bluuebunny/crossref_metadata_2025 huggingface dataset:
Endotoxin-induced serum factor that stimulates gamma interferon production
Authors: Nakamura K , Okamura H , Wada M , Nagata K , Tamura T
Published: January 2020

Serum from Mycobacterium bovis BCG-infected mice that had been challenged with lipopolysaccharide (LPS) exhibited a marked ability to induce gamma interferon (IFN-gamma) in cultures of spleen cells of normal mice in the presence of interleukin-2 (IL-2). The inducing activity became detectable in the circulatory system about 90 min after LPS challenge, disappeared at around 5 h, and was observable upon 640x dilution of the serum. Addition of monoclonal anti-IL-2 receptor antibody to the culture inhibited the induction by the serum. The activity induced high levels of IFN-gamma even in nylon wool-nonadherent cells, while concanavalin A failed to do so. Serum from uninfected mice challenged with LPS contained no such activity. The molecular weight of the active substance, estimated by gel filtration, was about 70,000. There were pronounced differences among mouse strains in the activities of the sera prepared, which paralleled the amounts of IFN-gamma produced in vivo. However, the levels of IFN-gamma produced in whole spleen cells and in nylon wool-nonadherent cells from mice of various strains were the same when stimulated with competent serum. These results indicate the existence of an unidentified factor that induces IFN-gamma in cooperation with IL-2 in macrophage-depleted splenocytes. They also suggest that IFN-gamma production in vivo is not genetically controlled at the lymphocyte level but, rather, at the level of synthesis of the unknown factor.
Isolation and characterization of a mutant of Haemophilus influenzae type b deficient in outer membrane protein P1
Authors: Munson R , Hunt A
Published: January 2020

The gene for outer membrane protein P1 of Haemophilus influenzae type b has been previously cloned and expressed in Escherichia coli. To investigate the physiologic role of the P1 protein, the cloned P1 gene was insertionally inactivated with the Tn5 derivative Tn5tac1, and an isogenic P1-deficient Haemophilus mutant was then generated by transformation with linearized plasmid DNA containing the insertionally inactivated gene. The P1-deficient strain grew normally in vitro and induced bacteremia in the infant rat model.
Expression of type 1 fimbriae and mannose-sensitive hemagglutinin by recombinant plasmids
Authors: Gerlach G F , Clegg S , Ness N J , Swenson D L , Allen B L , Nichols W A
Published: January 2020

Deletions within the cloned genes (fimA) encoding the type 1 major fimbrial subunits of two isolates of Klebsiella pneumoniae resulted in a nonfimbriate but hemagglutinating phenotype after transformation of Escherichia coli HB101 or ORN103. Phenotypic expression of type 1 fimbriae could be restored by transformation with plasmids containing the fimA genes of the fimbrial gene clusters from different strains. The surface fimbriae expressed were serologically identical to those of the polymerized product of the introduced fimA gene. The fimA gene products of Salmonella typhimurium and Serratia marcescens could utilize the accessory fimbrial genes of K. pneumoniae to produce surface-associated, hemagglutinating fimbriae. The relatedness of the type 1 fimbrial gene clusters from multiple isolates of members of the family Enterobacteriaceae was examined by DNA hybridization techniques. These analyses demonstrated little nucleotide sequence agreement among distinct genera of the enteric bacteria.
Purification and characterization of an extracellular protease from Pseudomonas cepacia
Authors: McKevitt A I , Bajaksouzian S , Klinger J D , Woods D E
Published: January 2020

An extracellular proteinase (PSCP) produced by Pseudomonas cepacia was purified from culture supernatants by ammonium sulfate precipitation, anion exchange chromatography on DEAE-Sephacel, and G200 gel filtration chromatography. The protease has an apparent Mr of 34,000 by electrophoresis. Substrates cleaved by the protease include gelatin, hide powder, and collagen but not human immunoglobulin G (IgG), IgM, secretory IgA, or IgA. The enzyme had the characteristics of a metalloprotease, a pH optimum of 6, and a temperature optimum of 45 degrees C. Intratracheal instillation of purified PSCP into rat lungs produced a bronchopneumonia characterized by polymorphonuclear cell infiltration and proteinaceous exudation into large airways. Rats responded immunologically to active immunization with PSCP, but this response was not protective against subsequent lung infection with P. cepacia. PSCP was shown to have antigenic similarity with Pseudomonas aeruginosa elastase by an immunoblotting technique. Sera from 10 cystic fibrosis patients, with and without a previous history of P. cepacia colonization, were shown to possess antibody reactive against PSCP.
Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes
Authors: Quakyi I A , Matsumoto Y , Carter R , Udomsangpetch R , Sjolander A , Berzins K , Perlmann P , Aikawa M , Miller L H
Published: January 2020

Erythrocytes containing mature gametocytes of Plasmodium falciparum circulate in the blood until they are ingested by a mosquito, an event that triggers gametogenesis and lysis of the infected erythrocyte. It was previously shown that a parasite protein (Pf155/RESA) accumulates in the erythrocyte cytoplasm next to the parasitophorous vacuolar membrane (S. Uni, A. Masuda, M. J. Stewart, R. Nussenzweig, and M. Aikawa, Am. J. Trop. Med. Hyg., 36:481-488, 1987). Using a monoclonal antibody to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we have studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after triggering gametogenesis, the parasitophorous membrane no longer surrounded the parasite, bringing the parasite membrane in contact with the erythrocyte cytoplasm. Clear spaces appeared throughout the hemoglobin-rich host cytoplasm; Pf155/RESA was now localized in the cytoplasm directly surrounding the spaces. No membrane existed between the spaces and the erythrocyte cytoplasm. The spaces with surrounding Pf155/RESA protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane (15 min after triggering gametogenesis), the protein was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane. The mechanism by which Pf155/RESA remained aggregated around the spaces and its role in erythrocyte lysis are unknown. It is of interest that the parasite appeared to use the same molecule during invasion of erythrocytes and during release of gametes from infected erythrocytes.
A 25-kilodalton fraction from Mycobacterium tuberculosis that inhibits hexose monophosphate shunt activity, lysozyme release, and H2O2 production: reversal by gamma interferon
Authors: Wadee A A , Clara A M
Published: January 2020

This study examined the effects of a 25-kilodalton (kDa) glycolipoprotein derived from Mycobacterium tuberculosis on phagocyte functions associated with antimicrobial activity. The 25-kDa fraction inhibited the ability of both polymorphonuclear cells and cultured monocytes to release lysozyme and produce hydrogen peroxide. In addition, the glycolipoprotein was capable of reducing hexose monophosphate shunt activity and interfered with the ability of polymorphonuclear cells to reduce Nitro Blue Tetrazolium. Inhibition of these antimicrobial systems was optimal at a 50-micrograms/ml concentration of the 25-kDa fraction. Gamma interferon, but not alpha interferon, partially reversed the inhibitory effect of the mycobacterial component in all of the systems assessed. These studies indicate important mechanisms in the understanding of the pathogenesis of tuberculosis and suggest that gamma interferon may have a therapeutic role in mycobacterial diseases.
DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes
Authors: Garfinkel L I , Giladi M , Huber M , Gitler C , Mirelman D , Revel M , Rozenblatt S
Published: January 2020

A number of DNA probes which hybridize to highly abundant DNA sequences of Entamoeba histolytica were developed. Variations in the hybridization patterns of different E. histolytica strains were detected with selected probes. Four types of restriction fragment length patterns were obtained. Of these, the first class belonged to E. invadens and E. histolytica-like var. Laredo. The next two classes consisted of various strains of E. histolytica which were originally isolated from symptomatic patients and possessed pathogenic patterns of isoenzymes (zymodemes), whereas the fourth group contained E. histolytica strains with nonpathogenic zymodemes obtained from asymptomatic carriers. DNA probes, based on DNA sequences specific to E. histolytica isolates with pathogenic and nonpathogenic zymodemes were isolated, and their nucleotide sequences were determined. These probes (P145 and B133) hybridized selectively to DNA of isolates possessing either pathogenic or nonpathogenic isoenzyme patterns. The newly developed probes could be useful for diagnostic purposes and could serve as tools to investigate the molecular basis of pathogenicity and the genetic mechanisms which regulate the variable aggressive behavior of the parasite.
Shedding of the immunodominant P20 surface antigen of Eimeria bovis sporozoites
Authors: Speer C A , Whitmire W M
Published: January 2020

P20 is an immunodominant surface antigen of Eimeria bovis sporozoites. As parasites underwent merogony within cultured bovine monocytes and Madin-Darby bovine kidney (MDBK) cells, P20 appeared to be shed gradually by meronts and was absent in type 1 and 2 first-generation merozoites. Meronts of E. bovis appeared to shed P20 into the parasitophorous vacuole of bovine monocytes, whereas MDBK cells evidently released P20 into the culture medium or destroyed its antigenic determinant.
Endotoxic-lipopolysaccharide-specific binding proteins on lymphoid cells of various animal species: association with endotoxin susceptibility
Authors: Roeder D J , Lei M G , Morrison D C
Published: January 2020

Endotoxic lipopolysaccharide (LPS), a common structural component of all gram-negative bacteria, is well recognized for its capacity to interact with and perturb immunologically relevant cells. Using a radioiodinated, photoactivatable LPS probe, we have recently identified an 80-kilodalton LPS-specific binding protein on murine B lymphocytes. We now have extended these studies to determine if other mammalian species, as well as representative endotoxin-resistant species (frog and chicken), have a similar LPS-binding protein. We have identified what appears to be a relatively conserved 80-kilodalton LPS-binding protein on mononuclear cells of all mammalian species tested. However, both frog and chicken leukocytes failed to show the presence of a similar LPS-binding protein. It is possible that the presence of specific LPS-binding proteins may be important for endotoxin sensitivity of most mammalian species.
Isolation and characterization of type IV group B Streptococcus capsular polysaccharide
Authors: Wessels M R , Benedí W J , Jennings H J , Michon F , DiFabio J L , Kasper D L
Published: January 2020

An antigenically distinct serotype, type IV, has recently been added to the recognized serotypes of group B streptococci (GBS). We isolated and purified the capsular polysaccharide antigen from a prototype type IV GBS strain. The type IV capsular polysaccharide formed a precipitin line with rabbit antiserum to type IV GBS organisms but not with antiserum to organisms of GBS serotype Ia, Ib, II, or III. Enzyme-linked immunosorbent assay inhibition experiments showed no cross-reaction between type IV antiserum and other GBS serotypes. Capsular polysaccharide released from the bacterial cells with mutanolysin and that isolated from the culture supernatant had similar elution profiles on Sepharose CL-6B, with a Kav of 0.30 and an estimated Mr of 200,000. The purified type IV polysaccharide was found to contain galactose, glucose, N-acetylglucosamine, and N-acetylneuraminic acid (sialic acid) as exclusive sugars. The polysaccharide contained 23% (by weight) sialic acid and galactose, glucose, and N-acetylglucosamine in a relative ratio of (1):1.10:0.55. These results are compatible with a repeating structure of six monosaccharide residues containing galactose, glucose, N-acetylglucosamine, and sialic acid in a molar ratio of 2:2:1:1. Unlike type Ia, II, and III GBS polysaccharides, desialylation of the type IV polysaccharide produced an antigen which formed a line of identity with the native type IV antigen in double diffusion in agar against homologous antiserum. This result suggests that sialic acid is not as critical to the immunodeterminant structure of the type IV antigen as it is for other GBS capsular types.
Inhibition of human neutrophil NADPH oxidase by Chlamydia serovars E, K, and L2
Authors: Tauber A I , Pavlotsky N , Lin J S , Rice P A
Published: January 2020

The effects of Chlamydia trachomatis (serovars E, K, and L2) on human neutrophil activation were examined with respect to the organisms both as primary agonists and as agents that modulate cell responses to a second stimulus. Unopsonized chlamydiae alone, at ratios of 1.5 to 100 organisms per cell, failed to elicit changes in intracellular calcium or membrane depolarization or to stimulate the respiratory burst or degranulation during 60 min of incubation. Each of these functions except the respiratory burst was also normally activated when chlamydia-infected neutrophils were subsequently stimulated with formylmethionyl leucine phenylalanine or phorbol myristate acetate; the respiratory burst was inhibited 30 to 65%. Inhibition was dependent on live organisms and was maximal within 5 min of incubation. The organisms had no effect on the superoxide (O2-) assay, and the site of chlamydial inhibition was determined at the level of the NADPH oxidase itself, not at an intermediate step in the activation cascade. The mechanism of enzyme inactivation could not be determined. These results show that unopsonized chlamydiae do not elicit responses from infected neutrophils and suggest that microbicidal mechanisms other than those dependent on elaboration of toxic oxygen-derived species are required to inactivate chlamydiae.
Protracted anemia associated with chronic, relapsing systemic inflammation induced by arthropathic peptidoglycan-polysaccharide polymers in rats
Authors: Sartor R B , Anderle S K , Rifai N , Goo D A , Cromartie W J , Schwab J H
Published: January 2020

Mild hypoproliferative anemia with abnormal iron metabolism frequently accompanies chronic inflammation and infection in humans. To determine whether anemia is associated with chronic relapsing arthritis induced by bacterial cell wall polymers, serial determinations of the hematocrit were measured in rats injected intraperitoneally with sonicated peptidoglycan-polysaccharide fragments from group A streptococci. Acute anemia peaked 5 to 10 days after injection, and chronic, spontaneously relapsing anemia persisted for 309 days. 51Cr labeling demonstrated decreased erythrocyte survival, i.e., a half-life of 8.4 days in cell wall-injected rats versus 11.8 days in controls. Erythrocytes were mildly microcytic, and leukocyte counts were elevated during early spontaneous reactivation of arthritis, 15 days after injection of peptidoglycan-polysaccharide. Bone marrow myeloid/erythroid precursor ratios were elevated in arthritic rats (P less than 0.0001). Purified peptidoglycan produced an acute anemia lasting 10 days, while injection of group A streptococcal polysaccharide and mutanolysin-digested cell wall did not affect the hematocrit. The minimal effective dose of peptidoglycan-polysaccharide was 5 micrograms of rhamnose per g (body weight). Serum iron and transferrin levels were decreased in cell wall-injected rats (P less than 0.005) and were closely correlated with hematocrit values and joint inflammatory scores. Stainable iron was increased in the liver, spleen, and mesenteric lymph nodes and unchanged in the bone marrow of cell wall-injected rats. Anemia accompanying chronic, relapsing systemic inflammation induced by peptidoglycan-polysaccharide polymers appears to be an excellent animal model of the anemia of chronic disease.
Correlation between macrophage activation and bactericidal function and Mycobacterium leprae antigen presentation in macrophages of leprosy patients and normal individuals
Authors: Desai S D , Birdi T J , Antia N H
Published: January 2020

The killing of Mycobacterium leprae by resting and gamma interferon (IFN-gamma)-activated macrophages in normal subjects and leprosy patients was assessed. Resting macrophages from normal individuals demonstrated the ability to kill M. leprae. For macrophages from tuberculoid patients, killing of M. leprae was only achieved in the presence of IFN-gamma, suggesting that initial T-cell activation occurs prior to the killing of M. leprae. In contrast, though activation with IFN-gamma rendered the lepromatous macrophages microbicidal, it failed to induce lymphocyte proliferation, suggesting a defect at either the antigen-presenting cell or the lymphocyte level or both. The concept that T-cell anergy is primarily due to lack of lymphokine generation was ruled out by our results, since responsiveness was restored in only a small proportion of lepromatous patients after exogenous lymphokine addition. In conclusion, this study demonstrated that killing and antigen presentation are two independent events. It appears that the ability of the macrophages per se to kill M. leprae may be of greater importance than lymphocyte-mediated activation for protection against M. leprae infection.
Inhibition by normal human serum of Mycobacterium avium multiplication in cultured human macrophages
Authors: Crowle A J , Poche P
Published: January 2020

Normal human serum used as a protein supplement in RPMI 1640 medium inhibited growth in blood-derived human macrophages (MP) of virulent Mycobacterium avium serovars 4 and 8, derived from patients with acquired immunodeficiency syndrome, but not virulent Mycobacterium tuberculosis. A defined serum substitute (SS) promoted the intramacrophage growth of M. avium but not of M. tuberculosis. The effects of serum or SS were measured by counting viable bacteria in lysates of the MP at 0, 4, and 7 days after their infection by the bacteria. Neither serum nor SS inhibited or enhanced M. avium growth in the absence of MP. The results suggest that a nutrient essential for intracellular replication of M. avium is made by MP from a pronutrient present in both SS and serum and that something in serum inhibits MP conversion of the pronutrient to nutrient. This inhibition may be an important mechanism of native resistance against M. avium in normal people.
Expression of the heat-modifiable major outer membrane protein of Haemophilus influenzae type b is unrelated to virulence
Authors: Hanson M S , Cope L D , Hansen E J
Published: January 2020

The heat-modifiable major outer membrane protein (P1) of Haemophilus influenzae type b (Hib) has been shown to be both exposed on the cell surface and capable of inducing the synthesis of antibodies protective against experimental Hib disease. Chemical mutagenesis of a recombinant plasmid containing the Hib gene encoding P1 resulted in inactivation of P1 expression by this plasmid. The mutated P1 gene was transformed into Hib to obtain an isogenic mutant lacking only the ability to synthesize this surface protein. In addition, the P1 gene was inserted into a plasmid shuttle vector and used to construct a recombinant Hib strain that overexpressed the P1 protein. Lack of P1 expression did not affect the ability of Hib to grow in vitro. Neither the absence nor the overproduction of P1 affected expression of capsular polysaccharide and lipooligosaccharide by Hib. The P1-negative mutant and the P1-overexpressing strain were both as susceptible to the bactericidal activity of pooled normal human serum as was the wild-type parent strain, while the P1-negative mutant was as resistant to the bactericidal activity of normal infant rat serum as was the wild-type parent strain. The P1-negative mutant was no less virulent than was the wild-type parent strain in an animal model system, such that both the numbers of animals infected by this mutant and the mean magnitudes of the resultant bacteremias were essentially identical to those obtained with challenge by the wild-type parent strain. Similarly, overexpression of P1 did not detectably affect the virulence of Hib. These data indicate that this protective protein antigen plays no detectable role in the expression of virulence by Hib, as assessed in an animal model system.
In vivo expression and variation of Escherichia coli type 1 and P pili in the urine of adults with acute urinary tract infections
Authors: Kisielius P V , Schwan W R , Amundsen S K , Duncan J L , Schaeffer A J
Published: January 2020

In vivo expression of pili by Escherichia coli in the urine of 41 adults with lower urinary tract infections was analyzed by immunostaining with polyclonal antiserum to type 1 and P pili. Type 1 pili were detected in 31 of 41 urine specimens, while P pili were detected in 6 of 18 specimens. The piliation status of bacterial populations in urine was heterogeneous, varying from predominantly piliated to a mixture of piliated and nonpiliated cells. Bacteria frequently adhered to exfoliated uroepithelial cells and leukocytes in urine. Expression of pili in vivo did not always correlate with the hemagglutination phenotype after growth in vitro. Strains isolated from different sites in the urogenital tract of two individuals showed phenotypic variation in the state of piliation. The results demonstrate that E. coli type 1 and P pili are expressed and are subject to variation in vivo during acute urinary tract infections in adults.
Plasmid-influenced changes in Mycobacterium avium catalase activity
Authors: Pethel M L , Falkinham J O
Published: January 2020

A virulent Mycobacterium avium strain, LR25, which carries three plasmids (18, 28, and 165 kilobases) and grows at 43 degrees C was compared with its plasmid-free, avirulent segregant, strain LR163, to identify the basis for the latter's inability to grow at 43 degrees C. The failure of mid-log-phase cultures of strain LR163 to grow at 43 degrees C was dependent on the presence of high levels of culture aeration. In addition, highly aerated cultures of strain LR163 failed to grow at 37 degrees C. Mid-log-phase cultures of strain LR163 had 30% of the catalase activity of strain LR25 and were more hydrogen peroxide (0.08%, wt/vol) susceptible. Catalase activity of strain LR25 was higher in cultures grown with high aeration than in those grown with almost no aeration. These data support the contention that plasmid-encoded genes influence M. avium catalase activity.
Lectinlike properties of pertussis toxin
Authors: Tyrrell G J , Peppler M S , Bonnah R A , Clark C G , Chong P , Armstrong G D
Published: January 2020

We have examined the lectinlike properties of pertussis toxin by binding-inhibition assays and affinity chromatography of goose erythrocyte membranes. Although pertussis toxin and wheat germ agglutinin apparently recognize similar sugar sequences on glycoproteins, the binding activities of the two lectins are not identical.
Comparison of the reactivities and immunogenicities of procholeragenoid and the B subunit of cholera toxin in Thai volunteers
Authors: Migasena S , Pitisuttitham P , Suntharasamai P , Prayurahong B , Supanaranond W , Desakorn V , Black R E
Published: January 2020

Procholeragenoid, a stable high-molecular-weight aggregate of cholera toxin derived by heat treatment, was evaluated for reactivity and immunogenicity in adult Thai volunteers. Since procholeragenoid is known to retain some residual activity of cholera toxin, increasing amounts were ingested until diarrhea occurred; 250 micrograms induced diarrhea, but 100 micrograms did not. Procholeragenoid and cholera toxin B subunit, both in 100-micrograms amounts, were then compared for systemic and intestinal antitoxin responses. When three peroral doses were given, these immunogens gave comparable responses. The secretory immunoglobulin A antitoxin responses to three doses of 100 micrograms of B subunit did not differ significantly from responses found in previous studies of Thai adults given 1 or 5 mg of B subunit, but serum antitoxin responses were less after 1 or 2 doses of 100 micrograms than after doses of 1 or 5 mg. Serum antitoxin levels were similar after 3 doses of B subunit. Procholeragenoid in the maximum safe dose of 100 micrograms does not offer any immunologic advantage over B subunit, although it may be less expensive and easier to produce. However, these studies suggest that higher amounts of B subunit are more immunogenic and may be preferable, if found to be sufficiently cost effective, when added to oral killed whole Vibrio cholerae vaccines.
Tumor necrosis factor alpha potentiates neutrophil antimicrobial activity: increased fungicidal activity against Torulopsis glabrata and Candida albicans and associated increases in oxygen radical production and lysosomal enzyme release
Authors: Ferrante A
Published: January 2020

The capacity of human tumor necrosis factor alpha (TNF-alpha) to modulate the killing of the opportunistic pathogens Torulopsis glabrata and Candida albicans by human neutrophils was studied. TNF-alpha significantly enhanced neutrophil fungicidal activity in a concentration-dependent manner and was evident in a range of neutrophil-fungus ratios. Enhanced killing of T. glabrata required much lower TNF-alpha concentrations than were required for enhancement of killing of C. albicans. Maximal enhancement of killing occurred with 20 and 100 U of TNF-alpha per 5 x 10(6) neutrophils for T. glabrata and C. albicans, respectively. The fungal killing kinetics demonstrated that TNF-alpha augmentation of fungicidal activity was evident within 1 h and persisted for an incubation period of at least 22 h. Preincubation of neutrophils with TNF-alpha was essential for the enhancement of killing. Maximal stimulation of killing was observed within 1 h of preincubation with TNF-alpha, and poor stimulation of killing was observed when TNF-alpha was added at time zero. Associated with the increase in fungicidal activity was an increased production of superoxide and an enhanced degranulation of enzymes and other proteins from azurophilic and specific granules in response to the fungi. The results demonstrate that TNF-alpha augments the neutrophil oxidative respiratory burst and the degranulation induced by opsonized fungi and that it increases the neutrophil fungicidal activity.
Identification and isolation of a major cell surface glycoprotein of Pneumocystis carinii
Authors: Radding J A , Armstrong M Y , Ullu E , Richards F F
Published: January 2020

Radioiodination of rat-derived Pneumocystis carinii obtained from an in vitro culture demonstrated the presence of a major surface glycoprotein (gp120). The glycoprotein was of the high mannose type. It exhibited adherence properties similar to those observed in the intact organism. Under nonreducing conditions, it existed as an aggregate with a molecular weight in excess of 2 x 10(6). Surface aggregating behavior and adherent quality prevented isolation of the glycoprotein by conventional methods. The glycoprotein was purified by chromatography on hydroxyapatite in the presence of sodium dodecyl sulfate under reducing conditions.
Cloning and sequencing of a Treponema pallidum gene encoding a 31.3-kilodalton endoflagellar subunit (FlaB2)
Authors: Pallesen L , Hindersson P
Published: January 2020

A library of Treponema pallidum DNA was established in the direct selection vector pUN121. Six clones carrying a gene coding for a 33-kilodalton T. pallidum flagellum subunit were identified by colony hybridization with an oligodeoxynucleotide probe based on the N-terminal amino acid sequence of this subunit. An open reading frame of 286 amino acids with the expected N-terminal sequence and absence of cysteine residues was identified. The deduced protein had a calculated molecular weight of 31.3 kilodaltons. We propose to name this flagellar subunit FlaB2. FlaB2 shows a significant amino acid homology with flagellins of several remotely related bacterial species. This homology was most pronounced corresponding to the C-terminal and N-terminal parts of the protein, whereas the central region was variable.
Purification of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies
Authors: Norris K A , Harth G , So M
Published: January 2020

Recent studies on the humoral immune response to Trypanosoma cruzi have shown that antibodies which are able to bind living parasites and lyse them in conjunction with complement are associated with host protection. Antibodies which support complement-mediated lysis (CML) of trypomastigotes are elicited as a result of an active infection and not after immunization with killed parasites. In spite of the requirement for immune antibodies, lysis proceeds mainly via the alternative complement pathway. We have purified a 160-kilodalton (kDa) glycoprotein from T. cruzi metacyclic trypomastigotes which appears to be a specific target for lytic antibodies. Rabbit antiserum to the purified 160-kDa protein was prepared, and we have determined that these antibodies will support CML of tissue-culture-derived trypomastigotes. The percentage of killing (65 to 70%) was consistent among three different T. cruzi strains tested. In order to examine the specificity of antibody-dependent CML, antibodies to T. cruzi neuraminidase, an unrelated trypomastigote membrane glycoprotein, were tested in the CML, assays and were not found lytic. Viable trypomastigotes bound anti-160-kDa antibodies uniformly as demonstrated by immunofluorescence, whereas antineuraminidase antibodies were extensively capped. The 160-kDa glycoprotein is specifically produced in infectious trypomastigotes (tissue culture derived and metacyclic) and was not detected in epimastigotes or amastigotes. The identification of the 160-kDa glycoprotein as a specific target for lytic antibodies, as well as its expression only in the infectious stage of the parasite, suggests an important role for this protein in eliciting host immunity.
Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis
Authors: Andersen A B , Hansen E B
Published: January 2020

Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule. None of the antibodies bound to the N-terminal 117-amino-acid peptide.
Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity
Authors: Saunders F K , Mitchell T J , Walker J A , Andrew P W , Boulnois G J
Published: January 2020

The role of the single cysteine residue in the activity of the thiol-activated toxin pneumolysin was investigated using oligonucleotide-mediated, site-directed mutagenesis. Three modified toxins in which the cysteine residue was changed to an alanine, a serine, or a glycine residue were purified to homogeneity and examined for activity. The Cys-428----Ala modified toxin was indistinguishable from the wild-type recombinant toxin in terms of hemolytic activity and lytic and inhibitory effects on human polymorphonuclear leukocytes (PMN), indicating that the cysteine residue is not essential for toxin activity. The Cys-428----Ser and Cys-429----Gly modified toxins had reduced activity on erythrocytes and polymorphonuclear leukocytes, being 6 and 20 times less active than the wild type, respectively. However, all the modified toxins formed oligomers in erythrocyte membranes to the same extent as the wild-type recombinant toxin. This suggests that the cysteine residue at position 428 is involved in neither the binding of toxin to membranes nor its insertion into the membrane, and also that the formation of oligomers is not by itself sufficient for toxin activity.
Dietary protein deficiency and Mycobacterium bovis BCG affect interleukin-2 activity in experimental pulmonary tuberculosis
Authors: McMurray D N , Mintzer C L , Bartow R A , Parr R L
Published: January 2020

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and response to exogenous recombinant IL-2 (rIL-2). Protein-malnourished guinea pigs responded only weakly to PPD skin tests, and their blood and lymph node lymphocytes exhibited impaired proliferation when cultured with PPD in vitro. IL-2 levels were consistently low in cultures of stimulated blood and spleen lymphocytes from protein-deprived animals. BCG vaccination of nutritionally normal guinea pigs, on the other hand, induced significantly more IL-2 production by PPD- and concanavalin A-stimulated lymphocytes. The addition of exogenous mouse rIL-2 (40 and 80 U/ml) in vitro to PPD-stimulated blood and lymph node cells from nonvaccinated, protein-deprived guinea pigs resulted in no improvement of the proliferative response. Previous vaccination of malnourished guinea pigs did not consistently enhance the response of PPD-stimulated lymphocytes to added rIL-2. Dietary protein deficiency and BCG vaccination appear to modulate antigen-driven cellular immunity in animals with tuberculosis by altering the production of, and the response to, IL-2 by PPD-stimulated lymphocytes.
Epitope specificities of murine monoclonal and rabbit polyclonal antibodies against enterobacterial lipopolysaccharides of the Re chemotype
Authors: Rozalski A , Brade L , Kosma P , Appelmelk B J , Krogmann C , Brade H
Published: January 2020

Murine monoclonal and rabbit polyclonal antibodies raised against the lipopolysaccharides (LPS) of Re mutants of Salmonella minnesota, Proteus mirabilis, and Escherichia coli were serologically characterized. Using natural Re LPS and natural and synthetic partial structures thereof, representing the 3-deoxy-D-manno-2-octulosonic acid (KDO) or lipid A region or both, the epitope specificities of four monoclonal antibodies were defined. Clones 20 (immunoglobulin M [IgM]) and 25 (IgG3) recognize a terminal alpha-pyranosidically linked KDO monosaccharide residue and the alpha-2,4-linked KDO disaccharide, respectively, as the immunodominant group. Therefore, these two antibodies are core antibodies which do not require the presence of lipid A constituents for binding. The minimal structure enabling binding of clone 17 (IgG2b) is a pseudotetrasaccharide of the sequence alpha-KDO-(2----4)-alpha-KDO-(2----6)-beta-glucosamine-(1----6)- glucosaminitol with two amide-linked 3-hydroxytetradecanoic acid residues. The smallest structure with which clone 22 (IgG3) reacted was de-O-acylated Re LPS. Therefore, clones 17 and 22 are LPS antibodies requiring both the lipid A and the KDO region for binding. Phosphoryl residues of the lipid A moiety in Re LPS are dispensable for the reaction with clone 17, whereas they are necessary for that with clone 22. These four different antibody types were also detected in polyclonal rabbit antisera and could be distinguished from each other by absorption experiments. It was found that type 20 and 25 antibodies either were not present or were present only in small amounts and that the majority of the antibodies were of types 17 and 22. From these data, we conclude that the immunodominant structures of Re LPS comprise both the KDO and lipid A domains.
Immunological characterization of a human homolog of the 65-kilodalton mycobacterial antigen
Authors: Dudani A K , Gupta R S
Published: January 2020

A human mitochondrial protein, designated P1 (63 kilodaltons [kDa], shows extensive sequence homology (47% identical residues and an additional approximately 20% conserved changes) to the 65-kDa mycobacterial antigen. To understand the relationship of these proteins, the cross-reactivity of several monoclonal antibodies directed against the 65-kDa Mycobacterium leprae antigen towards human, Chinese hamster, chicken, and bacterial cells has been examined. A number of antibodies (Y1-2, ML 30-A2, and F47-9-1) were found to cross-react with a 63-kDa antigen in vertebrate cell extracts and stained mitochondria in immunofluorescence studies. Some of these antibodies also reacted with a P1-beta-galactosidase fusion protein in recombinant Escherichia coli cells, expressing part of the human P1 protein. These results provide strong evidence that P1 is the mammalian homolog of the 65-kDa antigen. The human P1 protein also shows significant similarity (P less than 0.001) to a number of other bacterial and viral proteins including the pol polyprotein of human immunodeficiency viruses and the penicillin-binding protein of Neisseria gonorrhoeae. The observed similarity between human P1 protein and the major antigenic proteins of pathogenic organisms (e.g., 60- to 65-kDa mycobacterial antigen) suggests its possible involvement in autoimmune diseases (e.g., rheumatoid arthritis) by antigenic mimicry.
Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen
Authors: Derbyshire P , Baldwin T , Stevenson P , Griffiths E , Roberts M , Williams P , Hale T L , Formal S B
Published: January 2020

One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.
The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded
Authors: Schifferli D M , Beachey E H , Taylor R K
Published: January 2020

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.
Analysis of the proteinases of representative Trichomonas vaginalis isolates
Authors: Neale K A , Alderete J F
Published: January 2020

Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) were combined to evaluate the proteinases of both long-term-grown and fresh isolates of Trichomonas vaginalis. This two-dimensional substrate-SDS-PAGE resolved as many as 23 distinct proteinase activities in several isolates, and proteinases had relative molecular masses between 23 and 110 kilodaltons (kDa). Isoelectric points (pI) of proteinases ranged from 5.7 to 7.0. Overall, the various representative proteinase profiles were similar among those of long-term-grown and fresh isolates, although heterogeneity existed among several cysteine proteinase activities. Pattern changes were detected in fresh isolates passaged over several weeks, showing the ability of proteinases to be differentially expressed and to undergo phase variation. The two-dimensional proteinase patterns were very reproducible for isolates analyzed over a certain period of time before expression of some proteinases varied. The heterogeneity and differential expression of certain proteinases were not coordinated with phenotypic variation of already characterized immunogens and adhesins. Data suggesting that a 43-kDa proteinase resided on the parasite surface were obtained on the basis of removal of activity following pronase or proteinase K treatment of live organisms. Finally, immunized experimental animals produced antibody to many T. vaginalis proteinases, which indicates the immunogenic nature of trichomonad proteinases.
Receptors for human plasminogen on gram-negative bacteria
Authors: Ullberg M , Kronvall G , Karlsson I , Wiman B
Published: January 2020

A total of 188 strains representing 11 species of gram-negative bacteria were examined for the ability to interact with human plasminogen. Highly purified human plasminogen was labeled with 125I, and its uptake by different bacterial strains was measured. All 14 strains of Haemophilus influenzae and all 13 strains of Branhamella catarrhalis tested were positive with respect to plasminogen uptake. Also, eight species belonging to the family Enterobacteriaceae were tested, and of those, Proteus mirabilis demonstrated the most substantial uptake, with 28 of 39 strains taking up more than 10% of the plasminogen. Ten strains of Pseudomonas aeruginosa were also tested, of which seven showed uptake values higher than 10%. With H. influenzae and B. catarrhalis strains, Scatchard analysis indicated a two-phase receptor interaction, one more-avid receptor with a Kd of 6 to 8 nM and 2,000 to 2,500 sites per bacterium and a second receptor with a Kd of 50 to 80 nM and 9,000 sites per bacterium. With Pseudomonas aeruginosa strains, a single receptor interaction was detected with a Kd of 60 nM and the number of sites was estimated as 8,000 per bacterium. Scatchard analysis with strains of P. mirabilis indicated binding of a less-specific nature. However, plasminogen uptake by this species could be reduced by 50% by the addition of 2 mM unlabeled plasminogen. This estimate of Kd, as well as uptake studies with plasminogen fragments, suggests different properties of this receptor. With all receptor types, the addition of plasmin-aprotinin complex inhibited plasminogen uptake, which demonstrates that both forms of the molecule react with the same receptors. Plasminogen uptake could be eliminated by the addition of lysine or epsilon-aminocaproic acid, which suggests that the lysine-binding sites of the plasminogen molecule are involved in the receptor-ligand interaction.
Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes
Authors: Vismara D , Mezzopreti M F , Gilardini M S , Del Porto P , Lombardi G , Piccolella E , Damiani G , Rappuoli R , Colizzi V
Published: January 2020

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.
Comparative protection of mice against virulent and attenuated strains of Brucella abortus by passive transfer of immune T cells or serum
Authors: Araya L N , Winter A J
Published: January 2020

Passively transferred immune serum provided significantly greater protection to BALB/c mice against attenuated Brucella abortus 19 than against virulent strain 2308, whether serum donors had been infected with strain 19 or 2308. In contrast, immune T cells conferred better protection upon recipients challenged with the homologous strain of B. abortus. It is hypothesized that strain 2308, but not strain 19, can survive in macrophages after opsonization and that epitopes which induce protective cell-mediated immunity may differ between strains 19 and 2308.