Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates.

A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen. MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M. bovis, and M. kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb. A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M. tuberculosis clinical isolates. Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots. This suggests differences in PGL-Tb antigen production among M. tuberculosis strains even when they are grown in the same culture media and conditions. This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens. This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M. tuberculosis clinical isolates.     

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region

Summary Rabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80–103 but not in those to HTLV-II Env gp46, 171–196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80–103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1×10⁷ cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.     

Localization of efferent neurons innervating the pharyngeal constrictor muscles and the cervical esophagus muscle in the cat by means of the horseradish peroxidase method

Following horseradish peroxidase (HRP) injection into the cephalopharyngeal muscle (CeP), the hyopharyngeal muscle (HP), the thyropharyngeal muscle (TP), the cricopharyngeal muscle (CP) and the cervical esophagus muscle (CE) of the cat, labeled neurons were identified in the nucleus retrofacialis and the rostral part of the nucleus ambiguus ipsilaterally. The rostral end of the labeled cell column was located more rostrally for CeP and HP than for TP, CP and CE. No difference was noted within the former two or within the latter three. The level of the caudal end of the labeled cell column became more caudal in the order of CeP, HP, TP and CP. The caudal end was located more rostral for CE than for TP. The neurons for CE were located more ventro-laterally than those for the other muscles.     

Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation.

Pulmonary dysfunction is a significant complication following allogeneic hematopoietic stem cell transplantation (HSCT), and is associated with significant morbidity and mortality. Effective antimicrobial prophylaxis and treatment strategies have increased the incidence of noninfectious lung injury, which can occur in the early posttransplant period or in the months and years that follow. Late-onset noninfectious pulmonary complications are frequently encountered, but diagnostic criteria and terminology for these disorders can be confusing and therapeutic approaches are suboptimal. As a consequence, inaccurate diagnosis of these conditions may hamper the appropriate data collection, enrollment into clinical trials, and appropriate patient care. The purpose of this review is to clarify the pathogenesis and diagnostic criteria of representative conditions, such as bronchiolitis obliterans syndrome and bronchiolitis obliterans organizing pneumonia, and to discuss the appropriate diagnostic strategies and treatment options.     

A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering

Microporous, non-woven poly( epsilon -caprolactone) (PCL) scaffolds were made by electrostatic fiber spinning. In this process, polymer fibers with diameters down to the nanometer range, or nanofibers, are formed by subjecting a fluid jet to a high electric field. Mesenchymal stem cells (MSCs) derived from the bone marrow of neonatal rats were cultured, expanded and seeded on electrospun PCL scaffolds. The cell-polymer constructs were cultured with osteogenic supplements under dynamic culture conditions for up to 4 weeks. The cell-polymer constructs maintained the size and shape of the original scaffolds. Scanning electron microscopy (SEM), histological and immunohistochemical examinations were performed. Penetration of cells and abundant extracellular matrix were observed in the cell-polymer constructs after 1 week. SEM showed that the surfaces of the cell-polymer constructs were covered with cell multilayers at 4 weeks. In addition, mineralization and type I collagen were observed at 4 weeks. This suggests that electrospun PCL is a potential candidate scaffold for bone tissue engineering.     

Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay

The first step of enzyme linked immunosorbent assay (ELISA), namely, adsorption of antigen or antibody to the plastic microtiter well plate, was studied as a function of insolubility of IgG in water. Immobilization efficiency was assessed in terms of number of wells coated per milliliter of primary antiserum. We have compared different coating/immobilization protocols, i.e., direct and indirect immobilization of primary antibody to the plastic microtiter well plate using carbonate buffer and phosphate buffer with glutaraldehyde. We have observed efficient coating when the immobilization of primary antibody through an immunobridge technique was performed, where water was used as a coating medium. It gave a higher number of wells coated per milliliter of anti-serum (primary or secondary) than other compared coating protocols and it allowed the use of serum (non-immune) and anti-serum (primary and secondary antibody) dilutions, avoiding the need for gamma-globulin purification from normal and immunized serum.     

Role of the Nfa1 Protein in Pathogenic Naegleria fowleri Cocultured with CHO Target Cells

Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.     

Association analyses of DNA methyltransferase-1 (<Emphasis Type="Italic">DNMT1</Emphasis>) polymorphisms with systemic lupus erythematosus

The etiology of systemic lupus erythematosus (SLE) is very complex, and genetic factors appear to play a significant role in susceptibility to SLE, in determining the disease expression, and in the autoantibody profiles of individuals with SLE. DNA methyltransferase-1 (DNMT1) is a major enzyme that determines genomic methylation patterns and both maintains methyltransferase and exhibits de novo DNA methylation activity in vivo. In order to clarify the association of DNMT1 polymorphisms with SLE, we scrutinized the genetic polymorphisms in exons and their boundaries of DNMT1, including the –1,500 bp promoter region, by direct sequencing in 24 Korean individuals. Twenty-nine sequence variants were identified: two in 5UTR, six in exons, and 21 in introns. Eight of these polymorphisms were selected for a larger-scale genotyping (n=680) by considering their allele frequencies, haplotype-tagging status, and linkage disequilibrium coefficiencies (LDs) among polymorphisms. The associations between DNMT1 polymorphisms and the clinical profiles of SLE were analyzed. No significant associations with the risk of SLE were detected. However, further analyses of association with autoantibody production among SLE patients revealed that one nonsynonymous SNP, +14463G>C (V120L) in exon 4, was weakly associated with an increased risk of anti-La antibody production (P=0.04), although the significance could not be retained after correction of multiple tests. The DNMT1 variations and haplotypes clarified in this study would provide valuable information for future genetic studies of other autoimmune diseases.     

Diagnostic and public health dilemma of lactose-fermenting Salmonella enterica serotype Typhimurium in cattle in the Northeastern United States

The presence of lactose-fermenting Salmonella strains in clinical case materials presented to microbiology laboratories presents problems in detection and identification. Failure to detect these strains also presents a public health problem. The laboratory methods used in detecting lactose-fermenting Salmonella enterica serotype Typhimurium from six outbreaks of salmonellosis in veal calves are described. Each outbreak was caused by a multiply-resistant and lactose-fermenting strain of S. enterica serotype Typhimurium. The use of Levine eosin-methylene blue agar in combination with screening of suspect colonies for C8 esterase enzyme and inoculation of colonies into sulfide-indole-motility medium for hydrogen sulfide production was particularly effective for their detection. A hypothesis for the creation of lactose-fermenting salmonellae in the environment is presented. It is proposed that the environment and husbandry practices of veal-raising barns provide a unique niche in which lactose-fermenting salmonellae may arise.     

The reliability and validity of Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime Version- Korean version (K-SADS-PL-K)

In order to develop a structured and objective diagnostic instrument, authors completed: (1) the translation and back translation of the Korean version of the Kiddie-Schedule for Affective Disorders and Schizophrenia - Present and Lifetime Version (K-SADS-PL) and (2) the examination of its validity and reliability of the K-SADS-PL-Korean version (K-SADS- PL) when used with Korean children. A total of 91 study subjects were recruited from child and adolescent psychiatry outpatient clinics. Clinical diagnoses were used as a gold standard for the examination of validity of K-SADS-PL-K. Consensual validity of threshold and sub-threshold diagnoses were good to excellent for attention-deficit/hyperactivity disorder (ADHD), fair for tic and oppositional defiant disorders, and poor to fair for anxiety and depressive disorders. Inter-rater and test-retest reliabilities were fair to excellent for ADHD and tic disorder. The significant correlations between the K-SADS-PL-K and Korean Child Behavior Checklist (K-CBCL) were found, which provided additional support for the concurrent validity of the K-SADS-PL-K. Sensitivities varied according to the diagnostic categories, but specificities remained high over all diagnoses, suggesting that the K-SADS-PL-K is a desirable confirmatory diagnostic tool. The results of this study suggest that the K-SADS-PL-K is an effective instrument for diagnosing major child psychiatric disorders, including ADHD, behavioral disorders and tic disorders in Korean children. Future studies will examine the validity and reliability of the K-SADS-PL-K in larger samples, including adolescents and community samples on a variety of child and adolescent psychiatric disorders.