Betamethasone effects on fetal sheep cerebral blood flow are not dependent on maturation of cerebrovascular system and pituitary–adrenal axis

Synthetic glucocorticoids are administered to pregnant women in premature labour to accelerate fetal lung maturation at a time when fetal cerebrovascular and endocrine systems are maturing. Exposure to glucocorticoids at 0.8–0.9 of gestation increases peripheral and cerebrovascular resistance (CVR) in fetal sheep. We examined whether the increase of CVR and its adverse effect on cerebral blood flow (CBF) depend on the current level of maturation of the pituitary–adrenal axis and the cerebrovascular system. Using fluorescent microspheres, regional CBF was measured in 11 brain regions before and 24 h and 48 h after the start of 3.3 μg kg⁻¹ h⁻¹ betamethasone ( n = 8) or vehicle ( n = 7) infusions to fetal sheep at 0.73 of gestation. Hypercapnic challenges were performed before and 24 h after the onset of betamethasone exposure to examine betamethasone effects on cerebrovascular reactivity. Betamethasone exposure decreased CBF by approximately 40% in all brain regions after 24 h of infusion ( P < 0.05). The decline in CBF was mediated by a CVR increase of 111 ± 16% in the cerebral cortex and 129 ± 29% in subcortical regions ( P < 0.05). Hypercapnic cerebral vasodilatation and associated increase in CBF were blunted ( P < 0.05). Fetal CBF recovered after 48 h of betamethasone administration. There were no differences in glucocorticoid induced CBF and CVR changes compared with our previous findings at 0.87 of gestation. We conclude that the cerebrovascular effects of antenatal glucocorticoids are independent of cerebrovascular maturation and preparturient increase in activity of the fetal pituitary–adrenal axis.     

Increased in vivo frequency of IA-2 peptide-reactive IFNgamma+/IL-4- T cells in type 1 diabetic subjects

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls. When tested in a high resolution IFNgamma/IL-4 double color ELISPOT assay directly ex vivo, the number of IA-2-reactive IFNgamma producing cells was 17-fold higher in patients than in controls and IL-4 producing cells were not present. An average of 9 peptides was recognized in the patients vs. one in the controls. Determinant recognition primarily involved CD4+ cells and showed high variability among the patients. Furthermore, anti-CD28 antibody signal enhances quantitative assessment of effector T cells in T1D patients. In vitro expansion with peptides and IL-2 results in detection of responding cells in the controls and loss of disease specificity of the T cell response. Together these data provide strong evidence for the active targeting of IA-2 by Th1 memory effector cells in human type 1 diabetes.     

Schedule and quinine induced deprivation in feeding and refeeding

Twenty female albino rats were adapted to either 0 or 23 hr of food deprivation. Half of each group was then fed 0.125% quinine sulfate adulterated diet for seven days. Following the quinine feeding, ad lib feeding (refeeding) was instituted for 14 days. Several conclusions were drawn from the results: (1) rats on a deprivation schedule fail to show a predicted change to regulation on the basis of taste rather than calories; (2) rats on food deprivation actually increase their relative intake of water; (3) refeeding after a deprivation schedule does not lead to depression of initial intake below normal, but otherwise the process of recovery follows the same course as after total starvation.     

The osmotic behaviour of toad skin epithelium (Bufo viridis)

The effect of saline adaptation on the intracellular Na, K, Cl, P concentrations and dry weight content of the toad skin epithelium (Bufo viridis) was studied using the technique of electron microprobe analysis. The measurements were performed on isolated abdominal skins either directly after dissection or after additional incubation in Ussing-type chambers.Adaptations of the toads to increasing NaCl concentrations for 7 days resulted in increased blood plasma osmolarity and a parallel increase in the cellular electrolyte, P and dry weight concentrations of the epithelium, the K increase representing the most significant fraction of the intracellular osmolarity increase. No evidence was obtained to show that the nucleus and cytoplasm reacted differently from each other and all living epithelial cell types basically showed the same response.Incubation of the isolated skins under control conditions showed a drastic inhibition of the transepithelial Na transport after adaptation to high salinities. In spite of the large variations in the transport rate almost identical intracellular electrolyte concentrations were observed. In tap water adapted toads the average cellular concentrations were 8.8 mmole/kg wet weight for Na, 109.6 for K, 41.5 for Cl, and 135.3 for P, respectively. Incubation of the skin with Ringer's solution of different osmolarities demonstrated that the epithelial cells are in osmotic equilibrium with the inner bathing solution. The results are consistent with the view that the osmotic adaptation is mainly accomplished by the movement of water.This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk     

Detection of expansion regions in Portuguese bipolar families

We have studied 24 families with multiple affected members with bipolar disorder to test the hypothesis that in those families clinically showing genetic anticipation [Macedo et al., 1999] we would find large repeat expansions. The families meeting inclusion criteria had a minimum of two affected members over two generations and showed marked anticipation both in terms of age of onset and disease severity. We used the repeat expansion detection (RED) method to test patients (n = 24) and controls from these families and unrelated controls (n = 53). We also genotyped patients and family members from two families with large expansions at the known expansion loci on chromosomes 13, 17, and 18. The RED method revealed a higher number of large expansions in patients compared with controls (t-test; P < 0.0055: Mann-Whitney U; P = 0.02). The patients with the largest expansions were typed at the specific loci on chromosomes 13, 17, and 18 and the chromosome 18 expansion locus segregated with disease in one family, and a second family showed segregation with the expansion located at the SCA8 locus on chromosome 13. Genetic anticipation had been analyzed in this cohort of families, with correction for potential ascertainment bias, possible proband effects, cohort effects, regression to the mean, gender effects, and maternal vs. paternal transmission. None of these potential confounds appeared to account for the observed anticipation. We also identified that the presence of large expansions in affected family members derives primarily from two families from the genetically isolated Azores population. One family shows segregation with the chromosome 18 locus, whereas the other family segregates with expansions at the SCA8 locus. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:854-857, 2000.     

Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy

Dendritic cells (DC) are increasingly explored as cellular vaccines for tumor immunotherapy. In most reported DC-based cancer vaccine trials, DC have been pulsed with soluble tumor antigen-derived peptide ligands of MHC molecules. Considering that the half-life of peptide/MHC complexes on the cell surface is relatively short and that soluble exogenous protein antigens cannot be efficiently processed via the MHC class I-processing pathway, the current vaccination procedure is not optimal for the induction of strong T cell responses aiming at tumor rejection. Recently, we have shown that antigen presentation can be prolonged when synthetic peptides were encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS) for uptake by DC. In the present study, we investigated the phenotypic and functional consequences of MS uptake by human monocyte-derived dendritic cells (MoDC) in vitro. We found that immature MoDC that were prepared in serum free media suitable for clinical application were able to phagocytose high numbers of MS, while matured MoDC showed a reduced capacity for phagocytosis of MS. The ingestion of MS did not change the cell surface expression of CD80, CD83, CD86 and HLA-DR of immature and mature DC, suggesting that MS uptake did not induce DC maturation but that maturation by cytokines or LPS was unaltered in the presence of MS. Furthermore, MS-loaded mature MoDC expressed normal levels of the chemokine receptor CCR7 and migrated as efficiently towards CCL19 or CCL21 as unloaded MoDC. DC viability and the secretion of TNF-alpha and IL-12 was not significantly changed by MS loading. Taken together, our data indicate that PLGA-MS loading has no negative effects on the pivotal properties of MoDC in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections and tumors.