International multi-center evaluation of a novel chemiluminescence assay for the detection of anti-dsDNA antibodies.

OBJECTIVE: Anti-double stranded desoxyribonucleic acid (anti-dsDNA) antibodies are considered fairly specific for systemic lupus erythematosus (SLE) and their quantification is useful for the clinical management of SLE patients. We assessed the diagnostic performance of the QUANTA Flash dsDNA chemiluminescent immunoassay (CIA) in comparison to an ELISA, using patients from five participating countries. The main focus was to evaluate the correlation between anti-dsDNA antibody results from the CIA and global SLE disease activity, as measured by the SLE Disease Activity Index 2000 (SLEDAI-2K). PATIENTS AND METHODS: A total of 1431 samples (SLE, n = 843; disease controls, n = 588) from five countries (Canada, USA, Portugal, Sweden and Spain) were tested with QUANTA Flash dsDNA (Inova Diagnostics, San Diego, CA, USA). Data obtained with the QUANTA Lite dsDNA SC ELISA (Inova Diagnostics) were available for samples from three sites (Canada, USA and Sweden, n = 566). The SLEDAI-2K scores were available for 805 SLE patients and a cut-off of > 4 was used to define active disease. RESULTS: QUANTA Flash dsDNA had a sensitivity of 54.3% for the diagnosis of SLE, combined with 89.8% specificity. Anti-dsDNA antibody levels were significantly higher (p < 0.0001) in active SLE (SLEDAI-2K > 4; n = 232; median value 83.0 IU/mL) versus the inactive patients (n = 573; median value 22.3 IU/mL), and the SLEDAI-2K scoring correlated with their dsDNA antibody levels (Spearman's rho = 0.44, p < 0.0001). Similar but less pronounced findings were also found for the ELISA, in relation to disease activity. CONCLUSIONS: The QUANTA Flash dsDNA assay showed good clinical performance in a large international multi-center study. Additionally, the strong correlation between anti-dsDNA antibody results and SLEDAI-2K scores supported the potential utility of QUANTA Flash dsDNA for monitoring disease activity.

CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59fyn and p56lck in Jurkat T cell line.

The leukocyte differentiation antigen, CD50, has been recently identified as the intercellular adhesion molecule 3 (ICAM-3), the third counter-receptor of leukocyte function-associated antigen 1 (LFA-1). This molecule seems to be specially involved in the adhesion events of the initial phases of the immune response. To characterize the role of CD50 in leukocyte interactions, the different molecular events induced after cross-linking of CD50 on T cell-derived Jurkat cell line have been analyzed. When cells were incubated with anti-CD50 mAbs and cross-linked with polyclonal goat anti-mouse immunoglobulins, a rise in intracellular calcium concentration ([Ca2+]i) was observed. This increase in [Ca2+]i was mainly due to the uptake of extracellular Ca2+. This Ca2+ flux involved tyrosine phosphorylations and was further increased by CD3 costimulation. These data, together with those obtained by phosphotyrosine (P-Tyr) immunoprecipitation and in vitro kinase assays, suggested the involvement of protein-tyrosine kinases (PTK) in CD50 transduction pathways. By using specific antisera, the presence of p56lck and p59fyn protein tyrosine kinases (PTK) was clearly demonstrated in the CD50 immunoprecipitates. These findings suggest that the interaction of CD50 with its natural ligand (LFA-1) may result in T lymphocyte activation events, in which CD50 could play a very active role after antigen triggering.

Does medicine still show an unresolved discrimination against women? Experience in two European university hospitals.

Have invisible barriers for women been broken in 2007, or do we still have to break through medicine's glass ceiling? Data from two of the most prestigious university hospitals in Barcelona with 700-800 beds, Hospital Clínic (HC) and Hospital de la Santa Creu i Sant Pau (HSCSP) address this issue. In the HSCSP, 87% of the department chairs are men and 85% of the department unit chiefs are also men. With respect to women, only 5 (13%) are in the top position (department chair) and 4 (15%) are department unit chiefs. Similar statistics are also found at the HC: 87% of the department chairs and 89% of the department unit chiefs are men. Currently, only 6 women (13%) are in the top position and 6 (11%) are department unit chiefs. Analysis of the 2002 data of internal promotions in HC showed that for the first level (senior specialist) sex distribution was similar. Nevertheless, for the second level (consultant) only 25% were women, and for the top level (senior consultant) only 8% were women. These proportions have not changed in 2007 in spite of a 10% increase in leadership positions during this period. Similar proportions were found in HSCSP where 68% of the top promotions were held by men. The data obtained from these two different medical institutions in Barcelona are probably representative of other hospitals in Spain. It would be ethically desirable to have males and females in leadership positions in the medical profession.

Prognostic markers of clinical remission in early rheumatoid arthritis after two years of DMARDs in a clinical setting.

OBJECTIVE: To analyze the rate and baseline prognostic factors of clinical remission in a series of patients with early rheumatoid arthritis (RA) after 2 years of therapy based on a structured algorithm using disease-modifying anti-rheumatic drugs (DMARDs) in a clinical setting. To determine whether a good therapeutic response at 6 months of therapy is associated with remission at 2 years. METHODS: One hundred and five patients (81% female) with early RA (disease duration < 2 years) treated with the same therapeutic protocol using gold salts and methotrexate in a step-up strategy, together with methylprednisolone (4 mg/day), were followed up for 2 years. The outcome variable was clinical remission after 2 years of DMARD therapy using the 28-joint disease activity score (DAS28 < 2.6). Clinical, biological, immunogenetic and radiographic data (Larsen score) were analyzed at study entry and after 6, 12, 18 and 24 months of follow-up. Therapeutic response was analyzed using the ACR and EULAR criteria. RESULTS: Remission was observed in 34 patients (32.4%) after 2 years of follow-up. A baseline DAS28 score < 5.1 (p = 0.004), hemoglobin (p = 0.04) and male gender (p = 0.02) were associated with remission in the univariate analysis. In the multivariate logistic regression analysis, only a DAS28 < 5.1 was associated with remission at 2 years (OR 4.1, 95% CI: 1.56;10.77, p = 0.004). The percentage of ACR50 responses after 6 months was significantly higher in patients with remission at 2 years than in those without (66.7% vs 43.3%; p = 0.04). Similar results were obtained when analyzing the good EULAR response (50% vs 20.9%; p = 0.003). Furthermore, when the therapeutic response at 6 months was included in the logistic regression model, only an ACR50 response (OR 3.9, 95% CI 1.14;13.38, p = 0.03) and a good EULAR response (OR 6.23, 95% CI 1.61; 24.04, p = 0.008), but not an ACR20 response or a whole EULAR response were significantly associated with remission. CONCLUSION: In a series of early RA patients treated using a structured algorithm with DMARDs and very low doses of glucocorticoids, clinical remission was observed in one-third of patients after 2 years. Low or moderate disease activity (DAS28 < 5.1) at baseline and a good therapeutic response during the first months of therapy predicts clinical remission at 2 years.

Prognostic factors of radiographic progression in early rheumatoid arthritis: a two year prospective study after a structured therapeutic strategy using DMARDs and very low doses of glucocorticoids.

The objective of the study was to analyze the prognostic factors of radiographic progression in a series of patients with early rheumatoid arthritis (RA) after 2 years of therapy with a structured algorithm using disease-modifying antirheumatic drugs (DMARDs) and very low doses of oral glucocorticoids. One hundred and five patients (81% female) with early RA (disease duration <2 years) treated with the same therapeutic protocol using gold salts and methotrexate in a step-up strategy, together with methylprednisolone (4 mg/day), were followed up for 2 years. The outcome variable was radiographic progression after 2 years of DMARD therapy using the modified Larsen method. Clinical, biological, immunogenetic, and radiographic data were analyzed at study entry and after 1 and 2 years of follow-up. Radiographic progression (increase of four or more units in the Larsen score) was observed in 32% of patients after 2 years of follow-up. The percentage of erosive disease increased from 18.3% at baseline to 28.9% at 12 months and 44.6% at 24 months, in spite of a significant improvement in disease activity. New erosions appeared in 33% of patients after 2 years. Several baseline parameters were associated with radiographic progression in the univariate analysis: shared epitope (SE) homozygozity, HLA-DRB*04 alleles, female gender, hemoglobin, erythrocyte sedimentation rate, and anticyclic citrullinated peptide antibodies (anti-CCP). In the multivariate analysis, female gender [odds ratio (OR) 5.5, 95% confidence interval (CI): 1.1-28.2, p = 0.04], DRB1*04 alleles (OR 3.1, 95% CI 1.1-9, p = 0.03) and, marginally, anti-CCP antibodies (OR 3.6, 95% CI 0.9-14.5, p = 0.06), were associated with progression. Female patients with both DRB1*04 alleles and anti-CCP antibodies showed the highest scores in radiographic progression. The presence, but not the titer, of anti-CCP antibodies predicted progression. The positive predictive value of the multivariate model for progression was only 53.9% whereas the negative predictive value was 80.3%. In a series of early RA patients treated with a structured algorithm using DMARDs and very low doses of glucocorticoids, radiographic progression was observed in one third of patients after 2 years. Female gender, DRB1*04 alleles (rather than the SE), and the presence of anti-CCP antibodies at baseline (independently of the titer) were the most important predictors of progression. The utility of these parameters in clinical practice is limited by their relatively low positive predictive value.

Relevance of MICA antibodies in acute humoral rejection in renal transplant patients.

The presence of MICA antibodies was examined in eleven patients diagnosed with AHR. MICA typing was performed in both recipients and donors. Sera were collected sequentially: pre-transplant, at the AHR episode and at follow-up. Sera from 30 patients with functioning graft were also analysed. A stable MICA()008 transfected cell line was used as target to identify MICA antibodies. MICA antibodies were not detected pre-transplant nor post-transplant in patients receiving a compatible graft. MICA antibodies were detected post-transplant AHR in patients receiving an incompatible graft. The persistence of MICA antibodies was associated with chronic graft dysfunction in 3 of 4 patients in this series; although it was not always associated with the graft loss in treated AHR. None of the 30 patients in the control group with long-term functioning grafts showed antibodies to MICA()008. This report provides some insights of the relevance of MICA antibodies in AHR.

Subtyping obsessive-compulsive disorder: clinical and immunological findings in child and adult onset.

It has been suggested that certain kinds of childhood OCD with specific clinical, biological and immunological characteristics may form a subgroup of OCD. We study the presence of these characteristics in child onset OCD and propose that the disorder be considered as a subtype of adult OCD. Forty adult patients with OCD were divided in two groups according to time of disease onset: 18 early onset and 21 late. Both sets were compared with a control group of 14 psychiatric patients. Child onset OCD was associated with higher mean ASLO titers, higher frequencies of history of tic disorders and tonsillitis in childhood and compulsive symptoms. No differences were found in D8/17 antibody titers or in other autoimmune parameters. The findings suggest that child onset OCD can be considered as a subgroup of adult OCD, although more specific biological markers are needed to identify it.

Lipoprotein lipase mRNA expression in brown adipose tissue: translational and/or posttranslational events are involved in the modulation of enzyme activity.

Lipoprotein lipase mRNA abundance in rat brown adipose tissue increases during the first 24 h of cold exposure. Lipoprotein lipase mRNA levels do not change in brown fat throughout pregnancy and lactation, whereas enzyme activity is significantly lowered. After 5 h of acute cold or noradrenaline administration there is a 2-fold increase in lipoprotein lipase mRNA abundance, whereas lipoprotein lipase activity is stimulated to more than 6-fold the basal values. It is concluded that translational and/or posttranslational mechanisms are involved in the noradrenergic modulation of lipoprotein lipase activity in brown fat.

Activators of peroxisome proliferator-activated receptor-alpha induce the expression of the uncoupling protein-3 gene in skeletal muscle: a potential mechanism for the lipid intake-dependent activation of uncoupling protein-3 gene expression at birth.

The recently identified uncoupling protein-3 (UCP-3) gene, predicted to encode a new member of the family of uncoupling proteins, is preferentially expressed in skeletal muscle and has been related to phenotypes of obesity and type 2 diabetes. We have established that during mouse ontogeny, the expression of the UCP-3 gene is switched on in skeletal muscle just after birth. The induction of UCP-3 gene expression is dependent on the initiation of suckling and particularly on lipid intake. Treatment of newborn mice with activators of peroxisome proliferator-activated receptors (PPARs), such as clofibrate, bezafibrate, or (4-chloro-6-(2,3-xylidine)-pirimidinylthio)acetic acid (WY 14,643), mimics the action of food intake on UCP-3 gene expression. The specific ligand of PPAR-alpha WY 14,643 induces UCP-3 gene expression in a time- and dose-dependent manner, whereas the thiazolidinedione BRL 49653, specific for PPAR-gamma, has no effect. These treatments act without altering circulating free fatty acids. During development, skeletal muscle expresses constitutive levels of PPAR-delta mRNA, whereas expression of the PPAR-gamma gene is undetectable. PPAR-alpha gene expression is developmentally regulated in muscle as it is first expressed at birth, just before UCP-3 gene induction occurs. The induction of UCP-3 gene expression by WY 14,643 is impaired in skeletal muscle of premature neonates, which do not express PPAR-alpha. It is proposed that the UCP-3 gene is predominantly regulated in neonatal muscle by PPAR-alpha activation.

Impaired expression of the uncoupling protein-3 gene in skeletal muscle during lactation: fibrates and troglitazone reverse lactation-induced downregulation of the uncoupling protein-3 gene.

The expression of uncoupling protein (UCP)-3 mRNA in skeletal muscle is dramatically reduced during lactation in mice. The reduction in UCP-3 mRNA levels lowers the amount of the UCP-3 protein in skeletal muscle mitochondria during lactation. Spontaneous or abrupt weaning reverses the downregulation of the UCP-3 mRNA but not the reduction in UCP-3 protein levels. In lactating and virgin mice, however, fasting increases UCP-3 mRNA levels. Changes in UCP-3 mRNA occur in parallel with modifications in the levels of free fatty acids, which are reduced in lactation and are upregulated due to weaning or fasting. Modifications in the energy nutritional stress of lactating dams achieved by manipulating litter sizes do not influence UCP-3 mRNA levels in skeletal muscle. Conversely, when mice are fed a high-fat diet after parturition, the downregulation of UCP-3 mRNA and UCP-3 protein levels due to lactation is partially reversed, as is the reduction in serum free fatty acid levels. Treatment of lactating mice with a single injection of bezafibrate, an activator of the peroxisome proliferator-activated receptor (PPAR), raises UCP-3 mRNA in skeletal muscle to levels similar to those in virgin mice. 4-chloro-6-[(2,3-xylidine)-pirimidinylthio] acetic acid (WY-14,643), a specific ligand of the PPAR-alpha subtype, causes the most dramatic increase in UCP-3 mRNA, whereas troglitazone, a specific activator of PPAR-gamma, also significantly increases UCP-3 mRNA abundance in skeletal muscle of lactating mice. However, in virgin mice, bezafibrate and WY-14,643 do not significantly affect UCP-3 mRNA expression, whereas troglitazone is at least as effective as it is in lactating dams. It is proposed that the UCP-3 gene is regulated in skeletal muscle during lactation in response to changes in circulating free fatty acids by mechanisms involving activation of PPARs. The impaired expression of the UCP-3 gene is consistent with the involvement of UCP-3 gene regulation in the reduction of the use of fatty acids as fuel by the skeletal muscle and in impaired adaptative thermogenesis, both of which are major metabolic adaptations that occur during lactation.

Dominant negative regulation by c-Jun of transcription of the uncoupling protein-1 gene through a proximal cAMP-regulatory element: a mechanism for repressing basal and norepinephrine-induced expression of the gene before brown adipocyte differentiation.

The brown fat uncoupling protein-1 (ucp-1) gene is regulated by the sympathetic nervous system, and its transcription is stimulated by norepinephrine, mainly through cAMP-mediated pathways. Overexpression of the catalytic subunit of protein kinase A stimulated a chloramphenicol acetyltransferase expression vector driven by the 4.5-kb 5'-region of the rat ucp-1 gene. Mutant deletion analysis indicated the presence of the main cAMP-regulatory element (CRE) in the proximal region between -141 and -54. This region contains an element at -139/-122 able to confer enhancer and protein kinase A (PKA)-dependent activity to the basal thymidine kinase promoter. The potency of this element was much higher in differentiated than in nondifferentiated brown adipocytes. Gel shift analyses indicated that a complex array of proteins from brown fat nuclei bind to the -139/-122 element, among which CRE-binding protein (CREB) and Jun proteins were identified. In transfected brown adipocytes, c-Jun was a negative regulator of basal and PKA-induced transcription from the ucp-1 promoter acting through this proximal CRE region. A double-point mutation in the -139/-122 element abolished both PKA- and c-Jun-dependent regulation through this site, and overexpression of CREB blocked c-Jun repression. Thus, an opposite action of these two transcription factors on the -139/-122 CRE is proposed. c-Jun content in brown adipocytes differentiating in culture correlated negatively with both ucp-1 gene expression and the acquisition of the brown adipocyte morphology. These findings indicate that c-Jun provides a molecular mechanism to repress the basal and cAMP-mediated expression of the ucp-1 gene before the differentiation of the brown adipocyte.

Ontogeny of thyroid hormone receptors and c-erbA expression during brown adipose tissue development: evidence of fetal acquisition of the mature thyroid status.

We have determined the developmental changes in thyroid hormone receptor expression and thyroid hormone status in rat brown adipose tissue (BAT). The present study demonstrates that c-erbA alpha and -beta genes are expressed in the developing BAT. The 6.5-kilobase (kb) beta 1, 2.6-kb alpha 2, 5.5-kb alpha 1, and 6.6-kb alpha transcripts showed peak values on day 20 of fetal life. High affinity T3-binding sites were found in fetal BAT. Binding capacity was similar (18-day-old fetuses) or higher (20-day-old fetuses) than in postnatal or adult brown fat. In contrast, T3 receptors were scarce in fetal liver, and values in adult liver were similar to those in fetal BAT on day 20. The profiles of iodothyronine 5'-deiodinase, nuclear T3 content, and receptor occupancy also showed peak values in fetal BAT on day 20, but they were very low in fetal liver. Thus, BAT has already achieved complete maturation of its thyroid status on day 20 of gestational age. This highly tissue-specific developmental pattern is unique among other mammalian thyroid-sensitive tissues studied to date. Between days 18 and 20 of rat fetal development, there is a specific induction of the uncoupling protein gene expression, the main marker of differentiated adipocytes. The results suggest that thyroid hormones may be involved in the establishment of the differentiated phenotype of the brown fat cell in fetal life.

9-cis retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes.

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9-cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all-trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9-cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

Uncoupling protein-3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non-esterified fatty acids.

Uncoupling protein-3 gene expression in skeletal muscle is up-regulated during postnatal development of mice. A high-carbohydrate diet at weaning induces a decrease in uncoupling protein-3 mRNA levels that does not occur when mice were weaned onto a high-fat diet. Uncoupling protein-3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non-esterified fatty acids, uncoupling protein-3 mRNA is up-regulated by starvation. Over-nutrition or under-nutrition during lactation increases or decreases, respectively, uncoupling protein-3 mRNA expression in skeletal muscle. Regulation of uncoupling protein-3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non-esterified fatty acids.

Muscle/heart isoform of mitochondrial adenine nucleotide translocase (ANT1) is transiently expressed during perinatal development in rat liver.

A postnatal increase in the content of mitochondrial ANT in rat liver which is related to the maturation of mitochondrial function has previously been reported [Schönfeld et al., Biochim. Biophys Acta 1144 (1993) 353-358]. In order to define the contribution of the ANT isoforms to this postnatal increase we have studied the expression of ANT1 and ANT2 isoforms in the liver during this period. The results show that in contrast to adult liver, perinatal liver expressed the ANT1 isoform at the mRNA and protein level, and that during this period the expression of ANT1 increased to a similar extent as total ANT content. It is concluded that the postnatal increase in ANT is mainly due to the ANT1 isoform and therefore, a role for the ANT1 isoform in the postnatal maturation of mitochondrial respiration in rat liver is suggested.

Opposite regulation of PPAR-alpha and -gamma gene expression by both their ligands and retinoic acid in brown adipocytes.

The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: alpha, delta, and gamma. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARgamma and PPARdelta genes are already expressed in preadipocytes, the mRNA for PPARalpha appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARalpha or PPARgamma, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARalpha and down-regulate PPARgamma. The effects on PPARalpha mRNA are independent of protein synthesis, whereas inhibition of PPARgamma mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Respiratory chain activity and mitochondrial DNA content of nonpurified and purified pancreatic islet cells.

Considerable interest has recently focused on the possible role of alterations in mitochondrial activity and mutations in the mitochondrial genome for the development of non-insulin-dependent diabetes. Our study aimed at investigating the normal mitochondrial respiratory chain activity of nonpurified and purified islet cells to further explore whether some diabetic states are associated with alterations of mitochondrial oxidative processes. For this purpose, pancreatic islets were isolated from Wistar rats. Unpurified islet cells were obtained in the presence of trypsin and DNAse, and purified beta and non-beta cells were prepared by autofluorescence-activated sorting using a flowcytometer. Intact cell respiration and substrate oxidation in digitonin-permeabilized cells were measured polarographically with a Clark oxygen electrode in a micro-water-jacketed cell. Specific activity of the individual complexes of the respiratory chain was determined spectrophotometrically in unpurified islet cells. The relative amount of mitochondrial (mtDNA) and nuclear (nDNA) DNA in all three cell populations and in rat brain and skeletal muscle was estimated by dot blotting. The intact cell respiration of unpurified islet cells corresponds to the mean of values obtained for beta and non-beta islet cells. Oxidation rates of different substrates by permeabilized beta cells were lower than those for unpurified and non-beta cells. The amount of mtDNA relative to nDNA was similar in all three groups of cells, and was also similar to that obtained from brain and skeletal muscle. In summary, we have described mitochondrial respiratory chain activity in unpurified, beta, and non-beta islet cells. Our results represent an initial step in investigating the potential pathogenic role that alterations in oxidative phosphorylation could play in some diabetic states.

Follow-up study of the revascularization process of purified rat islet beta-cell grafts.

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3-5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10-14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland.

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and alpha-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the gamma-glutamyl cycle.

Changes in alanine and glutamine transport during rat red blood cell maturation.

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na(+)-dependent L-alanine transport were: Km 0.43 and 1.88 mM and Vmax 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na(+)-dependent L-glutamine transport in reticulocytes were Km 0.51 mM and Vmax 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.