Semi-automated digital quantification of cellular infiltrates for in vivo evaluation of transplanted islets of Langerhans encapsulated with bioactive materials.

BACKGROUND: In the field of xenotransplantation, digital image analysis (DIA) is an asset to quantify heterogeneous cell infiltrates around transplanted encapsulated islets. MATERIALS AND METHODS: RGD-alginate was used to produce empty capsules or to encapsulate neonatal porcine islets (NPI) with different combinations of human pancreatic extracellular matrix (hpECM), porcine mesenchymal stem cells (pMSC) and a chitosan anti-fouling coating. Capsules were transplanted subcutaneously in rats for one month and then processed for immunohistochemistry. Immunostainings for macrophages (CD68) and lymphocytes (CD3) were quantified by DIA in two concentric regions of interest (ROI) around the capsules. DIA replicability and reproducibility were assessed by two blind operators. Repeatability was evaluated by processing the same biopsies at different time points. DIA was also compared with quantification by point counting (PC). RESULTS: Methodology validation: different sizes of ROIs were highly correlated. Intraclass correlation coefficients confirmed replicability and reproducibility. Repeatability showed a very strong correlation with CD3 stains and moderate/strong for CD68 stains. Group comparisons for CD68 IHC at each time point proved internal consistency. Point counting and DIA were strongly correlated with both CD3 and CD68. Capsule biocompatibility: Macrophage infiltration was higher around capsules containing biomaterials than around empty and RGD-alginate-NPI capsules. Lymphocytic infiltration was comparable among groups containing cells and higher than in empty capsules. CONCLUSION: We validated a semi-automated quantification methodology to assess cellular infiltrates and successfully applied it to investigate graft biocompatibility, showing that neonatal porcine islets encapsulated in alginate alone triggered less infiltration than capsules containing islets and bioactive materials.

The Impact of the COVID-19 Pandemic and the Associated Belgian Governmental Measures on Cancer Screening, Surgical Pathology and Cytopathology.

INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 caused a pandemic of coronavirus disease 2019 (COVID-19). Unprecedented public health actions were introduced, including social distancing, travel restrictions and quarantine. The Belgian government announced a national emergency plan, thereby postponing all non-urgent medical consultations and operations. This report analyses the impact of these measures on cancer screening, through assessment of the workload of a laboratory for histopathology and cytopathology. METHODS: Data on monthly numbers of histological and cytological samples, immunohistochemistry and molecular tests were extracted from the laboratory information management system. RESULTS: The global histopathological and cytological workload was substantially reduced. The impact on oncology-related surgical procedures was rather limited. The anti-COVID-19 measures significantly diminished all screening-related samples, such as colon biopsies, breast biopsies and cervical cytology, and strongly reduced the number of samples related to "functional" pathology, such as thyroidectomies and gastric biopsies. CONCLUSIONS: Since many health care interventions are reflected in the workload of a pathology laboratory, this study enabled us to identify areas for "deconfinement" health care actions. Our findings indicate that various areas in medicine were affected, but the impact seemed largest for cancer screening. Health care professionals should assure that consultations related to cancer screening are postponed instead of cancelled.

Predictive markers for pathological complete response after neo-adjuvant chemotherapy in triple-negative breast cancer.

A combination of Sox10 and GATA3 was previously identified as a marker for metastatic triple-negative breast cancer (TNBC), but it is uncertain whether their expression is associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). This study investigates the predictive value of clinicopathological characteristics, as well as protein expression of Sox10, GATA3, p53 and p63, in a consecutive series of TNBC patients treated with NAC. Archived hematoxylin & eosin stained slides of core biopsies and resection specimens from 35 TNBC patients were reviewed. The following clinicopathological characteristics were determined at the biopsy level: age at diagnosis, cancer type, Nottingham grade, lympho-vascular invasion, syncytial growth, necrosis, clear cell differentiation, myxoid peritumor stroma, stromal tumor-infiltrating lymphocytes (sTILs) and presence of an in situ component. The MD Anderson residual cancer burden (RCB) score and corresponding RCB class were determined. Immunohistochemistry for Sox10, p53, GATA3 and p63 was performed at the biopsy level. sTILs, either as a continuous or as a dichotomous variable, were the only parameter that was significantly associated with pCR in univariable and multivariable analyses. Assessment of sTILs showed moderate to good interobserver agreement. High sTILs (≥40%) were significantly associated with increased pCR rates, and this association was observer-independent. This retrospective study of a consecutive community-based cohort of TNBC patients confirms that sTILs are a robust, observer-independent predictor for therapeutic response after NAC. The combination of Sox10, GATA3 and p53 immunoreactivity is unlikely to harbor any predictive value for pCR in TNBC.

Porcine pulmonary valve decellularization with NaOH-based vs detergent process: preliminary in vitro and in vivo assessments.

BACKGROUND: Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%). METHODS: Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2). RESULTS: Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm2 in the leaflets). In one animal, at three months we documented minimal calcification in the area of sinus leaflet and in one, microthrombi formation on the leaflet surface at 1 month. The endoluminal side of the valves showed partial reendothelialization. CONCLUSIONS: NaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis. Graphical abstract of the study.

Decellularized and Secured Porcine Arteries with NaOH-based Process: Proof of Concept.

BACKGROUND: There is a need for small caliber vascular prosthesis. Synthetic grafts are hindered by thrombogenicity and rapid occlusion. Decellularized matrices could be an alternative. We assessed in vitro and in vivo the biocompatibility of porcine artery treated with a chemical/physical process for decellularization and graft securitization with non/conventional pathogens inactivation. METHODS: Porcine carotid arteries (PCA) were treated. First, biopsies (n = 4/tissue) were performed before/after treatment to assess decellularization (hematoxylin and eosin/-4',6-diamidino-2-phenylindole/DNA/Miller). Second, 5 rats received an abdominal aortic patch of decellularized PCA (DPCA). Four pigs received subcutaneous DPCA implants (n = 2/pig). Half were explanted at day 15 and half at day 30. Finally, 2 pigs received DPCA (n = 2) and polytetrafluoroethylene prosthesis (n = 1), respectively, as carotid interposition. Implants were removed at day 30. Inflammation (CD3 and CD68 immunostaining) calcifications (von Kossa staining), remodeling (hematoxylin and eosin), and vascular characterization (CD31 and alpha-smooth muscle actin immunofluorescent staining) were investigated. RESULTS: Ninety-five percentage of decellularization was obtained without structural deterioration. No death occurred. Low inflammatory reaction was found in the 2 models for DPCA. Acquisition of vascular identity was confirmed in the rodent and porcine models. Similarity between native PCA and DPCA was observed after 30 days. In contrast, polytetrafluoroethylene graft showed severe calcifications, higher CD3 reaction, and higher intimal hyperplasia (P < 0.05). CONCLUSIONS: The physical and chemical process ensures decellularization of carotid porcine arteries and their in vivo remodeling with the presence of an endothelium and smooth-muscle-like cells as well as a low level of inflammatory cells.

Human Insulinomas Show Distinct Patterns of Insulin Secretion In Vitro.

Insulinomas are ß-cell tumors that cause hypoglycemia through inappropriate secretion of insulin. Characterization of the in vitro dynamics of insulin secretion by perifused fragments of 10 human insulinomas permitted their subdivision into three functional groups with similar insulin content. Group A (four patients with fasting and/or postprandial hypoglycemic episodes) showed qualitatively normal responses to glucose, leucine, diazoxide, tolbutamide, and extracellular CaCl2 omission or excess. The effect of glucose was concentration dependent, but, compared with normal islets, insulin secretion was excessive in both low- and high-glucose conditions. Group B (three patients with fasting hypoglycemic episodes) was mainly characterized by large insulin responses to 1 mmol/L glucose, resulting in very high basal secretion rates that were inhibited by diazoxide and restored by tolbutamide but were not further augmented by other agents except for high levels of CaCl2. Group C (three patients with fasting hypoglycemic episodes) displayed very low rates of insulin secretion and virtually no response to stimuli (including high CaCl2 concentration) and inhibitors (CaCl2 omission being paradoxically stimulatory). In group B, the presence of low-Km hexokinase-I in insulinoma ß-cells (not in adjacent islets) was revealed by immunohistochemistry. Human insulinomas thus show distinct, though not completely heterogeneous, defects in insulin secretion that are attributed to the undue expression of hexokinase-I in 3 of 10 patients.

Unambiguous detection of multiple TP53 gene mutations in AAN-associated urothelial cancer in Belgium using laser capture microdissection.

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Time course of pathogenic and adaptation mechanisms in cystinotic mouse kidneys.

Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodium-glucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.

Lanreotide treatment of metastatic hepatocellular carcinoma resulting in partial regression and more than 3 years of progression-free survival.

We describe the case of a 54 years old woman, with hepatitis B, in whom the diagnosis of a 6 cm hepatocellular carcinoma (HCC) in the left liver was made in 2001. Alpha-foeto-protein (AFP) was 63 ng/mL (NI < 10 ng/mL). After work-up including liver and tumor biopsy confirming HCC and only fibrosis in the nontumoral liver, left hepatectomy was performed. Final pathology showed a well differentiated HCC with tumoral portal vein thrombosis. Unfortunately, lung and mediastinal adenopathies were detected by CT scan 17 months later. Mediastinal nodes were punctured by endosonographic ultrasound, confirming HCC. The patient started treatment with Lanreotide 30 mg twice a month (Somatuline PR, Ipsen). Three months later, CT showed decrease in size of the mediastinal nodes and complete disappearance of the lung nodes. This objective response lasted for 42 months. The treatment was without any significant side effect. Retrospectively, immunohistochemistry was performed to detect somatostatine receptors (sstr) 2. Both the primary tumor and the node showed intense membranous and cytoplasmic staining for sstr2. In 2006, AFP rose and CT showed the appearance of a new mediastinal node. At that time, octreoscan was performed and showed uptake in the new node, although insufficient for metabolic radiotherapy. This case suggests that, although a number of randomized controlled trials did not show a benefit of somatostatin analogues in the treatment of advanced HCC, a subset of patients could benefit from treatment provided their tumor expresses sstr2, on which the existing drugs are efficient.

In vitro insulin secretion by pancreatic tissue from infants with diazoxide-resistant congenital hyperinsulinism deviates from model predictions.

Congenital hyperinsulinism (CHI) is the major cause of persistent neonatal hypoglycemia. CHI most often occurs due to mutations in the ABCC8 (which encodes sulfonylurea receptor 1) or KCNJ11 (which encodes the potassium channel Kir6.2) gene, which result in a lack of functional KATP channels in pancreatic ß cells. Diffuse forms of CHI (DiCHI), in which all ß cells are abnormal, often require subtotal pancreatectomy, whereas focal forms (FoCHI), which are characterized by localized hyperplasia of abnormal ß cells, can be cured by resection of the lesion. Here, we characterized the in vitro kinetics of insulin secretion by pancreatic fragments from 6 DiCHI patients and by focal lesion and normal adjacent pancreas from 18 FoCHI patients. Responses of normal pancreas were similar to those reported for islets from adult organ donors. Compared with normal pancreas, basal insulin secretion was elevated in both FoCHI and DiCHI tissue. Affected tissues were heterogeneous in their secretory responses, with increased glucose levels often producing a rapid increase in insulin secretion that could be followed by a paradoxical decrease below prestimulatory levels. The KATP channel blocker tolbutamide was consistently ineffective in stimulating insulin secretion; conversely, the KATP channel activator diazoxide often caused an unanticipated increase in insulin secretion. These observed alterations in secretory behavior were similar in focal lesion and DiCHI tissue, and independent of the specific mutation in ABCC8 or KCNJ11. They cannot be explained by classic models of ß cell function. Our results provide insight into the excessive and sometimes paradoxical changes in insulin secretion observed in CHI patients with inactivating mutations of KATP channels.

Morphologic analysis of focal and diffuse forms of congenital hyperinsulinism.

Congenital hyperinsulinism is clinically characterized by an inappropriate insulin secretion resulting in recurrent severe hypoglycemia. Nesidioblastosis, the proliferation of islet cells budding off from ducts, has been considered for years as the histologic lesion responsible for the syndrome. In our morphologic studies, we demonstrate that nesidioblastosis is not specific of the disease, which is actually not a single entity. Indeed, we recognize the existence of 2 different forms-a diffuse form and a focal form-and demonstrate that they can be differentiated by morphologic criteria, even on frozen sections during surgery. This histologic distinction directs the therapeutic approach because the patients experiencing the focal form of the syndrome can be completely cured by a very limited pancreatectomy. Molecular findings confirmed the reliability of this histologic distinction, showing a specific background for each form.

Changes in intestinal bifidobacteria levels are associated with the inflammatory response in magnesium-deficient mice.

Magnesium (Mg) deficiency is a common nutritional disorder that is linked to an inflammatory state characterized by increased plasma acute phase protein and proinflammatory cytokine concentrations. Recent studies have shown that changes in the composition of gut microbiota composition participate in systemic inflammation. In this study, therefore, we assessed the potential role of gut microbiota in intestinal and systemic inflammation associated with Mg deficiency in mice. For this purpose, mice were fed a control or Mg-deficient diet (500 mg vs. 70 mg Mg/kg) for 4 or 21 d. Compared with the mice fed the control diet, mice fed the Mg-deficient diet for 4 d had a lower gut bifidobacteria content (-1.5 log), a 36-50% lower mRNA content of factors controlling gut barrier function in the ileum (zonula occludens-1, occludin, proglucagon), and a higher mRNA content (by approximately 2-fold) in the liver and/or intestine of tumor necrosis factor-alpha, interleukin-6, CCAAT/enhancer binding protein homologous protein, and activating transcription factor 4, reflecting inflammatory and cellular stress. In contrast, mice fed the Mg-deficient diet for 21 d had a higher cecal bifidobacteria content compared with the control group, a phenomenon accompanied by restoration of the intestinal barrier and the absence of inflammation. In conclusion, we show that Mg deficiency, independently of any other changes in nutrient intake, modulates the concentration of bifidobacteria in the gut, a phenomenon that may time-dependently affect inflammation and metabolic disorders in mice.

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets.

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models.

BACKGROUND: In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin-producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre-clinical results and data in the pig-to-primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. METHODS: Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K+/Na+ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15,000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. RESULTS: By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P < 0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short-term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm2 (r = 0.708, P < 0.01) or with the percentage of large islets (r = 0.680, P < 0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm2 and more than 42% of large islets (>100 microm) thus enabled more than 120,000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C-peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post-transplantation, however, loss of islet function was associated with graft destruction and immune reaction. CONCLUSIONS: Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.

Noninvasive diagnosis of focal hyperinsulinism of infancy with [18F]-DOPA positron emission tomography.

Congenital hyperinsulinism of infancy (CHI) is characterized by severe hypoglycemia due to dysregulated insulin secretion, associated with either focal or diffuse pathology of the endocrine pancreas. The focal condition is caused by a paternally inherited mutation in one of the genes encoding the subunits of the beta-cell ATP-sensitive potassium channel (SUR1/ABCC8 or Kir6.2/KCNJ11) and somatic loss of maternal 11p15 alleles within the affected area. Until now, preoperative diagnostics have relied on technically demanding and invasive catheterization techniques. We evaluated the utility of fluorine-18 l-3,4-dihydroxyphenylalanine ([(18)F]-DOPA) positron emission tomography (PET) to identify focal pancreatic lesions in 14 CHI patients, 11 of which carried mutations in the ABCC8 gene (age 1-42 months). To reduce bias in PET image interpretation, quantitative means for evaluation of pancreatic [(18)F]-DOPA uptake were established. Five patients had a visually apparent focal accumulation of [(18)F]-DOPA and standardized uptake value (SUV) >50% higher (mean 1.8-fold) than the maximum SUV of the unaffected part of the pancreas. When these patients were operated on, a focus of 4-5 x 5-8 mm matching with the PET scan was found, and all were normoglycemic after resection of the focus. The remaining nine patients had diffuse accumulation of [(18)F]-DOPA in the pancreas (SUV ratio <1.5). Diffuse histology was verified in four of these, and pancreatic catheterization was consistent with diffuse pathology in four cases. In conclusion, [(18)F]-DOPA PET is a promising noninvasive method for the identification and localization of the focal form of CHI.

Focal and diffuse forms of congenital hyperinsulinism: the keys for differential diagnosis.

Congenital hyperinsulinism is clinically characterized by an inappropriate insulin secretion resulting in recurrent severe hypoglycemia. Nesidioblastosis, the proliferation of islets cells budding off from pancreatic ducts, has been considered for years as the histological lesion responsible for the syndrome. In our morphological studies, we demonstrated that nesidioblastosis is not specific for the disease, which is actually not a single entity. Indeed, we recognized the existence of two different forms--a diffuse form and a focal form--and demonstrated that they can be differentiated on the basis of morphological criteria, even on frozen sections during surgery. This histological distinction directs the therapeutic approach because the patients suffering from the focal form of the syndrome can be completely cured by a very limited pancreatectomy. Molecular findings confirmed the reliability of this histological distinction, showing a specific genetic background for each form.

The focal form of persistent hyperinsulinemic hypoglycemia of infancy: morphological and molecular studies show structural and functional differences with insulinoma.

Paternal mutation of ATP-sensitive K(+) (K(ATP)) channel genes and loss of heterozygosity (LOH) of the 11p15 region including the maternal alleles of ABCC8, IGF2, and CDKN1C characterize the focal form of persistent hyperinsulinemic hypoglycemia of infancy (FoPHHI). We aimed to understand the actual nature of FoPHHI in comparison with insulinoma. In FoPHHI, the lesion consists in clusters of beta-cells surrounded by non-beta-cells. Compared with adjacent islets, proinsulin mRNA is similar and proinsulin production higher (P < or = 0.02), indicating regulation at a translational level, with slightly lower insulin stock and lower ABCC8 peptide labeling (P<0.05). Insulinomas, composed of beta-cell nests or cords, have similar proinsulin mRNA compared with adjacent islets, highly variable proinsulin production, lower insulin stock (P < or = 0.02), and higher ABCC8 peptide labeling (P<0.05). Proinsulin mRNA is lower than in FoPHHI (P<0.001). Islets adjacent to FoPHHI appear to be resting, in contrast to those adjacent to insulinomas, evidencing intrapancreatic regulation of islet beta-cell activity. IGF2 peptide is present inside and outside both lesions, but IGF2 mRNA is restricted to the lesions. The 11p15 LOH and absence of CDKN1C peptide staining are demonstrated in all FoPHHI but also in three of eight insulinomas. Despite some molecular similarities, FoPHHI is thus fundamentally different from insulinoma.

SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca(2+) pumps for Ca(2+) homeostasis in pancreatic beta-cells.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.