In vivo genetic engineering of murine pancreatic beta cells mediated by single-stranded adeno-associated viral vectors of serotypes 6, 8 and 9.

AIMS/HYPOTHESIS: The genetic engineering of pancreatic beta cells could be a powerful tool for examining the role of key genes in the cause and treatment of diabetes. Here we performed a comparative study of the ability of single-stranded (ss) adeno-associated viral vectors (AAV) of serotypes 6, 8 and 9 to transduce the pancreas in vivo. METHODS: AAV6, AAV8 and AAV9 vectors encoding marker genes were delivered to the pancreas via intraductal or systemic administration. Transduced cells were analysed by immunostaining. AAV9 vectors encoding hepatocyte growth factor (HGF) were delivered intraductally to a transgenic mouse model of type 1 diabetes and glycaemia was monitored. RESULTS: AAV6, AAV8 and AAV9 mediated efficient and long-term transduction of beta cells, with AAV6 and AAV8 showing the highest efficiency. However, alpha cells were poorly transduced. Acinar cells were transduced by the three serotypes tested and ductal cells only by AAV6. In addition, intraductal delivery resulted in higher AAV-mediated transduction of the pancreas than did systemic administration. As proof of concept, intraductal delivery of AAV9 vectors encoding for the beta cell anti-apoptotic and mitogenic HGF preserved beta cell mass, diminished lymphocytic infiltration of the islets and protected mice from autoimmune diabetes. CONCLUSIONS/INTERPRETATION: Intraductal administration of AAV6, AAV8 and AAV9 is an efficient way to genetically manipulate the pancreas in vivo. This technology may prove useful in the study of islet physiopathology and in assessment of new gene therapy approaches designed to regenerate beta cell mass during diabetes.

Probiotics diminish the post-operatory pain following mandibular third molar extraction: a randomised double-blind controlled trial (pilot study).

The aim of this study was to investigate the effect of a probiotic in preventing infections after third molar surgery. Thirty-eight patients were consecutively enrolled to a double-blind randomised placebo-controlled trial. Patients were asked to take one tablet two times a day containing a mixture of Levilactobacillus brevis CECT7480 (KABP-052) and Lactoplantibacillus plantarum CECT7481 (KABP-051) or placebo for the first post-intervention week. The primary outcome was the postoperative infection rate. Secondary outcomes included swelling, eating difficulties and postoperative pain recorded by the patient using a visual analogue scale (VAS) during the first postoperative week. No statistically significant difference in the infection rate between the groups was found; with only three cases of infections reported (one in the probiotic group and two in the placebo group) on the first week. Compared to placebo, treatment with the probiotic showed a significantly higher reduction in pain and eating difficulties scores at 5, 6 and 7 days post-surgery. Swelling values were not significantly different between the groups at any time point. The findings of this pilot study justify a larger study to clarify the possible role of these bacterial strains on the post-operative pain management following third molar surgery.

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol.

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.

[Pancoast's syndrome secondary to neck metastasis with normal radiography of the thorax]. / Síndrome de Pancoast secundario a metástasis cervical con radiografía de tórax normal.

Pancoast's syndrome is characterized by pain in the shoulders and upper extremities, Horner's syndrome, bone loss and hand muscle atrophy. Bronchogenic carcinoma is the most common cause, although other neoplasms or lung infection are occasionally responsible. An apical mass on the chest film can be seen in over 90% of cases, although apical pleural thickening is sometimes the only radiographic finding. We describe a patient whose clinical picture was highly suggestive of Pancoast's syndrome but whose chest film was normal. Magnetic resonance imaging disclosed a cervical mass adjacent to the brachial plexus that proved to be cervical metastasis from an unknown primary tumor. We emphasize the need to consider the possibility of a metastatic cervical tumor compromising the brachial plexus in patients with a normal chest X-ray but clinical signs highly suggestive of Pancoast's syndrome.

IGF-I mediates regeneration of endocrine pancreas by increasing beta cell replication through cell cycle protein modulation in mice.

AIMS/HYPOTHESIS: Recovery from diabetes requires restoration of beta cell mass. Igf1 expression in beta cells of transgenic mice regenerates the endocrine pancreas during type 1 diabetes. However, the IGF-I-mediated mechanism(s) restoring beta cell mass are not fully understood. Here, we examined the contribution of pre-existing beta cell proliferation and transdifferentiation of progenitor cells from bone marrow in IGF-I-induced islet regeneration. METHODS: Streptozotocin (STZ)-treated Igf1-expressing transgenic mice transplanted with green fluorescent protein (GFP)-expressing bone marrow cells were used. Bone marrow cell transdifferentiation and beta cell replication were measured by GFP/insulin and by the antigen identified by monoclonal antibody Ki67/insulin immunostaining of pancreatic sections respectively. Key cell cycle proteins were measured by western blot, quantitative RT-PCR and immunohistochemistry. RESULTS: Despite elevated IGF-I production, recruitment and differentiation of bone marrow cells to beta cells was not increased either in healthy or STZ-treated transgenic mice. In contrast, after STZ treatment, IGF-I overproduction decreased beta cell apoptosis and increased beta cell replication by modulating key cell cycle proteins. Decreased nuclear levels of cyclin-dependent kinase inhibitor 1B (p27) and increased nuclear localisation of cyclin-dependent kinase (CDK)-4 were consistent with increased beta cell proliferation. However, islet expression of cyclin D1 increased only after STZ treatment. In contrast, higher levels of cyclin-dependent kinase inhibitor 1A (p21) were detected in islets from non-STZ-treated transgenic mice. CONCLUSIONS/INTERPRETATION: These findings indicate that IGF-I modulates cell cycle proteins and increases replication of pre-existing beta cells after damage. Therefore, our study suggests that local production of IGF-I may be a safe approach to regenerate endocrine pancreas to reverse diabetes.

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells.

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.

Resection of the buccal fat pad in the treatment of hypertrophy of the masseter muscle.

Partial resection of the buccal fat pad added to the resection of the masseter muscle clearly improves the results in hypertrophy of these muscles.

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone.

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.

Parotidectomy technique in the treatment of parotid tumours (an analysis of 70 cases).

A study of seventy different cases of parotid tumours, using subtotal as opposed to total parotidectomy in the treatment of benign tumours. In malignant tumours total parotidectomy was used, together with postoperative radiotherapy, as a standard procedure. The accurate meaning of subtotal parotidectomy is defined, comparing its indications as opposed to total parotidectomy. Preoperative biopsy is performed as a diagnostic procedure, and as a reference for the surgical decision. A new dissecting retractor is presented.

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes.

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.