Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110ß in the regulation of insulin secretion from rodent and human beta cells.

AIMS/HYPOTHESIS: Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and ß (p110α and -ß) in insulin granule recruitment and exocytosis in rodent and human islets. METHODS: The p110α and p110ß subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. RESULTS: Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85ß) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110ß blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kß (p110ß/p85ß) even when p110ß catalytic activity was inhibited. Conversely, both the wild-type p110ß and a catalytically inactive mutant directly facilitated exocytosis. CONCLUSIONS/INTERPRETATION: Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110ß is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

AIMS/HYPOTHESIS: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS: Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS: Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION: Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

Is colorectal surveillance indicated in patients with PTEN mutations?

AIM: Patients with germline phosphatase and tensin homologue (PTEN) mutations develop hamartomatous lesions in several organs and are at increased risk of various malignancies. We assessed the lifetime risk of benign and malignant gastrointestinal lesions in patients with a proven PTEN mutation. METHOD: Data on gender, mutation, dates of birth, last contact, and diagnosis, location and type of gastrointestinal lesions were collected from nine countries. The lifetime risk of gastrointestinal lesions was calculated by Kaplan-Meier methods. RESULTS: A total of 156 patients (67 men, 43%) from 101 families with a PTEN mutation were included. Patients were born between 1928 and 2008. Benign gastrointestinal polyps were reported in 49 (31%) patients at a mean age of 38 years (range 18-62 years) and were most often hamartomas. Twenty-two (44%) patients had upper as well as lower gastrointestinal lesions, 14 (29%) had only colonic lesions and 13 (27%) had gastrointestinal lesions at unknown sites. The cumulative risk of developing benign gastrointestinal polyps was 70% at age 60. Four patients (two men) developed colorectal carcinoma at 53, 57, 59 and 62 years, respectively. The cumulative risk of developing colorectal carcinoma was 18% at age 60. Except for one carcinoid in the small intestine, no upper gastrointestinal cancers were observed. CONCLUSION: Benign gastrointestinal lesions are common in PTEN mutation carriers, and a three- to four-fold increased lifetime risk of colorectal cancer compared with the general population may exist. Colorectal screening of patients with germline PTEN mutations is recommended, starting at age 40 years.

Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome?

Familial gastrointestinal stromal tumours (GISTs) are rare but otherwise well-characterized tumour syndromes, most commonly occurring on a background of germline-activating mutations in the tyrosine kinase receptor c-KIT. The associated clinical spectrum reflects the constitutive activation of this gene product across a number of cell lines, generating gain-of-function phenotypes in interstitial cells of Cajal (GIST and dysphagia), mast cells (mastocytosis) and melanocytes (hyperpigmentation). We report a three-generation kindred harbouring a c-KIT germline-activating mutation resulting in multifocal GISTs, dysphagia and a complex melanocyte hyperpigmentation and hypopigmentation disorder, the latter with features typical of those observed in Waardenburg type 2 syndrome (WS2F). Sequencing of genes known to be causative for WS [microphthalmia transcription factor (MITF), Pax3, Sox10, SNAI2 ] failed to show any candidate mutations to explain this complex cutaneous depigmentation phenotype. Our case report conclusively expands the clinical spectrum of familial GISTs and shows a hitherto unrecognized link to WS. Possible mechanisms responsible for this novel cause of WS2F will be discussed.

Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study.

OBJECTIVE: To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. PATIENTS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47·6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. CONCLUSIONS: The present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men.

Voltage-dependent K(+) channels are positive regulators of alpha cell action potential generation and glucagon secretion in mice and humans.

AIMS/HYPOTHESIS: The regulation of glucagon secretion from alpha cells is poorly understood. Since action potential firing at low glucose is required for glucagon secretion, we hypothesised that voltage-dependent K(+) (Kv) currents limit glucagon secretion under these conditions, similarly to their role in insulin secretion. METHODS: Kv currents and action potential firing of mouse and human alpha cells, identified by immunostaining, were examined by whole-cell patch-clamp. Glucagon secretion from mouse and human islets was measured by ELISA. RESULTS: Kv current density was 35% larger in alpha than in beta cells. Alpha cell Kv channels were sensitive to block by tetraethylammonium (TEA) and 4-aminopyridine. Surprisingly, Kv channel inhibition reduced glucagon release to the same extent as glucose. Robust action potential firing was observed in beta cells when ATP-sensitive K(+) channels were closed, but in alpha cells a negative current (-8 pA) injection was required for action potential firing. TEA (0.5 mmol/l) impaired alpha cell action potential firing, which could be restored by further hyperpolarising current injection (-16 pA). Kv currents were more sensitive to the Kv2 inhibitor stromatoxin (100 nmol/l) in mouse (80%) than in human (45%) alpha cells. Finally, the maxi-K (BK) channel inhibitor iberiotoxin (100 nmol/l) blocked 55% of the current in human alpha cells and inhibited glucagon release from human islets. CONCLUSIONS/INTERPRETATION: Kv currents in alpha cells are positive regulators of glucagon secretion. These currents, mediated by Kv2 and BK channels, limit membrane depolarisation, and prevent inactivation of alpha cell action potentials and suppression of glucagon release.

Low-level parental mosaicism in an apparent de novo case of Peutz-Jeghers syndrome.

We report the case of a female found to have mosaicism for mutation in the STK11 gene, with the mutant allele expressed in her gametes, evident by her affected offspring, and in her gastrointestinal tract demonstrated on an excised polyp analysed for diagnosis. Mosaicism for Peutz-Jeghers syndrome (PJS) has been reported in a small number of cases previously but a clinical presentation such as this has not previously been described. This finding of mosaicism was several years after initial investigations failed to identify the same STK11 mutation in this woman whose son was diagnosed with PJS at a young age. This case highlights the importance of considering mosaicism as an explanation for apparent de novo cases of PJS syndrome. It also has implications for genetic counselling, predictive testing and cancer screening.

STK11 status and intussusception risk in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. OBJECTIVE: To analyse the time to onset of intussusception in a large series of PJS probands. METHODS: STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. RESULTS: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, chi(2) = 0.58, with 1df; p = 0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. CONCLUSIONS: The risk of intussusception in PJS is not influenced by STK11 mutation status.

Upper intestinal surveillance in familial adenomatous polyposis.

Our understanding of the natural history of upper gastrointestinal (GI) involvement in familial adenomatous polyposis (FAP) is still evolving, although we know that the main cause of death after colectomy in FAP is upper GI malignancy, affecting 5% of patients. The aim of duodenal surveillance is to target high risk individuals and identify cancers early. We have screened 200 patients prospectively and have observed that duodenal polyposis progresses slowly, but there are some young people who have severe disease who merit close observation. We pay particular attention to endoscopic technique and histological detail, and use a duodenal staging system. Patients are offered randomisation to studies of chemopreventive agents, and those with advanced disease are considered for surgery. Successful management is inhibited by our deficient knowledge of the natural history of upper gastrointestinal polyposis, and by our inability to identify high risk individuals with histological markers rather than because of any technological deficiencies in endoscopic equipment.

Photodynamic therapy for polyps in familial adenomatous polyposis--a pilot study.

Photodynamic therapy (PDT) produces localised necrosis with light after prior administration of a photosensitising drug. As PDT lesions in the gastrointestinal tract heal so well, the technique is suitable for repeated endoscopic use. In this study, PDT was used to treat large polyps (four duodenal and two colorectal) unsuitable for surgery in 6 patients with familial adenomatous polyposis (FAP). Patients were sensitised with 60 mg/kg 5-aminolaevulinic acid (ALA) orally or intravenous (i.v.) 2.0 mg/kg Photofrin. Laser treatment was performed 6 h after ALA or 48 h after Photofrin using a gold vapour laser. Necrosis was only superficial (up to 1.8 mm) using ALA but much deeper using Photofrin. The one malignant polyp (8 mm diameter in the colon) showed a complete response using Photofrin. All healed safely with no complications. Photofrin worked better, but caused cutaneous photosensitivity lasting up to 3 months. ALA cleared within 2 days, but its use is limited by the superficial effect. Better results with ALA may be obtained using higher drug doses or modified light dosimetry. Fluorescence microscopy showed no evidence of selectivity of photosensitisation between neoplastic and normal tissue. PDT is a promising treatment for inoperable polyps in patients with FAP, but further work is required to optimise the treatment conditions.

Preventive measures in Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome is a rare genetic disorder characterized by mucocutaneous melanin deposition, intestinal polyposis and an increased risk of cancer, both intestinal and extra-intestinal. We describe the current status of diagnosis and the methods by which the consequences of this condition can be minimized. A surveillance program for those diagnosed is also included.

Quantitative assessment of postsurgical breakdown of the blood-aqueous barrier.

Breakdown and reestablishment of the blood-aqueous barrier (BAB) following experimental surgical trauma was evaluated by anterior segment slitlamp fluorophotometry. Substantially more fluorescein leakage was caused by 6-mm limbal incisions for at least the first four postoperative days than was produced by 3-mm incisions. Following 3-mm corneal incisions, leakage of fluorescein closely paralleled that of fluorescein-labeled dextran 70 (molecular weight, 70,000) for the first three postoperative days. On the fourth postoperative day, the BAB appeared reestablished to fluorescein-labeled dextran 70 but was still abnormally permeable to fluorescein. Thus, it appears that although fluorescein is the most sensitive technique of demonstrating breakdown of BAB, it may not always be an indicator of large-molecule permeability.

Upper gastrointestinal pathology in familial adenomatous polyposis: results from a prospective study of 102 patients.

Multiple gastric and duodenal biopsy specimens from 102 asymptomatic patients with familial adenomatous polyposis, taken during a prospective endoscopic screening programme were examined. One hundred patients had microscopic gastroduodenal pathology, often in the absence of macroscopic lesions. Adenomas were found in 94 patients in the duodenum, in the second and third parts. Hyperplasia of villous and crypt epithelium was also seen, sometimes in the absence of adenomas: this may be a precursor of neoplastic change. In the stomach fundic gland polyps were the commonest abnormality, seen microscopically in 44 patients. Chronic antral gastritis was common in patients without fundic polyps. Gastric adenomas were present in six patients, all of whom also had duodenal adenomas. If duodenal adenomas in familial adenomatous polyposis have a similar malignant potential to those in the colorectum sequential endoscopy and biopsy are necessary to detect cancer in these patients.

Evidence for adenoma-carcinoma sequence in the duodenum of patients with familial adenomatous polyposis. The Leeds Castle Polyposis Group (Upper Gastrointestinal Committee).

AIMS: To explore the association between duodenal adenoma and carcinoma in patients with familial adenomatous polyposis (FAP). METHODS: A multicentre survey of 1262 patients with FAP yielded 47 cases of duodenal cancer. The association between adenoma and cancer was assessed in these cases. RESULTS: Adenomatous tissue was found within duodenal cancer in 29 of 44 (66%) patients with FAP and in mucosa adjacent to duodenal cancer in 31 of 42 (73%) such patients. Adenomas were found as a component of, or adjacent to, duodenal cancer in 38 of 45 (84%) patients. CONCLUSIONS: These observations support the existence of the adenomacarcinoma sequence in the duodenum of patients with FAP. Factors associated with malignant change included villous histology, moderate or severe dysplasia, and the presence of stage IV duodenal polyposis.

The surgical management of overcorrection in myopic epikeratophakia.

Three patients underwent myopic epikeratophakia that resulted in overcorrection. The surgical management of these cases involved resuturing the myopic lenticule to flatten the peripheral curvature and reduce the amount of minus power. A reduction in the overcorrection was achieved in all cases.

Development of duodenal cancer in a patient with familial adenomatous polyposis.

A Japanese woman with familial adenomatous polyposis in whom a duodenal ampullary adenoma underwent malignant change during a 10-year follow-up period is reported. After restorative proctocolectomy in 1989, and extensive small bowel resection for desmoid disease in 1991, regular surveillance duodenoscopies, including three to nine biopsies (mean, 4.8) were performed annually or biannually. Until 1995, the endoscopic findings of duodenal polyposis (including an ampullary polyp) did not progress and the histopathology did not worsen. In 1996, there was an increase in the number and size of the duodenal polyps, and the ampulla of Vater looked enlarged. Open surgery was discussed but not proceeded with because of the risk for short bowel syndrome. In January 1998, she was admitted with a diagnosis of acute pancreatitis. Duodenoscopy and radiological examination revealed that an advanced ampullary cancer had developed, and histopathology revealed a well-differentiated adenocarcinoma. Multiple hepatic metastases and ascites led to her death, in June, 1998. This in-vivo demonstration of the adenoma-carcinoma sequence highlights current limitations in the surveillance and treatment of duodenal lesions.

Comparison of yellow dye, continuous wave Nd:YAG, and argon green laser on experimentally induced corneal neovascularization.

BACKGROUND: Corneal neovascularization is generally undesirable because it can lead to corneal scarring, lipid deposits, and corneal graft failure. To eliminate these vessels, several techniques are available including laser photocoagulation. METHODS: This prospective study was designed to compare the effectiveness of three laser wavelengths (continuous wave Nd:YAG, 1064 nm; argon green 514 nm; and yellow dye, 570 nm) to obliterate experimentally induced corneal neovascularization in the rabbit. Corneal vascularization was created in 12 rabbits by placing 7-0 silk sutures through two quadrants of the cornea. Once neovascularization was complete, the suture was removed and one of the three lasers was applied to occlude vessels at one of the neovascular sites. The other site was used as a control. RESULTS: The yellow wavelength, when compared with the green required fewer exposures to occlude corneal vessels. At no time during observation was any laser more effective than the control. In the continuous wave Nd:YAG group, tissue necrosis was needed to achieve closure of vessels. CONCLUSIONS: Yellow and green laser light are equally effective in eliminating the corneal vessels. Continuous wave Nd:YAG, as used here, appears to be a poor choice.

Four incision radial keratotomy.

Radial keratotomy is a constantly evolving procedure. This paper investigates the value of four incision radial keratotomy. The possible advantages of fewer incisions include increased corneal stability, lower risk of perforation, less potential of endothelial cell loss, decreased chance of overcorrection, and simplification of the procedure. We evaluated the results of four incision radial keratotomy in 55 eyes of 31 patients. Follow-up ranged from one to 18 months. Results in low myopia (-2.00 to -3.12 diopters) show 93% of the patients were 20/40 or better, 90% were within +/- 1 diopter of emmetropia, and no patients were overcorrected greater than 1 diopter. In moderate myopia (-3.25 to -4.37 diopters), 84% of the patients were 20/40 or better, 92% were within +/- 1 diopter of emmetropia, and no patients were overcorrected greater than 1 diopter. Regression analysis was performed and it was determined that the postoperative result was equal to .262 + [1.293 x preop spherical equivalent] - [1.166 x optical zone] + [0.56 x depth] + [.038 x age], R = .87.

Epikeratophakia: technique modifications and visual results compared to the national study.

Twenty-eight epikeratophakia procedures have been performed at the University of Minnesota since December 1985. This report describes several technique modifications and compares the refractive and visual outcome of these cases with the national results. There have been 17 aphakic cases (13 adult and four pediatric), seven myopic procedures, and four keratoconus cases. Of the adult aphakic cases, 80% were within 3 diopters of emmetropia at six months, with four cases showing an undercorrection. In the myopic group, five cases showed an overcorrection at six months and all patients were within two Snellen lines or better than their preoperative best corrected vision. The major complications included one graft removal for failure to epithelialize, an interface hematoma, and a severe overcorrection to +9.37 diopters in a myopic patient. A no-keratectomy technique was used in the aphakic and myopic groups. A variation on centering the Hessburg-Barron trephine and removing the epithelium was performed and allowed for more accurate trephine placement.

Visual results following vitreous loss and primary lens implantation.

Implantation of an intraocular lens following vitreous loss at cataract surgery is a controversial decision. To address this issue, we retrospectively identified all cases performed at the University of Minnesota in the last three years that had either a posterior or an anterior chamber lens placed following anterior vitrectomy. Twenty patients had a posterior chamber lens implant; 14 had follow-up longer than six months. All these patients achieved 20/40 or better visual acuity, although one patient had a retinal detachment. Of the six patients with an anterior chamber lens implant, four achieved 20/40 or better acuity and two achieved 20/50 acuity with follow-up of six months; one patient had a retinal detachment. This review demonstrates that with a meticulous anterior vitrectomy, good visual results can be achieved, although the risk of retinal detachment is higher than in uncomplicated cases.