bluuebunny/crossref_metadata_2025 huggingface dataset:
Astrovirus survival in drinking water
Authors: Abad F X , Pintó R M , Villena C , Gajardo R , Bosch A
Published: January 2020

A method based on infection of CaCo-2 cultured cell monolayers (CC) and reverse transcription-PCR (RT-PCR) was developed for the specific detection of infectious astrovirus. The procedure was validated by titrating poliovirus stocks in parallel in CaCo-2 cells by determining the most probable number of cytopathogenic units and by cell culture and subsequent RT-PCR (CC-RT-PCR). CC-RT-PCR was then employed to measure the persistence of astrovirus suspended in dechlorinated tap water. After 60 days, the decay of astrovirus infectivity was 2 log units at 4 +/- 1 degrees C and 3.2 log units at 20 +/- 1 degrees C, while after 90 days, the titer reduction was 3.3 and 5 log units at 4 +/- 1 degrees C and 20 +/- 1 degrees C, respectively. Astrovirus decay in the presence of free chlorine (FC) was monitored by CC-RT-PCR. Residual infectivity was found after 2 h in the presence of 1 mg of FC/liter. Under these conditions, astrovirus shows a log titer reduction (LTR) or 4, while 0.5 mg of FC/liter induced an LTR of 2.4. The possibility of acquiring data on the survival of fastidious viruses in the environment opens new perspectives on the epidemiology of some significant infections transmitted by the fecal-oral route.
The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs
Authors: McDonald I R , Murrell J C
Published: January 2020

The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these mxaF genes revealed strong sequence conservation. Of the 172 amino acid residues, 47% were conserved among all 22 sequences obtained in this study. Phylogenetic analysis of these MxaF sequences showed that those from type I and type II methanotrophs form two distinct clusters and are separate from MxaF sequences of other gram-negative methylotrophs. MxaF sequences retrieved by PCR from DNA isolated from a blanket bog peat core sample formed a distinct phylogenetic cluster within the MxaF sequences of type II methanotrophs and may originate from a novel group of acidophilic methanotrophs which have yet to be cultured from this environment.
Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. jegathesan
Authors: Cheong H , Gill S S
Published: January 2020

A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B. thuringiensis subsp. jegathasan was cloned employing a limited-growth PCR screening method with forward and reverse oligonucleotide primers designed from N-terminal amino acid sequences of native and trypsin-cleaved protein, respectively. The expressed protein showed little cross-reactivity to the antibody raised against the Cyt1Aa protein. Unlike Cyt1Aa and Cyt2Aa expression, there was little or no visible crystal inclusion formation under microscopic observation. The amino acid sequence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, respectively. The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions between alpha-helices and beta-sheets and one in the loop region between beta-sheets 5 and 6. beta-Blocks 4 to 7 are also conserved, not only structurally but also among the amino acids in the hydrophobic faces. Mosquitocidal activity assays indicated that the Cyt2Bb toxin had less toxicity than Cyt1Aa and had about 600-times-lower toxicity than the wild-type whole toxin crystal. However, both the Cyt2Bb and the Cyt1Aa toxin showed comparable levels of hemolytic activity.
Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscope slides and leaf surfaces
Authors: Li S , Spear R N , Andrews J H
Published: January 2020

A 21-mer oligonucleotide probe designated Ap665, directed at the 18S rRNA of Aureobasidium pullulans and labelled with five molecules of fluorescein isothiocyanate, was applied by fluorescence in situ hybridization (FISH) to populations of the fungus on slides and apple leaves from growth chamber seedlings and orchard trees. In specificity tests that included Ap665 and a similarly labelled universal probe and the respective complementary probes as controls, the hybridization signal was strong for Ap665 reactions with 12 A. pullulans strains but at or below background level for 98 other fungi including 82 phylloplane isolates. Scanning confocal laser microscopy was used to confirm that the fluorescence originated from the cytoplasmic matrix and to overcome limitations imposed on conventional microscopy by leaf topography. Images were recorded with a cooled charge-coupled device video camera and digitized for storage and manipulation. Image analysis was used to verify semiquantitative fluorescence ratings and to demonstrate how the distribution of the fluorescence signal in specific interactions (e.g., Ap665 with A. pullulans cells) could be separated at a given probability level from nonspecific fluorescence (e.g., in interactions of Ap665 with Cryptococcus laurentii cells) of an overlapping population. Image analysis methods were used also to quantify epiphytic A. pullulans populations based on cell number or percent coverage of the leaf surface. Under some conditions, leaf autofluorescence and the release of fluorescent compounds by leaves during the processing for hybridization decreased the signal-to-noise ratio. These effects were reduced by the use of appropriate excitation filter sets and fixation conditions. We conclude that FISH can be used to detect and quantify A. pullulans cells in the phyllosphere.
Acetyl Coenzyme A Acetyltransferase of Rhizobium sp. (Cicer) Strain CC 1192
Authors: Kim S A , Copeland L
Published: January 2020

To investigate why Rhizobium sp. (Cicer) strain CC 1192 cells accumulate poly-R-3-hydroxybutyrate in the free-living state but not as bacteroids in nodules on chickpea (Cicer arietinum L.) plants, we have examined the kinetic properties of acetyl coenzyme A (acetyl-CoA) acetyltransferase (also known as acetoacetyl-CoA thiolase and 3-ketothiolase [EC 2.3.1.9]) from both types of cells. The enzyme had a native molecular mass of 180 (plusmn) 4 kDa, and the subunit molecular mass was 44 (plusmn) 1 kDa. The seven amino acids from the N terminus were Lys-Ala-Ser-Ile-Val-Ile-Ala. Thiolysis and condensation activity of the enzyme from free-living CC 1192 cells were optimal at pHs 7.8 and 8.1, respectively. The relationship between substrate concentrations and initial velocity for the thiolysis reaction were hyperbolic and gave K(infm) values for acetoacetyl-CoA and CoA of 42 and 56 (mu)M, respectively. The maximum velocity in the condensation direction was approximately 10% of that of the thiolysis reaction. With highly purified preparations of the enzyme, a value of approximately 1 mM was determined for the apparent K(infm) for acetyl-CoA. However, with partially purified enzyme preparations or when N-ethylmaleimide was included in reaction mixtures the apparent K(infm) for acetyl-CoA was close to 0.3 mM. In the condensation direction, CoA was a potent linear competitive inhibitor with an inhibition constant of 11 (mu)M. The much higher affinity of the enzyme for the product CoA than the substrate acetyl-CoA could have significance in view of metabolic differences between bacteroid and free-living cells of CC 1192. We propose that in free-living CC 1192 cells, the acetyl-CoA/CoA ratio reaches a value that allows condensation activity of acetyl-CoA acetyltransferase, but that in CC 1192 bacteroids, the ratio is poised so that the formation of acetoacetyl-CoA is not favored.
Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS
Authors: Arfmann H , Timmis K N , Wittich R
Published: January 2020

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.
Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12
Authors: Lemoine J , Chirat F , Wieruszeski J M , Strecker G , Favre N , Neeser J R
Published: January 2020

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 x 10(6) Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: [formula: see text] Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: [formula: see text] This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.
Phosphorus effects on the mycelium and storage structures of an arbuscular mycorrhizal fungus as studied in the soil and roots by analysis of Fatty Acid signatures
Authors: Olsson P A , Baath E , Jakobsen I
Published: January 2020

The distribution of an arbuscular mycorrhizal (AM) fungus between soil and roots, and between mycelial and storage structures, was studied by use of the fatty acid signature 16:1(omega)5. Increasing the soil phosphorus level resulted in a decrease in the level of the fatty acid 16:1(omega)5 in the soil and roots. A similar decrease was detected by microscopic measurements of root colonization and of the length of AM fungal hyphae in the soil. The fatty acid 16:1(omega)5 was estimated from two types of lipids, phospholipids and neutral lipids, which mainly represent membrane lipids and storage lipids, respectively. The numbers of spores of the AM fungus formed in the soil correlated most closely with neutral lipid fatty acid 16:1(omega)5, whereas the hyphal length in the soil correlated most closely with phospholipid fatty acid 16:1(omega)5. The fungal neutral lipid/phospholipid ratio in the extraradical mycelium was positively correlated with the level of root infection and thus decreased with increasing applications of P. The neutral lipid/phospholipid ratio indicated that at high P levels, less carbon was allocated to storage structures. At all levels of P applied, the major part of the AM fungus was found to be present outside the roots, as estimated from phospholipid fatty acid 16:1(omega)5. The ratio of extraradical biomass/intraradical biomass was not affected by the application of P, except for a decrease at the highest level of P applied.
Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus
Authors: Sommer P , Bormann C , Götz F
Published: January 2020

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.
Chloroform Cometabolism by Butane-Grown CF8, Pseudomonas butanovora, and Mycobacterium vaccae JOB5 and Methane-Grown Methylosinus trichosporium OB3b
Authors: Hamamura N , Page C , Long T , Semprini L , Arp D J
Published: January 2020

Chloroform (CF) degradation by a butane-grown enrichment culture, CF8, was compared to that by butane-grown Pseudomonas butanovora and Mycobacterium vaccae JOB5 and to that by a known CF degrader, Methylosinus trichosporium OB3b. All three butane-grown bacteria were able to degrade CF at rates comparable to that of M. trichosporium. CF degradation by all four bacteria required O(inf2). Butane inhibited CF degradation by the butane-grown bacteria, suggesting that butane monooxygenase is responsible for CF degradation. P. butanovora required exogenous reductant to degrade CF, while CF8 and M. vaccae utilized endogenous reductants. Prolonged incubation with CF resulted in decreased CF degradation. CF8 and P. butanovora were more sensitive to CF than either M. trichosporium or M. vaccae. CF degradation by all three butane-grown bacteria was inactivated by acetylene, which is a mechanism-based inhibitor for several monooxygenases. Butane protected all three butane-grown bacteria from inactivation by acetylene, which indicates that the same monooxygenase is responsible for both CF and butane oxidation. CF8 and P. butanovora were able to degrade other chlorinated hydrocarbons, including trichloroethylene, 1,2-cis-dichloroethylene, and vinyl chloride. In addition, CF8 degraded 1,1,2-trichloroethane. The results indicate the potential of butane-grown bacteria for chlorinated hydrocarbon transformation.
Ecological implications of an improved direct viable count method for aquatic bacteria
Authors: Joux F , Lebaron P
Published: January 2020

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.
Application of microautoradiography to the study of substrate uptake by filamentous microorganisms in activated sludge
Authors: Andreasen K , Nielsen P H
Published: January 2020

Excessive growth of filamentous microorganisms in activated-sludge treatment plants is a major operational problem which causes poor settlement of activated sludge. An enhanced understanding of the factors controlling growth of different filamentous microorganisms is necessary in order to establish more successful control strategies. In the present study, the in situ substrate uptake was investigated by means of microautoradiography. It was demonstrated that the uptake of labeled organic substrates by the filamentous microorganisms, during short-term incubation, could be detected by microautoradiography. Viability and respiratory activity of the filaments were also detected by reduction of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and by incorporation of [(sup3)H]thymidine. Gram, Neisser, and fluorescence staining techniques were used for the localization and identification of the filaments. Activated-sludge samples from five wastewater treatment plants with bulking problems due to filamentous microorganisms were investigated. Microthrix parvicella, Nostocoida limicola, and Eikelboom's type 0041 and type 021N were investigated for their ability to take up organic substrates. A panel of six substrates, i.e., [(sup14)C]acetate, [(sup3)H]glucose, [(sup14)C]ethanol, [(sup3)H]glycine, [(sup3)H]leucine, and [(sup3)H]oleic acid, was tested. The uptake response was found to be very specific not only between the different filamentous types but also among filaments of the same type from different treatment plants. Interestingly, M. parvicella consistently took up only oleic acid among the tested substrates. It is concluded that microautoradiography is a useful method for investigation of in situ substrate uptake by filamentous microorganisms in activated sludge.
Coffea arabica L., a new host plant for Acetobacter diazotrophicus, and isolation of other nitrogen-fixing acetobacteria
Authors: Jimenez-Salgado T , Fuentes-Ramirez L E , Tapia-Hernandez A , Mascarua-Esparza M A , Martinez-Romero E , Caballero-Mellado J
Published: January 2020

Acetobacter diazotrophicus was isolated from coffee plant tissues and from rhizosphere soils. Isolation frequencies ranged from 15 to 40% and were dependent on soil pH. Attempts to isolate this bacterial species from coffee fruit, from inside vesicular-arbuscular mycorrhizal fungi spores, or from mealybugs (Planococcus citri) associated with coffee plants were not successful. Other acid-producing diazotrophic bacteria were recovered with frequencies of 20% from the coffee rhizosphere. These N2-fixing isolates had some features in common with the genus Acetobacter but should not be assigned to the species Acetobacter diazotrophicus because they differed from A. diazotrophicus in morphological and biochemical traits and were largely divergent in electrophoretic mobility patterns of metabolic enzymes at coefficients of genetic distance as high as 0.950. In addition, these N2-fixing acetobacteria differed in the small-subunit rRNA restriction fragment length polymorphism patterns obtained with EcoRI, and they exhibited very low DNA-DNA homology levels, ranging from 11 to 15% with the A. diazotrophicus reference strain PAI 5T. Thus, some of the diazotrophic acetobacteria recovered from the rhizosphere of coffee plants may be regarded as N2-fixing species of the genus Acetobacter other than A. diazotrophicus. Endophytic diazotrophic bacteria may be more prevalent than previously thought, and perhaps there are many more potentially beneficial N2-fixing bacteria which can be isolated from other agronomically important crops.
Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage
Authors: Ryser E T , Arimi S M , Donnelly C W
Published: January 2020

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.
Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation
Authors: Wang F , Lee S Y
Published: January 2020

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.
Benzylsuccinate Formation as a Means of Anaerobic Toluene Activation by Sulfate-Reducing Strain PRTOL1
Authors: Beller H R , Spormann A M
Published: January 2020

Permeabilized cells of toluene-mineralizing, sulfate-reducing strain PRTOL1 catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. Recent in vitro studies with two toluene-mineralizing, denitrifying bacteria demonstrated the same fumarate addition reaction and indicated that it may be the first step of anaerobic toluene degradation. This study with strain PRTOL1 shows that anaerobic toluene activation by fumarate addition occurs in bacteria as disparate as sulfate-reducing and denitrifying species (members of the delta and beta subclasses of the Proteobacteria, respectively).
Differential elevation of circulating interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 in AIDS-associated cachectic states
Authors: Bélec L , Meillet D , Hernvann A , Grésenguet G , Gherardi R
Published: January 2020

Elevation of serum interleukin-1 beta (IL-1 beta) levels, and to a lesser degree tumor necrosis factor alpha levels, was found in cachectic human immunodeficiency virus (HIV)-infected African patients without concurrent opportunistic infection or neoplasia (HIV wasting syndrome). A heterogeneous pattern of elevations of cytokine levels, including mild elevations of IL-1 beta and pronounced elevations of IL-6 levels, was found in other cachectic states.
Increased immunogenicity of inactivated influenza virus vaccine containing purified surface antigen compared with whole virus in elderly women
Authors: Powers D C
Published: January 2020

Thirty-eight elderly female subjects (aged 80 +/- 7 years, mean +/- standard deviation) were randomized to immunization with trivalent inactivated influenza virus vaccine containing either purified surface antigen (n = 18) or whole virus (n = 20) components from A/Texas/36/91 (H1N1), A/Beijing/353/89 (H3N2), and B/Panama/45/90 strains. Humoral and cellular immune responses were assessed by measuring serum hemagglutination inhibition antibodies and cytotoxic T lymphocyte (CTL) activity at 0 and 3 weeks postvaccination. Serological responses to both of the type A vaccine strains following immunization with surface antigen vaccine (SAV) were significantly more frequent and greater in magnitude than those induced by whole-virus vaccine. Antibody responses to the B/Panama component were modest and did not differ significantly between the two vaccines. Persons given SAV, but not those given whole-virus vaccine, had a small but significant increase in mean percent specific lysis of influenza A (H1N1) virus-infected autologous targets by peripheral blood mononuclear cells which were stimulated in vitro with influenza A (H1N1) virus. The H1N1-stimulated cytotoxic effectors induced by SAV were CD8+ and were not cross-reactive against H3N2-infected targets. Influenza B virus-specific CTL responses were not observed with either vaccine. These results suggest that currently available subunit influenza virus vaccines may offer an advantage over inactivated whole-virus preparations for inducing humoral and cellular immune responses in the elderly, although the CTL response may be too limited to be of physiological significance.
Immunological analysis of the components of the antigen complex A60 of Mycobacterium bovis BCG
Authors: Coetsier C , Baelden M C , Coene M , Cocito C
Published: January 2020

The antigen complex of A60 of Mycobacterium bovis BCG was analyzed by different immunological techniques to assess its relevance to tuberculosis and the involvement of its components in the immune reactions elicited in humans by tuberculous infection. A60 is composed of about 30 components, of which 8 were identified by available monoclonal antibodies (lipoarabinomannan, a glycolipid, and proteins of 65, 40, 38, 35, 19, and 14 kDa). The majority (87.5%) of anti-mycobacterial antibodies in sera from tuberculosis patients was directed against A60. Western blot (immunoblot) analysis indicated that the majority of the highly antigenic proteins present in mycobacterial homogenates were components of the A60 complex. A small percentage (7.8%) of A60 epitopes proved to be species specific. Thus, A60 proteins of 66, 41, 38, 37, 35, 34, 32, and 22 kDa were found to contain B-cell epitopes specific for M. bovis and not shared by Mycobacterium leprae oR Mycobacterium avium.
Clinical significance of serum 2,5-oligoadenylate synthetase and soluble interleukin-2 receptor in hemophiliacs positive and negative for human immunodeficiency virus type 1
Authors: Matsuda J , Gotoh M , Gohchi K , Tsukamoto M , Saitoh N , Kinoshita T
Published: January 2020

We measured serum 2,5-oligoadenylate synthetase (2,5-AS) levels and soluble interleukin-2 receptor (sIL-2R) levels in human immune deficiency virus type 1 (HIV-1)-positive and HIV-1-negative hemophiliacs in order to clarify the clinical significance of these parameters in hemophiliacs. Serum 2,5-AS levels were measured by a radioimmunosorbent assay, and sIL-2R levels were measured by an enzyme-linked immunosorbent assay. The mean serum 2,5-AS levels were higher in AIDS-related-complex and AIDS patients, asymptomatic carriers, and HIV-1-negative hemophiliacs than in hepatitis C virus-positive patients and healthy controls. Serial determinations showed that the 2,5-AS levels tended to increase in HIV-1-positive patients, especially those with AIDS-related complex or AIDS, although it showed a substantial decrease in the terminal stage. The serum sIL-2R levels were higher in HIV-1-positive patients, HIV-1-negative patients, and hepatitis C virus-positive patients than in controls. Serial studies showed little change in the HIV-1-positive and HIV-1-negative groups, although sIL-2R levels showed a tendency to decrease with zidovudine treatment. On the basis of the present results, we may well conclude that 2,5-AS and sIL-2R are not specific markers for hemophiliacs with HIV-1 infection. However, serial measurement of these markers can still be useful for assessing the progression of AIDS and the prognosis for patients with AIDS, as well as for monitoring the response to zidovudine.
Demonstration of human immunoglobulin G Fc-binding activity in oral bacteria
Authors: Grenier D , Michaud J
Published: January 2020

Nonimmune binding of immunoglobulins via the Fc fragment may reduce opsonization and phagocytosis of bacteria and is thus considered a virulence factor. The aim of this study was to investigate a wide range of oral bacterial strains for the presence of human immunoglobulin G (IgG) Fc-binding activity. A total of 132 strains representing 40 different gram-positive and gram negative bacterial species were tested for IgG Fc-binding activity by using a fast and simple dot blot procedure with horseradish peroxidase-conjugated Fc fragments from human IgG. Neither the human nor animal biotype of Porphyromonas gingivalis possessed IgG Fc-binding activity. The strongest positive reaction of gram-negative species with the IgG Fc fragments were obtained with strains of Prevotella intermedia and Fusobacterium nucleatum. Among the gram-positive bacteria tested, Peptostreptococcus micros, Lactobacillus spp., and several species of streptococci possessed IgG Fc-binding activity. In the present investigation, the ability of several oral bacterial species to bind IgG Fc fragments was demonstrated. This factor represents a potential virulence determinant as it may help pathogenic oral bacteria escape host defense mechanisms.
Elevated levels of total and Puumala virus-specific immunoglobulin E in the Scandinavian type of hemorrhagic fever with renal syndrome
Authors: Alexeyev O A , Ahlm C , Billheden J , Settergren B , Wadell G , Juto P
Published: January 2020

In a previous study, it was reported that the total immunoglobulin E (IgE) level was increased in patients with hemorrhagic fever with renal syndrome (HFRS). The aim of the present study was to investigate whether specific IgE is synthesized during the course of the disease. For this purpose, an epsilon-capture enzyme-linked immunosorbent assay was developed. A total of 72 patients with HFRS caused by Puumala virus were studied. Three different control groups were included: 20 blood donors, 20 patients with other viral diseases (influenza A and B virus, acute Epstein-Barr virus, and acute cytomegalovirus infections), and 5 subjects with high levels of total IgE (median, 1,070 kU/liter; range, 773 to 5,740 kU/liter). The levels of total IgE were significantly higher during the acute phase of HFRS than those of blood donors (P < 0.01) and of patients with other viral diseases (P < 0.001). All patients developed a specific IgE response (median, 55 arbitrary units; range 24 to 123 arbitrary units) in the acute phase of the disease, whereas in the different control groups no specific IgE was detectable. Both total and specific IgE levels decreased during convalescence compared with those during the acute phase of HFRS (P < 0.001 and P < 0.001, respectively). In conclusion, we have shown that both total and specific IgE levels are increased in patients with HFRS compared with levels in patients with other viral diseases. The possible pathogenetic role of the specific IgE response in HFRS is discussed.
Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines
Authors: Stiehm E R , Roberts R L , Ank B J , Plaeger-Marshall S , Salman N , Shen L , Fanger M W
Published: January 2020

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.
Levels of circulating fibronectin receptor in adult and pediatric patients with human immunodeficiency virus type 1 infection
Authors: Torre D , Zeroli C , Pugliese A , Giola M , Ferrario G , Bonetta G , Issi M , Martegani R , Speranza F , Fiori G P
Published: January 2020

We found a significant increase in fibronectin receptor (FNR) levels in the sera of adult human immunodeficiency virus type 1 (HIV-1)-infected patients, especially in those with AIDS (1,026.9 +/- 583.9 ng/ml; P < 0.0001). In contrast, AIDS patients with neurologic disorders and HIV-1-seropositive patients showed normal levels of FNR in serum. In addition, HIV-1-infected children showed increased levels of FNR in serum (824.4 +/- 333.5 ng/ml; P = 0.03). We suggest that an increase of FNR levels in AIDS patients is related to enhanced expression of FNR on HIV-1-infected cells.
Production of antisera against the enterotoxin of Bacteroides fragilis and their use in a cytotoxicity neutralization assay of HT-29 cells
Authors: Van Tassel R L , Lyerly D M , Wilkins T D
Published: January 2020

To study the enterotoxin of Bacteroides fragilis, the colon carcinoma cell line HT-29 was used in a standard cytotoxicity assay. We produced high-titer neutralizing antisera in rabbits and goats against both crude and purified toxin and developed a cytotoxicity neutralization assay for use in confirming enterotoxin activity in culture filtrates and stools. The neutralization titers of the antisera on the colon carcinoma cell line HT-29 ranged from 1,600 to 2,400. In an antibody screening enzyme-linked immunosorbent assay, titers ranged from 10(4) to 10(5). The antisera produced against the highly purified toxin also neutralized the enterotoxic activity of the toxin and were monospecific by immunoelectrophoresis.
Identification of antigens of pathogenic free-living amoebae by protein immunoblotting with rabbit immune and human sera
Authors: Powell E L , Newsome A L , Allen S D , Knudson G B
Published: January 2020

Prominent antigens of pathogenic and nonpathogenic free-living amoebae were identified by using polyclonal rabbit immune sera in immunoblot assays. The intent was to determine if prominent epitopes identified with rabbit immune sera could also be recognized by human sera. With rabbit sera, the development of immunoreactive bands was restricted to molecular masses of greater than 18.5 kDa for Naegleria, Hartmannella, and Vahlkampfia antigens. Two or more broad bands of less than 18.5 kDa were prominent features in three different Acanthamoeba species. Few cross-reactive antibodies could be detected between representative species of the three different subgroups of Acanthamoeba. Naegleria antigen was likewise serologically distinct, as were Hartmannella and Vahlkampfia antigens. The relative lack of cross-reacting antibodies between the pathogenic amoebae suggested that i would be desirable to use a panel of amoebic antigens to represent the range of serologically distinct antigens for assessing reactive antibodies in human sera. In pooled human sera (10 serum specimens per pool), the appearance of minimally reactive bands ranging from 32.5 to 106 kDa was a common feature of all six antigens. A prominent band of less than 18.5 kDa was identified in the Acanthamoeba culbertsoni antigen lane in 2 of the 10 human serum specimen pools. When sera from each of the two groups were tested individually by immunoblotting, the reaction with A. culbertsoni antigen could be associated with one individual. By using a panel of amoebic antigens, this method could prove useful in recognizing undiagnosed amoebic infections by revealing specific reactive antibodies.
Characterization of circulating CD4+ CD8+ lymphocytes in healthy individuals prompted by identification of a blood donor with a markedly elevated level of CD4+ CD8+ lymphocytes
Authors: Prince H E , Golding J , York J
Published: January 2020

During flow cytometric analysis of lymphocytes from healthy donors, we identified a donor (donor A) with 22% CD4+ CD8+ cells (versus values of < 4% for 65 other controls). To determine if CD4+ CD8+ cells from donor A and other controls were similar, we first defined the phenotypic profile of control CD4+ CD8+ cells. Enriched CD4+ CD8+ cell populations for 10 controls were prepared by a two-step positive selection scheme with anti-CD4-coated magnetic beads and anti-CD8-coated culture flasks; the selected population averaged 69% CD4+ CD8+ cells and 31% CD4+ CD8- cells. For all 10 controls, two subsets of CD4+ CD8+ cells, CD4dim CD8bright and CD4bright CD8dim, were observed. Phenotypic profiles of these two CD4+ CD8+ subsets were defined by pairing anti-CD8 with other monoclonal antibodies, and the profiles were compared with each other and with those of CD4+ CD8-, CD4- CD8bright, and CD4- CD8dim cells. CD8bright and CD4bright CD8dim cells differed in their proportions of CD62-L+ cells and in their levels of CD11a and CD2 expression. Both CD4+ CD8+ subsets resembled CD4+ CD8- cells in CD45RA, CD45RO, and CD25 expression; the comparable CD- CD8+ cells in CD62-L expression; and CD4- CD8bright cells in CD11b, CD11b, CD16/56, and CD28 expression. CD38 expression in both CD4+ CD8+ subsets was decreased compared with those of other cell subsets. Whereas control CD4+ CD8+ cells averaged 33% CD4dim CD8bright, CD4+ CD8+ cells from donor A were > 90% CD4dim CD8bright. Donor A CD4dim CD8bright cells exhibited proportional decreases in CD25 and CD62-L expression and increases in CD11b and CD54 expression compared with those of control CD4dim CD8bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Elevated levels of circulating tumor necrosis factor alpha in human immunodeficiency virus type 1-infected Africans living in Sweden
Authors: Sönnerborg A , Ayehunie S , Julander I
Published: January 2020

Elevated levels of tumor necrosis factor alpha in serum were found in human immunodeficiency virus type 1 (HIV-1)-infected Africans to a higher extent than in matched HIV-1-infected Caucasians, both groups living in Sweden. The results suggest that factors not related to the environment contribute to enhanced synthesis of tumor necrosis factor alpha in HIV-1-infected patients.
DNA enzyme immunoassay of the PCR-amplified HLA-DQ alpha gene for estimating residual leukocytes in filtered blood
Authors: Prati D , Rawal B D , Dang C , Capelli C , Vyas G N
Published: January 2020

Blood is filtered for selective removal of leukocytes (WBC) to reduce the immunological and virological risks of transfusion. Exceedingly low numbers of residual WBC in leukodepleted blood cannot be enumerated by conventional hematologic methods. Therefore, we investigated the application of a DNA enzyme immunoassay (DEIA) for detecting a region of the HLA-DQ alpha gene following amplification by PCR. After hybridization with a specific probe coated onto the wells of a microtiter plate, the PCR-amplified DNA was detected by adding monoclonal antibodies to double-stranded DNA, enzyme tracer, and chromogen substrate for colorimetric measurement. The sensitivities of DEIA and radioisotopic liquid hybridization were similar in five sets of experiments performed with a known number of human WBC. The optical density and the number of spiked human WBC in the range of 1.0 to 0.05 cells per microliter showed good correlation in five calibration experiments performed with human WBC suspended in heterologous blood. Using a calibration curve for DEIA, we estimated the concentration of residual WBC in five individual units of erythrocytes passed through blood filters. The postfiltration WBC count was 1.6 WBC per microliter in one unit, while in four other units it was below the lower detection limit (< 0.05 WBC per microliter) of the DEIA. DEIA obviates the use of radioisotopes in PCR for detection of exceedingly low numbers of residual WBC in filtered blood.
Differential effect of the immunomodulatory hormone somatostatin on replication of human immunodeficiency virus type 1 in CD4+ and CD8+ T lymphocytes
Authors: Mercure L , Phaneuf D , Wainberg M A
Published: January 2020

The long-acting somatostatin analog octreotide (SMS 201-995) possesses immunosuppressive properties and has been successfully used for the management of human immunodeficiency virus (HIV)-associated diarrhea, a condition commonly observed in the absence of known enteric pathogens. Since HIV type 1 (HIV-1) replication can occur in both CD4+ and CD8+ lymphocytes, we hypothesized that this benefit might be due to local effects on HIV-1 replication in these two T-cell subsets. As a model, we studied the effects of two synthetic molecules, SRIH 1-14 and SRIH 1-28, closely related to naturally occurring forms of somatostatin, as well as SMS 201-995 on HIV-1 replication in CD4+ and CD8+ cells derived from peripheral blood mononuclear cells (PBMC). We found that HIV-1 replication was inhibited in CD8+ cells but enhanced in infected CD4+ lymphocytes, as measured by p24 antigen levels in culture fluids. These differential effects were drug concentration dependent. We also observed that somatostatin inhibited the mitogen-induced proliferative responsiveness of both cell types. These effects on both HIV-1 replication and cell proliferation were independent of somatostatin gene expression, since somatostatin mRNAs were not detected in mitogen-stimulated PBMC, as determined by reverse transcription-PCR.
Flow cytometric analyses of the lymphocyte subsets in peripheral blood of children with untreated active juvenile dermatomyositis
Authors: O'Gorman M R , Corrochano V , Roleck J , Donovan M , Pachman L M
Published: January 2020

Juvenile dermatomyositis (JDMS) is a vasculopathy affecting primarily skin and muscle. Although the etiology is unknown, immunopathogenetic mechanisms appear to play a role in both the susceptibility to the disease and its progression. We measured the percentage and absolute numbers of B cells and T-cell subsets in the peripheral blood of untreated JDMS patients with active early disease and compared the results with those obtained from a study of peripheral blood obtained from a heatlhy age-related control group. The absolute number of total lymphocytes in the peripheral blood of the JDMS patients was significantly lower (P < 0.002) than that observed in the healthy control population, with an associated decrease in the absolute number of all T-cell subsets. No concomitant decrease in the absolute number of B lymphocytes was observed in the JDMS patients. In contrast, the percentage of B lymphocytes and the T-helper/T-suppressor cell ratio were significantly higher in the JDMS group than in the control group (P < 0.001 and P < 0.002, respectively). Retrospective analysis of JDMS patients' serum samples obtained within 1 month of the flow cytometric evaluation indicated that 79% of the sera contained an antinuclear antibody and 46% had immunoglobulin G values above age-adjusted reference ranges. The increased percentage of B cells, the increased T-helper/T-suppressor cell ratio, the positive antinuclear antibody results, and the increased concentration of serum immunoglobulin suggest that humoral immune dysregulation may contribute to the pathogenesis of JDMS.