Cerebral type 2 astroglia are heterogeneous with respect to their ability to respond to neuroligands linked to calcium mobilization.

Very little information is available concerning the pharmacology of type 2 astroglia. During the past decade it has become apparent that two distinct lineages of astroglial cells can be defined in vitro. These two lineages are commonly referred to as type 1 and type 2 and are distinguished from each other on the basis of their morphological features and antigenic phenotypes. In contrast to type 1 astroglia, very little is known about the pharmacology of type 2 astroglia. The lack of information concerning the responsiveness of these cells stems primarily from difficulties encountered in isolating large numbers of type 2 astroglia free of other cell types. In the present study video- and photometer-based imaging systems were used to monitor the influence of a series of neuroligands on the intracellular calcium levels of individual cerebral type 2 astroglia in order to assess their expression of calcium-mobilizing receptors. The responses of 85 immunocytochemically identified cerebral type 2 astroglia to bradykinin (BK), norepinephrine (NE), histamine (HIST), carbachol (CARB), 2-methyl-thio ATP (2MT-ATP), glutamate (GLUT), and serotonin (5-HT) were analyzed. Approximately 50% of cerebral type 2 astroglia responded to BK, NE, HIST, CARB, and 2MT-ATP whereas only 16% and 9% of the cells responded to GLUT and 5-HT, respectively. The number of neuroligands that increased calcium in individual cells ranged from 0 to 6. These responses are quite similar to those previously demonstrated in cultured cerebral type 1 astroglia. No pattern of receptor co-expression was observed for the different neuroligands tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

In VivoStudies of Adenovirus-Based p53 Gene Therapy for Ovarian Cancer

Objectives.To test the safety, efficacy, and toxicity of gene therapy using wild-type p53-expressing adenovirus (Ad-CMV-p53) in a nude mouse model with intraperitoneal (ip) 2774 human ovarian cancer cell line that contains a p53 mutation.Study design.An initial study of adenovirus tolerance was determined in nude mice by a single ip injection of increasing doses of Ad-CMV-p53. Nude mice were implanted with an LD₁₀₀dose of 1 × 10⁷cells. To study the efficacy and specificity of Ad-CMV-p53 treatment, the mice received treatment with different adenovirus constructs. One group received Ad-CMV-p53 and another group received a control adenovirus construct, Ad-CMV-βgal. To study the treatment response to Ad-CMV-p53, the mice were divided into groups and received various treatment schedules of 1 × 10⁸pfu of Ad-CMV-p53.Results.The mice tolerated Ad-CMV-p53 without adverse effects at doses of 1 × 10⁸pfu. The response to Ad-CMV-p53 showed significant survival duration in each dose regimen, with a survival time greater than that of untreated animals (P= 0.0173). However, no statistically significant survival advantage was observed between Ad-CMV-p53- and Ad-CMV-βgal-treated mice.Conclusions.These studies show that at the adenovirus dose and administration regimen used, there is effective but not specific 2774 tumor growth inhibitionin vivo.Efficient introduction of biologically active genes into tumor cells would greatly facilitate cancer therapy. Thus, although promising, these results caution that much effort will be required to realize the potential for clinical application of adenovirus-based ovarian cancer gene therapy.     

Structured treatment and teaching of patients with Type 2 diabetes mellitus and impaired cognitive function--the DICOF trial.

BACKGROUND: Patient education is integral part of any diabetes therapy in Germany, but elderly patients are not able to follow the variety of topics comprising standard treatment and teaching programmes (TTP), primarily due to impaired neuropsychological function. This leads to deficits in diabetes knowledge and hindered ability for diabetes self-management. AIM: To evaluate structured TTP for geriatric patients with impaired cognitive function. PATIENTS AND METHODS: A neuropsychological examination was performed on all patients over 54 years [n=102, age 68.6 +/- 8.7 years, diabetes duration 10.3 (0.03-35.4) years, HbA1c 10.3 +/- 1.7% (HPLC, Diamat, NR 4.5-6.3%), cognitive function 87.7 +/- 12.3 IQ points] who took part in TTP for insulin therapy. Patients with impaired cognitive function participated either in the standard TTP of Berger [n = 35, age 67.6 +/- 8.9 years, diabetes duration 9.9 (0.04-35.4) years, HbA1c 10.3 +/- 2.0%] or in the specialized structured geriatric DICOF-TTP [n=33, age 70.4 +/- 8.2 years, diabetes duration 10.4 (0.03-24.9) years, HbA1c 10.7 +/- 1.8%]. RESULTS: After TTP there were no differences in knowledge and ability for diabetes self-management (standard/DICOF: knowledge 11.0 +/- 2.6 vs. 12.2 +/- 2.7 points, P = 0.11; handling 14.9 +/- 3.3 vs. 15.9 +/- 2.5 points, P = 0.18). However, patients who took part in the DICOF programme showed better scores in satisfaction with the education programme [standard/DICOF 44.7 (31-57) vs. 52.5 (45-59) points, P < 0.001]. Six months later the DICOF participants showed better results regarding diabetes self-management (standard/DICOF: handling 12.5 +/- 4.1 vs. 15.9 +/- 3.1 points, P = 0.001). Both groups showed HbA1c decrease (8.3 +/- 1.4 vs. 8.5 +/- 1.3%, P=0.62) and similar incidence of acute complications. CONCLUSIONS: Elderly patients with impaired cognitive function should take part in specialized structured TTP. This leads to both better satisfaction with the education programme and an improved ability for diabetes self-management.     

Corpus callosotomy: a palliative therapeutic technique may help identify resectable epileptogenic foci.

Corpus callosotomy has a long history as a palliative treatment for intractable epilepsy. Identification of a single epileptogenic zone is critical to performing successful resective surgery. We describe three patients in which corpus callosotomy allowed recognition of unapparent seizure foci, leading to subsequent successful resection. We retrospectively reviewed our epilepsy surgery database from 2003 to 2005 for children who had a prior callosotomy and were candidates for focal resection. All underwent magnetic resonance imaging and scalp video electroencephalograph monitoring, and two had magnetoencephalography, electrocorticography and/or intracranial video electroencephalograph monitoring. The children were 8 and 9 years old, and seizure onset varied from early infancy to early childhood. One child had a history of head trauma preceding seizure onset, one had a large intracerebral infarct and dysplastic cortex in the contralateral frontal lobe, and the other had an anterior temporal lobe resection without improvement in seizure frequency. After medical management failed, callosotomy was performed with the expectation of decreasing the seizure types affecting both hemispheres. Following transection of the callosal fibers, a single focus was recognized and resected, with resultant dramatic improvement in seizure control. In medically refractory epilepsy, where rapid secondary bisynchrony is suspected but the electroencephalograph is non-localizing, callosotomy should be considered as a means of treating generalized seizure types, but may also assist in identifying potentially operable seizure foci. Study limitations include its retrospective nature and cohort size. The findings, however, suggest the need for prospective, systematic, well-controlled studies of the use of corpus callostomy in this intractable patient population.     

Pituitary responsiveness to GH-releasing hormone, GH-releasing peptide-2 and thyrotrophin-releasing hormone in critical illness

OBJECTIVE Protein hypercatabolism and preservation of fat depots are hallmarks of critical illness, which is associated with blunted pulsatile GH secretion and low circulating IGF-I, TSH, T4 and T3. Repetitive TRH administration is known to reactivate the pituitary-thyroid axis and to evoke paradoxical GH release in critical illness. We further explored the hypothalamic-pituitary function in critical illness by examining the effects of GH-releasing hormone (GHRH) and/or GH-releasing peptide-2 (GHRP-2) and TRH administration. PATIENTS AND DESIGN Critically ill adults (n=40; mean age 55 years) received two i.v. boluses with a 6-hour interval (0900 and 1500 h) within a cross-over design. Patients were randomized to receive consecutively placebo and GHRP-2 (n=10), GHRH and GHRP-2 (n=10), GHRP-2 and GHRH+GHRP-2 (n=10), GHRH+GHRP-2 and GHRH+GHRP-2+TRH (n=10). The GHRH and GHRP-2 doses were 1μg/kg and the TRH dose was 200μg. Blood samples were obtained before and 20, 40, 60 and 120 minutes after each injection. MEASUREMENTS Serum concentrations of GH, T4, T3, rT3, thyroid hormone binding globulin (TBG), IGF-I, insulin and cortisol were measured by RIA; PRL and TSH concentrations were determined by IRMA. RESULTS Critically ill patients presented a striking GH response to GHRP-2 (mean±SEM peak GH 51±9 μg/l in older patients and 102±2μg/l in younger patients; P=0.005 vs placebo). The mean GH response to GHRP-2 was more than fourfold higher than to GHRH (P=0.007). In turn, the mean GH response to GHRH+GHRP-2 was 2.5-fold higher than to GHRP-2 alone (P=0.01), indicating synergism. Adding TRH to the GHRH+GHRP-2 combination slightly blunted this mean response by 18% (P=0.01). GHRP-2 had no effect on serum TSH concentrations whereas both GHRH and GHRH+GHRP-2 evoked an increase in peak TSH levels of 53 and 32% respectively. The addition of TRH further increased this TSH response < ninefold (P=0.005), elicited a 60% rise in serum T3 (P=0.01) and an 18% increase in T4 (P=0.005) levels, without altering rT3 or TBG levels. GHRH and/or GHRP-2 induced a small increase in serum PRL levels. The addition of TRH magnified the PRL response 2.4-fold (P=0.007). GHRP-2 increased basal serum cortisol levels (531±29nmol/l) by 35% (P=0.02); GHRH provoked no additional response, but adding TRH further increased the cortisol response by 20% (P=0.05). CONCLUSIONS The specific character of hypothalamic-pituitary function in critical illness is herewith extended to the responsiveness to GHRH and/or GHRP-2 and TRH. The observation of striking bursts of GH secretion elicited by GHRP-2 and particularly by GHRH+GHRP-2 in patients with low spontaneous GH peaks opens the possibility of therapeutic perspectives for GH secretagogues in critical care medicine.     

The andrology laboratory in an Assisted Reproductive Technologies program. Quality assurance and laboratory methodology.

Assuring the highest possible quality of care to the patient is the first goal of the andrology laboratory. Quality control and quality assurance as they apply to the andrology laboratory are outlined, and special problems in quality control for sperm function assays are addressed. An example of the patient review process is presented. Quality of care also depends on the ability of the laboratory to perform appropriate tests of sperm function that can diagnose sperm defects, predict success or failure of in vitro fertilization, and lead to rational therapies for sperm dysfunctions. Methods for, and diagnostic value of, sperm swim-out, enhanced sperm penetration assays, acrosome reaction assessment, motility analysis of capacitating sperm, and sperm-zona binding assays are reviewed. No single test, other than in vitro fertilization itself, is capable of providing a complete diagnosis or a highly accurate prediction. A battery of sperm function tests, arranged in an algorithm, is presented as a theoretically better approach.     

Noninvasive in vivo 13C-NMR spectroscopy in the rat to study the pharmacokinetics of 13C-labeled xenobiotics.

Noninvasive NMR methodology has been developed to enable monitoring of 13C-labeled xenobiotics in the rat in vivo. 2,2-Dichloro-1-(2-chlorophenyl)-1-(4-chlorophenyl)-[3-13C]-propane can be detected in the liver of intact rats by in vivo 13C surface coil NMR spectroscopy after ip administration of the compound. The experiments were performed at 1.9 and 9.4 Tesla. The intrahepatic changes of the signal intensity of the labeled compound were followed as a function of time. In the days following administration, the concentration decreased and dropped to values below the detection limit after 12 days. The study demonstrates the feasibility of studies on pharmacokinetics of 13C-labeled compounds in the rat using noninvasive, in vivo surface coil NMR spectroscopy in animals. The sensitivity allows the detection of a single dose of the drug of 200 mg/kg, but can be improved.     

Production and certification of an enzyme reference material for gamma-glutamyltransferase (CRM 319). Part 2: Certification campaign

We describe the process of certification for a gamma-glutamyltransferase reference material (CRM no. 319). Fifteen laboratories participated to this interlaboratory evaluation. All steps of the measurements were controlled in an effort to locate potential sources of variations. In particular, the exclusion of some data was strictly documented or justified by the non-observance of the IFCC method and (or) discrepancies in instrumentation, reconstitution of the lyophilized samples, or measurement technique. Inaccuracy in the reconstitution of the lyophilized material was +/- 0.68%, and the molar absorptivity of the 5-amino-2-nitrobenzoate reported by each laboratory was within +/- 2% limits of the value reported by the IFCC. Calculated from the sets of accepted results, the total CV among samples was 2.6% and the overall CV was 3.2%. Within-day and between-day CVs were 1.1% and 1.4%, respectively. The greatest variation for a single component was the between-laboratory variability (CV 3.1%); the within-laboratory CV, including the day effect, was 1.8%. Finally, the certified value for the catalytic concentration of this enzyme in the reconstituted lyophilized reference material was 86.8 U/L with an uncertainty of +/- 2.1 U/L (0.95 confidence interval). The uncertainty appeared to be compatible with the end-use of this reference material.