Enterovirus infection of human islets of Langerhans affects ß-cell function resulting in disintegrated islets, decreased glucose stimulated insulin secretion and loss of Golgi structure.

AIMS/HYPOTHESIS: In type 1 diabetes (T1D), most insulin-producing ß cells are destroyed, but the trigger is unknown. One of the possible triggers is a virus infection and the aim of this study was to test if enterovirus infection affects glucose stimulated insulin secretion and the effect of virus replication on cellular macromolecules and organelles involved in insulin secretion. METHODS: Isolated human islets were infected with different strains of coxsackievirus B (CVB) virus and the glucose-stimulated insulin release (GSIS) was measured in a dynamic perifusion system. Classical morphological electron microscopy, large-scale electron microscopy, so-called nanotomy, and immunohistochemistry were used to study to what extent virus-infected ß cells contained insulin, and real-time PCR was used to analyze virus induced changes of islet specific genes. RESULTS: In islets infected with CVB, GSIS was reduced in correlation with the degree of virus-induced islet disintegration. The expression of the gene encoding insulin was decreased in infected islets, whereas the expression of glucagon was not affected. Also, in islets that were somewhat disintegrated, there were uninfected ß cells. Ultrastructural analysis revealed that virus particles and virus replication complexes were only present in ß cells. There was a significant number of insulin granules remaining in the virus-infected ß cells, despite decreased expression of insulin mRNA. In addition, no typical Golgi apparatus was detected in these cells. Exposure of islets to synthetic dsRNA potentiated glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: Glucose-stimulated insulin secretion; organelles involved in insulin secretion and gene expression were all affected by CVB replication in ß cells.

Tropism Analysis of Two Coxsackie B5 Strains Reveals Virus Growth in Human Primary Pancreatic Islets but not in Exocrine Cell Clusters In Vitro.

Human Enteroviruses (HEVs) have been implicated in human pancreatic diseases such as pancreatitis and type 1 diabetes (T1D). Human studies are sparse or inconclusive and our aim was to investigate the tropism of two strains of Coxsackie B virus 5 (CBV-5) in vitro to primary human pancreatic cells. Virus replication was measured with TCID50 titrations of aliquots of the culture medium at different time points post inoculation. The presence of virus particles or virus proteins within the pancreatic cells was studied with immunohistochemistry (IHC) and electron microscopy (EM). None of the strains replicated in the human exocrine cell clusters, in contrast, both strains replicated in the endocrine islets of Langerhans. Virus particles were found exclusively in the endocrine cells, often in close association with insulin granules. In conclusion, CBV-5 can replicate in human endocrine cells but not in human exocrine cells, thus they might not be the cause of pancreatitis in humans. The association of virus with insulin granules might reflect the use of these as replication scaffolds.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane.

AIMS/HYPOTHESIS: Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. METHODS: Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. RESULTS: Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. CONCLUSIONS/INTERPRETATION: The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.

BACKGROUND: Intraportal transplantation of pancreatic islets offers improved glycaemic control and insulin independence in type 1 diabetes mellitus, but intraportal thrombosis remains a possible complication. The thrombotic reaction may explain why graft loss occurs and islets from more than one donor are needed, since contact between human islets and ABO-compatible blood in vitro triggers a thrombotic reaction that damages the islets. We investigated the possible mechanism and treatment of such thrombotic reactions. METHODS: Coagulation activation and islet damage were monitored in four patients undergoing clinical islet transplantation according to a modified Edmonton protocol. Expression of tissue factor (TF) in the islet preparations was investigated by immunohistochemistry, immunoprecipitation, electron microscopy, and RT-PCR. To assess TF activity in purified islets, human islets were mixed with non-anticoagulated ABO-compatible blood in tubing loops coated with heparin. FINDINGS: Coagulation activation and subsequent release of insulin were found consistently after clinical islet transplantation, even in the absence of signs of intraportal thrombosis. The endocrine, but not the exocrine, cells of the pancreas were found to synthesise and secrete active TF. The clotting reaction triggered by pancreatic islets in vitro could be abrogated by blocking the active site of TF with specific antibodies or site-inactivated factor VIIa, a candidate drug for inhibition of TF activity in vivo. INTERPRETATION: Blockade of TF represents a new therapeutic approach that might increase the success of islet transplantation in patients with type 1 diabetes, in terms of both the risk of intraportal thrombosis and the need for islets from more than one donor.

The transplanted fetal endocrine pancreas undergoes an inherent sequential differentiation similar to that in the native pancreas. An ultrastructural study in the pig-to-mouse model.

This study examines, at the ultrastructural level, whether the fetal porcine endocrine pancreas (insulin, glucagon, somatostatin, and pancreatic polypeptide [PP]- and islet amyloid polypeptide [IAPP]-containing cells) develops normally after transplantation under the kidney capsule in athymic mice. We have thus used an in vivo pig-to-mouse model for the differentiation of the endocrine pancreas removed from its normal milieu. Islet-like cell clusters (ICCs) were prepared from the fetal porcine pancreas as previously described and transplanted under the renal capsule of athymic mice. At various times after transplantation, the endocrine pancreas was removed and the level of differentiation was compared with the native pancreas of the same biological age. At the ultrastructural level, several sequential steps could be identified based on the morphology and hormone content of the secretory granules of the endocrine cell examined. Applying this approach, we could demonstrate that the ontogeny of the transplanted fetal pig pancreas follows the same sequential differentiation as the native pancreas. The process seems to be under stringent control, apparently directly related to the biological age of the tissue, and independent not only of the new environment under the kidney capsule but also of the adult and xenogeneic milieu provided after transplantation to the athymic nude mouse. Therefore, all four major hormone-producing cells seem to develop normally after transplantation when compared with the development in the native pancreas. IAPP was produced by the pluripotent fetal endocrine cells as well as the adult alpha-, beta-, and delta-cell granules in the native pancreas; however, in the transplanted pancreas, IAPP expression was demonstrated only in beta-cells, delta-cells, and PP cells. No IAPP was found in granules of the alpha-cell lineage. The results suggest a sequential differentiation of all four major types of islet cells from a common pluripotent progenitor cell, which seems to be located in the pancreatic ducts. Therefore, the results presented strongly suggest that the ontogeny of the four major endocrine islet cells is determined by genetic information carried by the progenitor cells and not by the systemic or local environment.

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells.

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

Synaptic vesicle protein 2, A new neuroendocrine cell marker.

Synaptic vesicle protein 2 (SV2) is a glycoprotein identified in the nervous system of several species, including man, but its occurrence in the human neuroendocrine (NE) cell system has not been investigated. By using a monoclonal antibody to SV2, immunoreactivities were demonstrated in NE cell types in human gastrointestinal tract, pancreas, anterior pituitary gland, thyroid, parathyroid, and adrenal medulla, and also in chief cells of gastric oxyntic mucosa. Immunoelectron microscopy of pancreatic islets revealed SV2 immunoreactivity in secretory granules. Comparison of SV2, synaptophysin, and chromogranin A immunoreactivity showed more SV2- and synaptophysin- than chromogranin A-immunoreactive cells in the antrum and pancreas. In the other gastrointestinal regions and in the other endocrine organs more SV2- than synaptophysin-immunoreactive cells were seen. More chromogranin A- than SV2-immunoreactive cells were observed in duodenum, colon, and parathyroid. Various NE tumors were examined and all contained SV2-immunoreactive cells. The staining patterns with the three markers agreed well, except in hindgut carcinoids, which showed strong SV2 immunoreactivity, weak synaptophysin but no chromogranin A immunostaining. In pituitary adenomas more cells were immunoreactive to SV2 than to the other two antibodies. In conclusion, SV2 is recognized as a further broad marker for NE cells and widens the arsenal of diagnostic tools for NE tumors. It is of special importance for identifying hindgut carcinoids.

Nuclear localization of 111In after intravenous injection of [111In-DTPA-D-Phe1]-octreotide in patients with neuroendocrine tumors.

UNLABELLED: Treatment with tumor-targeting substances is currently being evaluated in clinical trials. For patients with neuroendocrine tumors expressing somatostatin receptors, the 111In-labeled somatostatin analog [diethylenetriaminepentaacetic acid (DTPA)-DPhe1]-octreotide has been used with promising results. To further investigate the clinical effect of the injected conjugate, we analyzed the cellular distribution of 111In by ultrastructural autoradiography. METHODS: Seven patients with somatostatin receptor-expressing midgut carcinoid tumors scheduled for abdominal surgery were investigated by somatostatin receptor scintigraphy. During operation, tumor tissue samples and samples of normal intestine were collected, fixed, and processed for electron microscopy. A thin layer of film emulsion was applied on sections and after the exposure film was developed. The cellular distribution of silver precipitations indicating the presence of isotope was evaluated. RESULTS: Cell surface receptor binding and internalization of [111In-DTPA-D-Phe1]-octreotide in the tumor cells was easily revealed by silver precipitations in the film. Multiple silver grains were seen at the plasma membrane, in the cytoplasmic area among secretory granules and vesicular compartments, and in the perinuclear area. Silver grains were also regularly located in the nucleus. For all patients, the silver precipitation patterns from 111In decay were identical in all examined cells from removed tumors, and in most cells 111In could be seen in the nucleus. The specificity of the silver reaction products is supported by the observation that enterocytes in intestinal tissue specimens from near the tumor did not show any silver grains and no background labeling was seen in the plastic. CONCLUSION: After internalization through the somatostatin receptor system, 111In is translocated to the perinuclear area and into the nucleus. Whether the nuclide is still conjugated to the intact somatostatin analog or to part of it cannot be evaluated in this study. Despite the short irradiation range of 111In, the nuclear localization can explain its clinical effectiveness. The results from this study suggest that [111In-DTPA-D-Phe1]-octreotide may act as a powerful tumor cell-targeting substance.

A method to study apoptosis in eosinophils by flow cytometry.

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

Subcellular distribution of phospholipase C isoforms in rodent pancreas and gastric mucosa.

Phosphoinositide-specific phospholipase C (PLC) has been implicated as a participant in cell proliferation as well as enzyme and hormone secretion. Defining the subcellular distribution of PLC isoforms would possibly contribute to further understanding of their function. We investigated the intracellular distribution of four PLCs (beta1, beta2, beta3, and gamma1) in mouse pancreatic cells as well as mouse and rat gastric mucosa cells by ultrastructural immunocytochemistry. In pancreatic acinar cells, PLCbeta1 and PLCgamma1 were demonstrated in the zymogen granules while PLCbeta2 was present in the granulae as well as the endoplasmic reticulum (ER), and PLCbeta3 was prominent in the ER. In the endocrine pancreas, PLCbeta2 immunolabeling was expressed in the secretory granulae of alpha, beta, delta, and pancreatic polypeptide cells. PLCbeta3 showed a slight labeling in the nucleus and ER of all four pancreatic endocrine cell types while PLCgamma1 was prominent in alpha cell granulae. In the gastric mucosa cells, PLCbeta2 was highly expressed in the heterochromatin areas and in the ER of parietal, chief, mucous, and enterochromaffin-like cells. PLCbeta3 were expressed in a manner similar to PLCbeta2 in those cells; however, no immunoreaction was seen in the ER of parietal cell. PLCgamma1 was demonstrated in the chief cell granulae. One possible, although yet speculative, interpretation of our results is that the studied PLC isoforms may be involved in processing in pancreatic secretory granulae and that nuclear PLCbeta2 and PLCbeta3 signaling pathways may be operative in the cells of the gastric mucosa.

A mechanism behind the antitumour effect of 6-diazo-5-oxo-L-norleucine (DON): disruption of mitochondria.

6-diazo-5-oxo-L-norleucine (DON) exerts a growth inhibitory effect selectively on the neuroendocrine tumour cell line BON and is proposed as an antitumour drug. The mechanism behind this has not yet been clarified. In the present study, transmission electron microscopy was used for the assessment of changes in cellular organelles. Furthermore, the methylthiazolyldiphenyl tetrazolium (MTT) assay for mitochondrial enzymatic activity, a fluorescent marker (rhodamine 123) for mitochondrial integrity and [2-(11)C]-acetyl-carnitine which is a substrate of the tricarboxylic acid cycle of mitochondria were employed. The studies were performed in parallel in BON and in a neuroblastoma cell line LAN, with the cells grown as monolayers or as multicellular aggregates. Severe morphological changes of intracellular organelles were observed in BON aggregates treated with low-doses of DON. Especially striking was the disruption of mitochondrial internal membrane structures. Other features included the swelling of endoplasmic reticulum, autophagocytosis of secretory granules and nuclear condensation (apoptosis). In LAN cells, no ultrastructural changes were seen after DON treatment. The MTT assay indicated inhibition of mitochondrial enzymatic activity in BON cells but not in LAN cells after 5 h treatment with DON. The mitochondrial damage was also demonstrated as a reduced metabolism of [2-(11)C]-acetyl-carnitine. The observations revealed mitochondrial damage by DON treatment and suggest that the mitochondria might be a primary target for the antitumour effect in neuroendocrine cells.

In vivo cellular distribution and endocytosis of the somatostatin receptor-ligand complex.

Radioactive tumor targeting agents are highly interesting and for treatment of neuroendocrine tumors expressing somatostatin receptors, radiolabeled somatostatin analogues (including [(111)In-DTPA-D-Phe1]-octreotide) has been tried in a small number of patients with encouraging results. To increase our knowledge about the in vivo processing of administered [(111)In-DTPA-D-Phe1]-octreotide we have examined tumor and normal tissue material from a patient with a midgut carcinoid tumor. By ultrastructural autoradiography, silver grains indicating the presence of [(111)In-DTPA-D-Phe1]-octreotide could be identified within tumor cells, both in the primary tumor and in the mesenteric metastases. Silver grains were also found in leukocytes and in blood vessels. However, normal enterocytes did not show any specific radioligand uptake. This study indicates that the binding and endocytosis of [(111)In-DTPA-D-Phe1]-octreotide is a specific process that takes place in cells expressing somatostatin receptors. However, the importance of the number of somatostatin receptors and subtypes expressed will have to be further studied.

Molecular cloning and characterization of a cDNA encoding mouse phospholipase C-beta3.

A cDNA encoding mouse PLC-beta3 (mPLC-beta3) was identified by screening a mouse kidney cDNA library and using the rapid amplification of cDNA ends (RACE) method. The predicted open reading frame was 3705 bp in length. The deduced 1235 amino acid (aa) sequence shares 95.3% and 92% homology with the sequences of rat and human PLC-beta3, respectively. The corresponding mRNA is highly expressed in kidney, skeletal muscle, liver, lung, heart and brain. In spleen, mPLC-beta3 mRNA was not detectable, which is in contrast to humans where there is a distinct expression. Using ultrastructural immunocytochemistry, mPLC-beta3 expression was detected in the heterochromatin of the nucleus in mouse brain neurons. The observation of PLC-beta3 nuclear localization suggests that PLC-beta3 may have intranuclear functions.

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

The synthesis and intracellular localization of the putative hormone islet amyloid polypeptide (IAPP) and its relation to insulin and glucagon during ontogenesis was investigated in fetal and adult porcine and human pancreatic islets. By means of ultrastructural immunogold immunocytochemistry, it was revealed that IAPP is produced by the hormonally pluripotent endocrine stem cells from the earliest time point studied. IAPP was colocalized with insulin and glucagon in the immature and nondifferentiated cell granules in both species. In adult man, highly intense IAPP immunoreactivity was found in beta-cell granules and, at lower intensity, in delta-cell granules. Some alpha-cells also contained a small amount of IAPP in their granules, and among these occasional granules displayed an intense immunoreactivity. In adult pig, IAPP was stored in quantity in beta-cell granules and in small amounts in granules of alpha- and delta-cells. It was difficult to determine the presence of IAPP in pancreatic polypeptide cells, because they were so seldom seen in this material. It is concluded that, in both man and pig, fetal pancreatic islet stem cells synthesize and store IAPP together with insulin and glucagon. The storage in different types of cells and granules was not as predictable as that of the classical islet hormones. The substance is more widely distributed within the pancreatic islet cell types than are any of the other islet hormones, which presumably has functional implications.

Induction of apoptosis in neuroendocrine tumors of the digestive system during treatment with somatostatin analogs.

The extent of apoptosis identified by in situ DNA nick end labelling (TUNEL) on tissue samples obtained from patients with neuroendocrine tumors was correlated with the clinical outcome in patients treated with high-dose somatostatin analog (lanreotide 12 mg/day), n = 8, or other biotherapy including interferon-alpha (IFN-alpha), n = 4, low-dose somatostatin analog (octreotide or lanreotide), n = 3, or a combination of both, n = 1. Biopsies were obtained before the start of treatment and/or after 6 months and 12 months. After 6 months of treatment, 5 patients receiving high-dose somatostatin analog showed a biochemical response (decrease in different neuroendocrine tumor markers) and 4 of these showed an increase in apoptotic index (AI: percentage of apoptotic cells) by 1.94 +/- 1.71%. At 12 months, AI was also increased in patients with a biochemical response (4.22 +/- 3.93%). However, none showed a decrease in tumor size on computerized tomography (CT) and none of the patients treated with low-dose somatostatin analog or IFN-alpha showed any significant increase in AI during treatment. In an experimental model, nude mice were xenografted with the neuroendocrine cell line (BON-1). From the 2nd day of tumor implantation, they received treatment with either placebo, high-dose octreotide, IFN-alpha, or a combination of both, for 28 days. In mice receiving treatment with high-dose octreotide (300 microg/kg, t.i.d) there was a threefold increase in apoptotic cells as compared to the placebo group (p = 0.0084), while the combination group had few cells with ultra-structural changes indicating apoptosis and the IFN-alpha treated group showed no significant changes. However, tumor growth inhibition was more pronounced in the combination group (p = 0.0011). This probably denotes that tumor growth inhibition could be achieved more efficiently by blocking the cell cycle than by inducing apoptosis. We concluded that treatment with high-dose somatostatin analogs may induce apoptosis in neuroendocrine tumors, while this is not found during treatment with low-dose somatostatin analogs or IFN-alpha. We also found that an increase in AI during high-dose somatostatin analog treatment was correlated with the biochemical response, but not with the tumor size as detected by CT in patients or with the tumor mass in the experimental model.

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

The synthesis and intracellular localization of the putative hormone islet amyloid polypeptide (IAPP) and its relation to insulin and glucagon during ontogenesis was investigated in fetal and adult porcine and human pancreatic islets. By means of ultrastructural immunogold immunocytochemistry, it was revealed that IAPP is produced by the hormonally pluripotent endocrine stem cells from the earliest time point studied. IAPP was colocalized with insulin and glucagon in the immature and nondifferentiated cell granules in both species. In adult man, highly intense IAPP immunoreactivity was found in beta-cell granules and, at lower intensity, in delta-cell granules. Some alpha-cells also contained a small amount of IAPP in their granules, and among these occasional granules displayed an intense immunoreactivity. In adult pig, IAPP was stored in quantity in beta-cell granules and in small amounts in granules of alpha- and delta-cells. It was difficult to determine the presence of IAPP in pancreatic polypeptide cells, because they were so seldom seen in this material. It is concluded that, in both man and pig, fetal pancreatic islet stem cells synthesize and store IAPP together with insulin and glucagon. The storage in different types of cells and granules was not as predictable as that of the classical islet hormones. The substance is more widely distributed within the pancreatic islet cell types than are any of the other islet hormones, which presumably has functional implications.

Prostasomes are neuroendocrine-like vesicles in human semen.

BACKGROUND: Prostasomes are prostate-derived organelles that exist extracellularly in human seminal plasma. METHODS: In this study, we have investigated and characterized human prostasomes with regard to their contents of synaptophysin, members of the chromogranin family, and some neuropeptides. RESULTS: By radioimmunoassay measurement and electron microscopy we show the presence of the neuroendocrine markers chromogranin B, neuropeptide Y, and vasoactive intestinal polypeptide in about equimolar amount in human prostasomes and chromogranin A in about 2% of that amount. To our knowledge, such a high ratio of chromogranin B to chromogranin A has never before been observed. The membrane-bound protein synaptophysin, a well-established immunocytochemical marker for neuroendocrine cells and neurones, was also detected. Hence, we show that synaptophysin could be used as a marker for intact prostasomes. CONCLUSIONS: The presence of synaptophysin has recently been shown in the serotonincontaining vesicles in platelets. A protein with a similar structure denoted granulophysin has been found in granulocytes and prostasomes. It is suggested that synaptophysin and granulophysin molecules are members of a family of proteins, maybe expressed in all cells that have regulated release of granule content. Our presented data indicate a neurotransmittor function of the prostasomes. The target cells are however not known but could be either the spermatozoa, the epithelial mucous cells of the uterus or tubas or perhaps the ovum.

Ultrastructural localization of endothelin-1 in nonneoplastic, hyperplastic, and neoplastic adrenal gland.

Endothelin (ET)-1 is a 21-amino acid peptide with potent vasopressor and vasoconstrictive properties. Biochemical and recent histochemical studies have shown that this peptide is present in human adrenal cortex. This study was intended to determine ET-1 immunoreactivity in human adrenal cortex and cortical adenoma, and hyperplasia ultrastructurally. Light microscopical examination confirmed recent findings of ET-1 immunoreactivity in the three cortical zones (but not in the medulla) as well as in cortical adenoma and cortical hyperplasia. The immunoreactivity in the cortex and adenoma appeared in the cytoplasm in the form of vacuolar structures and grains. Focally, the cell membranes also showed immunoreactive staining. Electronmicroscopical investigation revealed ET-1 immunoreactive products adjacent to the outer surface of the membrane of lipid bodies, in mitochondria and rough endoplasmic reticulum and focally on the cell membrane, but no immunolabeling was seen in the medulla. The localization of ET-1 in the endoplasmic reticulum indicates that this peptide is synthesized in the cortical cells. Its localization in the membrane of the lipid bodies and in the mitochondria suggests that it takes part in synthesis and/or secretion of steroid hormones. The focally immunolabeled cell membranes may be dependent on ET-1 binding to ET receptors.

Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets.

The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re-endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants.

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.