bluuebunny/crossref_metadata_2025 huggingface dataset:
Technical Basis for Flaw Acceptance Criteria for Cast Austenitic Stainless Steel Piping
Authors: Shim Do Jun , Cofie Nathanial , Dedhia Dilipkumar , Griesbach Tim , Amberge Kyle
Published: December 2019

According to the current ASME Code Section XI, IWB-3640 and Appendix C flaw evaluation procedure, cast austenitic stainless steel (CASS) piping with ferrite content (FC) less than 20% is treated as wrought stainless steel. For CASS piping with FC equal or greater than 20%, there was no flaw evaluation procedure in the ASME Code prior to the 2019 Edition. In this paper, the technical basis for the recently approved Code change containing flaw acceptance criteria for CASS piping is presented. The procedure utilizes the current rules in ASME Code Section XI, IWB 3640/Appendix C and the existing elastic-plastic correction factors (i.e., Z-factors) for other materials in the Code. The appropriate Z-factor to use for the CASS piping is determined based on the FC (using Hull's equivalent factor). Experimentally measured fully saturated fracture toughness and tensile data of the three most common grades of CASS material in the U.S. (CF3, CF8, and CF8M) were used to determine the flaw acceptance criteria in the Appendix C Code method. As described here, the method is conservative since it utilizes the fully saturated condition of CASS materials. In addition, it is simple and consistent with the current regulatory guidance on aging management of CASS piping.
Experimental Study of Flow Structure in Converging–Diverging Axisymmetric Geometry
Authors: Volent Eirik , Sagmo Kristian , Solemslie Bjørn Winther , Dahlhaug Ole Gunnar
Published: December 2019

Control valves, nozzles, and other mechanical flow constricting constructions often introduce highly turbulent flow downstream of vena contracta. When solid particles are present in the fluid stream erosion can occur due to the high fluid velocity and turbulence, both in the converging and diverging section of a constriction. This paper presents an experimental study of such flow structure in a converging–diverging valve geometry. The purpose of the study is to obtain velocity profiles downstream of vena contracta by the use of particle image velocimetry measurements. Profiles and flow structure, with and without introduced swirl component, is investigated and discussed with respect to erosion in such specific geometries.
Near Wake Development Behind Marine Propeller Model in Presence of Freestream Turbulence
Authors: Hermsen Bennitt L. , Bornemeier Matthew , Luznik Luksa
Published: December 2019

Three-dimensional particle image velocimetry (PIV) experiments were conducted in the immediate near wake and up to seven diameters downstream of a three-bladed marine propeller model operating in two different inflow conditions: one with imposed freestream turbulence with intensity of 7% and streamwise integral length scale comparable to propeller geometry, and the second experiment with a quiescent inflow conditions as a reference. The resulting Reynolds number based on propeller chord and relative velocity is Re0.7R = 4.7 × 105. All components of radial transport of mean flow kinetic energy are analyzed and the largest contributor to the fluxes is found to be correlated to Reynolds shear stresses, resulting in radially outward flux in the wake. Two regions of the near wake are distinguishable with downstream extent dependent on the level of external turbulence. In the first region, immediately behind the propeller, shed tip vortices are very coherent and undergo grouping and roll-up around each other and the second region where the vortex merger process is complete and characterized by breakdown of vortices into small-scale turbulence. The latter region was found to occur earlier in the experiment with external turbulence. Conditional statistics of velocity fluctuations were employed and they show that outward interactions and sweep events contribute the most to the transfer of mean flow kinetic energy from the inner wake to the freestream.
Tarsocheylidae, a newly recorded family with description of a new species from China

Authors: Xu Yun , Zhu Yu-zhen , Wu Jie-Qin , Zhang Fei-Ping
Published: December 2019

A new species of the genus Hoplocheylus Atyeo and Baker (Acariformes: Heterostigmata: Tarsocheylidae), H. lindquisti Xu & Zhang sp. nov., is described based on larva, deutonymph, adult male and female specimens collected from the bark of dead Pinus massoniana Lamb. infested with Monochamus alternatus (Coleoptera: Cerambycidae) in Fuzhou city, China. This is the first report of the genus Hoplocheylus and the family Tarsocheylidae in China.
An order-aware scheme for robust direction of arrival estimation in the spherical harmonic domain
Authors: Gao Weixia , Chen Huawei
Published: December 2019

Direction of arrival (DOA) estimation of sound sources using a spherical microphone array is usually performed in the spherical harmonic (SH) domain. In a non-noisy environment, it suffices to use only the zeroth- and first-order spherical harmonic beams (SHBs) in the SH domain for DOA estimation. One such method is based on the pseudo-intensity vector (PIV), which is attractive due to its low computational complexity. To improve the performance of the PIV method in reverberant environments, some methods have been proposed recently to further exploit high-order SHBs. However, these methods ignore the effect of noise on high-order SHBs, which may lead to poor performance in low signal-to-noise ratio (SNR) environments. To address the problem, this paper proposes an order-aware scheme that is able to select the high-order SHBs reliable for robust DOA estimation of multiple speech sources. Simulation and real-world experimental results demonstrate that the order-aware scheme based methods outperform their existing counterparts with less computational complexity in terms of both accuracy and robustness of DOA estimation. Moreover, the performance improvement is more significant in low SNR environment and in a scenario with small angular separation of sources.
Coaching Positive Youth Development in Brazil: Recommendations for Coach Education Programs
Authors: Gaion Patricia , Milistetd Michel , Santos Fernando , Contreira Andressa , Arantes Luciane , Caruzzo Nayara
Published: December 2019

Coaching positive youth development (PYD) represents a challenge for many participation and high-performance coaches across the globe, including in Brazil. Coach education has been acknowledged as a formal learning context that may help prepare coaches to effectively foster PYD outcomes and provide high-quality developmental experiences for athletes across different sport contexts. Therefore, the purpose of this paper is to describe the key concepts and existing guidelines for coaching and coach education in Brazil, and provide context-specific recommendations for coach education to include PYD materials. Coaching in Brazil includes a long preparation period that includes diversified opportunities for coach learning. However, there are some discrepancies between the objectives and outcomes prioritized by governing bodies and sport organizations and how learning contexts are framed. In other words, although PYD is considered to be a necessary endeavor, it is not explicitly included in any coach education program. Moving forward, we provide several recommendations, through a bottom-up approach, in order to embed PYD within the Brazilian sport system.
What the Empirical Studies on Sport Coach Education Programs in Higher Education Have to Reveal: A Review
Authors: Trudel Pierre , Milestetd Michel , Culver Diane M.
Published: December 2019

Facing the increase in the number of publications, often spread across many journals, researchers have developed different approaches to review the literature. In the first part of this article we present the result of an “overview” type of review of literature on sport coach education programs in higher education (HE) between 2000–2018. By sharing the list of the 38 articles found, along with some general characteristics of these, our hope is, as suggested in many reviews of literature, that researchers will use the review to more easily frame their studies and discuss their results. However, there are very few examples of how this is done. We would argue that how researchers use the same review of literature varies depending of their context, research interests, and research paradigm. Therefore, in the second part of the article we highlight what we took from our review of the literature based on our specific research context—Brazilian HE sport coach education programs. The three key topics presented and discussed are: (a) the importance of considering the student-coaches’ biography, (b) how to prepare student-coaches for reflective practice, and (c) the complexity of internships.
Effects of lysine analogs on Penicillium chrysogenum
Authors: Friedrich C G , Demain A L
Published: December 2019

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.
trans
-Monounsaturated Acids in a Marine Bacterial Isolate
Authors: Gillan F. T. , Johns R. B. , Verheyen T. V. , Volkman J. K. , Bavor H. J.
Published: December 2019

A sedimentary bacterial isolate has been shown to contain
trans
-monounsaturated fatty acids (6% of the total fatty acids). The ratio of
trans
- to
cis
-acids in this isolate was in the range 3.2 to 7.6. The identification of
trans
-monounsaturated acids in a marine bacterium implied that the
trans
-acids which have been reported in recent sediments could derive, in whole or part, from direct bacterial input.
Effect of Soybean Casein Digest Agar Lot on Number of
Bacillus stearothermophilus
Spores Recovered
Authors: Pflug I. J. , Smith Geraldine M. , Christensen Ronald
Published: December 2019

In recent years it has become increasingly apparent that
Bacillus stearothermophilus
spores are affected by various environmental factors that influence the performance of the spores as biological indicators. One environmental factor is the recovery medium. The effect of different lots of commercial soybean casein digest agar on the number of colony-forming units per plate was examined in two series of experiments: (i) several lots of medium from two manufacturers were compared in single experiments, and (ii) paired media experiments with four lots of medium were carried out and yielded three-point survivor curves. The results demonstrate that commercial soybean casein digest agar is variable on a lot-to-lot basis. The variation was lowest when recovering unheated or minimally heated spores and increased greatly with the severity of heating.
Metabolism of ochratoxin A by rats
Authors: Støren O , Holm H , Størmer F C
Published: December 2019

Albino rats were given ochratoxin A (6.6 mg/kg body weight) intraperitoneally or per os. Independent of route administration, 6% of a given dose was excreted as the toxin, 1 to 1.5% as (4R)-4-hydroxyochratoxin A, and 25 to 27% as ochratoxin alpha in the urine. The metabolite (4S)-4-hydroxyochratoxin A, which is formed by rat liver microsomes in the presence of NADPH, was not detected. Only traces of ochratoxins A and alpha were found in feces. Identical experiments were carried out with brown rats, since the Km value for the formation of the 4S epimer was considerably lower when brown rat microsomes were used. About the same ratios of metabolites and metabolite recoveries as those found for albino rats were found for brown rats. Brown rats were also given the two hydroxylated metabolites and ochratoxin alpha (0.66 mg/kg body weight) intraperitoneally. The three compounds were excreted in the urine; within 48 h, 90% recovery of ochratoxin alpha and 54 and 35%, respectively, of the 4R and 4S isomers were observed.
Lipoteichoic acids in Lactobacillus strains that colonize the mouse gastric epithelium
Authors: Sherman L A , Savage D C
Published: December 2019

Extracts of cells of 22 strains of Lactobacillus species, and of fluids from cultures in a defined medium of two of the strains, were assayed by hemagglutination inhibition for lipoteichoic acids. A total of 10 of the preparations gave positive tests for the acids, including those from eight strains known to adhere to the keratinized squamous epithelium of the mouse stomach. These findings are consistent with an hypothesis that lactobacilli colonize the epithelial surface via adhesive interaction with the keratinized cells mediated by macromolecular complexes containing lipoteichoic acids.
Aerobic Metabolism of Trichloroethylene by a Bacterial Isolate
Authors: Nelson Michael J. K. , Montgomery S. O. , O'Neill E. J. , Pritchard P. H.
Published: December 2019

A number of soil and water samples were screened for the biological capacity to metabolize trichloroethylene. One water sample was found to contain this capacity, and a gram-negative, rod-shaped bacterium which appeared to be responsible for the metabolic activity was isolated from this sample. The isolate degraded trichloroethylene to CO
2
and unidentified, nonvolatile products. Oxygen and water from the original site of isolation were required for degradation.
Measuring genetic stability in bacteria of potential use in genetic engineering
Authors: Walter M V , Porteous A , Seidler R J
Published: December 2019

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.
Incidence of coliphage in potable water supplies
Authors: el-Abagy M M , Dutka B J , Kamel M , el Zanfaly H T
Published: December 2019

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.
Variation in Microbial Biomass and Community Structure in Sediments of Eutrophic Bays as Determined by Phospholipid Ester-Linked Fatty Acids
Authors: Rajendran Narasimmalu , Matsuda Osamu , Imamura Norifumi , Urushigawa Yoshikuni
Published: December 2019

The distribution of phospholipid ester-linked fatty acids (PLFA) in sediments of eutrophic bays (Hiroshima Bay and Aki Nada) was studied to quantify the microbial biomass, community structure, and nutritional status. A total of 63 fatty acids in the range of C
10
to C
24
were determined. They consist of saturated fatty acids, branched fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, and variation was revealed in the relative proportions of these fatty acids in sediments. On the basis of the PLFA concentration in sediments, the calculated microbial biomass showed variation (mean ± standard deviation = 0.70 × 10
8
± 0.53 × 10
8
cells per g [dry weight] of sediment) in the eutrophic bays. In sediments, a higher amount of biomass was observed in the coastal area of Hiroshima Bay than that observed in the rest of the bay and adjacent Aki Nada. The microbial community structure of the present study area, as characterized by the PLFA profiles, showed very low percentages of polyunsaturated fatty acids and long-chain fatty acids characteristic of microeukary-otes and terrestrial input, respectively, and high percentages of fatty acids characteristic of bacteria. The distribution of PLFA profiles also showed the relative contribution of both aerobic and anaerobic bacteria, especially sulfate-reducing bacteria, in the study area. The relative proportions of PLFA revealed distinctive differences among the stations of the study area, as is evidenced from six clusters obtained for the PLFA profiles. The results of Tukey's honestly significant difference test further confirmed that the sediments in the coastal area of Hiroshima Bay were significantly enriched by a number of fatty acids when compared with other areas investigated where relatively few fatty acids were present in significant quantities. No marked variation in environmental parameters in the surface- and bottom-water samples was observed, indicating the absence of any water movement in the study area. Furthermore, low redox potential and the levels of sulfide in the sediment revealed the reduced condition of the sediment. The existing environmental conditions and pollution of the study area were attributed to the observed microbial community structure in the sediments.
A Novel Approach for Detecting an Immunodominant Antigen ofPorphyromonas gingivalisin Diagnosis of Adult Periodontitis
Authors: Kawai Toshihiasa , Ito Hiro-O , Sakato Nobuo , Okada Hiroshi
Published: December 2019

In the course of long-term infection withPorphyromonas gingivalisin adult periodontitis, a specific antibody response to this organism is generated. We describe a potential novel approach for identifying an immunodominant antigen in human periodontitis patients. First, various monoclonal antibodies (MAbs) were established from mice immunized with crude antigen preparations ofP. gingivalisFDC 381. The antigen specificities of these MAbs were compared with those of serum antibodies of 10 periodontitis patients in a competitive enzyme-linked immunosorbent assay. The binding of one MAb (termed PF18) was readily inhibited by sera from all patients but not by sera from healthy volunteers. The antigen recognized by PF18 existed on the cell surface, presumably in the capsule layer, shown by immunoelectron microscopic analysis. Purification of the antigenic substance, termed PF18-Ag, was performed by immunoaffinity chromatography with the MAb. Characterization of PF18-Ag suggested that the epitope was composed of carbohydrates but not peptides and that the substance was different from lipopolysaccharide. Measurement of levels of serum antibody to PF18-Ag better discriminated periodontitis patients from healthy individuals than measurement of antibodies to crude antigen preparations ofP. gingivalis. Immunoglobulin G2 was the predominant isotype among the antibodies to PF18-Ag in the patients’ sera. These results suggest that PF18-Ag, which is possibly a novel substance, is an important antigenic substance and is potentially useful for the clinical diagnosis of adult periodontitis. The approach that was used would also be relevant to detecting immunodominant antigens of other infectious microorganisms.
Age-Stratified Seroprevalence of Neutralizing Antibodies to Astrovirus Types 1 to 7 in Humans in The Netherlands
Authors: Koopmans M. P. G. , Bijen M. H. L. , Monroe S. S. , Vinjé J.
Published: December 2019

Astroviruses are a new family of positive-stranded RNA viruses that cause gastroenteritis in a wide range of animals and in humans. Seven types of astrovirus, tentatively considered serotypes, have been distinguished by enzyme-linked immunosorbent assays (ELISA) or immunoelectron microscopy, but it is unclear whether the serotype designation is used properly. To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests (NT) using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed. In rabbits, neutralizing antibodies were predominantly serotype specific, with the exception of low-level cross-reactivity in astrovirus serotype 4 reference serum with astrovirus serotype 1 virus. Similarly, in humans, no evidence of cross-reactivity was found for the serotype combinations tested (all except the combination 1 and 7 and the combination 6 and 7). Typing by NT was concordant with typing by ELISA and genotyping, with one exception. The seroprevalence rates of neutralizing antibodies in an age-stratified sample of the population in Utrecht Province (
n
= 242) were 91% for astrovirus serotype 1, 69% for astrovirus serotype 3, 56% for astrovirus serotype 4, 36% for astrovirus serotype 5, 31% for astrovirus serotype 2, 16% for astrovirus serotype 6, and 10% for astrovirus serotype 7. Acquisition of antibodies was slower among persons seropositive for astrovirus serotype 5 than among those seropositive for astrovirus serotypes 1 to 4, suggesting that the epidemiology of serotype 5 astrovirus is different from that of astrovirus serotypes 1 to 4.
Chemotactic Activities in Nonmastitic and Mastitic Mammary Secretions: Presence of Interleukin-8 in Mastitic but Not Nonmastitic Secretions
Authors: Barber Michele R. , Yang T. J.
Published: December 2019

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.
Reactivity of Sera from Systemic Lupus Erythematosus and Sjögren’s Syndrome Patients with Peptides Derived from Human Immunodeficiency Virus p24 Capsid Antigen
Authors: Deas Jane E. , Liu Leonita G. , Thompson James J. , Sander David M. , Soble Sara S. , Garry Robert F. , Gallaher William R.
Published: December 2019

We have previously demonstrated that about one-third of patients with either Sjögren’s syndrome (SS) or systemic lupus erythematosus (SLE) react to human immunodeficiency virus (HIV) p24 core protein antigen without any evidence of exposure to, or infection with, HIV itself. Herein, we further characterize the specificity of this reaction using enzyme-linked immunosorbent assay to peptides representing fragments of p24. Characteristic epitope-specific profiles were seen for SS and SLE patients. SS patients had significantly increased responses to peptides F (p24 amino acids 69 to 86) and H (amino acids 101 to 111) and diminished reactivity to peptides A (amino acids 1 to 16) and P (amino acids 214 to 228). SLE patients had increased reactivity to peptides E (amino acids 61 to 76), H, and P. Utilization of peptide P hyporeactivity as the criterion to select for SS patients results in a screen that is moderately sensitive (64%) and specific (79.3%). Adding hyperreactivity to one other peptide (F or H) as an additional criterion yields an expected decrease in sensitivity (to 41%) while increasing specificity (to 93.1%). All sera-reactive peptides from regions of known structure of HIV p24 were located in the apex of the p24 molecule. Thus, the specificity of the peptide reactivities described here indicates a specific pattern of a nonrandom cross-reactivity between HIV type 1 p24 and autoimmune sera which may be partially syndrome specific. The future focus of our work will be to optimize assays of the peptide as diagnostic tools.
Association of Low Concentrations of Serum Mannose-Binding Protein with Recurrent Infections in Adults
Authors: Kakkanaiah Vellalore N. , Shen Guo Qiu , Ojo-Amaize Emmanuel A. , Peter James B.
Published: December 2019

Low concentrations of mannose-binding protein (MBP; also known as mannose-binding lectin) are associated with common opsonic defect in immunodeficient children. We compared the concentrations of MBP in the sera of 47 adults with non-human immunodeficiency virus-related recurrent infections (group I) and 50 healthy adult controls. Mean serum MBP concentrations in the patient group did not differ significantly from those in the control group (
P
< 0.4). Nevertheless, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in the patient group (21%,
P
= 0.01) than in the control group (4%). Group II consisted of 73 pediatric and 56 adult patients with recurrent infections. Pediatric patients had significantly lower mean concentrations of serum MBP than their controls (
P
< 0.005), and there was no significant difference between the concentrations in sera of adult patients and adult controls (
P
< 0.4). Again, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in both pediatric (22%,
P
= 0.045) and adult (38%,
P
= 0.000016) patients than in their respective controls (4%). Our results demonstrate that, as in children, low concentrations of serum MBP can be associated with recurrent infections in adults.
The Polysaccharide Fucoidin Inhibits the Antibiotic-Induced Inflammatory Cascade in Experimental Pneumococcal Meningitis
Authors: Granert Carl , Raud Johan , Lindquist Lars
Published: December 2019

There is evidence that the treatment of bacterial meningitis with antibiotics liberates harmful bacterial products in the subarachnoid space (SAS). This enhances meningeal inflammation and in particular the recruitment of leukocytes into the cerebrospinal fluid (CSF), which has been shown to be more harmful than beneficial in this disease. In this study, we used a rabbit meningitis model based on intracisternal injection of live
Streptococcus pneumoniae
. Ampicillin (40 mg/kg of body weight given intravenously [i.v.] 16 h after induction of meningitis) caused a fivefold increase in CSF leukocytes over a 4-h period. Inhibition of leukocyte rolling by treatment with the polysaccharide fucoidin (10 mg/kg, i.v.) prevented the enhanced leukocyte extravasation into the SAS and attenuated the leakage of plasma proteins over the blood-brain barrier. These results suggest that certain polysaccharides that block leukocyte rolling have the potential to reduce leukocyte-dependent central nervous system damage in bacterial meningitis.
Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Monocytic Cells Stimulated with Lipopolysaccharide of Oral Microorganisms
Authors: Baqui A. A. M. A. , Meiller Timothy F. , Chon Jennifer J. , Turng Been-Foo , Falkler William A.
Published: December 2019

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1β and TNF-α production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms,Porphyromonas gingivalisandFusobacterium nucleatum. LPS ofP. gingivalisorF. nucleatumwas prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS ofP. gingivalisorF. nucleatumand/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1β and TNF-α in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1β was not detected in untreated THP-1 cells. IL-1β production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1β production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1β-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1β mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1β transcriptional activation time. GM-CSF plusF. nucleatumorP. gingivalisLPS activated THP-1 cells to produce a 1.6-fold increase in TNF-α production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1β and TNF-α.
Inducible Nitric Oxide Synthase and Nitrotyrosine Are Found in Monocytes/Macrophages and/or Astrocytes in Acute, but Not in Chronic, Multiple Sclerosis
Authors: Oleszak Emilia L. , Zaczynska Ewa , Bhattacharjee Meena , Butunoi Catalin , Legido Agustin , Katsetos Christos D.
Published: December 2019

We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.
Inhibition ofMycobacterium bovisBCG-Induced Tumor Necrosis Factor Alpha Secretion in Human Cells by Transforming Growth Factor β
Authors: Méndez-Samperio Patricia , Hernandez-Garay Marisol , Vazquez Angela Nuñez
Published: December 2019

The effect of exogenous transforming growth factor β (TGF-β) onMycobacterium bovisBCG-induced tumor necrosis factor alpha (TNF-α) production by human mononuclear cells was studied. It was found that TNF-α production by human cells stimulated with BCG was significantly inhibited by TGF-β. The specificity of the observed inhibition was demonstrated, since the addition of an anti-TGF-β neutralizing monoclonal antibody completely reversed the inhibitory effect. Furthermore, the suppressive effect of TGF-β on TNF-α secretion in this system was not due to a direct cytotoxic effect, since cell viability was comparable in the presence or absence of TGF-β. Interestingly, our results demonstrated comparative suppressive effects of TGF-β and interleukin-10 on BCG-induced TNF-α secretion. Together, the data demonstrate, for the first time, that TGF-β inhibits BCG-induced TNF-α secretion by human cells.
Expression of the Extracellular Domain of the Human Immunodeficiency Virus Type 1 Envelope Protein and Its Fusion with β-Galactosidase in
Saccharomyces cerevisiae
Authors: Liu Wei-Feng , Gao Dong , Wang Zu-Nong
Published: December 2019

Two envelope glycoprotein gene fragments were cloned from the proviral genome of the HXB2 isolate of human immunodeficiency virus (HIV). For the production of the two domains of the envelope gene product these cloned gene fragments were inserted into an
Escherichia coli
-yeast inducible shuttle vector fused to the galactokinase (GAL1) promoter. Cell extracts from strains of
Saccharomyces cerevisiae
harboring these two vectors (pYENV1 and pYENV2) were found to contain a specific protein with a size of 50 kDa when induced by galactose, while the protein could not be detected in extracts from control cells containing only the
E. coli
-yeast vector in the presence of galactose. Furthermore, another expression plasmid coding for fusion proteins from the majority of the external envelope glycoprotein (gp120) moiety and a large part of the β-galactosidase was constructed. Antibodies from HIV type 1-positive sera could react with recombinant fusion polypeptides. Transformants could produce this fusion protein to a level of about 1.6% of the total protein content, as deduced from β-galactosidase activity.
Use of Highly Encapsulated
Streptococcus pneumoniae
Strains in a Flow-Cytometric Assay for Assessment of the Phagocytic Capacity of Serotype-Specific Antibodies
Authors: Jansen W. T. M. , Gootjes J. , Zelle M. , Madore D. V. , Verhoef J. , Snippe H. , Verheul A. F. M.
Published: December 2019

A phagocytosis assay for
Streptococcus pneumoniae
based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of
S. pneumoniae
grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (
r
= 0.98,
P
< 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 μl per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.
Immune Response against the Exp-1 Protein of
Plasmodium falciparum
Results in Antibodies That Cross-React with Human T-Cell Lymphotropic Virus Type 1 Proteins
Authors: Porter Kevin R. , Aguiar Joao , Richards Allen , Sandjaya B. , Ignatias H. , Hadiputranto H. , Ridley Robert G. , Takacs Bela , Wignall F. Stephen , Hoffman Stephen L. , Hayes Curtis G.
Published: December 2019

To examine the role of the
Plasmodium falciparum
Exp-1 blood-stage protein in producing antibodies that cross-react with human T-cell lymphotropic virus type I (HTLV-I) proteins, we studied sera from Indonesian volunteers who seroconverted to malaria after transmigrating to an area where malaria is hyperendemic. Samples from Philippine volunteers, that were used in a prior study that examined malaria antibodies that cross-react with HTLV-I proteins, were also used. Eighty-three percent of the Indonesian transmigrants developed antibodies against the malaria Exp-1 protein by 6 months postmigration. Of these malaria seroconverters, 27% developed false-positive HTLV-I enzyme immunoassay (EIA) immunoreactivity, as indicated by indeterminate HTLV-I Western blot banding patterns. Five of the six Philippine samples tested were HTLV-I EIA false positive and Western blot indeterminate. When a recombinant Exp-1 protein was used in blocking experiments, the HTLV-I Western blot immunoreactivity of sera from both groups was either completely eliminated or greatly reduced. No effect on the Western blot immunoreactivity of truly HTLV-I-positive sera was seen. To determine if immunization with the recombinant Exp-1 protein could elicit the production of HTLV-I antibodies, six mice were inoculated with the recombinant protein. Following administration of three 50-μg doses of the protein, four of the six mice developed antibodies that cross-reacted with HTLV-I proteins on Western blot. These results indicate that the immune response against the malaria Exp-1 protein may result in HTLV-I-cross-reacting antibodies that can lead to false-positive EIA and indeterminant Western blotting results.
A Serotyping Assay for Hepatitis C Virus in Southeast Asia
Authors: Songsivilai Sirirurg , Kanistanon Duangjit , Dharakul Tararaj
Published: December 2019

A serotyping assay for hepatitis C virus (HCV) was evaluated with samples from Thailand, where the distribution of HCV genotypes was different from that in Western countries where the assay was designed and validated. The sensitivity of the assay was low (58%) for HCV RNA-positive samples compared to that of the genotyping assay (95%,
P
< 0.01). In addition, only 36% of anti-HCV-positive but HCV RNA-negative samples could be serotyped. The serotypes and genotypes were identical in 96% of the samples that could be typed by both methods. Most of the samples with genotype 6, which was common in Southeast Asia, were nontypeable by this serotyping assay.
Analysis of Mumps Vaccine Failure by Means of Avidity Testing for Mumps Virus-Specific Immunoglobulin G
Authors: Narita Mitsuo , Matsuzono Yoshihiro , Takekoshi Yasuo , Yamada Satoshi , Itakura Osamu , Kubota Mitsuru , Kikuta Hideaki , Togashi Takehiro
Published: December 2019

To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution–6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of ≦31% was determined to be low, and ≧32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.
Use of the 27-Kilodalton Recombinant Protein fromParacoccidioides brasiliensisin Serodiagnosis of Paracoccidioidomycosis
Authors: Ortiz B. L. , Díez S. , Urán M. E. , Rivas J. M. , Romero M. , Caicedo V. , Restrepo A. , McEwen J. G.
Published: December 2019

Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent,Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation ofEscherichia coliDH5α, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 μg/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic diagnosis of PCM and, by so doing, achieve high coverage rates. Furthermore, antigen reproducibility can now be ensured, thus facilitating inter- and intralaboratory standardization.
Rotavirus G-Type Restriction, Persistence, and Herd Type Specificity in Swedish Cattle Herds
Authors: de Verdier Klingenberg K. , Nilsson M. , Svensson L.
Published: December 2019

G-typing of rotavirus strains enables the study of molecular epidemiology and gathering of information to promote disease prevention and control. Rotavirus strains in fecal specimens from neonatal calves in Swedish cattle herds were therefore characterized by using G1 to -4-, G6-, G8-, and G10-specific primers in reverse transcription (RT)-PCR. Fecal samples were collected from one dairy herd (herd A) for 4 consecutive years and from 41 beef and dairy herds (herd B) experiencing calf diarrhea outbreaks. Altogether, 1,700 samples were analyzed by group A rotavirus enzyme-linked immunosorbent assay, and 98 rotavirus-positive specimens were selected for G-typing by RT-PCR. The effect of herd type, time, geographic region, and clinical symptoms on the G-type distribution was evaluated. Altogether (herds A and B), G10 was found in 59 (60.2%) fecal specimens, G6 was found in 30 (30.6%) specimens, G3 was found in 1 (1.0%) specimen, and G8 was found in 1 (1.0%) specimen. Seven (7.1%) fecal specimens were not typeable. Herd type specificity in the G-type distribution was demonstrated in the herds in herd B. In the 6 beef suckler herds, only G6 was detected, while rotavirus strains from the 35 dairy herds were predominantly (54%) G10. The G-type distribution was restricted in herds A and B. Twenty-nine of 30 strains from herd A were characterized as G10. In the vast majority of herds in herd B, a single G-type was identified. The serotype G10 and the electropherotype persisted over time in herd A. No characteristic G-type variation in the geographic distribution of cattle herds in herd B was obvious. There was no difference in the G-type distributions between the strains from clinically and subclinically rotavirus-infected calves in dairy herd A. The results from this study strongly indicate a pronounced stability in the rotavirus G-type distribution in Swedish cattle herds, which emphasizes the importance of continuous preventive measures for control of neonatal calf diarrhea. A future bovine rotavirus vaccine in Sweden should contain G10 and G6 strains.
Performance of Competitive and Indirect Enzyme-Linked Immunosorbent Assays, Gel Immunoprecipitation with Native Hapten Polysaccharide, and Standard Serological Tests in Diagnosis of Sheep Brucellosis
Authors: Marín C. M. , Moreno E. , Moriyón I. , Díaz R. , Blasco J. M.
Published: December 2019

Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera fromBrucella-free,Brucella melitensis-infected, andB. melitensisRev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis.