Surface markers of lymphocytes in the snake, Spalerosophis diadema. I. Investigation of lymphocyte surface markers.

A specific antiserum was raised in rabbits against thymocytes from snakes, Spalerosophis diadema, and was absorbed repeatedly with snake erythrocytes and kidney cells. In complement-dependent cytotoxicity assays, the absorbed anti-thymocyte serum (ATS) was, at any given dilution, cytotoxic to Sp. diadema thymocytes > peripheral blood lymphocytes (PBL) > spleen cells and could be titrated to a plateau defining a population of about 98% of thymocytes, 80% of PBL and 72% of spleen cells. Antiserum directed against snake immunoglobulins was obtained by injecting rabbits with gamma-globulins separated from snake serum by DEAE-cellulose filtration. the anti-gamma globulin serum was absorbed with snake erythrocytes, and in indirect membrane immunofluorescence stained no thymocytes while reacted with about 15% of PBL and 29% of spleen lymphocytes up to a 1:8 dilution. Fluorescence of positive cells was distributed in spots, patches or caps; cap formation could be inhibited by maintaining the immunofluorescence test at +4 degrees. In each of six separate experiments performed during spring, the percentage of lymphocytes which reacted with anti-snake gamma-globulin serum complemented the percentage of cells recognized by ATS. It was shown, furthermore, that about 3%, 8% and 21% of lymphocytes from thymus, peripheral blood and spleen, respectively, possess a receptor for 2-mercaptoethanol-insensitive antibody-sheep erythrocyte complexes. The results indicate that lymphocyte structural heterogeneity exists in reptiles.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Exposure to extremely low-frequency electromagnetic fields improves social recognition in male rats

The effect of exposure to low-frequency electromagnetic fields (ELF EMFs) on social recognition was studied. The test was based upon a comparison between two encounters of an adult rat and a conspecific juvenile, separated by an interexposure interval (IEI). The exposure to ELF EMF of 1 mT intensity during 2 h for 9 days increased the duration of short-term memory of adult male Wistar rats up to 300 min. These data indicate, for the first time, that ELF EMF improves social recognition memory in rats.     

Optimization of Ga-67 imaging for detection and estimation tasks: dependence of imaging performance on spectral acquisition parameters

We have compared the use of two (93 and 185 keV) and three (93, 185, and 300 keV) photopeaks for Ga-67 tumor imaging and optimized the placement of each energy window. METHODS: The bases for optimization and evaluation were ideal and Bayesian signal-to-noise ratios (SNR) for the detection of spheres embedded in a realistic anthropomorphic digital torso phantom and ideal SNR for the estimation of their size and activity concentration. Seven spheres of radii ranging from 1 to 3 cm, located at several sites in the torso, were simulated using a realistic Monte Carlo program. We also calculated the ideal SNR for the detection from simple phantom acquisitions. RESULTS: For detection and estimation tasks, the optimum windows were identical for all sphere sizes and locations. For the 93 keV photopeak, the optimal window was 84-102 keV for the detection and 87-102 keV for estimation; these windows are narrower than the 20% window often used in the clinic (83-101 keV). For the 185 keV photopeak, the optimal window was 170-220 keV for the detection and 170-215 keV for estimation; these are substantially different than the 15% window used in our clinic (171-199 keV). For the 300 keV photopeak, the optimal window for detection was 270-320 keV, and for estimation, 280-320 keV. Using the three optimized, rather than only the two lower-energy, windows yielded a 9% increase in the SNR for the detection of the 3 cm diam sphere (a 12% increase for a 2 cm diam sphere) and a 7% increase in the SNR for estimation of its size. For the acquired phantom data, detection also increased by 9%-12% when using three, rather than two, energy windows.     

Wiedemann‐Rautenstrauch (neonatal progeroid) syndrome: New case with normal telomere length in skin fibroblasts

Wiedemann‐Rautenstrauch (neonatal progeroid) syndrome is an autosomal recessive condition with characteristic appearance of premature aging present at birth (aged face, natal teeth, and wrinkled skin). Other features of the syndrome are generalized lipoatrophy with specific fat accumulation in the lateral suprabuttock region, hypotrichosis, macrocephaly (pseudohydrocephalus), and mental retardation. We report on a new case that demonstrates all typical features of the syndrome. The girl is now 16 years and 10 months old and has had follow‐up from birth. We measured terminal restriction fragment (TRF) length to evaluate whether the patient's premature aging process is accompanied by shortening of telomere length in her cultured fibroblasts. Mean TRF of 13.5 kb found in our patient's fibroblasts is not shortened as compared to that of normal fibroblasts. Our results differ from those observed in Hutchinson‐Gilford progeria. © 2001 Wiley‐Liss, Inc.     

Molecular characterization of a 1p36 chromosomal duplication and in utero interference define ENO1 as a candidate gene for polymicrogyria

While chromosome 1p36 deletion syndrome is one of the most common terminal subtelomeric microdeletion syndrome, 1p36 microduplications are rare events. Polymicrogyria (PMG) is a brain malformation phenotype frequently present in patients with 1p36 monosomy. The gene whose haploinsufficiency could cause this phenotype remains to be identified. We used high-resolution arrayCGH in patients with various forms of PMG in order to identify chromosomal variants associated to the malformation and characterized the genes included in these regions in vitro and in vivo. We identified the smallest case of 1p36 duplication reported to date in a patient presenting intellectual disability, microcephaly, epilepsy, and perisylvian polymicrogyria. The duplicated segment is intrachromosomal, duplicated in mirror and contains two genes: enolase 1 (ENO1) and RERE, both disrupted by the rearrangement. Gene expression analysis performed using the patient cells revealed a reduced expression, mimicking haploinsufficiency. We performed in situ hybridization to describe the developmental expression profile of the two genes in mouse development. In addition, we used in utero electroporation of shRNAs to show that Eno1 inactivation in the rat causes a brain development defect. These experiments allowed us to define the ENO1 gene as the most likely candidate to contribute to the brain malformation phenotype of the studied patient and consequently a candidate to contribute to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.Subject terms: Gene regulation, Genetics research     

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Identification ofSchistosoma haematobium soluble egg antigens that elicit human granuloma formation in vitro

Schistosoma haematobium soluble egg antigens (SH SEAs) induce intense granulomas in human hosts that often culminate in severe disease. In an attempt to identify the SH SEA fractions that are responsible for pathology, we combined T-cell Western blotting and an in vitro model of granuloma formation. Whole SH SEAs were dotted onto nitrocellulose pieces or were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose paper. Horizontal strips bearing the separated antigens were solubilized in dimethylsulfoxide and precipitated in carbonate/bicarbonate buffer. Antigen-free and antigen-bearing particles were used to stimulate peripheral blood mononuclear cells (PBMCs) obtained fromS. haematobium-infected patients and sex- and agematched healthy controls to form granulomas in vitro. Whole SH SEA-bearing nitrocellulose particles elicited in vitro formation of granulomas by PBMCs from infected donors. The response was similar in sensitivity, specificity, and reproducibility to that evoked by SH SEA-bound polyacrylamide beads. The results obtained in samples from 30 patients and 10 controls tested with SH SEA-separated fractions revealed that SEA bands of 84 000, 63 000, 57 000, 55 000, 40 000, 30 000, and 28 000 Da elicited in vitro granuloma reactions by PBMCs of almost all infected patients. Conversely, separated soluble adult-worm antigens failed to stimulate PBMCs of infected patients to form granulomas. This study is the first to identify the SH SEA fractions that evok in vitro granuloma formation and represents an initial step toward the development of an anti-urinary schistosomiasis pathology vaccine.