Circulating endothelial microparticles in diabetes mellitus.

BACKGROUND: Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. METHODS AND RESULTS: EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/microL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. CONCLUSION: Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

Effect of a bone marrow microenvironment on the ex-vivo expansion of umbilical cord blood progenitor cells.

Progenitor cells (CD34(+)) can be isolated from umbilical cord blood and used to correct or reconstitute various cell lines within the haematopoietic and endothelial cell lineage. The main disadvantage of this procedure relates to the low volume of blood that can be collected after the umbilical cord has been clamped, which limits the number of progenitor cells available for treatment. This limitation, however, can be overcome by expanding CD34(+) cells ex vivo. Our aim was to perform a controlled study to determine if the ex-vivo proliferation of umbilical cord CD34(+) cells is enhanced when they are placed in a system that mimics the bone marrow microenvironment. For this purpose, CD34(+) cells were isolated from umbilical cord blood using a magnetic cell sorting kit and seeded in platforms containing different cocktails of cytokines with and without a three-dimensional (3D) biomatrix. Results from this study suggest that the number of viable cells can double after 1 week in any of the culture platforms and that the 3D biomatrix does not enhance cell proliferation.

Role of reactive oxygen species in IL-1 beta-stimulated sustained ERK activation and MMP-9 induction.

We have recently demonstrated that interleukin-1 beta (IL-1 beta) stimulates matrix metalloproteinase-9 (MMP-9) induction. In this study we have investigated the roles of superoxide and extracellular signal-regulated kinase (ERK) activation in MMP-9 induction following exposure to IL-1 beta. IL-1 beta stimulated biphasic ERK activation in vascular smooth muscle (VSM) cells, a transient activation that reached a maximum at 15 min and declined to baseline levels within 1 h, and a second phase of sustained ERK activation lasting up to 8 h. To determine the role of ERK in IL-1 beta-stimulated MMP-9 induction, we treated cells with the specific ERK pathway inhibitor PD-98059 at different time intervals after IL-1 beta stimulation. Addition of PD-98059 up to 4 h after IL-1 beta stimulation significantly inhibited MMP-9 induction, suggesting a role for sustained ERK activation in MMP-9 induction. IL-1 beta treatment stimulated superoxide production in VSM cells that was inhibited by pretreatment of cells with the superoxide scavenger N-acetyl-L-cysteine (NAC) and also by overexpression of the human manganese superoxide dismutase (MnSOD) gene. Treatment of VSM cells with NAC selectively inhibited the sustained phase of ERK activation without influencing the transient phase, suggesting a role for reactive oxygen species in sustained ERK activation. In addition, both NAC treatment and MnSOD overexpression significantly inhibited IL-1 beta-stimulated MMP-9 induction (P < 0.05). The results demonstrate that IL-1 beta-dependent MMP-9 induction is mediated by superoxide-stimulated ERK activation.

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.

Comparison of direct and concentrated acid-fast smears to identify specimens culture positive for Mycobacterium spp.

Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in the initial diagnosis of tuberculosis, monitoring of treatment, and determination of eligibility for release from isolation. The objective of this study was to compare the sensitivity obtained with smears for detection of AFB (AFB smears) made directly from respiratory specimens (direct AFB smears) to that obtained with parallel smears made from concentrates of the specimens (concentrated AFB smears). A total of 2,693 specimens were evaluated; 1,806 were from the University of California Irvine Medical Center Medical Microbiology Laboratory (UCIMC), which serves a tertiary-care hospital with outpatient clinics, and 887 were from the Microbial Disease Laboratory at the California Department of Public Health (MDL), which receives specimens from outpatient facilities and clinics on Pacific islands. Of the 353 AFB culture-positive specimens at UCIMC, there was a statistically significant difference in the sensitivity of the direct AFB smear (34%) and that of the smear made from the concentrated specimen (58%) (P < 0.05). This was also true for the 208 specimens positive for Mycobacterium tuberculosis, for which the sensitivity of the direct smear was 42% (87 of 208) and that for the smear made from the concentrated specimen was 74% (154 of 208). At MDL, where all but 1 of the 45 culture-positive specimens grew M. tuberculosis, the sensitivity of the smear made from the concentrated specimen was 93% (42 of 45) and was not significantly higher than the sensitivity of the direct smear, which was 82% (37 of 45). By combining the results from both laboratories, 42 patients from whom at least three specimens were received were culture positive for M. tuberculosis. The cumulative results for the initial three specimens from these patients showed that the direct smear detected M. tuberculosis in 81% of these patients, whereas the smear made from the concentrate detected M. tuberculosis in 91% of these patients. In summary, when all culture-positive specimens are considered, the sensitivity of the direct smear compared to that of a smear made from the concentrated specimen was significantly different overall in the two different laboratory settings. However, this difference was reduced only if the cumulative results for the initial three specimens received from patients who were culture positive for M. tuberculosis were evaluated.

Emotion recognition and social competence in chronic schizophrenia.

This study evaluated (a) whether chronic, medicated schizophrenia patients show deficits in emotion recognition compared to nonpatients, and (b) whether deficits in emotion recognition are related to poorer social competence. Two emotion recognition tests developed by S. L. Kerr and J. M. Neale (1993) and Benton's Test of Facial Recognition (A. Benton, M. VanAllen, K. Hamsher, & H. Levin, 1978) were given to patients with chronic schizophrenia and nonpatient controls. Patients' social skills, social adjustment, and symptomatology were assessed. Like Kerr and Neale's unmedicated patients, these patients performed worse than controls on both emotion recognition tests and the control test. For patients, facial perception was related to the chronicity of illness and social competence. Chronicity of illness may contribute to face perception deficits in schizophrenia, which may affect social competence.

Clozapine withdrawal effects and receptor profiles of typical and atypical neuroleptics.

Withdrawal effects of neuroleptics have not received much attention. Clozapine withdrawal phenomena have been attributed to psychosis arising from D2 supersensitivity, which is unlikely since it has minimal action on D2 receptors. The time course and clinical features of this phenomenon suggest that cholinergic overdrive and gamma-aminobutyric acid (GABA) supersensitivity occurs after withdrawal, since it is strongly anticholinergic and has a GABAergic action. Recently, a number of patients showed marked decompensation when they were switched from clozapine to risperidone, especially when they were rapidly tapered off clozapine. This was probably more due to withdrawal effects than the primary psychosis or a lack of efficacy of risperidone. A slow withdrawal schedule would facilitate homeostatic mechanisms; anticholinergics would be useful in clozapine withdrawal. This area has not received any attention from researchers, nor are there any guidelines for clinicians. This will be particularly important with the widespread use of atypical agents in the future.

Direct and telemetered lead impedance.

OBJECTIVES: We undertook this study to determine whether telemetered lead impedance measurements (LIM) can be correlated with direct LIM and to determine the stability of LIM over time when measured directly and via telemetry. METHODS: Direct LIM and telemetered LIM were measured in 91 patients; 101 leads during initial implantation and 40 leads during pulse generator replacement. Differences in direct LIM measured during initial implant and pulse generator replacement (direct-direct) were compared in 41 patients (28 atrial leads and 37 ventricular leads). The stability of telemetered LIM obtained immediately postoperatively, at 1 month and 1 year, postimplantation was assessed in 50 patients (23 atrial and 49 ventricular leads). RESULTS: In atrial leads acute direct LIM was 633.9 +/- 18.4 omega versus 575.8 +/- 18.5 omega for telemetered LIM (r = 0.58), and chronic direct LIM was 670.9 +/- 49.3 omega versus 607.0 +/- 36.3 omega for telemetered LIM (r = 0.87). In ventricular leads acute direct LIM was 747.3 +/- 16.9 omega and 684.7 +/- 16.4 omega for telemetered LIM (r = 0.69), and chronic direct LIM was 674.8 +/- 29.9 omega and 625.2 +/- 28.5 omega for telemetered LIM (r = 0.68). The mean direct-direct LIM rose 124 omega (P < 0.001) in atrial leads and 10 omega (P = NS) in ventricular leads. Telemetered LIM for atrial leads was 581.0 +/- 27.6 omega immediately postimplantation compared to 625.7 +/- 34.8 omega at 1 month and 754.1 +/- 43.0 omega at 1 year. Telemetered LIM for ventricular leads was 661.3 +/- 17.5 omega at implant, 684.6 +/- 20.7 omega at 1 month and 724.7 +/- 22.7 omega at 1 year. CONCLUSIONS: There is a good but limited correlation between direct and telemetered LIM. Mean direct LIM obtained at initial implantation is similar to that measured at pulse generator replacement. The telemetered LIM is stable over the first month postimplantation but tends to rise during the first year of follow-up and substantial changes in impedance are not uncommon in individuals with normal function. There is a tendency for LIM to rise with lead maturation. If telemetered LIM is to be followed over time, a baseline telemetered value should be obtained immediately postoperatively.

Hybridoma stability.

Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product. MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones. The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated. Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks. Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non-reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N-terminal sequencing, carbohydrate composition and binding assays. Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed. Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs. Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process.

Metabolic, molecular genetic and toxicological aspects of the acetylation polymorphism in inbred mice.

Over the past 10 years, much fascinating information has been obtained concerning the biochemistry, genetics, toxicological implications and molecular genetics of the N-acetylation polymorphism in mice. Using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, it has been shown that the polymorphism observed in N-acetyltransferase (NAT) activity in liver also occurs in kidney, bladder, blood, and other tissues. The development of congenic acetylator mouse lines derived from B6 and A, have provided the necessary tools to study the role of the acetylation polymorphism, on either the B6 or A genetic background, free of nearly all other genetic differences between these strains. Eliminating genes which modify and complicate the differences due to the acetylator genes make the congenic lines very useful in toxicology studies, particularly those involving carcinogenesis. The molecular genetic basis of the acetylator polymorphism in B6 and A mice involves two Nat genes. Nat-1 encodes a protein termed NAT1 which is identical in rapid and slow acetylator strains. Nat-2, however, differs between rapid and slow strains by a single nucleotide change in the coding region. The corresponding NAT2 proteins differ by a single change at amino acid 99: an hydrophilic asparagine in rapid acetylator NAT2 to an hydrophobic isoleucine in NAT2 from slow acetylators. The mechanistic basis for the differences between rapid and slow acetylation in mice appears to be that NAT2 from the rapid B6 strain is 15-fold more stable at 37 degrees C and is transcribed/translated with a maximal efficiency twice that of the enzyme from slow acetylator A mice. Results discussed in this review indicate that mice provide an excellent system for studying the N-acetyltransferase polymorphism and also are useful for modelling several aspects of the human N-acetyltransferase polymorphism.