[Clinical and imaging features of acute histoplasmosis].

Objective: To investigate the clinical and imaging characteristics of acute histoplasmosis. Methods: The clinical and imaging data of 10 patients with acute histoplasmosis were studied. Their clinical and imaging characteristics were analyzed. All the patients returned from a South American republic in April 2019 and were treated at the Chongqing public health medical treatment center. Results: All the 10 patients were male, aged 30-56 years old, with an average age of 43.8 years old. Four of them were engaged in soil clearing, 2 in gas cutting, 2 in moving tools, and 2 in inspection. The disease in all the 10 patients was caused by inhaling a large amount of bacteria-bearing dust in a short time, with an incubation period of 9-13 days, and the main clinical manifestations were fever, insomnia, dizziness, headache, cough, poor appetite, rash and diarrhea. One patient's head CT showed extensive thickening and increased density of bilateral frontotemporal, parietal and occipital meninges, while the other 9 patients showed no obvious abnormalities. Chest CT findings were as follows: (1) Multiple nodular shadow: the chest CT findings of 4 patients were miliary nodular shadow with diffuse distribution in both lungs. Most of the nodules were less than 5 mm in diameter and distributed evenly or unevenly. CT findings of 6 cases showed scattered nodular shadows in both lungs, with diameters ranging from 2 to 15 mm, and obvious distribution in subpleural and inferior lobes of both lungs. (2) Consolidation shadow: in 2 cases, the size of the shadow was uneven and the density increased, mainly distributed in the subpleura and the lower lobe of both lungs. (3) Ground glass density shadow: mainly distributed around nodules, halo signs can be seen around some nodules. (4) Mediastinum and/or hilar lymph nodes were enlarged. (5) Pleural effusion: a small amount of pleural effusion was found in 4 cases. (6) Pericardial effusion in 3 cases. Abdominal CT showed splenomegaly in 8 cases and hepatomegaly in 1 case. Conclusions: Acute histoplasmosis has no specificity in clinical manifestations. However, there are still some features in CT manifestations, including multiple nodules in both lungs accompanied by halo, enlarged liver, spleen and mediastinal lymph nodes, and multiple serous cavity effusions.

[Therapeutic effect of IGF-1R-targeting inhibitor (TAE226) on malignant pleural effusion in nude mice].

OBJECTIVE: To study the therapeutic effect of IGF-1R inhibitor TAE226 on malignant pleural effusion (MPE) in nude mice. METHODS: Human lung carcinoma A549 cells were injected into the pleural cavity of nude mice to establish MPE model. The mice were randomly divided into model group and treatment group, and were orally administered with distilled water and TAE226 (20 mg/kg) in the same volume, respectively. The volume of pleural effusion and tumor weight of the two groups were observed. HE staining was used to reveal the histological changes and enzyme-linked immunosorbent assay (ELISA) was used to detect the IGF-1R protein expression. IGF-1R mRNA level in the tumor tissue was determined by RT-PCR. Microvessel density (MVD) and cell proliferation index (PI) were assessed by immunohistochemical analysis. The protein expression levels of IGF-1R, p-IGF-1R, PI3K and p-PI3K in the tumor tissue were determined by Western blotting. RESULTS: The volumes of pleural effusion were (241.4±89.7) µl and (121.7±78.8) µl in the model and treatment groups, respectively (P<0.05). The tumor weight of treatment group was (316.7±186.3) mg, significantly lower than that of the model group (671.4±281.4) mg (P<0.05). RT-PCR analysis showed that IGF-1R mRNA level was 0.914±0.029 in the treatment group, significantly lower than that of the model group (1.152±0.037, P<0.01). The ELISA data revealed that IGF-1R protein expression level of the model group was significantly higher than that of the treatment group [(41.0±4.7) µg/L vs. (24.0±3.1) µg/L, P<0.01]. Immunohistochemical analysis showed that there were significant differences between MVD and PI in the model and treatment groups [MVD, 34.75±3.49 vs. 22.25±3.63; PI, (75.25±7.15)% vs. (45.75±5.12)%; P<0.01 for both). Western blot data showed that IGF-1R and PI3K protein expression levels were not significantly different between the model and treatment groups (1.03±0.33 vs. 0.98±0.37 and 1.05±0.28 vs. 0.98±0.19), respectively (P>0.05), but p-IGF-1R and p-PI3K protein expression levels had significant differences between the two groups (1.08±0.10 vs. 0.51±0.08 and 1.12±0.09 vs. 0.86±0.09), respectively (P<0.01 for both). CONCLUSIONS: The IGF-1R inhibitor can effectively inhibit the formation of malignant pleural effusion. Its mechanism may be related to the suppression of tumor cell proliferation, invasion and angiogenesis through inhibition of PI3K signaling. TAE226 treatment may be a potential therapeutic regimen of treating malignant pleural effusion.

A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells.

CD8 T cells specific for islet autoantigens are major effectors of ß cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114-123 (VMNILLQYVV), with studies demonstrating both 114-123 and 114-122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) ß cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114-122 alone or both 114-122 and 114-123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114-123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201(+) human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114-123 as the predominant epitope in this region. These studies highlight the importance of understanding ß cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells.

Modification of human islet preparation: an effective approach to improve graft outcome after islet transplantation?

Revascularisation of transplanted islets is an essential prerequisite for graft survival and function. However, current islet isolation procedures deprive the islets of endothelial tubulets. This may have a detrimental effect on the revascularisation process of islets following transplantation. We hypothesise that modification of the isolation procedure that preserves islet endothelial vessels may improve the islet revascularisation process following transplantation. Here, we present a modified islet isolation method by which a substantial amount of endothelial cells still attached to the islets could be preserved. The islets with preserved endothelial cells isolated by this method were revascularised within 3 days, not observed in islets isolated by standard methods. Further, we observed that grafts of islets isolated by standard methods had more patches of dead tissue than islet grafts obtained by the modified method, indicating that attached endothelial cells may play an important role in the islet revascularisation process and potentially help to improve the transplantation outcome.

Attainment of metabolic goals in the integrated UK islet transplant program with locally isolated and transported preparations.

The objective was to determine whether metabolic goals have been achieved with locally isolated and transported preparations over the first 3 years of the UK's nationally funded integrated islet transplant program. Twenty islet recipients with C-peptide negative type 1 diabetes and recurrent severe hypoglycemia consented to the study, including standardized meal tolerance tests. Participants received a total of 35 infusions (seven recipients: single graft; 11 recipients: two grafts: two recipients: three grafts). Graft function was maintained in 80% at [median (interquartile range)] 24 (13.5-36) months postfirst transplant. Severe hypoglycemia was reduced from 20 (7-50) episodes/patient-year pretransplant to 0.3 (0-1.6) episodes/patient-year posttransplant (p < 0.001). Resolution of impaired hypoglycemia awareness was confirmed [pretransplant: Gold score 6 (5-7); 24 (13.5-36) months: 3 (1.5-4.5); p < 0.03]. Target HbA1c of <7.0% was attained/maintained in 70% of recipients [pretransplant: 8.0 (7.0-9.6)%; 24 (13.5-36) months: 6.2 (5.7-8.4)%; p < 0.001], with 60% reduction in insulin dose [pretransplant: 0.51 (0.41-0.62) units/kg; 24 (13.5-36) months: 0.20 (0-0.37) units/kg; p < 0.001]. Metabolic outcomes were comparable 12 months posttransplant in those receiving transported versus only locally isolated islets [12 month stimulated C-peptide: transported 788 (114-1764) pmol/L (n = 9); locally isolated 407 (126-830) pmol/L (n = 11); p = 0.32]. Metabolic goals have been attained within the equitably available, fully integrated UK islet transplant program with both transported and locally isolated preparations.

Investigation of intracellular signalling cascades mediating stimulatory effect of a Gymnema sylvestre extract on insulin secretion from isolated mouse and human islets of Langerhans.

AIM: Traditional plant-based remedies such as Gymnema sylvestre (GS) extracts have been used to treat diabetes mellitus for many centuries. We have shown previously that a novel GS extract, OSA®, has a direct effect on insulin secretion but its mode of action has not been studied in detail Thus this study investigated the possible underlying mechanism(s) by which OSA® exerts its action. METHODS: The effects of OSA® on [Ca(2+)]i and K(+) conductances were assessed by Ca(2+) microfluorimetry and electrophysiology in dispersed mouse islets and MIN6 ß-cells, respectively. Isolated mouse (from 20 to 25 mice) and human (from 3 donors) islets, and MIN6 ß-cells, were used to investigate whether the stimulatory effect of OSA® on insulin secretion was dependent on the presence of extracellular calcium and protein kinase activation. RESULTS: OSA ®-induced insulin secretion from mouse islets and MIN6 ß-cells was inhibited by nifedipine, a voltage-gated Ca(2+) channel blocker, and by the removal of extracellular Ca(2+), respectively. OSA® did not affect the activities of KATP channels or voltage-dependent K(+) channels in MIN6 ß-cells but it caused an increase in intracellular Ca(2+) ([Ca(2+)]i) concentrations in Fura-2-loaded mouse islet cells. The insulin secretagogue effect of OSA® was dependent, in part, on protein kinase activation since incubating mouse or human islets with staurosporine, a general protein kinase inhibitor, resulted in partial inhibition of OSA®-induced insulin secretion. Experiments using permeabilized, Ca(2+)-clamped MIN6 ß-cells revealed a Ca(2+)-independent component action of OSA® at a late stage in the stimulus-response coupling pathway. OSA®-induced insulin secretion was unexpectedly associated with a decrease in intracellular cAMP levels. CONCLUSIONS: These data indicate that the GS isolate OSA® stimulates insulin secretion from mouse and human islets in vitro, at least in part as a consequence of Ca(2+) influx and protein kinase activation.

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans.

AIMS: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. METHODS: Human islets were isolated from pancreases retrieved from heart-beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT-PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. RESULTS: RT-PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co-localized with insulin-expressing ß-cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and CB2 (N-(1,3-Benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. CONCLUSIONS: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor-independent pathways in the antagonist-induced stimulatory effects.

A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

Inability to process and store proinsulin in transdifferentiated pancreatic acinar cells lacking the regulated secretory pathway.

Generation of new beta-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true beta-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. beta-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the beta-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted beta-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new beta-cells.

Human islets derived from donors after cardiac death are fully biofunctional.

Islets from brain-dead donors (BDDs) are being used in the treatment of Type 1 diabetes. However, both donor numbers and islet survival are limited. We explored the clinical potential for islets from non-heart-beating donors (NHBDs), who have lower circulating cytokines, by comparing islets from 10 NHBDs against 12 identically-isolated islets from BDDs over the same time period. The quantity and quality of islets from NHBDs was good. NHBD yielded approximately 12.6% more islets than those of BDDs (505,000 +/- 84,230 vs. 400,970 +/- 172,430 islet equivalent number [IEQ]/pancreas, p = 0.01) with comparable viability. ATP and GTP contents were lower (6.026 +/- 3.076 vs. 18.105 +/- 7.8 nM/mg protein, p = 0.01 and 1.52 +/- 0.87 vs. 3.378 +/- 0.83 nM/mg protein, p = 0.04) and correlated negatively to warm ischemia time (R(2)= 0.8022 and R(2)= 0.7996, respectively). Islets from NHBDs took longer to control hyperglycemia in diabetic mice, but were equally able to sustain euglycemia. With a warm ischemia time (WIT) of

Gene expression heterogeneity in human islet endocrine cells in vitro: the insulin signalling cascade.

AIMS/HYPOTHESIS: Insulin secretion is a highly regulated mechanism involving a complex insulin-dependent network of communication between alpha, beta and delta cells. However, whereas the role of insulin in beta cells has been well documented, very little is known about its role in alpha and delta cells. Having recently demonstrated heterogeneity of insulin receptor (INSR) isoform expression in these three endocrine cell types, our current study aimed to characterise the expression pattern of the multiple isoforms involved in the insulin signal transduction cascade in human alpha, beta and delta cells in vitro. MATERIALS AND METHODS: cDNA samples prepared from single human islet cells were subjected to nested PCRs. RESULTS: Of 706 cells analysed, 15% were alpha cells, 28% beta cells, 8% delta cells and 46% non-endocrine cells. Profiling of expression of the insulin signalling cascade elements showed a heterogeneity between islet cell types, although at least one member of each protein family was expressed in the three populations of endocrine cells. Thus, the mRNAs coding for INSR-B, phosphoinositide-dependent protein kinase-1 and the human homologue of v-akt murine thymoma viral oncogene homologue 1 (AKT1) could not be detected in alpha cells, but were expressed by beta and delta cells. In addition, while the insulin receptor substrates IRS1 and IRS2, phosphoinositide-3-kinase, catalytic, beta polypeptide (PIK3CB) and AKT2 were expressed with relatively low frequencies in alpha and delta cells (<17% for IRS1, IRS2, PIK3CB; <25% for AKT2), their frequencies of expression in beta cells were 50, 33, 33 and 100%, respectively. CONCLUSIONS/INTERPRETATION: Our results suggest that insulin signalling cascade elements in human alpha, beta and delta cells have distinct expression patterns.

Islet cell transplantation.

People with type 1 diabetes have normal exocrine pancreatic function, making islet cell rather than whole organ transplantation an attractive option. Achieving insulin independence in type 1 diabetes was the perceived goal of islet cell transplantation. The success of the Edmonton group in achieving this in a selected group of type 1 patients has led to renewed optimism that this treatment could eventually replace whole organ pancreas transplantation. However the long-term results of this treatment indicate that insulin independence is lost with time in a significant proportion of patients, although they may retain glycaemic stability. In this context, the indications for islet cell transplantation, which have evolved over the last 5 years, indicate that the patients who benefit most are those who experience severe hypoglycaemic reactions despite optimal insulin therapy. This review will summarise the history of islet cell transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, indications for islet cell transplantation, current clinical trials, the early UK islet cell transplant experience using the Edmonton protocol, and some of the challenges that lie ahead.

Successful in vitro priming of EBV-specific CD8+ T cells endowed with strong cytotoxic function from T cells of EBV-seronegative children.

Epstein-Barr virus (EBV)-seronegative transplant recipients are at high risk of developing EBV-associated post-transplant lymphoproliferative disorder (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. So far, efforts to activate CD8+ EBV-specific cytotoxic T lymphocytes (CTL) endowed with high specific cytotoxicity from EBV-seronegative children have failed. We compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on lymphoblastoid cell lines (LCL) stimulation potentially enhanced by either LCL presentation through dendritic cells, or selection of IFN-gamma+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, according to the standard protocol for reactivation of EBV-specific CTL. We found that only specific LCL stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children. The lines thus activated, which included specificities toward EBV latent and lytic proteins, showed high percentage CD8+ T cells, with <10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7 T-cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, it is feasible to activate EBV-specific CD8+ CTL with suitable characteristics for in vivo employment from EBV-seronegative children.

Direct regulation of insulin secretion by angiotensin II in human islets of Langerhans.

AIMS/HYPOTHESIS: This study aimed to identify the expression of angiotensin II receptors in isolated human islets and beta cells and to examine the functional consequences of their activation. MATERIALS AND METHODS: Single-cell RT-PCR was used to identify whether human islet cells express mRNA for type 1 angiotensin II receptors (AT(1)), and western blotting was used to determine AT(1) protein expression by human islets and MIN6 beta cells. We measured changes in intracellular calcium by microfluorimetry using Fura 2-loaded MIN6 cells and human islet cells. Dynamic insulin secretory responses were determined by RIA following perifusion of human islets and MIN6 cells. RESULTS: Human islets expressed mRNAs for both the angiotensin precursor, angiotensinogen, and for angiotensin-converting enzyme. In addition, human and mouse beta cells expressed AT(1). These were functionally coupled to increases in intracellular calcium, which occurred at least in part through phospholipase-C-sensitive mechanisms and calcium influx through voltage-operated calcium channels. Short-term exposure of human islets and MIN6 cells to angiotensin II caused a rapid, short-lived initiation of insulin secretion at 2 mmol/l glucose and potentiation of insulin secretion induced by glucose (at 8 and 16.7 mmol/l). CONCLUSIONS/INTERPRETATION: These data demonstrate that the AT(1) is expressed by beta cells and that angiotensin II effects a short-lived and direct stimulation of human and mouse beta cells to promote insulin secretion, most probably through elevations in intracellular calcium. Locally produced angiotensin II may be important in regulating a coordinated insulin secretory response from beta cells.

Hydroxyurea and splenic irradiation-induced tumour lysis syndrome: a case report and review of the literature.

Therapeutic agents for chronic myeloid leukaemia (CML) in the chronic phase include hydroxyurea, interferon alpha, allogeneic stem cell transplantation and the tyrosine kinase inhibitor imatinib (STI 571, Gleevec). For elderly patients, oral hydroxyurea is suitable for the relief of symptoms caused by hyperleukocytosis, and splenic irradiation would be considered if abdominal discomfort or fullness induced by splenomegaly were present. Tumour lysis syndrome (TLS) is seldom seen in the treatment for CML, and TLS caused by hydroxyurea or splenic irradiation is rarely observed. Herein, we report an elderly CML patient who received treatment with hydroxyurea, allopurinol, hydration and splenic irradiation. After 3 days, acute TLS developed. Aggressive supportive treatment, including haemodialysis, stabilized the condition.

Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation.

AIMS/HYPOTHESIS: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. MATERIALS AND METHODS: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. RESULTS: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. CONCLUSIONS/INTERPRETATION: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

Insulin-producing cells derived from human pancreatic non-endocrine cell cultures reverse streptozotocin-induced hyperglycaemia in mice.

AIMS/HYPOTHESIS: The aim of the study was to investigate the potential of human pancreatic non-endocrine cells to transdifferentiate into endocrine cells that would be capable of secreting insulin in response to glucose and ameliorating insulin-deficient diabetes after transplantation. MATERIALS AND METHODS: Cell fractions enriched with exocrine cells after human islet isolation were treated with streptozotocin to remove residual beta cells, grown in monolayer culture to allow de-differentiation, transferred to cluster culture for redifferentiation in the presence of activin A, betacellulin, nicotinamide and glucose, supplemented with 10% FCS, and administered to streptozotocin-induced diabetic SCID mice. A subset of cells was transfected with the IPF1 gene (also known as PDX1) before transdifferentiation. RESULTS: No insulin was detectable in cell preparations after 5 days of treatment with streptozotocin. In monolayer culture, 90% of the streptozotocin-treated pancreatic cells co-expressed cytokeratin-19 and vimentin at 2 weeks and 60% expressed nestin at 4 weeks. Cell cultures with a high proportion of nestin-expressing cells had greater plasticity for transdifferentiation into cells with phenotypic and functional markers of beta cells, this property being significantly enhanced by transfection with IPF1 gene and leading to 15+/-6.7% insulin-positive cells after transplantation vs. 0.01% of cells transplanted after streptozotocin treatment alone. These cells improved glucose control in all of 42 diabetic mice after transplantation, restoring normoglycaemia in 40%. CONCLUSIONS/INTERPRETATION: Human pancreatic cells are a potential source of new glucose-responsive insulin-producing cells that may be developed further for clinical use.

Expression of nephrin by human pancreatic islet endothelial cells.

AIMS/HYPOTHESIS: The islet microcirculation has morphological characteristics resembling those of renal glomeruli. Transcription of the nephrin gene, a highly specific barrier protein of the slit diaphragm of podocyte foot processes, has been reported in the pancreas, although its cellular localisation and function remain to be defined. In this study, we purified and characterised microvascular endothelial cells (MECs) isolated from human islets and investigated the expression and distribution of nephrin on these cells. METHODS: Human islet MECs were extracted and purified using anti-CD105-coated immunomagnetic beads and their endothelial characteristics were confirmed by expression of classical endothelial markers and basal high-level expression of intercellular adhesion molecule-1 and TNF-alpha-inducible vascular cell adhesion molecule-1. Nephrin expression was assessed by immunofluorescence, flow cytometric analysis and western blotting on cell lysates, as well as by RT-PCR. RESULTS: Immunofluorescence studies detected nephrin in a fine, punctate, diffuse pattern on cultured islet MECs, and also in human pancreatic islet sections. In both cases nephrin colocalised with endothelial markers. TNF-alpha treatment induced a marked reduction and redistribution of the protein in one or multiple aggregates. Nephrin expression was confirmed by flow cytometry, western blotting and RT-PCR studies. In contrast, nephrin could not be detected at the protein or mRNA level in human macro- and microvascular cells from other sites. CONCLUSIONS/INTERPRETATION: Nephrin is expressed at protein and mRNA levels in islet microendothelium, supporting the hypothesis that islet MECs exhibit distinctive morphological characteristics that indicate functional specialisation of potential pathophysiological importance.

ACTH stimulates insulin secretion from MIN6 cells and primary mouse and human islets of Langerhans.

It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tIssue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tIssue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH(1-24) (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH(1-24) (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 +/- 13.6% basal secretion; 187 +/- 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH(1-24) at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into beta-cells.