Morphological study of the anatomical variations of anterior belly of digastric muscle in Brazilian cadavers.

BACKGROUND: Cases of variations in anterior belly of the digastric muscle must be carefully identified to avoid misinterpretations and assist in the correct surgical or aesthetic procedure and help in the teaching of anatomy. The aim of this study was to describe the anatomical variations of anterior belly of digastric muscle in Brazilian cadavers. MATERIALS AND METHODS: Thirty-one human heads were selected, from adult cadavers (18-80 years, 29 males and 2 females). The morphology of the anterior belly of the digastric muscle was observed, identifying the possible anatomical variations that were characterised and classified according to the amount of muscle bellies, fibre direction and place of origin and insertion. The morphometric measurements were performed using a digital calliper. To analyse the data obtained, photographic documentation, anatomical description and individual morphometric description of each muscle belly were performed. The incidence of anatomical variation was obtained in percentage (%). RESULTS: The anatomical variation of the anterior belly of the digastric muscle was present in 6 cadavers (19.31%; 1 female and 5 male). All anatomical variations presented an accessory belly to the anterior belly. However, these accessory bellies were configured differently in the location, direction of muscle fibres and in their dimensions (length and width). CONCLUSIONS: The gross anatomy of the anterior belly of the digastric muscle and their variations is important to assist in surgical procedures, pathological or diagnostic function. In addition, asymmetrical variations in the submental region must be carefully identified to avoid misinterpretations.

In Vivo Pulp Temperature Changes During Class V Cavity Preparation and Resin Composite Restoration in Premolars.

OBJECTIVE: This in vivo study evaluated the influence of the sequence of all restorative steps during Class V preparation and restoration in human premolars on pulp temperature (PT). METHODS AND MATERIALS: Intact premolars with orthodontic extraction indication of 13 volunteers received infiltrative anesthesia and isolation with rubber dam. An occlusal preparation was made with a high-speed diamond bur under air-water spray until the pulp was minimally exposed, then a thermocouple probe was inserted within the pulp. A deep, 2.0-mm depth Class V preparation was made using a high-speed diamond bur under air-water spray. Three restorative techniques were performed (n=7): Filtek Z250 placed in two increments (10-second exposure, shade:A2, 3M ESPE, St. Paul, MN, USA), Filtek Z350 XT (40-second exposure, shade:A3D, 3M ESPE) and Tetric N Ceram Bulk Fill (10-second exposure, shade:IVA, Ivoclar Vivadent, Schaan, Liechtenstein), both placed in a single layer. Bonding layer and resin composite were exposed to light from the same Polywave LCU (Bluephase 20i, Ivoclar Vivadent). The peak PT and the difference between peak PT and baseline (ΔT) values were subjected to two-way, repeated measures analysis of variance (ANOVA), followed by the Bonferroni post-hoc test (α=0.05). RESULTS: Cavity preparation and etch & rinse procedures decreased the PT values (p<0.001). The 40-second exposure of Filtek Z350 caused the highest peak PT values (38.7±0.8°C) and the highest ΔT values (3.4±0.8°C), while Tetric N Ceram Bulk Fill showed the lowest values (-1.6±1.3°C; p=0.009). CONCLUSION: None of the evaluated procedures resulted in a PT rise near to values that could offer any risk of thermal damage to the pulp.

Obesity and surgical complications of pancreaticoduodenectomy: An observation study utilizing ACS NSQIP.

BACKGROUND: An estimated 38% of US adults are obese. Obesity is associated with socioeconomic disparities and increased rates of comorbidities, and is a known risk factor for development of pancreatic cancer. As a fourth leading cause of death in the United States, pancreatic cancer is commonly treated with a pancreatico-duodenectomy (PD), or Whipple procedure. Data regarding the effects of obesity on post-operative complication rate primarily comes from specialized centers, however the results are mixed. Our aim is to elucidate the effects that obesity has on outcomes after PD for pancreatic head cancer using a national prospectively maintained clinical database. METHOD: The 2010-2015 American College of Surgeons National Surgical Quality Improvement Project (ACS NSQIP) Participant Use Files (PUF) were used as the data source. We identified cases in which PD was performed (CPT code 48150) in the setting of a postoperative diagnosis of pancreatic cancer (ICD9 code 157.0). We excluded cases that had emergency admissions, BMI ≤18.5 kg/m2, intraoperative wound classification of III or IV, and disseminated cancer. Cases with missing BMI, preoperative albumin, operative time, LOS data were also excluded. Multiple imputation for missing sex, race, functional status, and ASA classification using chained equations was performed.16 Patients that had BMI ≥30 kg/m2 were considered obese, and patients with BMI <30 kg/m2 were used as control. RESULTS: 3484 patients underwent pancreaticoduodenectomy for pancreatic cancer. 860 patients were identified as obese. Propensity score analysis was performed matching age, sex, race, functional status, presence of dyspnea, diabetes, hypertension, acute renal failure, dialysis dependence, ascites, steroid use, bleeding disorders, history of chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF), weight loss, American Society of Anesthesiologists (ASA) classification, and preoperative albumin levels. After matching, obese patients had higher risk of 30-day postoperative complications compared to control, including organ space wound infections (OR 1.38, 95% CI 1.07-1.79, p = 0.0128), returning to the operating room (OR 1.39, 95% CI 1.01-1.91, p = 0.0461), failure to extubate for greater than 48 h (OR 1.60, 95% CI 1.09-2.34, p = 0.0153), death (OR 1.68, 95% CI 1.01-2.78, p = 0.0453), septic shock (OR 2.22, 95% CI 1.46-3.38, p = 0.0002), pulmonary embolism (OR 2.42, 95% CI 1.07-5.45, p = 0.0332), renal insufficiency (OR 2.67, 95% CI 1.33-5.38, p = 0.0058). Sensitivity analysis yielded similar results with the exception of risk for return to the operating room, death, and pulmonary embolism, P > .05. CONCLUSION: In this large observational study using a national clinical database, obese patients undergoing PD for head of pancreas cancer had increased risk of postoperative complications and mortality in comparison to controls.

Outcomes in video-assisted thoracoscopic surgery lobectomies: challenging preconceived notions.

BACKGROUND: Most thoracic surgical procedures in the United States are being performed by general surgeons (GSs) without any advanced training. With the recent approval of computed tomography screening for lung malignancy in high-risk populations, the number of thoracic oncologic resections is expected to rise. Previous literature has demonstrated consistently worsened outcomes for patients undergoing thoracic surgical procedure when done by nonthoracic fellowship-trained surgeons. Using the American College of Surgeons National Surgical Quality Improvement Project database, we examined short-term outcomes in patients undergoing video-assisted thoracoscopic surgery (VATS) lobectomy for malignancy. MATERIALS AND METHODS: Data were obtained from the American College of Surgeons National Surgical Quality Improvement Project from 2010-2015. We identified patients who had an International Classification of Disease 9 diagnosis of lung cancer (162) who underwent VATS lobectomy (current procedural terminology 32663). We included only adults (≥18y) and elective cases. We excluded patients who had preoperative diagnosis of sepsis, contaminated wound class, or those patients with missing American Society of Anesthesiologists classification, morbid obesity, functional status, length of stay (LOS), or sex, and race information. We identified two groups by specialty: GS versus cardiothoracic (CT) surgeon. We then performed univariate analysis. We then performed propensity score analysis using a 1:3 ratio of general surgery patients to CT patients. Outcomes of interest included 30-d postoperative mortality, 30-d postoperative morbidity, and LOS. RESULTS: A total of 4105 patients were identified, 607 performed by GSs, 3508 performed by CT surgeons. The mean age for patients who underwent lobectomies by GSs was 68.6 versus 67.8 in the CT surgeon group (P < 0.05). The majority were female (58.09% GS versus 57.74% CT surgeon). There was a statistically significant difference in race between groups; patients were more likely to be African American in the CT surgeon group. Operative time was lower in the GS group as opposed to the CT surgeon group 179 min versus 196 (P < 0.01). Univariate analysis (mortality <0.1 CT surgeon and GS) and 1:3 propensity score matched analysis (0.08 GS% versus 0.08% CT surgeon) failed to demonstrate a significant difference in mortality. There was a statistically significant difference in median LOS between groups (6.2 GS versus 5.1 CT surgeon). Univariate and propensity matched analyses of pneumonia, sepsis, wound infection, deep vein thrombosis, transfusion requirement, myocardial infarction stroke, postoperative renal insufficiency, failure to wean, pulmonary embolism, reintubation, and deep organ space infection all failed to demonstrate a statistically significant difference between our groups of interest. Urinary tract infection was noted to be higher in the GS group operating room 2.29 as compared to the CT surgeon group (P value 0.02). CONCLUSIONS: In this large observational study, we found that VATS lobectomies performed by GS compared to the matched CT surgeon cohort had shorter operative time, and there was no difference in major postoperative morbidity or mortality. However, LOS was higher and there was increased risk of urinary tract infection in the GS compared to matched CT surgeon cohort.

Hepatic intra-arterial therapies in metastatic neuroendocrine tumors: lessons from clinical practice.

BACKGROUND: Liver metastases are common in patients with neuroendocrine tumors (NETs), having a negative impact on disease prognosis. The options for selective therapy in patients with unresectable multiple liver metastases are limited to TACE (transarterial chemoembolization), TAE (transarterial embolization), or SIRT (selective internal radiation therapy). AIM: To explore the clinical outcome, survival and safety of these therapies in NETs patients. METHODS: Retrospective case series of consecutive patients (mean age 56.6 years, 59% male) treated at two tertiary university medical centers from 2005 to 2015. RESULTS: Fifty-seven patients with G1, G2, and low G3 NETs with liver metastases were investigated (pancreatic NET (pNET), 24; small bowel, 16; unknown origin (UKO), 9; rectal, 3; lung, 3; and gastric, 2). Fifty-three patients underwent TACE, three patients underwent TAE, and one patient underwent SIRT. Clinical improvement and tumor response were observed in 54/57 patients (95%), together with marked decreased in tumor markers. The median time to tumor progression following the first treatment was 14 ± 16 months. The median overall survival was 22 ± 18 months, more pronounced in the pNET, followed by small bowel and UKO subgroups. There was a trend for a better survival in patients with disease limited to the liver and in whom the primary tumor was resected. CONCLUSION: Hepatic intra-arterial therapies are well tolerated in the majority of patients with NETs and liver metastases and associated with both clinical improvement and tumor stabilization for prolonged periods. These therapies should be always considered, irrespective of the presence of extrahepatic metastasis.

Efficacy of everolimus plus octreotide LAR in patients with advanced neuroendocrine tumor and carcinoid syndrome: final overall survival from the randomized, placebo-controlled phase 3 RADIANT-2 study.

Background: In the phase 3 RADIANT-2 study, everolimus plus octreotide long-acting repeatable (LAR) showed improvement of 5.1 months in median progression-free survival versus placebo plus octreotide LAR among patients with advanced neuroendocrine tumors associated with carcinoid syndrome. The progression-free survival P-value was marginally above the prespecified threshold for statistical significance. Here, we report final overall survival (OS) and key safety update from RADIANT-2. Patients and methods: The RADIANT-2 trial compared everolimus (10 mg/day, orally; n = 216) versus placebo (n = 213), both in conjunction with octreotide LAR (30 mg, intramuscularly, every 28 days). Patients, unblinded at the time of progression or after end of double-blind core phase following primary analysis, were offered open-label everolimus with octreotide LAR (open-label phase). In the open-label phase, patients had similar safety and efficacy assessments as those in the core phase. For OS, hazard ratios (HRs) with 95% CIs using unadjusted Cox model and a Cox model adjusted for prespecified baseline covariates were calculated. Results: A total of 170 patients received open-label everolimus (143 crossed over from the placebo arm; 27 in the everolimus arm continued to receive the same treatment after unblinding). The median OS (95% CI) after 271 events was 29.2 months (23.8-35.9) for the everolimus arm and 35.2 months (30.0-44.7) for the placebo arm (HR, 1.17; 95% CI, 0.92-1.49). HR adjusted for baseline covariates was 1.08 (95% CI, 0.84-1.38). The most frequent drug-related grade 3 or 4 AEs reported during the open-label phase were diarrhea (5.3%), fatigue (4.7%), and stomatitis (4.1%). Deaths related to pulmonary or cardiac failure were observed more frequently in the everolimus arm. Conclusion: No significant difference in OS was observed for the everolimus plus octreotide LAR and placebo plus octreotide LAR arms of the RADIANT-2 study, even after adjusting for imbalances in the baseline covariates. Clinical Trial Number: NCT00412061, www.clinicaltrials.gov.

The role of hepatic trans-arterial chemoembolization in metastatic medullary thyroid carcinoma: a specialist center experience and review of the literature.

BACKGROUND: Liver metastases are relatively common in patients with metastatic medullary thyroid carcinoma (MTC), carrying a negative impact on disease prognosis. The options for selective therapy of liver metastases in MTC patients are limited to catheter-guided procedures such as trans-arterial chemoembolization (TACE). Data regarding the effectiveness and safety of this procedure in MTC are limited. AIM: To explore the clinical outcome, survival and safety profile of TACE for liver metastases in a group of MTC patients. METHODS: Retrospective case series of patients treated at a single tertiary University Medical Center from 2005 to 2015. RESULTS: Seven consecutive patients (mean age 64.5 ± 10.9 years, 5 females) with histologically confirmed MTC with liver metastases were included. Metastatic involvement of the liver was less than 50% of the liver volume in all patients. The median size of the largest liver lesion was 40 ± 6.9 mm. The patients underwent in total 20 sessions of TACE. Clinical improvement as well as tumor response (PR) were observed in all patients. The median time to tumor progression was 38 months (range 8-126). Three patients were still alive at the end of the follow-up period (a median overall survival rate of 57 ± 44 months). CONCLUSION: TACE in MTC patients with hepatic metastases is usually well tolerated and induces both clinical improvement and tumor response for prolonged periods of time in the majority of patients. This therapeutic option should always be considered, irrespective of the presence of extrahepatic metastasis.

Everolimus therapy for progressive adrenocortical cancer.

Patients with advanced adrenocortical carcinoma (ACC) have limited treatment options after failure of chemotherapy. Tumor IGF2 expression has been shown to be amplified in the majority of cases of ACC and autocrine/paracrine activation of the IGF receptor (IGF-R) is thought to play a major role in the pathogenesis of ACC. It has been shown in vitro that inhibition of the IGF-R inhibits ACC cell proliferation. mTOR is a downstream effector of the IGFR signaling pathway; therefore, the rapamycin analog everolimus could prove to be useful for treatment of patients with ACC. Four women with ACC (ages 25-60 years) developed stage IV disease after surgery. All had progressive disease (PD) despite treatment with mitotane and other treatment modalities (etoposide, doxorubicin, cis-platinum in 3/4 patients, further streptozotocin + 5-FU in 1/4 patients, further thalidomide therapy in 2/4 patients; 1 patient progressed on an IGF-R antagonist). The patients were started on everolimus 10 mg/day orally and 2/4 patients also continued mitotane. Disease progression was monitored monthly by CT in 3/4 and after 3 months in 1/4. In all patients everolimus was well tolerated. In the three patients monitored monthly, PD was evident after 1, 3, and 4 months; in the patient evaluated after 3 months PD was also evident. In this small exploratory study, no clinically meaningful response was observed with everolimus in four patients with advanced ACC. The failure of efficacy could be related to an interaction with mitotane, multiple signaling pathways, and/or other downstream IGF-R effectors operative in the pathogenesis of ACC.

Current size criteria for the management of neuroendocrine tumors of the appendix: are they valid? Clinical experience and review of the literature.

We evaluated the latest pathological criteria for completion right hemicolectomy (RHC) in patients with appendiceal neuroendocrine tumors (ANETs) with emphasis on the size of the primary tumor. Data of 28 consecutive patients who underwent RHC for ANETs in three tertiary hospitals were reviewed retrospectively to assess the indications for completion RHC. 10/28 patients were found to have residual disease (36%). In 8/28 patients (29%), the tumor diameter was <1 cm (mean 0.7 ± 0.2 cm, range 0.5-0.9 cm); the indications for RHC included: tumor presence in surgical margins (1 patient), extensive mesoappendiceal invasion (EMI) (1 patient), vascular invasion (VI) (3 patients), Ki-67 ≥2% (3 patients); residual disease was present in 1 patient (3.5%). In 13/28 patients (46%), the tumor diameter was ≥1 and <2 cm (mean 1.30 ± 0.2 cm, range 1.0-1.8 cm); the indications for RHC were: EMI (2 patients), VI (2 patients), Ki-67 ≥2% (2 patients); residual disease was present in 5 patients (18%). In 7/28 patients (25%), the tumor diameter was ≥2 cm (mean 2.5 ± 0.7 cm, range 2.0-4.0 cm). In this final subgroup, RHC was an accepted practice irrespective of other pathologic findings: the tumor was present in surgical margins in 2 patients, in 5 patients VI was demonstrated, and Ki-67 ≥2% was found in 5 patients; residual disease was present in 4 patients (14%). Using the latest European Neuroendocrine Tumor Society criteria for RHC, residual disease may be missed in 18% of ANET patients.

New drugs in the therapy of neuroendocrine tumors.

Neuroendocrine tumors (NET) are a rare and heterogeneous group of neoplasms of a relatively indolent nature whose incidence and prevalence are increasing. Despite the advances made in the field of NET over the past years, these tumors eventually progress to metastatic disease in most of the patients, with a fatal outcome in the majority. Traditional cytotoxic agents remain of limited efficacy; however, recently, a better understanding of molecular pathways has provided clues to potential molecular targets for new therapeutic strategies. Somatostatin analogs are well known to be useful for the control of symptoms in functioning tumors, and it was recently demonstrated that they can inhibit tumor progression in certain disease settings. Moreover, the recently published randomized trials with the multi-TKI sunitinib and with the mTOR-inhibitor everolimus have demonstrated, for the first time, their ability to positively impact the natural history of pancreatic NET (PNET). In this short review, we will discuss available data on newer molecular targeted agents for the treatment of advanced well-differentiated gastro-entero- pancreatic NET (GEP-NET). A possible algorithm for the use of these treatments in the context of the extreme heterogeneity of GEP-NET presentation will be proposed.

Clinically silent chromaffin-cell tumors: Tumor characteristics and long-term prognosis in patients with incidentally discovered pheochromocytomas.

BACKGROUND: Chromaffin-cell tumors (CCT), a rare group of catecholamine producing endocrine neoplasms, are traditionally suspected and diagnosed in patients presenting with episodic hypertension, together with the classic triad of headache, sweating, and tachycardia. Asymptomatic CCT are increasingly diagnosed, frequently as "incidentalomas". We have conducted a multicenter retrospective study, to assess the characteristics of a group of patients with clinically silent CCT, compared with a group of patients with typical CCT. METHODS: Forty-three consecutive patients with CCT (24 with silent and 19 with typical tumors) have been retrospectively studied for a period of up to 20 yr (between 1989 and 2009); clinical picture, biochemical tests, as well as topographic and functional assessment were analyzed at diagnosis and periodically following treatment. Surgical samples were reviewed for neuroendocrine markers and for signs of invasiveness. RESULTS: Patients with clinically silent CCT were significantly older than the typical ones (56.3±3.4 vs 48.0±4.8 yr; p<0.05); 15 of them (63%) were completely asymptomatic, and 9 patients (37%) complained of non-specific abdominal symptoms. Hypertension was present in only 6 silent CCT patients (25%), it was well controlled [mean blood pressure (BP) 134/84 mmHg], and persisted after surgery in only 2 patients. Fourteen out of twenty-four silent CCT patients (58%) were managed pre-operatively with prophylactic combination of α and ß blockade, despite normal BP values. Clinically silent CCT were larger than typical CCT (mean diameter of 5.2±2.3 cm vs 4.6±1.5 cm, p<0.05) and secreted higher a mounts of normeta neph rines. All clinically silent CCT patients were defined as "cured" after surgery. CONCLUSION: Clinically silent CCT are more prevalent than previously reported. With an adequate pre-surgical diagnosis and patient preparation, the prognosis of silent tumors is usually excellent.

Novel and evolving therapies in the treatment of malignant phaeochromocytoma: experience with the mTOR inhibitor everolimus (RAD001).

Phaeochromocytoma and paraganglioma are rare neuroendocrine tumours (NETS). They may be benign or malignant but the pathological distinction is mainly made when metastases are present. Available treatments in the form of surgery, chemotherapy, and radionuclide therapy may improve symptoms and biochemical markers, but the results for the control of tumour bulk are less favourable. Furthermore, responses to treatment are frequently short-lived. This short review outlines the main molecular and histological features of malignant phaeochromocytoma and the difficulties in differentiating between benign and malignant disease. We list current therapies used for malignant pheochromocytoma; however, these generally achieve relatively low success rates. Hence, there is a need for new and more effective therapies. In vitro studies have implicated the PI3/Akt/mTOR pathway in the pathogenesis of malignant NETS, including phaeochromocytoma. Everolimus (RAD001, Novartis UK) is a compound that inhibits mTOR (mammalian Target Of Rapamycin) signalling. We have used RAD001 in four patients with progressive malignant paraganglioma/phaeochromocytoma in addition to other therapies (with institutional approval for compassionate use), and evaluated the effects of this treatment. We outline these four cases and review the theoretical background for this therapy, although the outcomes were relatively disappointing.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin.

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.

Proinsulin processing in the diabetic Goto-Kakizaki rat.

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes.

The homeodomain transcription factor IPF1/PDX1 is required in beta-cells for efficient expression of insulin, glucose transporter 2, and prohormone convertases 1/3 and 2. Psammomys obesus, a model of diet-responsive type 2 diabetes, shows markedly depleted insulin stores when given a high-energy (HE) diet. Despite hyperglycemia, insulin mRNA levels initially remained unchanged and then decreased gradually to 15% of the basal level by 3 weeks. Moreover, insulin gene expression was not increased when isolated P. obesus islets were exposed to elevated glucose concentrations. Consistent with these observations, no functional Ipf1/Pdx1 gene product was detected in islets of newborn or adult P. obesus using immunostaining, Western blot, DNA binding, and reverse transcriptase-polymerase chain reaction analyses. Other beta-cell transcription factors (e.g., ISL-1, Nkx2.2, and Nkx6.1) were expressed in P. obesus islets, and the DNA binding activity of the insulin transcription factors RIPE3b1-Act and IEF1 was intact. Ipf1/Pdx1 gene transfer to isolated P. obesus islets normalized the defect in glucose-stimulated insulin gene expression and prevented the rapid depletion of insulin content after exposure to high glucose. Taken together, these results suggest that the inability of P. obesus islets to adapt to dietary overload, with depletion of insulin content as a consequence, results from IPF1/PDX1 deficiency. However, because not all animals become hyperglycemic on HE diet, additional factors may be important for the development of diabetes in this animal model.

Missense polymorphism in the human carboxypeptidase E gene alters enzymatic activity.

Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. One of the features of type 2 diabetes mellitus (T2DM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio, suggesting that mutations in proinsulin processing enzymes may contribute to the development of T2DM. We scanned CPE for mutations in a collection of Ashkenazi T2DM families and identified five novel single nucleotide polymorphisms (SNPs). An SNP in the 283(rd) codon, c.847C>T, changes arginine to tryptophan (R283W). The residue Arg283 is conserved among CPE orthologs as well as most enzymatically active metallocarboxypeptidases. Of the 272 Ashkenazi T2DM pedigrees screened, we found four families segregating R283W. Within these four families, patients who inherited one copy of this variant had much earlier age of onset for T2DM. The R283W CPE protein cleaves peptide substrates with substantially lower efficiencies and is less stable at elevated temperature. In addition, the R283W CPE variant has a narrower pH optimum and is much less active at pH 6.0-6.5, indicating that the R283W CPE variant would be substantially less active than wild type CPE in the trans-Golgi network and immature secretory vesicles where the enzyme functions in vivo. To summarize, we uncovered a rare non-conservative missense mutation in CPE and demonstrated that the mutant protein has altered enzymatic properties. We predict that this mutant could cause hyperproinsulinism and diabetes in the homozygous state.

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells.

Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells.

The immunomodulator Linomide: role in treatment and prevention of autoimmune diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is considered to be an autoimmune disorder characterized by destruction of the pancreatic beta-cells by auto-reacting lymphocytes. An attractive therapeutic approach to this disease would be to abrogate the autoimmune process at an early stage, thus preserving a critical mass of pancreatic beta-cells necessary for maintenance of normal glucose tolerance. Linomide (quinoline-3-carboxamide, Roquinimex, LS 2616), is a novel, orally absorbed, immunomodulatory drug that has been shown to be effective in various models of autoimmunity without causing non-specific immunosuppression. In this review, we describe the efficacy of Linomide for ameliorating the autoimmune process and diabetes in the non-obese diabetic (NOD) model of IDDM when administered at early stages of the disease. We also show that advanced disease in the NOD mouse can be treated effectively by combining Linomide with therapeutic modalities designed to increase pancreatic beta-cell mass. Subsequent clinical studies have shown that Linomide preserves beta-cell function in individuals with new-onset IDDM. Based on these data, Linomide or derivatives thereof might be useful for treatment of human IDDM.

beta-cell glucotoxicity in the Psammomys obesus model of type 2 diabetes.

Deficient insulin secretion and relative hyperproinsulinemia are characteristic features of type 2 diabetes. The gerbil Psammomys obesus appears to be an ideal natural model of the human disease because it shows increased tendency to develop diet-induced diabetes, which is associated with moderate obesity. The disease is characterized by initial hyperinsulinemia, progressing to hypoinsulinemia associated with depleted pancreatic insulin stores and an increased proportion of insulin precursor molecules in the blood and islets. Although the proinsulin translational efficacy was found to be increased in hyperglycemic animals, insulin mRNA levels were not augmented and exhibited a gradual decrease with disease progression. The development of hyperglycemia was associated with a transient increase in beta-cell proliferative activity, as opposed to a prolonged increase in the rate of beta-cell death, culminating in disruption of islet architecture. The hypothesis that glucotoxicity is responsible in part for these in vivo changes was investigated in vitro in primary islet cultures. Islets from diabetes-prone P. obesus cultured at high glucose concentrations displayed changes in beta-cell function that mimic those observed in diabetic animals. These changes include deficient insulin secretion, depleted insulin content, an increased proportion of insulin precursor molecules, a progressive increase of DNA fragmentation, and a transient proliferative response. Furthermore, insulin mRNA was not increased by short-term exposure of P. obesus islets to elevated glucose in vitro. It is proposed that beta-cell glucotoxicity in P. obesus results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. The resulting depletion of the insulin stores may be related to deficient glucose-regulated insulin gene transcription, possibly due to defective PDX-1 (pancreatic duodenal homeobox factor-1) expression in the adult P. obesus. An additional glucotoxic effect involves the loss of beta-cell mass in hyperglycemic P. obesus as a result of progressive beta-cell death without an adequate increase in the rate of beta-cell proliferation.