bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of a Chlamydia trachomatis L3 DNA fragment that codes for an antigenic region of the major outer membrane protein and specifically hybridizes to the C- and C-related-complex serovars
Authors: Carlson E J , Peterson E M , de la Maza L M
Published: January 2020

Chlamydia trachomatis L3 DNA was cloned and expressed in lambda gt11. A recombinant plaque that expressed an antigen that reacted with rabbit polyclonal antichlamydial L3 serum and with two monoclonal antibodies specific for serovars L3 and I was selected from this Chlamydia genomic library. The beta-galactosidase Chlamydia fusion protein was purified by immunoaffinity chromatography and injected into mice to produce monoclonal antibodies. These monoclonal antibodies reacted by Western (immuno-) blot with both the fusion protein and the major outer membrane protein from purified L3 elementary bodies. The chlamydial DNA fragment was shown by DNA sequence analysis to be 168 base pairs in length and to correspond to the constant regions 1 and 2 and the variable segment 1 of the major outer membrane protein gene. The recombinant chlamydial DNA fragment hybridized under stringent conditions by Southern and dot blot analysis exclusively with the DNA from the C- and C-related-complex C. trachomatis serovars.
Capacity of recombinant gamma interferon to activate macrophages for Salmonella-killing activity
Authors: Kagaya K , Watanabe K , Fukazawa Y
Published: January 2020

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.
Purification, cyanogen bromide cleavage, and amino terminus sequencing of class 1 and class 3 outer membrane proteins of meningococci
Authors: Poolman J T , Timmermans H A , Teerlink T , Seid R C
Published: January 2020

Meningococcal class 1 and 3 outer membrane proteins (OMPs) were subjected to cyanogen bromide treatment. The class 3 OMP was found to be resistant to cyanogen bromide, while the class 1 OMP was cut into two main fragments of 25 and 17 kilodaltons. The N-terminal sequences were determined for class 1 and class 3 proteins, which exhibit similarities to one another and to OMP I of gonococci. The C-terminal class 1 OMP fragment bound the bactericidal monoclonal antibodies tested.
Binding sites of attachment-inhibiting monoclonal antibodies and antibodies from patients on peptide fragments of the Mycoplasma pneumoniae adhesin
Authors: Jacobs E , Gerstenecker B , Mader B , Huang C H , Hu P C , Halter R , Bredt W
Published: January 2020

The adherence protein (P1 protein) of Mycoplasma pneumoniae was purified by electroelution and cleaved with cyanogen bromide. The resulting peptides were separated by two-dimensional electrophoresis. Spots reacting in Western immunoblots with two attachment-inhibiting monoclonal antibodies were isolated, and the amino-terminal ends of these peptides were microsequenced. The two monoclonal antibodies had different binding sites. One was associated with the amino-terminal region of the whole P1 protein beginning at amino acid position 237, and the other was associated with amino acid position 702, which was localized approximately in the middle of the P1 amino acid sequence. Serum samples from three M. pneumoniae-infected patients were tested by Western blotting against the cyanogen bromide peptide pattern. All three serum samples reacted with peptide fragments beginning at amino acid position 702, but the serum of only one patient also had antibodies against the oligopeptides beginning at amino acid position 237. These results indicate that the corresponding epitopes of the P1 protein are also immunogenic if they are presented at the surface of the infecting organism.
Two types of suppressor T cells that inhibit delayed-type hypersensitivity to Mycobacterium intracellulare in mice
Authors: Nakamura R M , Goto Y , Kitamura K , Tokunaga T
Published: January 2020

Mycobacterium intracellulare Mino grows progressively in the organs of C57BL/6 mice. When C57BL/6 mice were injected with strain Mino, very weak or almost no delayed-type hypersensitivity (DTH) to the specific antigen, M. intracellulare purified protein derivative, was observed. The DTH response was enhanced by cyclophosphamide pretreatment, indicating that some suppression mechanism(s) might be involved in DTH. In cell transfer experiments it was shown that two types of suppressor T cells, afferent type (TS-Aff) and efferent type (TS-Eff), are induced in the spleens of Mino-infected mice. TS-Aff were detected from the early stage to the late stage of infection. TS-Eff were detected only in the late stage of infection. These two types of suppressor T cells were both Thy-1 positive and Lyt-2 positive; however, TS-Aff seemed to be I-J positive, and TS-Eff seemed to be I-J negative. Cross-reactivity in suppression was observed between Mino and Mycobacterium bovis BCG but not between Mino and sheep erythrocytes. DTH to Mino and BCG was not cross-reactive. The possible role of suppressor T cells in M. intracellulare infection is discussed.
Stimulation by fibronectin of macrophage-mediated phagocytosis of Pseudomonas aeruginosa
Authors: Kluftinger J L , Kelly N M , Hancock R E
Published: January 2020

In a previous investigation it was determined that Pseudomonas aeruginosa cells taken directly from a mouse in vivo growth system were significantly more susceptible to nonopsonic phagocytosis by macrophages than were similar cells after being washed in buffer (N. M. Kelly, J. L. Battershill, S. Kuo, J. P. Arbuthnott, and R. E. W. Hancock, Infect. Immun. 55:2841-2843, 1987). It was demonstrated that a phagocytosis-promoting factor was found in the supernatant obtained from chambers incubated in the peritoneal cavities of laboratory mice or rats. The phagocytosis-promoting factor was effective with both strains of P. aeruginosa tested, using both unelicited mouse peritoneal macrophages and the P388D1 mouse macrophage cell line as the phagocytic cells. Phagocytosis enhancement was observed with in vivo-grown bacteria and with bacteria grown in vitro on agar plates, but not with bacteria grown in vitro with rapid agitation. Supernatants from mice and rats were fractionated using a fast pressure liquid chromatography gel exclusion column. The phagocytosis-promoting factor copurified with fibronectin. Furthermore, antifibronectin sera negated the phagocytosis-promoting activities of in vivo chamber supernatant, while commercial bovine fibronectin was itself capable of promoting phagocytosis. The concentrations of fibronectin increased in both rat and mouse peritoneal chambers with time, coincident with the ability of chamber supernatants to promote phagocytosis. It was concluded that fibronectin was the phagocytosis-promoting factor of chamber supernatants. Bacterial presence in the peritoneal chambers was not required to elicit fibronectin uptake into the chambers.
Influence of Lsh, H-2, and an H-11-linked gene on visceralization and metastasis associated with Leishmania mexicana infection in mice
Authors: Roberts M , Alexander J , Blackwell J M
Published: January 2020

Visceral infection and metastatic lesion development following intravenous or subcutaneous inoculation of Leishmania mexicana amastigotes were examined in different B10 congenic mouse strains carrying alternative alleles at Lsh, H-2, or H-11. The results show that, despite a failure to observe any differences in rates of expansion of primary lesions in mice inoculated subcutaneously, each of these genes could be shown to exert some influence during visceralization and metastatic spread of L. mexicana infection. Of particular interest were (i) the continuous advantage observed throughout 160 to 200 days of infection in Lshr versus Lshs mice, (ii) the association between structural gene polymorphism at H-2 and profound visceral and metastatic spread of the parasite producing disease phenotypes akin to diffuse cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis in humans, and (iii) similar effects observed in mice differing at H-11, the functional basis for which involves modified expression of major histocompatibility complex class II molecules. The results are discussed in relation to the human disease and the possibility that homologs for each of these genes regulate leishmanial infections in humans.
Tumor necrosis factor alpha-induced pulmonary vascular endothelial injury
Authors: Goldblum S E , Hennig B , Jay M , Yoneda K , McClain C J
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) mediates components of the acute-phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied whether human recombinant TNF-alpha (rTNF-alpha) could increase pulmonary edema formation and pulmonary vascular permeability. Rabbits preinfused with 125I-albumin were administered rTNF-alpha or saline. Animals were sacrificed, and lung wet/dry weight ratios as well as bronchoalveolar lavage fluid and plasma 125I activities were determined. rTNF-alpha increased lung wet/dry weight ratios by 151% (P less than 0.02) and bronchoalveolar lavage fluid/plasma 125I activity ratios by 376% (P less than 0.01) compared with values for saline controls. Electron microscopy of lung sections demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural permeability tracer. To demonstrate that rTNF-alpha could directly increase pulmonary vascular endothelial permeability in vitro, we studied albumin transfer across cultured porcine pulmonary artery endothelial cell monolayers. rTNF-alpha induced time-dependent dose-response increments in transendothelial albumin flux in the absence of granulocyte effector cells. These observations suggest that rTNF-alpha can provoke acute pulmonary vascular endothelial injury in vivo as well as in vitro.
Bacteriologic and serologic findings in experimental pyelonephritis caused by Ureaplasma urealyticum
Authors: Pickering W J , Birch D F
Published: January 2020

A challenge procedure based on reflux of Ureaplasma urealyticum to the rat kidney was used to examine the disease-causing potential of this species for the upper urinary tract. Renal infection was detected 8 days after challenge in 90 to 100% of animals exposed to 10(7) ureaplasmas but was not detected in animals exposed to fewer than 10(6) microorganisms. Viable microorganisms were recoverable from more than 60% of infected animals throughout a 24-week follow-up period of study. Animals with persistent infection exhibited a humoral response involving immunoglobulin M and immunoglobulin G antibody. These results show that U. urealyticum is capable of giving rise to chronic infection in the kidneys of rats following challenge via the ascending route.
Detection of Mycoplasma pulmonis in arthritic joints of rats by indirect immunoperoxidase staining
Authors: Kohn D F , Chinookoswong N
Published: January 2020

Neonatal and 8-week-old rats were inoculated with Mycoplasma pulmonis. A portion of the animals developed polyarthritis. Indirect immunoperoxidase staining was used to identify the localization of M. pulmonis within arthritic joints. M. pulmonis antigen was most often observed within cartilage in the neonatal group and in synovial tissue in the 8-week-old group.
Population genetics of human and animal enteric Campylobacter strains
Authors: Aeschbacher M , Piffaretti J C
Published: January 2020

A total of 125 strains of Campylobacter jejuni and Campylobacter coli from human and animal hosts were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized by the electrophoretic mobilities of their products, and all were found to be polymorphic. The nine loci considered for genetic analysis had an average allele number of 6.9 and a mean genetic diversity of 0.634. The strains were grouped into 64 electrophoretic types (ETs) forming two principal clusters; the first cluster consisted of 50 ETs represented by 104 isolates of C.jejuni, and the second included 14 ETs represented by 21 isolates of C. coli. The genetic distance between the two species was 0.939. The data support the conclusions that (i) campylobacteriosis caused by C. jejuni and C. coli is a zoonosis, (ii) human and animal strains do not constitute subpopulations, and (iii) every animal strain may be considered a potential human pathogen. The frequency of intraspecies gene transfer seems to be moderately high whereas interspecies gene transfer is very rare at best, which suggests the existence of a biological barrier between C. jejuni and C. coli.
Experimental Campylobacter jejuni infection in Macaca nemestrina
Authors: Russell R G , Blaser M J , Sarmiento J I , Fox J
Published: January 2020

Experimental infection of four specific-pathogen-free Macaca nemestrina monkeys (aged 3.5 and 4.5 months) with Campylobacter jejuni 81-176 caused acute diarrheal illness, characterized by fluid diarrhea, bloody stools, and fecal leukocytes, which lasted for approximately 7 to 11 days. Histologic examination of intestinal biopsies showed acute colitis characterized by infiltration of the mucosa with neutrophils and lymphocytes, and cryptitis. There were no histologic changes in the small intestine. Excretion of C. jejuni was demonstrated for 2 to 4 weeks postchallenge. Plasma antibodies to C. jejuni group antigen were elevated after challenge. Only mild diarrhea occurred after rechallenge with the same strain or with a heterologous C. jejuni strain (79-168) followed by further elevation in specific immunoglobulins A, M, and G. Four 1-year-old juvenile M. nemestrina monkeys which had experienced multiple infections with Campylobacter spp. did not exhibit illness when challenged with C. jejuni 81-176. All had elevated immunoglobulin A, M, and G plasma antibodies prior to challenge, and these humoral antibody levels were indicative of the immunity to challenge. The results demonstrate that C. jejuni infection in M. nemestrina caused colitis with clinical and pathologic results similar to those found in humans and indicate that prior infection protects against subsequent challenge.
Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains
Authors: Zhang Y X , Morrison S G , Caldwell H D , Baehr W
Published: January 2020

We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.
Immunological and nonimmunological control of severity of Trypanosoma musculi infections in C3H and C57BL/6 inbred mice
Authors: Albright J W , Albright J F
Published: January 2020

Studies concerned with the mechanisms responsible for relative resistance or susceptibility of strains of inbred mice to Trypanosoma musculi infections are presented. Treatment with 400 rads of ionizing radiation, silica dust, or trypan blue (reticuloendothelial blocking agents) rendered C3H mice unable to control the initial maximum level of parasite growth, and the mice died of overwhelming infections. In contrast, similarly treated C57BL/6 (relatively resistant) mice controlled initial trypanosome growth as well as controls; however, the duration of infection, preceding eventual cure, was approximately doubled. Combined treatment with trypan blue and 400 rads of radiation resulted in much higher initial levels of infection in C57BL/6 mice, and about half of the mice died; the remaining mice eventually recovered after a prolonged course of infection. These results indicate that a nonimmunological mechanism, which controls initial infection, and an immunological mechanism cooperate to limit T. musculi infections in normal mice. We present results that suggest that both mechanisms are less effective in C3H than in C57BL/6 mice. The initial control of infection presumably reflects the activity of some type(s) of phagocytic effector cell; we show, however, that the initial control of infection is not an attribute of the liver Kupffer cells. Identification and characterization of the cells capable of controlling initial infection could lead to procedures for enhancing their function and, thus, to enhanced resistance to, and elimination of, trypanosome infections.
Heterogeneity of the L-rhamnose residue in the outer core of Pseudomonas aeruginosa lipopolysaccharide, characterized by using human monoclonal antibodies
Authors: Yokota S , Ochi H , Ohtsuka H , Kato M , Noguchi H
Published: January 2020

Hybridoma cell lines producing human monoclonal antibodies (MAbs) MH-4H7 and KN-2B11 [immunoglobulin M (lambda)] which bound to the outer core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) were established by cell fusion of human peripheral lymphocytes with human-mouse heteromyeloma SHM D-33. Both binding specificity experiments with a series of LPS-defective mutants derived from P. aeruginosa PAC1R (P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem.132:329-337, 1983) and competitive enzyme immunoassay experiments with monosaccharides demonstrated that alpha-L-rhamnose residues in the outer core of LPS might be in part an epitope. The MAbs specifically bound to clinical isolates belonging to Homma serotypes A, F, G, and K at a frequency of 70 to 86% and to serotypes H and M isolates at about 50%. They did not bind to any isolates of serotype B, E, and I tested. This evidence indicates that L-rhamnose and probably its neighboring residues in the other core of P. aeruginosa are heterogeneous in some association with the O serotype.
Serotype 8- and serocluster-specific surface-expressed antigens of Ureaplasma urealyticum
Authors: Thirkell D , Myles A D , Russell W C
Published: January 2020

The polypeptides of all 14 serotypes of Ureaplasma urealyticum were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. The electrophoretic patterns did not allow ready discrimination of individual serotypes or seroclusters. The analysis of the antigens of serotype 8 was reported previously (B. L. Precious, D. Thirkell, and W. C. Russell, J. Gen. Microbiol. 133:2659-2676, 1987). In this study, three of the surface-expressed membrane antigens of 16, 17, and 96 kilodaltons were further investigated, and monoclonal antibodies were raised against these three polypeptides. The major 96-kilodalton polypeptide was serotype 8 specific, and the 16-kilodalton polypeptide was present only in the larger serocluster. We describe monoclonal antibody probes that unequivocally differentiate serotype 8 from the other serotypes and that separate the two seroclusters of the organism.
Enhancement of immunoglobulin G responses in mice against hepatitis B virus surface antigen, influenza virus hemagglutinin vaccine, and tetanus toxoid by 6-O-acylated muramyl dipeptides
Authors: Furuya T , Kumazawa Y , Takimoto H , Nagumo T , Watanabe M , Aizawa C , Kiso M , Hasegawa A , Nomoto K
Published: January 2020

The adjuvant activity of chemically synthesized 6-O-acylated muramyl dipeptides (MDP) was tested in aqueous form. The activity was assessed by determining immunoglobulin G (IgG) titers in sera of mice immunized with hepatitis B virus surface antigen, influenza virus hemagglutinin (HA) vaccine, or tetanus toxoid with an enzyme-linked immunosorbent assay. Administration of 6-O-acyl-MDP analogs with antigens induced marked enhancement of primary and secondary IgG antibody responses and maintained high antibody levels for at least 7 weeks. Among the analogs tested, an MDP methyl ester carrying a 6-O-3-hexadecanoyl-oxytetradecanoyl group (compound 309) exhibited the most intensive adjuvant activity. Its activity was stronger than that of 6-O-2-tetradecylhexadecanoyl (B3O)-MDP used as a positive control. However, accumulation of peritoneal cells and activation of peritoneal macrophages by compound 309 was weaker than that by 6-O-B30-MDP, suggesting that 309 as an immunoadjuvant is more suitable for vaccination in terms of its stronger enhancement of antibody formation and lower induction of inflammatory response than 6-O-B30-MDP.
Participation of yeast cell surface hydrophobicity in adherence of Candida albicans to human epithelial cells
Authors: Hazen K C
Published: January 2020

Recent studies have revealed that hydrophobic cells of the opportunistic pathogenic fungus Candida albicans are more virulent than hydrophilic cells. One critical step in the pathogenic process is adherence to host tissues. Adherence of C. albicans to epithelial tissues is mediated primarily by specific adhesin-receptor interactions, but whether cell surface hydrophobicity (CSH) of the yeast cells may also contribute has not been definitively demonstrated. Nineteen isolates of C. albicans were grown in Sabouraud dextrose broth at either 23 or 37 degrees C and tested for CSH by a polystyrene microsphere assay and for the ability to adhere to HeLa cells, a human cervical epithelioid carcinoma cell line. For 13 isolates, growth at 23 degrees C resulted in significantly higher levels of CSH than did growth at 37 degrees C. Three isolates were hydrophobic and two were hydrophilic regardless of growth temperature. One isolate was more hydrophobic after growth at 37 degrees C. Of the isolates that were more hydrophobic after growth at 23 degrees C, 86.5% (11 of 13) were also more adherent to HeLa cells. Growth temperature did not appear to determine adherence ability, as all isolates that did not differ in CSH after growth at either temperature also did not differ in ability to adhere. No correlation (r = 0.44) was obtained between CSH and adherence when the isolates grown at 23 degrees C were evaluated as a group. Higher correlation (r = 0.65) was obtained when the isolates were grown at 37 degrees C. Interestingly, a significantly positive correlation between CSH and adherence was obtained when individual isolates were analyzed. To accomplish this analysis, the isolates were allowed to vary in CSH over time in tissue culture medium without serum, and the corresponding adherence values determined. Only isolates that varied in CSH by greater than 10% were used. Correlation statistical analysis in which the coefficient of determination (r2) was calculated indicated that poor correlation between CSH and adherence for the isolates evaluated as a group was likely due to the fact that CSH had little effect on adherence once a moderately high level of CSH was attained. These results indicate that CSH is involved in adherence but is not the predominant mechanism and that the effect of CSH on adherence is isolate dependent.
Macrophage activation by an ornithine-containing lipid or a serine-containing lipid
Authors: Kawai Y , Akagawa K
Published: January 2020

alpha-N-(3-Acyloxyacyl)-ornithine (or -serine) is the structure of lipoamino acids obtained by us previously from some gram-negative bacteria (Y. Kawai and I. Yano, Eur. J. Biochem. 136:531-538, 1983; Y. Kawai, I. Yano, and K. Kaneda, Eur. J. Biochem. 171:73-80, 1988; Y. Kawai, I. Yano, K. Kaneda, and E. Yabuuchi, Eur. J. Biochem. 175:633-641, 1988). The 3-acyloxyacylamide structure is present in both the lipoamino acids and lipid A of lipopolysaccharide (endotoxin). The efficacy of lipoamino acids (an ornithine-containing lipid and a serine-containing lipid) in activating C3H/HeSlc mouse peritoneal exudate macrophages was compared with that of bacterial lipopolysaccharide, because the two types of substances were expected to exhibit similar biological activities and physiological functions on the basis of their structural similarities. Actually, the lipoamino acids, as well as lipopolysaccharide, strongly activated the macrophages to generate the immunoregulatory substances prostaglandin E2 and interleukin-1, but their effect on the induction of L929 cell cytolytic factor (a possible tumor necrosis factor), another immunoregulatory substance, was weaker than that of lipopolysaccharide. The effect of lipoamino acids on the cytotoxicity of macrophages for EL-4 leukemia cells was very weak. However, all of these activities, as far as tested, were strongly enhanced by synergistic action with gamma interferon. Only the serine-containing lipid killed both C3H/HeSlc and C3H/HeJ macrophages to almost the same degree as endotoxin killed C3H/HeSlc macrophages. On the other hand, lethal toxicity for mice was not found with either the ornithine-containing lipid or the serine-containing lipid, even when 7 mg of compound was injected into a mouse. These studies suggest that the lipoamino acids are nontoxic characteristic immunoactivators.
Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1
Authors: Bhatnagar R , Singh Y , Leppla S H , Friedlander A M
Published: January 2020

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.
Conservation of the biologically active portions of staphylococcal enterotoxins C1 and C2
Authors: Bohach G A , Schlievert P M
Published: January 2020

We determined the primary sequence of staphylococcal enterotoxin (SE) C2 by sequencing its cloned structural gene, entC2. The entC2 structural gene contains an 801-base-pair open reading frame which encodes a 266-amino-acid precursor with a molecular weight of 30,608. Mature SE C2, produced by removal of the signal peptide, contains 239 amino acids with a molecular weight of 27,531. A sequence comparison between SE C2 and SE C1 showed that the 167 carboxyl amino acids in both toxins were 100% conserved. In contrast, the 72 N-terminal residues were 10% divergent. This provides additional evidence that carboxyl regions of staphylococcal and streptococcal pyrogenic toxins determine shared biological activities and cross-reactive epitopes.
Altered virulence and vaccination properties of Leishmania parasites grown in infected vaccinated mice
Authors: Gorczynski R M
Published: January 2020

BALB/c mice, which are normally highly susceptible to growth of Leishmania mexicana parasites in vivo, can be vaccinated with avirulent temperature-sensitive mutants of L. mexicana so that challenge with virulent organisms results in markedly diminished growth of the latter. Parasites extracted from the lesions which do appear in these mice are able to produce active infection in secondary hosts, although the rate of progression of these lesions is slower than that seen with the original virulent cloned organism. Interestingly, when irradiated parasites from the secondary hosts are themselves used to vaccinate naive BALB/c mice, less protection is seen than when irradiated virulent organisms from the initial infecting clone are used. These data suggest that when infection does take place in mice vaccinated with avirulent clones of parasite, the organisms which develop in lesions in these animals are substantially modified from those present in the initial infecting inoculum.
Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protection against coccidiosis
Authors: Kim K S , Jenkins M C , Lillehoj H S
Published: January 2020

Inoculation of chickens with live Escherichia coli N6405 transformants containing a plasmid which encodes ampicillin resistance and an immunodominant p250 surface antigen of Eimeria acervulina merozoites induced partial protection against challenge with live coccidia. The inoculation with E. coli transformants induced antigen-specific immunoglobulin and cell-mediated immune responses. Challenge with infective oocysts of Eimeria acervulina enhanced both immune parameters, indicating that administration of live E. coli transformants served to prime the immune system for recognition of specific epitopes on the 250-kilodalton protein. Although the mechanism of antigen presentation is unclear, the data suggest that in vivo expression of recombinant merozoite antigen is operative. After administration, no E. coli N6405 transformants could be recovered from intestinal or fecal materials of inoculated chickens, as assessed by enumeration on selective medium. However, ampicillin-resistant E. coli originating from the normal flora and harboring the gene sequences for both antibiotic resistance and Eimeria acervulina merozoite surface protein could be recovered from these chickens. Furthermore, normal-flora E. coli transformants were capable of generating functional beta-lactamase product, as evidenced by their resistance to ampicillin, and immunoreactive E. acervulina merozoite recombinant antigen, as revealed by immunofluorescence staining with p250-specific antiserum.
Hexagonal surface layer of Campylobacter fetus isolated from humans
Authors: Fujimoto S , Umeda A , Takade A , Murata K , Amako K
Published: January 2020

Electron microscopic studies of the surface structure of Campylobacter fetus by the freeze-etching method showed two different types of S layer. One was in a hexagonal array, and the other was in a tetragonal array. A high-passage-number strain lost its S layer during cultivation on culture media but regained it after a single animal passage.
Cloning of a hemolysin gene from Leptospira interrogans serovar hardjo
Authors: del Real G , Segers R P , van der Zeijst B A , Gaastra W
Published: January 2020

A DNA fragment encoding both hemolysin and sphingomyelinase C activity was cloned from the pathogenic bacterium Leptospira interrogans serovar hardjo. Initial clones were obtained by screening a genomic library in EMBL3 for hemolytic activity. Both hemolytic and sphingomyelinase C activities were coded for by a 3.9-kilobase BamHI fragment. The hemolysin was expressed from its own promoter in Escherichia coli K-12. Similar DNA sequences were also present in the serovars tarassovi and ballum.
Fine structure of A and M antigens from Brucella biovars
Authors: Meikle P J , Perry M B , Cherwonogrodzky J W , Bundle D R
Published: January 2020

Brucella A and M epitopes were found on single O-polysaccharide chains of all biotype strains of this species. Lipopolysaccharides from the type and reference strains of five of the six Brucella species, B. abortus, B. melitensis, B. suis, B. canis, and B. neotomae, were extracted and purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in conjunction with silver staining and immunoblotting developed by monoclonal antibodies, showed bands characteristic of A, M, or mixed A and M antigens. The A antigen previously described as an exclusively alpha 1,2-linked homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranose was shown by 1H and 13C nuclear magnetic resonance spectroscopy to possess a fine structure consistent with the low-frequency occurrence of alpha 1, 3-linked 4,6-dideoxy-4-formamido-D-mannopyranose residues. This feature was previously attributed only to the M antigen, which is also a homopolymer of the same sugar. B. melitensis biotype 3 and B. suis biotype 4 lipopolysaccharides showed characteristics of mixed A and M antigens. Immunoabsorption of these O polysaccharides on a column of immobilized A-antigen-specific monoclonal antibody enriched polymer chains with A-antigen characteristics but did not eliminate M epitopes. Composite A- and M-antigen characteristics resulted from O polysaccharides in which the frequency of alpha 1,3 linkages, and hence, M-antigen characteristics, varied. All biotypes assigned as A+ M- expressed one or two alpha 1,3-linked residues per polysaccharide O chain. M antigens (M+ A-) also possessed a unique M epitope as well as a tetrasaccharide determinant common to A-antigen structures. B. canis and B. abortus 45/20, both rough strains, expressed low-molecular-weight A antigen.
Identification of genus-specific epitopes on the outer membrane complexes of Chlamydia trachomatis and Chlamydia psittaci immunotypes 1 and 2
Authors: Mondesire R R , Maclean I W , Shewen P E , Winston S E
Published: January 2020

Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.
Structural and antigenic analysis of Chlamydia pneumoniae
Authors: Campbell L A , Kuo C C , Grayston J T
Published: January 2020

Several isolates of Chlamydia pneumoniae were compared with each other and to Chlamydia trachomatis and Chlamydia psittaci by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein profiles of the C. pneumoniae isolates appeared to be the same and were distinct from the other Chlamydia species. A 39.5-kilodalton (kDa) protein, similar in molecular weight to the major outer membrane proteins (MOMP) of C. trachomatis and C. psittaci, was found in the Sarkosyl-insoluble fraction, demonstrating its association with the outer membrane complex. In the outer membrane complex, the MOMP was shown to exist in disulfide-linked protein complexes. Electron microscopy of the Sarkosyl-extracted elementary bodies showed that the structural rigidity and pear-shaped morphology remained intact. Rabbit immune sera prepared against C. pneumoniae demonstrated immunoreactive proteins of 98-, 77-, 75-, 66-, 60-, 39.5-, 28-, and 17.5-kDa proteins. Cross-reactivity experiments revealed that most of the antigenic reactivities shared between C. psittaci and C. trachomatis extend to C. pneumoniae and that the 98-kDa protein recognition appeared to be C. pneumoniae specific. In contrast to the other Chlamydia spp., the recognition of the C. pneumoniae MOMP by homologous immune sera was weak and was cross-reactive with the MOMPs of the other Chlamydia species. These results suggest that the C. pneumoniae MOMP is less immunogenic and antigenically complex than are the MOMPs of C. trachomatis and C. psittaci.
Functional and phenotypic changes in human lymphocytes after coincubation with Leishmania donovani in vitro
Authors: Hviid L , Sørensen A L , Kharazmi A , Theander T G
Published: January 2020

In this paper we describe functional and phenotypic changes in T cells after in vitro coincubation of peripheral blood mononuclear cells (PBMC) and Leishmania donovani parasites at different parasite/peripheral blood mononuclear cell ratios. The phytohemagglutinin (PHA)-induced lymphoproliferative response was reduced by the coincubation, and at the maximal parasite/peripheral blood mononuclear cell ratio used (7.5:1), the average response was less than 40% of the response in the absence of parasites. The cause of the reduction in lymphoproliferation is not clear, but it requires live parasites. Interleukin-1 production was unaffected, the levels of soluble interleukin-2 receptor in supernatants were not changed by the coincubation, and the addition of exogenous interleukin-2 failed to revert the suppressive effect of the parasites. In addition to the reduction in lymphocyte proliferation, phenotypic lymphocyte changes were observed. Cell surface expression of the CD3 antigen, which is part of the CD3-T-cell receptor complex, was significantly reduced with increasing parasite/peripheral blood mononuclear cell ratios; the reduction was general in the sense that the parasites caused a shift in the fluorescent intensities of anti-CD3 labeled cells toward lower values, without affecting the distribution pattern. In contrast, the parasites altered the CD25 (interleukin-2 receptor) expression on PHA-stimulated cells from a homogenous CD25-positive population to two populations, one small and without CD25 expression and the other, larger population with only a slight reduction in size and CD25 expression. In addition to the changes in expression of surface antigens, a general reduction in the size of PHA-stimulated lymphocytes after coincubation with the parasites was observed. The data presented thus suggest that the inhibition of the proliferative response to PHA by live L. donovani in vitro is associated with early processes in lymphocyte activation. Further studies on the inhibitory phenomena described may be of potential significance in the investigation of the suppressive mechanisms in human visceral leishmaniasis.
Immunization with Theileria parva parasites from buffaloes results in generation of cytotoxic T cells which recognize antigens common among cells infected with stocks of T. parva parva, T. parva bovis, and T. parva lawrencei
Authors: Kariuki T M , Grootenhuis J G , Dolan T T , Bishop R P , Baldwin C L
Published: January 2020

Immunity to infection by the protozoan parasite Theileria parva in cattle is partially attributable to cytotoxic T cells, which kill lymphocytes infected with the schizont stage of the parasite. Here we evaluated five stocks of buffalo-derived T. parva lawrencei parasites and two stocks of cattle-derived T. parva parva parasites for their ability to induce in vivo cytotoxic T cells which can kill lymphocytes infected with a wide variety of strains of T. parva parasites. A group of seven full-sibling cattle, produced by embryo transfer and matched for at least one major histocompatibility complex class I haplotype, were immunized by infection and treatment with the parasite stocks. Target cells used in in vitro cytotoxicity assays were infected with five buffalo-derived parasite stocks and five cattle-derived parasite stocks, including T. parva parva and T. parva bovis. Immunization with any of the seven parasite stocks resulted in the generation of cytotoxic T cells which recognized parasite antigens on most if not all of the target cell lines tested, although the T. parva bovis stock was the least effective at doing so. Further in-depth analyses performed with peripheral blood mononuclear cells from one of the cattle immunized with T. parva lawrencei parasites showed that the pattern of killing of the panel of target cells was altered when either cells infected with different parasite stocks or clones of infected cells were used as stimulator cells in vitro, suggesting the presence of more than one population of parasite-specific cytotoxic effector cells in the peripheral blood mononuclear cells. However, clones of these cytotoxic effector cells recognized common or cross-reactive antigen epitopes expressed by the entire panel of infected target cells. These T-cell clones will be useful for identifying common T-cell antigen epitopes of T. parva and the parasite genes encoding them.
Identification and partial characterization of a cytolytic toxin produced by Gardnerella vaginalis
Authors: Rottini G , Dobrina A , Forgiarini O , Nardon E , Amirante G A , Patriarca P
Published: January 2020

Generation and release into the culture medium of a cytolytic toxin by Gardnerella vaginalis has been demonstrated. Addition of starch and of the nonionic detergent Tween 80 to the culture medium was essential to recover cytolytic activity. A protein with an apparent molecular mass of 61 to 63 kDa was purified from the culture supernatants showing lytic activity towards erythrocytes and nucleated cells, such as human endothelial cells and human neutrophils. The protein had marked selectivity for human erythrocytes, while erythrocytes from other species were not lysed or were lysed at much higher concentrations of the protein than those needed for human erythrocytes. The cytolytic activity was remarkably unstable in polar media, but was stabilized by nonionic detergents, by binding, or by insertion into the target cell membrane, suggesting its amphiphilic nature.
Combined inheritance of epithelial and erythrocyte receptors for Haemophilus influenzae
Authors: van Alphen L , Levene C , Geelen-van den Broek L , Poole J , Bennett M , Dankert J
Published: January 2020

Haemophilus influenzae type b expressing fimbriae showed no adherence to buccal epithelial cells and no agglutination of erythrocytes from three AnWj-negative siblings in one family. Hemagglutination of erythrocytes from 13 AnWj-positive members of the same family and from 24 controls was normal, and H. influenzae adhered well to buccal epithelial cells from them. These data indicate that the expression of epithelial and erythrocyte receptors for H. influenzae is inherited concomitantly. Combined with previous data (L. van Alphen, J. Poole, L. Geelen, and H. Zanen, Infect. Immun. 55:2355-2358, 1987), the results show that the receptor molecules on the surfaces of the epithelial cell and the erythrocyte are different but that the binding sites for the fimbriae of H. influenzae are similar.
Growth of Chlamydia trachomatis in enucleated cells
Authors: Perara E , Yen T S , Ganem D
Published: January 2020

Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells. Little is known about the role of the host in supporting chlamydial replication beyond the facts that host cells provide ATP and that de novo host protein synthesis is not required for bacterial growth. To further explore potential contributions of host nuclear function to chlamydial development, we questioned whether murine C. trachomatis could grow in mouse L cells that had been enucleated with cytochalasin B. Following enucleation, cells were infected with chlamydiae and analyzed morphologically and biochemically. Late in infection, substantial numbers of chlamydiae of all developmental stages were seen within large cytoplasmic inclusions that were indistinguishable from those seen in infected intact cells. Normal numbers of infectious progeny particles were produced from enucleated cultures. We conclude that active host cell nuclear function is not required to support the growth of chlamydiae.
Hemin levels in culture medium of Porphyromonas (Bacteroides) gingivalis regulate both hemin binding and trypsinlike protease production
Authors: Carman R J , Ramakrishnan M D , Harper F H
Published: January 2020

Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.
Molecular cloning and nucleotide sequence analysis of cholera toxin genes of the CtxA- Vibrio cholerae strain Texas Star-SR
Authors: Brickman T J , Boesman-Finkelstein M , Finkelstein R A , McIntosh M A
Published: January 2020

The ctx operons from the Vibrio cholerae El Tor strain 3083 and its CtxA- derivative Texas Star-SR were cloned, and their nucleotide sequences were compared. A single missense mutation in the Texas Star-SR ctxA cistron which results in the substitution of threonine for alanine at position 191 of the 258-amino-acid CtxA precursor was identified. Immunoblot analysis detected the mutant CtxA intracellularly early in the culture cycle but not extracellularly at any growth stage.