bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Thiobacillus thioparus LV43 and its distribution in a chemoautotrophically based groundwater ecosystem
Authors: Vlasceanu L , Popa R , Kinkle B K
Published: January 2020

Bacterial strain LV43 was previously isolated from a floating microbial mat located in Movile Cave, the access point to a chemoautotrophically based groundwater ecosystem in southern Romania. This gram-negative, rod-shaped organism grows autotrophically through the oxidation of thiosulfate and sulfide, but it does not grow heterotrophically. Strain LV43 grows over a pH range of 5.0 to 9.0, with an optimum near 7.5 at 28 degrees C. The pH of the medium decreased from 7.5 to 6.5 during growth on thiosulfate. Carbon isotope fractionation values for strain LV43 were within the previously reported range of fractionation values for the overall floating microbial mat in Movile Cave and were similar to values reported for chemoautotrophic sulfur-oxidizing strains of Thiobacillus neapolitanus and Thiomicrospira sp. The 16S rRNA gene sequence of strain LV43 was determined, and phylogenetic analysis indicated that strain LV43 was most closely related to Thiobacillus thioparus and the uncultured bacterial strain Strip2, which is represented by a 16S rRNA clone obtained by direct PCR from the Stripa research mine in Sweden. This identification of strain LV43 is supported by its G+C content of 62%, which is within the range reported for strains of T. thioparus. Fluorescently labeled polyclonal antibodies specific for strain LV43 were used to locate and enumerate this strain at different locations in Movile Cave and in nearby surface-water and groundwater sources. Strain LV43 was found only at aerobic, neutral-pH sites within the cave. Strain LV43 was also found outside Movile Cave in surface waters and in groundwater believed to intercept the same sulfurous aquifer as Movile Cave.
Identification and detection of Bacillus sporothermodurans spores in 1, 10, and 100 milliliters of raw milk by PCR
Authors: Herman L M , Vaerewijck M J , Moermans R J , Waes G M
Published: January 2020

A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively.
Phosphorus and bacterial growth in drinking water
Authors: Miettinen I T , Vartiainen T , Martikainen P J
Published: January 2020

The availability of organic carbon is considered the key factor to regulate microbial regrowth in drinking water network. However, boreal regions (northern Europe, Russia, and North America) contain a large amount of organic carbon in forests and peatlands. Therefore, natural waters (lakes, rivers, and groundwater) in the northern hemisphere generally have a high content of organic carbon. We found that microbial growth in drinking water in Finland is highly regulated not only by organic carbon but also by the availability of phosphorus. Microbial growth increased up to a phosphate concentration of 10 micrograms of PO4-P liter-1. Inorganic elements other than phosphorus did not affect microbial growth in drinking water. This observation offers novel possibilities to restrict microbial growth in water distribution systems by developing technologies to remove phosphorus efficiently from drinking water.
Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation
Authors: Kosono S , Maeda M , Fuji F , Arai H , Kudo T
Published: January 2020

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.
Competition for Dimethyl Sulfide and Hydrogen Sulfide by Methylophaga sulfidovorans and Thiobacillus thioparus T5 in Continuous Cultures
Authors: De Zwart J , Sluis J , Kuenen J G
Published: January 2020

Pure and mixed cultures of Methylophaga sulfidovorans and Thiobacillus thioparus T5 were grown in continuous cultures on either dimethyl sulfide, dimethyl sulfide and H(inf2)S, or H(inf2)S and methanol. In pure cultures, M. sulfidovorans showed a lower affinity for sulfide than T. thioparus T5. Mixed cultures, grown on dimethyl sulfide, showed coexistence of both species. M. sulfidovorans fully converted dimethyl sulfide to thiosulfate, which was subsequently further oxidized to sulfate by T. thioparus T5. Mixed cultures supplied with sulfide and methanol showed that nearly all the sulfide was used by T. thioparus T5, as expected on the basis of the affinities for sulfide. The sulfide in mixed cultures supplied with dimethyl sulfide and H(inf2)S, however, was used by both bacteria. This result may be explained by the fact that the H(inf2)S-oxidizing capacity of M. sulfidovorans remains fully induced by intracellular H(inf2)S originating from dimethyl sulfide metabolism.
Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians
Authors: Humanes L , López-Ruiz A , Merino M T , Roldán J M , Diez J
Published: January 2020

Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.
Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis
Authors: Verhaert R M , Riemens A M , van der Laan J M , van Duin J , Quax W J
Published: January 2020

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.
Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures
Authors: Keeton T P , Bulla L A
Published: January 2020

The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule.
Expression of a plant protein by Neurospora crassa
Authors: Rasmussen-Wilson S J , Palas J S , Wolf V J , Taft C S , Selitrennikoff C P
Published: January 2020

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.
A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila
Authors: Hughes D S , Felbeck H , Stein J L
Published: January 2020

The uncultivated bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a central role in providing their host with fixed carbon. While this intimate association between host and symbiont indicates tight integration and coordination of function via cellular communication mechanisms, no such systems have been identified. To elucidate potential signal transduction pathways in symbionts that may mediate symbiont-host communication, we cloned and characterized a gene encoding a histidine protein kinase homolog isolated from a symbiont fosmid library. The gene, designated rssA (for Riftia symbiont signal kinase), resembles known sensor kinases and encodes a protein capable of phosphorylating response regulators in Escherichia coli. A second open reading frame, rssB (for Riftia symbiont signal regulator), encodes a protein similar to known response regulators. These results suggest that the symbionts utilize a phosphotransfer signal transduction mechanism to communicate external signals that may mediate recognition of or survival within the host. The specific signals eliciting a response by the signal transduction proteins of the symbiont remain to be elucidated.
Isolation and Characterization of Strain MMB-1 (CECT 4803), a Novel Melanogenic Marine Bacterium
Authors: Solano F , Garcia E , Perez D , Sanchez-Amat A
Published: January 2020

A novel marine melanogenic bacterium, strain MMB-1, was isolated from the Mediterranean Sea. The taxonomic characterization of this strain indicated that it belongs to the genus Alteromonas. Under in vivo conditions, L-tyrosine was the specific monophenolic precursor for melanin synthesis. This bacterium contained all types of activities associated with polyphenol oxidases (PPOs), cresolase (EC 1.18.14.1), catecholase (EC 1.10.3.1), and laccase (EC 1.10.3.2). These activities were due to the presence of two different PPOs. The first one showed all the enzymatic activities, but it was not involved in melanogenesis in vivo, since amelanogenic mutant strains obtained by nitrosoguanidine treatment contained levels of this PPO similar to that of the wild-type MMB-1 strain. The second PPO showed cresolase and catecholase activities but no laccase, and it was involved in melanogenesis, since this enzyme was lost in amelanogenic mutant strains. This PPO was strongly activated by sodium dodecyl sulfate below the critical micelle concentration, and it is a tyrosinase-like enzyme showing a lag period in its tyrosine hydroxylase activity that could be avoided by small amounts of L-dopa. This is the first report of a bacterium that contains two PPOs and also the first report of a pluripotent PPO showing all types of oxidase activities. The bacterium and the pluripotent PPO may be useful models for exploring the roles of PPOs in cellular physiology, aside from melanin formation. On the other hand, the high oxidizing capacity of the PPO for a wide range of substrates could make possible its application in phenolic biotransformations, food processing, or the cosmetic industry, where fungal and plant PPOs are being used.
Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme
Authors: Dong G , Vieille C , Savchenko A , Zeikus J G
Published: January 2020

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).
Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6
Authors: Keck A , Klein J , Kudlich M , Stolz A , Knackmuss H J , Mattes R
Published: January 2020

The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.
Lignin Degradation and Modification by the Soil-Inhabiting Fungus Fusarium proliferatum
Authors: Regalado V , Rodriguez A , Perestelo F , Carnicero A , De La Fuente G , Falcon M A
Published: January 2020

A soil-inhabiting Fusarium proliferatum strain was capable of transforming or degrading nonlabeled and (sup14)C-labeled industrial, natural, and synthetic lignin. The mineralization rate per day (expressed as the percentage of added radioactivity recovered as to (sup14)CO(inf2)) was maximal during primary metabolism.
Enzymatic removal and disinfection of bacterial biofilms
Authors: Johansen C , Falholt P , Gram L
Published: January 2020

Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata. Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite. The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured. Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata. A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity. Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.
Transforming growth factor beta in Alzheimer's disease
Authors: Chao C C , Hu S , Frey W H , Ala T A , Tourtellotte W W , Peterson P K
Published: January 2020

Alzheimer's disease (AD) has been hypothesized to be an inflammatory condition. We hypothesized that anti-inflammatory cytokines, such as transforming growth factor beta (TGF-beta), counteract the inflammatory process. In the present study, we found that TGF-beta levels were elevated in both cerebrospinal fluid and serum samples obtained from AD patients < 6 h after death. Serum TGF-beta levels were also markedly elevated before death. These results suggest that elevated TGF-beta levels in AD may represent a protective host response to immunologically mediated neuronal injury.
Laboratory diagnosis of congenital syphilis by immunoglobulin M (IgM) and IgA immunoblotting
Authors: Schmitz J L , Gertis K S , Mauney C , Stamm L V , Folds J D
Published: January 2020

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants.
Detection of beta-2 transferrin in otorrhea and rhinorrhea in a routine clinical laboratory setting
Authors: Normansell D E , Stacy E K , Booker C F , Butler T Z
Published: January 2020

A simple, straightforward, and rapid method for the detection of beta-2 transferrin in otorrhea and rhinorrhea that can be used in a routine clinical laboratory is described. The beta-2 transferrin was detected by agarose gel electrophoresis of the fluid on Beckman Paragon equipment, followed by pressure transfer to a nitrocellulose membrane and then incubation with enzyme-labeled antitransferrin antibody and substrate. The procedure was fast (3.5 h) and sensitive (detected as little as 1 microgram/ml) and required only 3 microliters of fluid. Beta-2 transferrin was detected in cerebrospinal fluid diluted up to eightfold. No special training or expertise was needed, and all equipment and procedures used are commonly available in a routine clinical laboratory.
Detection of antibodies to trans-activator protein (p40taxI) of human T-cell lymphotropic virus type I by a synthetic peptide-based assay
Authors: Rudolph D L , Coligan J E , Lal R B
Published: January 2020

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) trans-activator protein (p40taxI) were determined in serum specimens from individuals infected with HTLV-I (n = 138) and HTLV-II (n = 19). Western blot (immunoblot) analysis using recombinant tax demonstrated the presence of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-I-associated myelopathy, 43% of those (20 of 46) with adult T-cell leukemia, and 61% of asymptomatic HTLV-I blood donors (40 of 66); only one of the HTLV-II specimens reacted with the recombinant tax protein. Synthetic peptides (Tax8(106-125), Tax22(316-335), Tax-23(331-350), and Tax-24(336-353) representing the immunodominant epitopes of p40taxI detected anti-tax antibodies in 66 (48%), 50 (36%), 66 (48%), and 64 (46%) of 138 HTLV-I-positive specimens, respectively. An enzyme immunoassay using an equimolar ratio of these four peptides allowed sensitive detection of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-1-associated myelopathy, 52% of adult T-cell leukemia patients (24 of 46), and 62% of asymptomatic HTLV-1-infected donors (41 of 66). The synthetic peptide-based cocktail assay was HTLV-I specific, since none of the HTLV-II-infected specimens reacted with these peptides. Interestingly, the corresponding regions from the HTLV-II tax protein, Tax8II(106-125), and Tax-22II(312-331) did not react with either HTLV-II or HTLV-I specimens. Thus, a synthetic peptide-based assay composed of immunodominant epitopes located towards the amino terminus and the C terminus of p40taxI provides a reliable and sensitive assay for the detection of anti-tax antibodies in seroepidemiologic studies.
Immunochemical identification of Brucella abortus lipopolysaccharide epitopes
Authors: Rojas N , Freer E , Weintraub A , Ramirez M , Lind S , Moreno E
Published: January 2020

Sera from Brucella abortus-infected and -vaccinated bovines recognized four lipopolysaccharide (LPS) determinants: two in the O-polysaccharide (A and C), one in the core oligosaccharide from rough Brucella LPS (R), and one in lipid A (LA). From 46 different hybridomas secreting monoclonal antibodies (MAbs) against various LPS moieties, 9 different specificities were identified. Two epitopes, A and C/Y, were present in the O-polysaccharide. Two epitopes were found in the core oligosaccharide (R1 and R2) of rough Brucella LPS. MAbs against R1 and R2 epitopes reacted against LPS from different rough Brucella species; however, MAbs directed to the R2 epitope also reacted against enterobacterial LPS from deep rough mutants. Three epitopes (LA1, LA2, and LA3) were located in the lipid A backbone. Different sets of MAbs recognized two epitopes in the lipid A-associated outer membrane protein (LAOmp3-1 and LAOmp3-2). LPS preparations from smooth brucellae had small amounts of rough-type LPS. Although LPS from rough brucellae did not show smooth-type LPS in western blots (immunoblots), two hybridomas generated from mice immunized with rough B. abortus produced antibodies against smooth B. abortus LPS. Results are discussed in relation to the structure and function of B. abortus LPS and to previous findings on the epitopic density of the molecule.
Occurrence, in Crohn's disease, of antibodies directed against a species-specific recombinant polypeptide of Mycobacterium paratuberculosis
Authors: Vannuffel P , Dieterich C , Naerhuyzen B , Gilot P , Coene M , Fiasse R , Cocito C
Published: January 2020

Sera from patients with Crohn's disease and control were analyzed by an enzyme-linked immunosorbent assay based on the Mycobacterium paratuberculosis-specific recombinant polypeptide a362. Anti-a362 immunoglobulin G (IgG) (P < 0.05) and IgA (P < 0.001) titers were higher in patients with Crohn's disease than in controls. A monomodal Gaussian distribution of anti-a362 IgA levels were found for controls, and a bimodal distribution was found for patients with Crohn's disease. An M. paratuberculosis etiology is suggested for the 36% of patients with Crohn's disease who had an anti-a362 IgA level higher than that of controls.
Correlation between serological and mucosal inflammatory responses to Helicobacter pylori
Authors: Pérez-Pérez G I , Brown W R , Cover T L , Dunn B E , Cao P , Blaser M J
Published: January 2020

In 82 patients who underwent gastroduodenoscopy, acute and chronic gastric mucosal inflammation was scored for severity, and systemic humoral immune responses to Helicobacter pylori antigens were assessed by enzyme-linked immunosorbent assays. On the basis of culture, gastric histology, and serologic evaluation, 33 patients were classified as H. pylori infected and 36 were classified as uninfected. Thirteen patients had negative cultures and stains but were seropositive and were analyzed separately from the other two groups. Specific serum immunoglobulin G (IgG) subclass responses to H. pylori whole-cell antigens and specific IgG responses to the 54-kDa heat shock protein homolog (Hp54K) and vacuolating cytotoxin were significantly greater in infected than in uninfected patients as were specific IgA responses to whole-cell antigens and cytotoxin (P < 0.001). Among the H. pylori-infected persons, serum IgG responses to Hp54K and to the vacuolating cytotoxin were correlated with acute mucosal inflammatory scores. In contrast, serum IgA responses to whole-cell sonicate and to vacuolating cytotoxin were inversely related to chronic inflammatory scores. By multivariant regression analysis, only specific serum IgG responses to Hp54K correlated with severity of inflammation (both acute and chronic; P < 0.001); these responses may be markers of inflammation or these antibodies could play a direct role in the pathogenesis of H. pylori-induced inflammation.
Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method
Authors: Zancopé-Oliveira R M , Bragg S L , Reiss E , Wanke B , Peralta J M
Published: January 2020

The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen.
Markers of hepatitis C and B virus infections among blood donors in Ho Chi Minh City and Hanoi, Vietnam
Authors: Song P , Duc D D , Hien B , Nakata S , Chosa T , Watanabe J , Tsuda F , Murata K , Okamoto H
Published: January 2020

Blood donors in two cities in Vietnam were tested for markers of hepatitis C virus (HCV) and hepatitis B virus infections. Antibody to HCV was detected by passive hemagglutination with antigens of the second generation in 101 (20.6%) of 491 donors in Ho Chi Minh City; it was detected less frequently (P < 0.001) in donors in hanoi (4 [0.8%] of 499). HCV RNA was tested for in donors with antibody by PCR with nested primers from the 5'-noncoding region and detected in 79 donors in Ho Chi Minh City and 4 donors in Hanoi. HCV RNA was genotyped by PCR with type-specific primers from the core gene. Of 83 HCV carriers from Vietnam, 24 (29%) were infected with HCV of genotype I/1a 19 (23%) were infected with II/1b, 4 (5%) were infected with III/2a, and 2 (2%) were infected with mixed genotypes (I/1a and II/1b); HCV genotypes in the remaining 34 (41%) donors, including all 4 donors in Hanoi, were not classifiable into I/1a, II/2a, IV/2b, or V/3a. Of the 10 isolates with unclassifiable genotypes, 2 showed substantial sequence divergence within the 5'-noncoding region from reported isolates with known genotypes (I/1a to 6a). An analysis of part of the core gene sequence indicated that six of the remaining isolates most likely represented new HCV genotypes. Hepatitis B surface antigen and the corresponding antibody, respectively, were detected in 15 (3.1%) and 234 (47.7%) donors in Ho Chi Minh City as well as 15 (3.0%) and 248 (49.7%) donors in Hanoi. These results indicate an extensive spread of HCV among Ho Chi Minh City donors and HCV of novel genotypes in vietnam.
Antineurofilament and antiretinal antibodies in AIDS patients with cytomegalovirus retinitis
Authors: Rosberger D F , Tshering S L , Polsky B , Heinemann M H , Klein R F , Cunningham-Rundles S
Published: January 2020

Sera obtained from AIDS patients with cytomegalovirus (CMV) retinitis before and after treatment with foscarnet, AIDS patients with human immunodeficiency virus (HIV) retinopathy, AIDS patients without retinal disease, and normal healthy controls with and without positive CMV serologies were assayed for the presence of antibodies against the 200-kDa outer, 160-kDa middle, and 68-kDa core subunits of the neurofilament triplet. Additional studies were performed to determine the presence of antibodies reactive with proteins extracted from crude human retinal antigen preparations. Antibodies against the 200-, 260-, and 68-kDa proteins of the neurofilament triplet were detected in 15 of 15 AIDS patients with CMV retinitis. The expression of these antibodies was unaffected, qualitatively, by successful treatment with foscarnet. In contrast, only 30% of patients with HIV retinopathy unrelated to CMV, fewer than 35% of AIDS patients with positive CMV titers but without evident retinitis, and fewer than 25% of healthy controls with positive or negative CMV titers possessed antibodies against any of the triplet proteins (P < 0.001). Antibodies against several clusters of retinal antigens were also identified in the sera of patients with CMV retinitis. In summary, the data indicate that retinal elements damaged by CMV infection induce an antibody response against the 200-, 160-, and 68kDa components of the neurofilament triplet as well as other, as yet undefined retinal antigens.
Epitope-specific antibody response against glycoprotein E of pseudorabies virus
Authors: Jacobs L , Kimman T G
Published: January 2020

In this study we investigated the epitope-specific antibody response against glycoprotein E (gE) of pseudorabies virus. Epitope-specific antibody responses were investigated by enzyme-linked immunoperoxidase monolayer assays. In a vaccinated crossbred pig population, most pigs responded to antigenic domain E and to a lesser degree to antigenic domains C and D. Only few pigs responded to antigenic domains F, A, and B. Using vaccinated pigs, we investigated the influence of two different pseudorabies virus strains and the genetic background of the host on the epitope-specific antibody response. More pigs infected with the virulent NIA-3 strain had a detectable antibody response against antigenic domains C, F and A than did pigs infected with the mildly virulent Sterksel strain (P < or = 0.05; Fisher's exact test). No differences in the epitope-specific antibody responses of two genetically different pig breeding lines were observed (P > or = 0.1; Fisher's exact test). In both breeding lines the incidence of the epitope-specific antibody response was comparable to that in the crossbred pig population. In addition, we studied the epitope-specific antibody responses were strikingly different and indicated that genetic background influenced the epitope-specific antibody response. Of the serum samples of mice with C57BL and a BALB background, 40 and 17% respectively, were positive in the one of the epitope-specific immunoassays. In contrast to pigs, mice responded predominantly to antigenic domain D and to a lesser degree to antigenic domains E and B. Only few mice had a detectable antibody response against antigenic domains C and A, and none had a detectable antibody response against antigenic domain F.
DNA-based HLA typing of nonhematopoietic tissue used to select the marrow transplant donor for successful treatment of transfusion-associated graft-versus-host disease
Authors: Friedman D F , Kwittken P , Cizman B , Argyris E , Kearns J , Yang S Y , Zmijewski C , Bunin N , Douglas S D , Monos D
Published: January 2020

Transfusion-associated graft-versus-host disease (TAGVHD) is a rare and usually fatal complication of blood transfusion which can arise when immunocompetent lymphocytes from the donor of a cellular blood product are transfused into a severely immunocompromised recipient. We describe the case of an 8-month-old male with a severe combined immunodeficiency syndrome who developed TAGVHD after receiving an unirradiated transfusion. Serologic HLA typing of the parents, the patient, and the blood donor demonstrated the foreign origin of circulating lymphocytes, confirming the diagnosis of TAGVHD. The manifestations of TAGVHD did not respond to medical immunosuppressive therapy, and bone marrow transplantation was planned to treat the underlying immunodeficiency as well as the TAGVHD. By using DNA-based class I and class II HLA typing, the child's HLA type was determined from nonhematopoietic tissues. This information proved critical in selecting the bone marrow donor. The child received immunosuppression, myeloablation, and a T-depleted, maternal bone marrow graft mismatched at one HLA class II allele. Trilineage hematopoietic engraftment occurred within 3 weeks, and the child remains clinically stable with no evidence of TAGVHD more than 2 years after the transplant. This case illustrates that TAGVHD can be successfully treated by allogeneic bone marrow transplantation and that DNA-based HLA typing can play a unique role in the diagnosis and management of TAGVHD.
Major antigenic region on the integrase (IN) protein of human immunodeficiency virus type 1 determined by reactivity of human sera and a monoclonal antibody to IN protein
Authors: Tachibana Y , Yasuda A , Kurata T , Oka S , Shimada K , Kojima A
Published: January 2020

The gene encoding the integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) was expressed in vaccinia virus and Escherichia coli, and sera from 55 HIV-1-infected individuals were examined for immunoreactivity to the recombinant IN proteins by Western immunoblot. Approximately 98% (54 of 55) of the HIV-1-infected individuals showed reactivity to both the full-length IN protein of 32 kDa (IN32 protein) and the carboxy-terminal portion of the IN protein (IN17 protein). Serum samples from only 6 of the 54 antibody-positive individuals and a monoclonal antibody against the IN protein, 6F4, reacted with the amino-terminal portion of the IN protein (IN15 protein). The eight AIDS patients tested were seronegative to IN15 protein. The magnitude of reactivity to the recombinant IN proteins decreased slightly in the progression of the course of HIV-1 infection. These results suggest that a B-cell immunodominant epitope(s) on the IN protein is located on the C-terminal IN17 portion and that a minor epitope(s) recognizable by 6F4 and by rare patients is on the N-terminal IN15 portion.
Impairment of monocytic function during Trypanosoma cruzi infection
Authors: Louie M J , Cuna W R , Rodriguez De Cuna C , Mayer L , Sperber K
Published: January 2020

During acute infection, Trypanosoma cruzi, the etiologic agent of Chagas' disease, causes immunosuppression by mechanisms that are not fully delineated. Since mononuclear phagocytes are major target cells in trypanosomiasis, we investigated monocytic function during acute T. cruzi infection. A series of human monocyte and macrophage hybridomas, which represent clonal expansions of subpopulations of human macrophages and possess many normal monocytic functions, were successfully infected with T. cruzi. Clones 63 and 53, chosen for stability in long-term culture, were studied extensively after infection with T. cruzi. Following infection of clone 63, the trypomastigote did not transform into the amastigote multiplicative form, suggesting that clone 63 did not support the entire T. cruzi life cycle. The typical life cycle was completed in clone 53, and thus, clone 53 was used in subsequent studies. Following infection, clone 53 lost expression of class II antigens compared with uninfected cells (DR of 2.2% versus 29.3% and mean channel fluorescence intensity [mean channel] of 4.1 versus 30.5, DQ of 2.3% versus 15.6% and mean channel of 5.4 versus 11.4, and DP of 6.3% versus 27.2% and mean channel of 10.3 versus 33.4). The expression of Class I antigens (87.9% versus 82.8%; mean channel, 20 versus 120) and the adhesion molecules LFA-1 (72.9% versus 28.7%; mean channel, 50.7 versus 23.7) and LFA-3 (10.8% versus 0.7%; mean channel, 20.7 versus 15.1) was increased in infected cells compared with that in uninfected cells. Production of interleukin-1 alpha was decreased and interleukin-6 production was increased in infected clone 53 compared with those in the uninfected cells, while production of tumor necrosis factor alpha was increased.(ABSTRACT TRUNCATED AT 250 WORDS)
Development, characterization, and biological properties of meningococcal immunotype L3,7,(8),9-specific monoclonal antibodies
Authors: Verheul A F , Kuipers A J , Braat A K , Dekker H A , Peeters C C , Snippe H , Poolman J T
Published: January 2020

In this study, we characterize the properties of nine monoclonal antibodies (MAbs) that recognize meningococcal lipopolysaccharides (LPS). The following three specific MAbs that had not been described previously were elicited in BALB/c mice by using an immunotype L3,7,9 oligosaccharide-tetanus toxoid conjugate in combination with Quil A: 4D1-B3, 3A12-E1, and 4A8-B2. These MAbs reacted with L3,7,9 LPS on immunoblots and in the LPS enzyme-linked immunosorbent assay (ELISA) and recognised strains containing L3, L3,7, L8 (except 3A12-E1), or L9 LPS in the whole-cell ELISA. The six other MAbs have been described in previous studies (K. Saukkonen, M. Leinonen, H. Abdillahi, and J.T. Poolman, Vaccine 7:325-328, 1989; R.J.P.M. Scholten, B. Kuipers, H.A. Valkenburg, J. Danjert, W.D. Zollinger, and J.T. Poolman, J. Med. Microbiol., in press) and were obtained after immunization with outer membrane protein complexes containing LPS: MN15A11, MN15A8-1, MN15A17-1, MN11A11G, MN14F20-11, and MN14F21-11. MN15A11 was specific for L3,7,9 LPS and displayed properties similar to those of 3A12-E1. MN15A17-1, MN14F20-1, and MN11A11G were cross-reactive, and MN14F21-11 was specific for the L1,8 immunotype. Epitope specificities of MAbs reacting with L3,7,(8),9 strains were analyzed. MAbs 4D1-B3, 3A12-E1, and 4A8-B2 recognized phosphoethanolamine group-containing oligosaccharide-specific epitopes. MN15A11 and MN15A17-1 were probably directed against a conformational epitope, although for MN5A11 recognition of an unknown L3,7,9-specific epitope in the 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A region cannot be excluded. MN15A8-1, a strongly cross-reactive MAb, recognized a determinant which included the KDO-lipid A region and the more terminal saccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum expressed in Escherichia coli
Authors: Theisen M , Vuust J , Gottschau A , Jepsen S , Høgh B
Published: January 2020

A recombinant Plasmodium falciparum glutamate-rich protein (GLURP) was produced in Escherichia coli as a nearly full-length protein. In order to map immunodominant regions on GLURP, the nonrepetitive amino-terminal region (R0) as well as the central repeat region (R1) and the carboxy-terminal repeat region (R2) were also produced as separate products. All four purified gene products reacted specifically with serum samples from adults living in an area of Liberia where malaria is holoendemic. It appears that the human immune response against GLURP is primarily directed against the R2 region because 94% of the serum samples reacted with this region in an immunoassay. Antibody reactivity against the R0 region was also observed in 75% of the serum samples, while the R1 region showed only weak antibody-binding activity. When the nearly full-length GLURP molecule was adsorbed to Al(OH)3 it was found to be immunogenic in mice. In these experiments, the antibody response was almost exclusively directed against the R2 region. When anti-GLURP sera were obtained from rabbits immunized with the three regions, R0, R1, and R2, respectively, they recognized in immunoprecipitation experiments authentic GLURP from P. falciparum grown in vitro. These results demonstrate that GLURP produced in E. coli can induce a humoral immune response against GLURP derived from blood-stage parasites.
Changes in CD45 isoform expression vary according to the duration of T-cell memory after vaccination
Authors: McElhaney J E , Pinkoski M J , Meneilly G S
Published: January 2020

Healthy young (< 40 years) and elderly (< 60 years) adults were immunized with the 1992-1993 preparation of trivalent influenza vaccine, and changes in CD45 isoform expression on peripheral blood lymphocytes were measured in the pre- and postvaccination periods. Fluorescence-activated cell sorter analysis was used to study T-cell subsets in fresh peripheral blood lymphocytes (day 0) and after 6 days of culture with live influenza virus. We have reported previously that the interleukin-2 response to the stimulating strain of virus, A/Texas/16/89, did not decline until 26 weeks postvaccination. In ex vivo CD4+ subsets, this interleukin-2 response was paralleled by a > 10% increase in the proportion of cells expressing the CD45RO+ phenotype following vaccination (p < 0.0001). In vitro stimulation had no effect on CD4+ subsets prior to vaccination but, after vaccination, was associated with a > 10% increase in CD45RA+RO+ cells (P < 0.0001). In addition, we have identified a change in the population of cells that express a CD45 isoform that is neither CD45RA nor CD45RO (CD45RA-RO-). At 26 weeks postvaccination, the proportion of CD45RA-RO- cells in ex vivo CD4+ peripheral blood mononuclear cells increased by approximately 15% from that measured at the earlier postvaccination time points (P < 0.0001). In vitro stimulation with influenza virus resulted in a further 20% increase in the proportion of CD45RA-RO- cells (P < 0.0001). The CD45RA-RO- phenotype may identify a population of cells undergoing apoptosis (programmed cell death) that limits the duration of helper T-cell (CD4+) memory after vaccination.
Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis
Authors: Waag D M , McKee K T , Sandstrom G , Pratt L L , Bolt C R , England M J , Nelson G O , Williams J C
Published: January 2020

The specific humoral and cell-mediated immune responses of human volunteers vaccinated with the Francisella tularensis live vaccine strain (LVS) were evaluated. In the search for an optimal antigen to measure the immunogenicity of the vaccine in an enzyme-linked immunosorbent assay, we tested irradiation-killed LVS, an aqueous ether extract of the LVS (EEx), lipopolysaccharide (LPS) from LVS, and a virulent strain (SCHU4). Volunteers were immunized with LVS by scarification. Immunoglobulin G (IgG) responses to LVS and LPS gave the highest background titers when tested with sera from unimmunized volunteers, whereas IgA, IgG, and IgM background titers to EEx and SCHU4 were low. Vaccination caused a significant rise (P < 0.01) in IgA, IgG, and IgM titers to all antigens tested, except for the IgG response to LPS. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx 14 days postvaccination, while 50% were positive to LVS. By day 14 after vaccination, 70% of immunized volunteers exhibited a positive response to EEx in an in vitro peripheral blood lymphocyte proliferation assay. EEx, a specific and sensitive antigen for evaluating immune responses of vaccinated volunteers, may be a superior antigen for the diagnosis of tularemia.