressively declined. The mice infected with C. trachomatis had no detectable levels of gamma interferon (IFN-gamma) in their sera. However, stimulation of their spleen cells with either concanavalin A or heat-killed C. trachomatis resulted in the release of high levels of IFN-gamma (600 to 900 IU/ml) at 5 to 8 days postinfection. The increased release of IFN-gamma from the spleen cells paralleled the clearance of chlamydia from the liver and lungs. Sera and spleen cells from animals immunized with live C. trachomatis were transferred to recipient mice that were subsequently challenged with C. trachomatis. Transfer of spleen cells resulted in a reduction of the infection in the recipient animal as measured by the yield of chlamydia from the spleen, but transfer of the sera did not confer protective immunity. In addition, mice infected with C. trachomatis serovar L1 were treated with a hamster neutralizing monoclonal antibody to recombinant murine IFN-gamma (MAb-MuIFN-gamma). In the animals receiving the MAb-MuIFN-gamma, the yield of chlamydia from the lungs, spleen, and liver was significantly higher than from the control groups of mice. Histopathological analysis of tissues from the chlamydia-infected mice showed that the animals treated with the MAb-MuIFN-gamma had a significantly more extensive inflammatory reaction in their lungs, liver, and spleen.
Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG
Authors: Yamaguchi R , Matsuo K , Yamazaki A , Abe C , Nagai S , Terasaka K , Yamada T
Published: January 2020

The gene for immunogenic protein MPB64 found in culture filtrates of only Mycobacterium tuberculosis and some strains of Mycobacterium bovis BCG was cloned by using a single-probe method and was sequenced. The gene analysis revealed that the structural gene for MPB64 consisted of 618 base pairs, and its deduced molecular weight was 22,400. Twenty-two amino acids for a putative signal peptide and 205 amino acids for the MPB64 protein were observed. In the coding region, the third letter of the codon showed a biased codon and a high G+C content (78.5%). The gene was expressed in Escherichia coli by using an E. coli expression vector. The product showed migration similar to that of the authentic MPB64 protein by electrophoresis and reacted with the polyclonal and the monoclonal antibodies raised against the MPB64 protein. The strict specificity of MPB64 could be applied to immunodiagnosis of tuberculosis.
Protein 20, an immunodominant surface antigen of Eimeria bovis
Authors: Whitmire W M , Kyle J E , Speer C A
Published: January 2020

Autoradiography identified six 125I-labeled proteins, ranging in molecular weight (Mr) from 20,000 to approximately 110,000, on the plasmalemma of Eimeria bovis sporozoites. Immunoblotting with bovine antiserum generated by intravenous inoculations of sporozoites and with immune serum generated by per os inoculations of oocysts revealed that protein 20 (i.e., 20,000 Mr) was an immunodominant antigen on the surface of E. bovis sporozoites.
Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase I activity are resistant to Entamoeba histolytica-mediated cytotoxicity
Authors: Li E , Becker A , Stanley S L
Published: January 2020

To study the relationship between carbohydrate-specific amebic cytoadherence and ameba-mediated cytotoxicity, we measured Entamoeba histolytica trophozoite-mediated cytolysis directed against a panel of four Chinese hamster ovary (CHO) cell lines that have defined alterations in their glycosylation patterns. We recently measured amebic trophozoite adherence to this panel of CHO cells and showed that trophozoites bind variant cells (RICR 15B), which are deficient in Asn-linked N-acetyllactosamine units, at 12% of the level observed for wild-type cells (E. Li, A. Becker, and S. L. Stanley, J. Exp. Med 167:1725-1730, 1988). Using a 51Cr release assay to measure trophozoite-mediated cytolysis, we demonstrate in this study that RICR 15B cells are less susceptible to trophozoite-mediated cytolysis than are wild-type cells. In addition, we found that N-acetyllactosamine, which inhibits trophozoite adherence to CHO cells, also inhibited trophozoite-mediated cytolysis of wild-type cells. These studies indicate that surface carbohydrates on target cells can influence susceptibility to ameba-mediated cytotoxicity. This panel of CHO cells provides a useful model system for investigating the role of glycoconjugates in mediating amebic interactions with mammalian cells.
Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis
Authors: Grenier D , Chao G , McBride B C
Published: January 2020

Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. Sulfhydryl group reagents and N-alpha-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50 degrees C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75 degrees C for 30 min.
Cloning and expression of Staphylococcus saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence
Authors: Gatermann S , Marre R
Published: January 2020

The urease gene of Staphylococcus saprophyticus CCM883 was cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned DNA sequences revealed six polypeptides (of 70, 47, 29, 27, 20, and 17 kilodaltons) that were associated with enzyme activity. Introduction of the cloned genes into a urease-negative mutant of S. saprophyticus restored the virulence of this strain, confirming our previous suggestion (S. Gatermann, J. John, and R. Marre, Infect. Immun. 57:110-116, 1989) that this enzyme is a major virulence factor of the organism and contributes mainly to cystopathogenicity.
Intraperitoneal host cellular responses and in vivo killing of Bacteroides fragilis in a bacterial containment chamber
Authors: Onderdonk A B , Cisneros R L , Crabb J H , Finberg R W , Kasper D L
Published: January 2020

A bacterial containment chamber was used to evaluate the peritoneal cellular response to Bacteroides fragilis during intraperitoneal challenge. This containment system was also used to determine the fate of bacteria within the peritoneal cavities of animals immunized, either actively or through adoptive transfer of cells or cell lysates, with the capsular polysaccharide of B. fragilis. This system demonstrated that the dominant cell types in the peritoneal cavities within 48 h of implantation of the containment chambers containing B. fragilis were neutrophils and macrophages. However, the early cellular response in immunized animals included an increase in the lymphocyte population within 4 h of challenge which was not detected in naive animals. In immunized animals, a later dramatic increase in the lymphocyte population at approximately 4 to 6 days following implantation of the containment chambers occurred. This increase in the lymphocyte population in immunized animals coincided with a decline in the viable bacterial counts within the chambers from 10(8) to 10(9) CFU/ml to less than 10(2) CFU/ml. A similar decline was not seen in naive animals challenged in the same manner. Killing of B. fragilis within containment chambers occurred when spleen cells, T cells, or lysates of T cells from actively immunized animals were passively transferred to naive recipient animals. It was shown that the factor responsible for bacterial killing was not antibody mediated, since bacteria contained within dialysis sacs with an exclusion of 50 kilodaltons were still killed in this model. Moreover, removal of T cells from adoptively transferred cell populations before transfer abrogated the decline in viable bacterial populations. The postulated mechanisms by which this bacterial killing occurred are discussed.
Comparison of T-cell responses in self-limiting versus progressive visceral Leishmania donovani infections in golden hamsters
Authors: Gifawesen C , Farrell J P
Published: January 2020

Leishmania donovani infection in golden hamsters was studied as a model for human kala-azar. After intradermal inoculation of L. donovani amastigotes, hamsters developed positive skin reactions (delayed-type hypersensitivity [DTH]) to parasite antigens and lymphoid cells from these hamsters proliferated to parasite antigens in vitro and transferred DTH reactivity to normal recipients. In contrast, hamsters infected by the intracardial route developed progressive visceral infections and failed to respond to skin test antigens. Spleen cells, lymph node cells, and peripheral blood lymphocytes (PBLs) from these hamsters were unresponsive to parasite antigens in vitro, and spleen cells failed to transfer DTH to normal recipients. Spleen cells, but not PBLs, displayed depressed responses to T-cell mitogens and also suppressed the proliferative response of cells from hamsters inoculated intradermally. Removal of adherent cells restored the capacity of spleen cells, but not PBLs, to respond to parasite antigens. The nonadherent population of these spleen cells also transferred DTH to normal recipients. The adherent suppressor cells, which have the characteristics of macrophages, appear to be localized to the spleen and are apparently not responsible for the failure of peripheral lymphoid cells to respond to antigen. These studies suggest that hamsters with visceral infections develop a population of antigen-reactive cells and that in the absence of suppression these cells may express functional activities, including the capacity to elicit DTH responses.
Cloning and sequencing of the immunoglobulin A1 protease gene (iga) of Haemophilus influenzae serotype b
Authors: Poulsen K , Brandt J , Hjorth J P , Thøgersen H C , Kilian M
Published: January 2020

Secretion of immunoglobulin A1 (IgA1) proteases is a characteristic of Haemophilus influenzae and several other bacterial pathogens causing infectious diseases, including meningitis. Indirect evidence suggests that the proteases are important virulence factors. In this study, we cloned the iga gene encoding immunoglobulin A1 (IgA1) protease from H. influenzae serotype b into Escherichia coli, in which the recombinant H. influenzae iga gene was expressed and the resulting protease was secreted. Sequencing a part of a 7.5-kilobase DNA fragment containing the iga gene revealed a large open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,541 amino acids and a molecular mass of 169 kilodaltons. Putative promoter and terminator elements flanking the open reading frame were identified. Comparison of the deduced amino acid sequence of this H. influenzae IgA1 protease with that of a similar protease from Neisseria gonorrhoeae revealed several domains with a high degree of homology. Analogous to mechanisms known from the N. gonorrhoeae IgA protease secretion, we propose a scheme of posttranslational modifications of the H. influenzae IgA1 protease precursor, leading to a secreted protease with a molecular mass of 108 kilodaltons, which is close to the 100 kilodaltons reported for the mature IgA1 protease.
Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens
Authors: Oaks E V , Rice R M , Kelly D J , Stover C K
Published: January 2020

The genetic and antigenic relatedness of eight antigens in three strains of Rickettsia tsutsugamushi has been studied by using recombinant organisms expressing epitopes of the 150-, 110-, 72-, 58-, 56-, 49-, 47-, and 20-kilodalton (kDa) polypeptide antigens of the Karp strain. Southern blot analysis of Karp, Kato, and Gilliam strain genomic DNA by using probes specific for each antigen class indicated that while strong homology exists between each of the corresponding antigen genes in these three strains, some restriction fragment length polymorphism exists. Antibodies affinity purified against each recombinant antigen class reacted with a comparably sized polypeptide in the Karp, Kato, and Gilliam strains in Western blots (immunoblots). Against more recent human isolates of R. tsutsugamushi, the affinity-purified antibodies against the 58-kDa recombinant antigen (anti-58-kDa) reacted with all nine isolates, anti-56-kDa reacted with eight of nine isolates, anti-47-kDa reacted with eight of nine isolates, anti-72-kDa reacted with eight of nine isolates, and anti-110-kDa reacted with four of nine isolates. Additional analysis indicated that the 110-kDa antigen may contain strain-specific epitopes similar to those previously reported for the 56-kDa polypeptide. Evidently, the strain heterogeneity among scrub typhus rickettsiae is a result of multiple components that exhibit variability in a background of strong homology.
Evidence for direct regulation of diphtheria toxin gene transcription by an Fe2+-dependent DNA-binding repressor, DtoxR, in Corynebacterium diphtheriae
Authors: Fourel G , Phalipon A , Kaczorek M
Published: January 2020

Previous studies provided indirect evidence that in Corynebacterium diphtheriae regulation of diphtheria toxin gene (tox) transcription by iron is mediated by a bacterial repressor. By performing in vitro protein-DNA binding experiments, we establish that a corynebacterial Fe2+-sensitive protein, named DtoxR, can bind to a palindromic motif present in the tox promoter region. Binding of this factor prevents the interaction of the transcription initiation machinery with presumptive critical promoter elements, providing evidence that DtoxR is responsible for the repression of toxinogenesis observed in iron-containing growth medium.
Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi
Authors: Torii M , Adams J H , Miller L H , Aikawa M
Published: January 2020

We used immunoelectron microscopy to study the fate of dense granules during the invasion of erythrocytes by Plasmodium knowlesi merozoites. When merozoites entered host cells, dense granules moved to the pellicle, released their contents into the parasitophorous vacuole space, and then moved into fingerlike channels of the vacuole membrane. This is the first report showing that the content of dense granules of P. knowlesi is different from the contents of rhoptries and micronemes and is associated with the formation of channels from the parasitophorous vacuole.
Efficacy of interleukin-1 beta against systemic Candida albicans infections in normal and immunosuppressed mice
Authors: Pecyk R A , Fraser-Smith E B , Matthews T R
Published: January 2020

Prophylactic treatments with either recombinant human interleukin-1 beta (rHuIL-1 beta) or a muramyl dipeptide analog ([Abu1]MDP) enhanced the resistance of mice to systemic infection with Candida albicans. The optimum treatment regimen in both normal and cyclophosphamide-treated mice was intraperitoneal administration of 100 ng of rHuIL-1 beta or 1.6 mg of [Abu1]MDP per mouse once daily for 3 consecutive days before infection. Neither rHuIL-1 beta nor [Abu1]MDP was efficacious when started after the infection or when given before cyclophosphamide to mice infected subsequently. Continuing to treat after the infection with either drug neither enhanced nor antagonized the efficacy of prophylactic treatments.
DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus
Authors: Ficht T A , Bearden S W , Sowa B A , Adams L G
Published: January 2020

The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When the promoter-containing fragment was inverted, a 33-kDa protein was expressed. These results were consistent with the predicted sizes based on the nucleotide sequences of the open reading frames in omp 2b and omp 2a. Whether this locus contains one active and one silent or cryptic porin gene, or two active Brucella porin genes expressed under different environmental conditions, is discussed.
Cloning and expression in Escherichia coli of the genes of the arginine deiminase system of Streptococcus sanguis NCTC 10904
Authors: Burne R A , Parsons D T , Marquis R E
Published: January 2020

The common oral bacterium Streptococcus sanguis can degrade arginine via the arginine deiminase (AD) system. The three enzymes of this system, AD, ornithine carbamyltransferase (OTC), and carbamate kinase (CK), catalyze the breakdown of arginine to ornithine, CO2, and two molecules of ammonia, with the production of ATP from ADP. The genes of the AD system, which are subject to complex regulation in the oral streptococci, have been isolated in bacteriophage lambda by screening for AD activity. The AD gene, designated arcA, was expressed from recombinant bacteriophage or in cells harboring plasmid subclones from this phage at a level up to 1,000-fold lower than the level in fully derepressed S. sanguis but apparently under the control of its own promoter. By subcloning in Escherichia coli mutants defective in anabolic OTC (argF argL) and CK (carB), it was demonstrated that the genes for S. sanguis OTC and CK were located adjacent to the AD gene. The levels of expression of the OTC and CK genes (arcB and arcC, respectively) were also very low in E. coli, although arcC expression was not as poor as arcA and arcB expression when compared with the levels found in S. sanguis. Also, arcB and arcC were unable to complement the defects in their anabolic counterparts. Introduction of the entire AD system or subclones which encoded only the AD gene into E. coli harboring defects in arginine and pyrimidine biosynthesis resulted in a 10- to 15-fold decrease in the level of AD activity, suggesting that arginine or its metabolites may regulate AD expression. Transposon mutagenesis was utilized to construct defined mutants of S. sanguis with mutations in the AD gene cluster. AD gene expression in these mutants indicated that the expression of the AD genes in this organism is strongly interrelated. The isolation and partial characterization of the arc genes represents the first step in the genetic manipulation of the AD system in the oral streptococci for analysis of the regulation of AD, analysis of the role of the system in plaque ecology, and utilization of the system to modulate the cariogenicity of dental plaque.
Isolation, purification, and characterization of fragment B, the NH2-terminal half of the heavy chain of tetanus toxin
Authors: Matsuda M , Lei D L , Sugimoto N , Ozutsumi K , Okabe T
Published: January 2020

Fragment B, the N-terminal half of the heavy chain, an important domain of the tetanus neurotoxin molecule, was isolated for the first time. Tetanus toxin (composed of three domains, A, B, and C) was prepared from culture filtrates. Fragment A-B, derived from the toxin treated mildly with papain, was used for the isolation of fragment B. Fragment A-B obtained was dissociated into fragments A and B by reduction with 100 mM dithiothreitol and treatment with 2 M urea. Fragment B was separated from fragment A by ion-exchange column chromatography on a Mono Q column equilibrated with 20 mM Tris hydrochloride buffer (pH 7.6), containing 1 mM dithiothreitol and 2 M urea, in a fast-protein liquid chromatography system by elution with a linear gradient of 0 to 0.5 M NaCl. Fragment B was obtained in two forms having molecular weights of 48,000 +/- 2,000, which were indistinguishable by sodium dodecyl sulfate-gel electrophoresis or antigenic specificity, but distinguishable on polyacrylamide gel electrophoresis without sodium dodecyl sulfate and on isoelectric focusing (pI 6.7 and 7.3). The recovery of fragment B was 50 to 72% of that of fragment A-B on a molar basis. Purified fragment B was not toxic to mice on intravenous or intramuscular injection at doses of up to 100 micrograms, but was found to form channels (ca. 2.3 pS) in a lipid bilayer membrane by a patch clamp technique. The role of domain B of the tetanus toxin molecule in the mechanism of action of the toxin is discussed.