Effect of Pseudomonas aeruginosa cytotoxin on thymidine incorporation by murine splenocytes.

The interaction of highly purified Pseudomonas aeruginosa cytotoxin (PAC) with murine splenocytes was examined. Added at culture initiation, PAC (0.1 to 0.5 microgram/ml) inhibited subsequent [3H]deoxythymidine incorporation measured between 42 to 48 h. Incorporation of [3H]deoxythymidine was inhibited 50% in lipopolysaccharide-, phytohemagglutinin-, and concanavalin A-stimulated cultures by 0.20, 0.32, and 0.39 microgram of PAC per ml, respectively. It is concluded that PAC exhibits a narrow inhibitory concentration response range of 0.1 to 0.5 microgram/ml which, secondarily, is affected by the presence of mitogens. Antitoxin added at splenocyte culture initiation, directly after PAC, yielded greater than or equal to 86% protection against PAC inhibition of [3H]deoxythymidine incorporation. Addition of antitoxin to cultures at different times after PAC demonstrated a time-dependent loss of antitoxin protective effect over a 12-h period, indicating that PAC became cell associated and refractory to antitoxin within this time period. PAC preincubated with splenocytes at 4 degrees C for less than or equal to 1 h could not be removed by washing of cells and was fully inhibitory to [3H]deoxythymidine incorporation when these cells were cultured at 37 degrees C. This finding was confirmed by demonstrating that 125I-labeled PAC bound immediately to cells. It is concluded that PAC action on splenocytes is dose- and time-dependent and consists of a two-phase process: (i) a very rapid binding of PAC to the cell surface available to antitoxin, and (ii) a slower toxicity development phase of ca. 12 h, during which PAC becomes refractory to antitoxin.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Patients with erythrophobia (fear of blushing) show abnormal autonomic regulation in mental stress conditions

OBJECTIVE: The objective of this study was to analyze the autonomic functions of patients with erythrophobia. METHODS: Forty patients with a diagnosis of erythrophobia (female/male ratio 18/22) without any other organic lesions and 20 healthy volunteers (female/male ratio 10/10) were assessed. Clinical evaluation was performed using a modified version of semistructured interviews. Autonomic testing was performed by means of spectral analysis of heart rate and continuous blood pressure by sparse discrete Fourier transformation at rest and under mental stress. RESULTS: There were no significant difference between the two samples in age, sex distribution, BMI, resting systolic, or diastolic blood pressure, nor was there a difference in autonomic baseline functioning between the 40 patients with erythrophobia and the control subjects. On the other hand, patients with erythrophobia consistently showed higher pulse rates (88 +/- 20 vs. 78 +/- 9 bpm, p <.05), higher total heart rate power values (8.40 +/- 0.63 vs. 8.07 +/- 1.02 p <.05), higher midfrequency spectral values (7.38 +/- 0.66 vs. 7.02 +/- 1.18, p <.01), higher high-frequency spectral values (6.89 +/- 0.86 vs. 6.48 +/- 1.44, p <.05), and lower baroreceptor sensitivity (8.62 +/- 8.16 vs. 11.65 +/- 4.42, p <.005) than the healthy subjects. ANOVA showed a significant group interaction (p <.0001) between the samples. CONCLUSIONS: This study provides evidence for abnormal autonomic functioning in patients with erythrophobia when under mental stress.     

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.     

Relationship of different serum levels of theophylline on methacholine sensitivity

With an increased awareness of the importance of bronchial hyperreactivity on the clinical state of asthma, efforts at establishing effective treatment for asthma have been aimed at trying to reduce the level of bronchial hyperreactivity. This study investigated whether or not theophylline at different serum concentrations had any effect on bronchial hyperreactivity, as measured by methacholine challenges. Twelve adult subjects with mild asthma participated. All subjects were atopic and were nonsmokers. Subjects were studied on three separate occasions according to a double-blind, randomized, crossover study design with one of the following medications: two placebo capsules in the morning and evening, a placebo and 250 mg of sustained-release theophylline in the morning and evening, or two 250 mg sustained-release theophylline capsules each morning and evening. Subjects were administered their medication between 5 and 15 days before performing methacholine challenges in the morning at the presumed trough level, and in the afternoon at the presumed peak serum theophylline level. Despite statistically significant differences in the pulmonary function and theophylline levels between the two active dosages (p less than 0.05 for both), there was no difference in methacholine sensitivity at the time of ingestion of the two dosage forms. There was also no correlation between a particular theophylline level and degree of methacholine sensitivity. It was concluded that, although theophylline has significant bronchodilator capability, it has little, if any, effect on bronchial hyperreactivity, as measured by methacholine challenges. Thus, other forms of long-term therapy should be used in reducing bronchial hyperreactivity.     

Cognitive and motor skills in achondroplastic infants: Neurologic and respiratory correlates

Thirteen infants with achondroplasia underwent psychometric testing as part of a comprehensive neurologic assessment. As a group, mental development was average and motor development was delayed, although a wide range of scores was obtained. Foramen magnum measurements were correlated with respiratory dysfunction, abnormal so-matosensory evoked potentials, and delayed motor development. Abnormal polysomno-gram outcome was associated with reduced mental capacity. In light of the reported increased frequency of respiratory dysfunction in achondroplasia, these findings warrant careful attention and further study.     

Infection with HIV type 1 group M non-B subtypes in individuals living in New York City

OBJECTIVE: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. DESIGN: From October 1999 through April 2003, HIV-1-seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. METHODS: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. RESULTS: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE-like (n = 7; 13%), CRF02_AG-like (n = 19; 34%), an intersubtype recombinant form G/A (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). CONCLUSION: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.     

T-cell receptor variable region of the β-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis

In order to look for a site-specific T-cell response in RA SM, PCR analyses using oligonucleotide primers specific for 24 TCRBV (Vβ) families were performed to compare the respective usage of each TCRBV gene by T cells present in PB and SM of 13 patients with RA. In four patients, SM cells from two or three sites of inflammation were subjected to analysis. In one patient, synovial tissue was studied at two different phases of the disease, resulting in a total number of 19 samples of SM cells, which were compared with paired samples of PB cells. The results showed that whereas all 24 TCRBV gene families could be detected in both PB and SM cells, there was some skewing of increased or decreased usage frequencies of particular TCR Vβ genes among SM cells. Three TCRBV families were often overexpressed in SM: Vβ3, Vβl7, and Vβ22. Moreover, Vβ4 was often decreased in SM (7 out of 13). This decrease was statistically significant in the RA population studied. SM from different joints of a given patient showed similar variations of T-cell repertoire compared to PB, even 6 months later in the course of the disease. These results demonstrate a biased TCRBV gene utilization in RA SM. This bias appears to be similar in different joints and at different times in the course of the disease. No correlation was found between the bias of TCR repertoire in SM and the HLA typing of these patients.