Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.

AIMS/HYPOTHESIS: Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect. METHODS: Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague-Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n = 4; adult, n = 3). RESULTS: Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults. CONCLUSIONS/INTERPRETATION: The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells.

Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells.

AIM/HYPOTHESIS: Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. METHODS: By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). RESULTS: At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. CONCLUSIONS/INTERPRETATION: In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor.

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection.

Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration.

BACKGROUND: After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study. METHODS AND RESULTS: Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm(2) for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-beta (PDGF-Rbeta) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-Rbeta. CONCLUSIONS: SS inhibits SMC migration and invasion via diminished PDGF-Rbeta expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

A model for glucose control of insulin secretion during 24 h of free living.

The aim of this work was to develop a mathematical model describing the functional dependence of insulin secretion on plasma glucose concentrations during 24 h of free living. We obtained hourly central venous blood samples from a group of healthy volunteers who spent 24 h in a calorimetric chamber, where they consumed standardized meals. Insulin secretory rates were reconstructed from plasma C-peptide concentrations by deconvolution. The relationship between insulin release and plasma glucose concentrations was modeled as the sum of three components: a static component (describing the dependence on plasma glucose concentration itself, with an embedded circadian oscillation), a dynamic component (modeling the dependence on glucose rate of change), and a residual component (including the fraction of insulin secretion not explained by glucose levels). The model fit of the individual 24-h secretion profiles was satisfactory (within the assigned experimental error of glucose and C-peptide concentrations). The static component yielded a dose-response function in which insulin release increased quasi-linearly (from 40 to 400 pmol/min on average) over the range of 4-9 mmol/l glucose. The dynamic component was significantly different from zero in coincidence with meal-related glucose excursions. The circadian oscillation and the residual component accounted for the day/night difference in the ability of glucose to stimulate insulin release. Over 24 h, total insulin release averaged 257+/-58 nmol (or 43+/-10 U). The static and dynamic component together accounted for approximately 80% of total insulin release. The model proposed here provides a detailed robust description of glucose-related insulin release during free-living conditions. In nondiabetic subjects, non-glucose-dependent insulin release is a small fraction of total insulin secretion.

Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study.

The contribution of gluconeogenesis (GNG) to endogenous glucose output (EGO) in type 2 diabetes is controversial. Little information is available on the separate influence of obesity on GNG. We measured percent GNG (by the 2H2O technique) and EGO (by 6,6-[2H]glucose) in 37 type 2 diabetic subjects (9 lean and 28 obese, mean fasting plasma glucose [FPG] 8.3 +/- 0.3 mmol/l) and 18 control subjects (6 lean and 12 obese) after a 15-h fast. Percent GNG averaged 47 +/- 5% in lean control subjects and was significantly increased in association with both obesity (P < 0.01) and diabetes (P = 0.004). By multivariate analysis, percent GNG was independently associated with BMI (partial r = 0.27, P < 0.05, with a predicted increase of 0.9% per BMI unit) and FPG (partial r = 0.44, P = 0.0009, with a predicted increase of 2.7% per mmol/l of FPG). In contrast, EGO was increased in both lean and obese diabetic subjects (15.6 +/- 0.5 micromol x min(-1) x kg(-1) of fat-free mass, n = 37, P = 0.002) but not in obese nondiabetic control subjects (13.1 0.7, NS) as compared with lean control subjects (12.4 +/- 1.4). Consequently, gluconeogenic flux (percent GNG x EGO) was increased in obesity (P = 0.01) and markedly elevated in diabetic subjects (P = 0.0004), whereas glycogenolytic flux was reduced only in association with obesity (P = 0.05). Fasting plasma glucagon levels were significantly increased in diabetic subjects (P < 0.05) and positively related to EGO, whereas plasma insulin was higher in obese control subjects than lean control subjects (P = 0.05) and unrelated to measured glucose fluxes. We conclude that the percent contribution of GNG to glucose release after a 15-h fast is independently and quantitatively related to the degree of overweight and the severity of fasting hyperglycemia. In obese individuals, reduced glycogenolysis ensures a normal rate of glucose output. In diabetic individuals, hyperglucagonemia contributes to inappropriately elevated rates of glucose output from both GNG and glycogenolysis.

Effect of physiological hyperinsulinemia on gluconeogenesis in nondiabetic subjects and in type 2 diabetic patients.

Gluconeogenesis (GNG) is enhanced in type 2 diabetes. In experimental animals, insulin at high doses decreases the incorporation of labeled GNG precursors into plasma glucose. Whether physiological hyperinsulinemia has any effect on total GNG in humans has not been determined. We combined the insulin clamp with the (2)H(2)O technique to measure total GNG in 33 subjects with type 2 diabetes (BMI 29.0 +/- 0.6 kg/m(2), fasting plasma glucose 8.1 +/- 0.3 mmol/l) and in 9 nondiabetic BMI-matched subjects after 16 h of fasting and after euglycemic hyperinsulinemia. A primed-constant infusion of 6,6-(2)H-glucose was used to monitor endogenous glucose output (EGO); insulin (40 mU. min(-1). m(-2)) was then infused while clamping plasma glucose for 2 h (at 5.8 +/- 0.1 and 4.9 +/- 0.2 mmol/l for diabetic and control subjects, respectively). In the fasting state, EGO averaged 15.2 +/- 0.4 micromol. min(-1). kg(-1)(ffm) (62% from GNG) in diabetic subjects and 12.2 +/- 0.7 micromol. min(-1). kg(-1)(ffm) (55% from GNG) in control subjects (P < 0.05 or less for both fluxes). Glycogenolysis (EGO - GNG) was similar in the two groups (P = NS). During the last 40 min of the clamp, both EGO and GNG were significantly (P < 0.01 or less, compared with fasting) inhibited (EGO 7.1 +/- 0.9 and 3.6 +/- 0.5 and GNG 7.9 +/- 0.5 and 4.5 +/- 1.0 respectively) but remained significantly (P < 0.05) higher in diabetic subjects, whereas glycogenolysis was suppressed completely and equally in both groups. During hyperinsulinemia, GNG micromol. min(-1). kg(-1)(ffm) in diabetic and control subjects, was reciprocally related to plasma glucose clearance. In conclusion, physiological hyperinsulinemia suppresses GNG by approximately 20%, while completely blocking glycogenolysis. Resistance of GNG (to insulin suppression) and resistance of glucose uptake (to insulin stimulation) are coupled phenomena. In type 2 diabetes, the excess GNG of the fasting state is carried over to the insulinized state, thereby contributing to glucose overproduction under both conditions.

Effect of vitamin C on forearm blood flow and glucose metabolism in essential hypertension.

In 9 patients with essential hypertension, we tested whether a high-dose (12 mg. min(-1)) vitamin C infusion into the brachial artery, by improving endothelium-dependent vasodilatation, would also attenuate the insulin resistance of deep forearm tissues. We measured the effect of vitamin C on acetylcholine (Ach)-induced vasodilatation and on forearm glucose uptake during systemic hyperinsulinemia; in all studies, the contralateral forearm served as the control. Intrabrachial Ach infusion produced a stable increase in forearm blood flow, from 2.6+/-0.3 to 10.6+/-2.1 mL. min(-1). dL(-1); when vitamin C was added, a further rise in forearm blood flow (to 13.4 mL. min(-1). dL(-1); P<0.03 vs Ach alone) was observed. In response to insulin, blood flow in both the infused and control forearms did not significantly change from baseline values (+10+/-16% and +2+/-11%, respectively). In contrast, when vitamin C was added, blood flow in the infused forearm increased significantly (to 3.7+/-0.7 mL. min(-1). dL(-1); P<0.02 vs 2.8+/-0.6 mL. min(-1). dL(-1) in the control forearm). Insulin stimulated whole-body glucose disposal to 20+/-2 micromol. min(-1). kg(-1), compatible with the presence of marked insulin resistance. Forearm glucose uptake was similarly stimulated after 80 minutes of insulin infusion (to 2.11+/-0.42 and 2.06+/-0.43 micromol. min(-1). dL(-1), infused and control, respectively). When intrabrachial vitamin C was added, no difference in glucose uptake was observed between the 2 forearms (infused, 2.37+/-0.44 micromol. min(-1). dL(-1)and control, 2.36+/-0. 53 micromol. min(-1). dL(-1)). Forearm O(2) uptake at baseline was also similar in the 2 forearms (infused, 9.7+/-0.7 micromol. min(-1). dL(-1) and control, 9.6+/-1.1 micromol. min(-1). dL(-1)) and was not changed by either insulin or vitamin C. We conclude that in the deep forearm tissues of patients with essential hypertension and insulin resistance, an acute improvement in endothelial function, obtained with pharmacological doses of vitamin C, restores insulin-mediated vasodilatation but does not improve insulin-mediated glucose uptake. Thus, the endothelial dysfunction of essential hypertension is unlikely to be responsible for their metabolic insulin resistance.

Wild-type p53 gene transfer inhibits invasion and reduces matrix metalloproteinase-2 levels in p53-mutated human melanoma cells.

The tumor suppressor gene p53 has inhibitory effects on cell growth and angiogenesis and induces apoptosis when overexpressed in melanoma and in a variety of tumor cells by adenovirus-mediated gene transfer. The invasive ability of tumor cells, facilitating local infiltration and metastasis, is related to matrix metalloproteinase levels. In melanoma, matrix metalloproteinase-2 and matrix metalloproteinase-9 have a prominent role in this process. The aim of this study was to evaluate whether wild-type p53 overexpression, obtained by a recombinant adenovirus vector (AdCMV.p53), affects cell invasiveness through modulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Two human melanoma cell lines were used in this study: the SK-MEL-110, carrying a mutated p53 gene, and the SK-MEL-147, carrying the wild-type p53 gene. SK-MEL-110 cells infected with AdCMV.p53 exhibited decreased invasion capability from day 1 after infection, compared with cells not infected or infected with the control vector AdCMV.Null. This reduced invasiveness was associated with decreased matrix metalloproteinase-2 levels in conditioned media whereas no changes were detected in matrix metalloproteinase-9 secreted levels. No modulation in matrix metalloproteinase-2 mRNA levels was detectable, however, after wild-type p53 gene transfer. Furthermore, protein expression of secreted tissue inhibitor of metalloproteinase-2 was not altered by AdCMV.p53 treatment. In contrast, in SK-MEL-147 cells, AdCMV.p53 did not affect cell invasiveness and levels of secreted matrix metalloproteinase-2. Gene transfer of wild-type p53 inhibited proliferation of both cell lines, showing that also SK-MEL-147 cells respond to wild-type p53 overexpression. This novel mechanism of action of wild-type p53 gene transfer may contribute to its antitumor effect by downregulating cell invasion and matrix metalloproteinase-2 secreted levels in mutated p53 human melanoma cell lines.

Vasodilation with sodium nitroprusside does not improve insulin action in essential hypertension.

The vasodilation induced by systemic insulin infusion is mediated by nitric oxide and is impaired both in obese subjects and patients with essential hypertension. Whether this vascular defect explains the metabolic resistance to insulin action is uncertain. In 8 overweight male patients with essential hypertension, we used the double forearm (ie, infused versus control) technique, combined with the euglycemic hyperinsulinemic clamp, to test whether sustained vasodilation (induced by intra-arterial sodium nitroprusside infusion) improves insulin-mediated glucose uptake. During the clamp, whole-body glucose disposal rose to 24.4+/-2.9 micromol x min(-1) x kg(-1). Forearm blood flow in the control forearm was stable (3.1+/-0.4 versus 2.9+/-0.3 mL x min[-1] x dL[-1]), while in the infused forearm it increased from 3.4+/-0.5 to 10.6+/-1.3 mL x min(-1) x dL(-1) in response to sodium nitroprusside. During insulin administration, tissue glucose extraction rose from 2+/-1% to 21+/-4% (P<.001) in the control forearm and from 2+/-1% to 8+/-3% in the infused forearm (P<.02 versus baseline for both); the calculated net glucose uptake reached similar plateaus in the two forearms (3.5+/-0.7 versus 3.7+/-0.6 micromol x min(-1) x kg(-1), control versus infused, P=.6). We conclude that in overweight male patients with essential hypertension, increasing forearm perfusion with sodium nitroprusside does not attenuate the insulin resistance of forearm tissues.

Structural features of latex gloves in dental practice.

The aim of this study was to define from a morpho-structural point of view, using scanning electron microscopy, the features of various types of disposable latex gloves commonly used in Italian dental practice (Biogel D, Trend, Pagni, J&J, Latechnics, Pehasoft, Bantex). None of the brands examined was free from morphological flaws; however, while in some of these only slight depressions were found (Biogel D, Trend), in others (Latechnics, Bantex) there was a marked lack of homogeneity in the latex structure or real holes (Pehasoft). This study emphasizes the current difficulties faced by dentists in the search for safe working conditions.

Role of thyroglobulin measurement in fine-needle aspiration biopsies of cervical lymph nodes in patients with differentiated thyroid cancer.

The identification of metastatic neck lymph nodes in patients awaiting surgery for differentiated thyroid tumor permits their excision during thyroidectomy. In order to detect thyroid cancer lymphatic metastasis before surgery, we measured thyroglobulin (Tg) in the needle wash-out of fine-needle aspiration biopsy (FNAB). Ultrasound-guided FNAB on enlarged neck nodes was performed in 23 patients awaiting surgery for differentiated thyroid tumor (n = 33 lymph nodes), 47 patients previously thyroidectomized for thyroid tumor (n = 89 lymph nodes), and 60 patients without thyroid disease (n = 94 lymph nodes). Immediately after aspiration biopsy, the needle was rinsed with 1 mL of normal saline solution and Tg levels were measured on the needle wash-out (FNAB-Tg). FNAB-Tg levels were markedly elevated in metastatic lymph nodes both in patients awaiting thyroidectomy (metastatic vs. negative lymph nodes, mean +/- SEM, 16,593 +/- 7,050 ng/mL vs. 4.91 +/- 1.61 ng/mL; p < 0.001) and in thyroidectomized patients (11,541 +/- 7,283 ng/mL vs. 0.45 +/- 0.07 ng/mL; p < 0.001). FNAB-Tg sensitivity, evaluated through histological examination in 69 lymph nodes, was 84.0%. The combination of cytology plus FNAB-Tg increased FNAB sensitivity from 76% to 92.0%. In conclusion, FNAB-Tg measurement is a useful technique for early diagnosis of lymph node metastasis originating from differentiated thyroid cancer.

[Acute suppurative thyroiditis in a patient with prior subacute thyroiditis]. / Tiroidite acuta suppurativa in un paziente con pregressa tiroidite subacuta.

Acute suppurative thyroiditis is an uncommon thyroid disorder usually caused by bacterial infection. The most common route of infection is a fistula that originates from the fundus of the pyriform sinus. Pre-existing thyroid disease, most commonly nodular goiter, has been reported to be present in acute suppurative thyroiditis. A 44 year old man presented a subacute thyroiditis, resolved by nonsteroidal antiinflammatory treatment. One year later, the patient abruptly complained of fever and painful swelling in the thyroid region. A relapse subacute thyroiditis was diagnosed and prednisone treatment was started. A few days later owing to a worsening of the pain and of the clinical features the patient was referred to our department. He presented dysphagia and he was feverish, the overlying skin of the neck swelling was erythematous and warm. There was a neutrophilia (83.7%). Plasma FT4, FT3 and TSH were normal. Anterior neck region ultrasonography showed an enlargement of the left thyroid lobe with poorly defined shapes and inhomogeneous parenchyma while the right lobe of the gland was normal. The 131-I thyroid scan showed a large cold area in the upper part of the left thyroid lobe and preserved radionuclide uptake in the residual parenchyma. The RAIU was normal. We diagnosed acute suppurative thyroiditis and started antibiotics treatment. The day after the patient was still feverish and he gave out from the mouth a great quantity of sero-purulent material with a swelling reduction and improvement of the neck pain. Barium swallow examination did not show any fistula in the cervical esophagus. The fistula opening was demonstrated by indirect laryngoscopy in the postero-lateral side of hypopharynx.(ABSTRACT TRUNCATED AT 250 WORDS)

[Exacerbation of autoimmune hypothyroidism after hemi-hypophysectomy in a patient with Cushing's disease]. / Esacerbazione di ipotiroidismo autoimmune dopo emi-ipofisectomia in una paziente con morbo di Cushing.

A 41 year old woman affected by Cushing's disease underwent hemi-hypophysectomy with removal of an ACTH secreting microadenoma. Forty days later, when normal ACTH, cortisol plasma levels and urinary cortisol levels were restored, features of primary autoimmune hypothyroidism developed. While cortisol levels were elevated serum thyroid hormone levels were normal, serum hormone TSH was at the upper limit of the normal range and serum antimicrosomal antibodies were slightly elevated. It is likely that hypothyroidism already present before surgery was not clinically evident due to the immunosuppressive effect of high cortisol levels. The need to assess thyroid function in patient with hypercortisolism is emphasized with the aim to identify the possible onset of autoimmune thyroid disease when cortisol levels are normalized.

[Hyperprolactinemia in hypothyroidism: effects of L-thyroxine therapy]. / Iperprolattinemia nell'ipotiroidismo: effetti della terapia con L-tiroxina.

It is well known that the pituitary PRL secretion is modified in patients with primary hypothyroidism. The serum PRL is elevated in approximately one third of patients, the others presenting with enhanced PRL release after TRH intravenous (i.v.) administration. The objective of this study was to verify how treatment with L-thyroxine modifies the enhanced PRL response to TRH administration in a group of patients with primary hypothyroidism. Eight female patients aged 28 to 64 (mean age +/- SD = 17.2 +/- 6.0) with primary hypothyroidism were studied. Diagnosis was based on clinical features and plasma FT4 (mean +/- SD = 5.2 +/- 0.9 pmol/l; n.r. 7.7-19.3 pmol/l) and TSH (mean +/- SD = 87.3 +/- 20 mUI/l; n.r. 0.2-5 mUI/l) levels. As controls eleven normal age-matched female subjects were also studied. After an overnight fasting an indwelling catheter was inserted into an antecubital vein of the forearm and kept patient by slow infusion of normal saline solution. After 60' the basal blood sample was collected and 200 mcg of TRH was injected intravenously (0'), further blood samples were taken at 30', 60', 90', 120' and 180'. PRL determinations (n.r. 3-19 ng/ml) of the blood samples obtained were made using fluoroimmunometric assay. Hypothyroid patients underwent a second TRH test after L-thyroxine replacement therapy (100 mcg/day) had led to euthyroidism for at least three months.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hyperplasia of the coronoid processes causing limitation of mouth opening. A report of 4 cases]. / L'iperplasia dei processi coronoidi causa di limitazione dell'apertura della bocca. Presentazione di 4 casi.

Four cases of subtotal clamping of the jaws caused by bilateral hyperplasia of the coronoid processes are reported. The aetiopathogenetic theories are examined and clinical cases and therapy described. It is concluded that treatment can only be surgical because the mechanical obstacle between the lengthened coronoid apophysis and the internal surface of the malar bone has to be removed.

[The marginal leakage of amalgam and Vitrebond restorations after an occlusal load test]. / Infiltrazione marginale di restauri in amalgama e Vitrebond dopo test da carico occlusale.

The use of adhesive liners in amalgam fillings and restorations is a recent form of conservative dentistry. The aim of our study was to assess marginal microleakage in amalgam and Vitrebond restorations after occlusal load test. Using extracted teeth, we prepared 16 class II amalgam restorations, (Valiant ICQ, Caulk). Adhesive liner, (Vitrebond, 3M USA), was used in half of these. The samples then had first a cyclic load test, (27 kg per 7,000 cycles), followed by a microleakage test, (sample immersed in 2% erythrosin solution for 24 hours). The results showed that, in samples with adhesive liner, color penetrated to a statistically lesser extent than in the control group without Vitrebond.

Changes in gene expression in beta cells after islet isolation and transplantation using laser-capture microdissection.

AIMS/HYPOTHESIS: The process of islet isolation can cause chemical and mechanical injury to beta cells. In addition, hyperglycaemia after islet transplantation can compromise beta cell function. The aim of this experiment was to evaluate changes in gene expression in endogenous islets using laser-capture microdissection (LCM). MATERIALS AND METHODS: Islets from B6AF1 mice were studied in situ in the pancreas as well as those freshly isolated or cultured for 24 h. Fresh islets were transplanted under the kidney capsule of syngeneic diabetic (streptozocin-induced) and non-diabetic mice. Frozen sections from all the samples were prepared for LCM to obtain beta cell-enriched tissue; RNA was extracted and amplified using T7 polymerase. RT-PCR was used to assess expression of selected genes critical for beta cell function (Ins, Ipf1 [previously known as Pdx1], Slc2a2 [previously known as GLUT2] and Ldha) and the stress response (Hmox1 [previously known as HO-1], Gpx1, Tnfaip3 [previously known as A20] and Fas). Immunostaining was also performed. RESULTS: In freshly isolated and cultured islets, insulin and Ipf1 mRNA levels were decreased by 40% (compared with islets in situ), while stress genes were upregulated. Comparison between in situ pancreatic islets and engrafted beta cells of cured mice showed declines in Ipf1 expression. CONCLUSIONS/INTERPRETATION: Our experiment, the first report to investigate changes in gene expression in endogenous islets using LCM, indicate that beta cells following islet isolation and residing in a foreign graft environment have decreased expression of genes involved in insulin production and increased expression of stress genes. Our data suggest that an islet graft, even in successful transplantation, may be different from endogenous islets in gene expression.

Oxalate solution and collagen-oxalate solution as protective liners for dentine.

The aim of the present study was to evaluate under scanning electron microscopy the ability of different chemically reactive solutions to form a protective layer on dentine. The materials selected were an oxalate solution, a collagen-oxalate solution, a collagen-oxalate solution plus glutaraldehyde and a saline control solution. Small dentine fragments, obtained from extracted human teeth, were treated with the various chemical solution for 60 seconds. All samples were then examined in a Jeol scanning electron microscope. The preliminary observations suggest that dentine may be covered by a mixture of oxalate crystals and collagen producing a homogeneous layer of reactive agents.