Preparation of Recombinant Islet Cell Autoantigen 69 kD Fusion Protein

To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.

Antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocin-induced diabetes in rats

AIM:To investigate the antioxidant activity of chitooligosaccharides(COSs)on pancreatic islet cells in diabetic rats induced by streptozotocin. METHODS:The antioxidant effect of COSs on pancreatic islet cells was detected under optical microscopy and with colorimetric assay and gel electrophoresis.The activities of glutathione peroxidase and superoxide dismutase,total antioxidant capacity,and content of malondialdehyde in serum and tissue slices of pancreas were examined after 60 d to determine the effect of COSs in streptozotocin-induced diabetes in rats. RESULTS:COSs can prohibit the apoptosis of pancreatic islet cells.All concentrations of COSs can improve the capability of total antioxidant capacity and activity of superoxide dismutase and decrease the content of malondialdehyde drastically.Morphological investigation in the pancreas showed that COSs have resulted in the reduction of islets,loss of pancreatic cells,and nuclear pyknosis of pancreatic cells. CONCLUSION:COSs possess various biological activities and can be used in the treatment of diabetes mellitus.

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

AIM: To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats.METHODS: In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS: Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides (100 mg/L) had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance.CONCLUSION: Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.

Surgical treatment of nonfunctioning islet cell tumor: report of 41 cases

BACKGROUND: Nonfunctioning islet cell tumor (NIT)as a rare pancreatic endocrine neoplasm is characterized byunspecific clinical symptoms and is hard to diagnose. InChina, NIT accounts for 15%-41% in pancreatic endocrineneoplasms just next to insulinoma. In this study, weevaluated the surgical modalities of NIT.METHODS: From January 1978 through February 2002, 41patients with NIT were treated at the Department of Sur-gery of the First Affiliated Hospital, China Medical Univer-sity, Shenyang, China. Tumors in the head of the pancreaswere noted in 28 patients, and in the body or in the tail in13 patients. The mean diameter of the tumors was 10. 7cm. Fifteen patients underwent enucleation and 21 receivedpancreatectomy. Tumors were unresectable in 5 patientsbecause of extensive infiltration. The mean diameter was9.6 cm in patients treated by enucleation, 13.1 cm in thoseby pancreaticoduodenectomy, 9.9 cm in those by distalpancreatectomy, and 11.6 cm in those with unresectabletumors.RESULTS: The curative resection rate was 88% (n =36),and the complication rate after enucleation and pancreatec-tomy was 33% ( n = 5 ) and 14% (n=3), respectively. Nolocal recurrence was found after both enucleation and pan-createctomy. Liver metastases occurred in 3 patients treatedby enucleation.CONCLUSIONS: Both enucleation and pancreatectomy areeffective for NIT of the pancreas. No local recurrence hasbeen found in patients treated by the two surgical proce-dures. The complication rates of the two modalities arecomparable.

Spiral CT localization of pancreatic function-ing islet cell tumors

BACKGROUND: Surgeons are always concerned about the localization of pancreatic functioning islet cell tumor. If the tumor is accurately localized before operation, resection of the pancreatic body and tail without intention can be avoided. The purpose of this study was to evaluate spiral CT localization of pancreatic functioning islet cell tumors and CT techniques. METHODS: CT manifestations in 6 patients with clinically and pathologically proved pancreatic functioning islet cell tumors ware analyzed retrospectively. RESULTS: In 4 patients with insulinomas and 2 patients with glucagonomas, 5 were localized accurately by CT be- fore surgery and 1 was detected retrospectively. The en- hancement of tumors was greater than that of normal pan- creas in arterial phase and pancreatic parenchymal phase. Four patients showed mild high-density and 2, iso-density in the portal venous phase. CONCLUSION: Spiral CT multi-phase enhanced scan with 1.5 ml/kg contrast agent and 2-5 mm slice width can loca- lize functioning islet cell tumors accurately.

Effects of High Concentration Glucose on the Expression of NF-κB,Bax and Cytochrome C and Apoptosis of Islet Cells in Mice

The roles of NF-kappaB （NF-κB） expression, Bax activity and cytochrome C （Cyt C） release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups： G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-rd3 expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in GI, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-κB expression was also increased （P〈0.05）, but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-rd3 expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups （P〈0.05）. It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-κB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-κB expression could be an effective strategy for protecting pancreatic islet cells.

SPECTRUM OF FUNCTIONING ISLET CELL TUMOR ON MULTISLICE COMPUTED TOMOGRAPHY:EXPERIENCE ON 70 PATIENTS

Objective To review experience in preoperative detection of islet cell tumors using multislice computed tomography （MSCT） and summarize various imaging features of functioning islet cell tumors on enhanced MSCT. Methods Seventy patients with clinical or pathological diagnosis of functioning pancreatic islet cell tumor between October 2003 and February 2007 were included in this retrospective study. Seventy-four enhanced MSCT scans in these patients were identified. All MSCT scans were interpreted by two experienced radiologists by consensus interpretation. Surgery and pathology reports were used to confirm the diagnosis, localization, and size of tumors. Results Totally, 73 functioning islet cell tumors including 65 benign insulinomas, 2 benign glucagonomas, 3 malignant insulinomas, and 3 malignant glucagonomas were pathologically diagnosed. Tumors in only two cases were not found by MSCT. In 67 benign lesions, 32 showed typical enhancement style, 21 showed prolonged enhancement in portal venous phase, 4 showed delayed enhancement, 4 had iso-dense enhancement with normal pancreatic parenchyma, 2 had no enhancement at all in arterial phase and portal venous phase, and 4 had inhomogeneous enhancement with necrosis or cyst-formation. Patchy or spotty calcifications were found in 3 of the 67 tumors. In 6 malignant islet cell tumors, vessel invasion （2/6） and bowel invasion （1/6） were seen. Different enhancement patterns were shown. All hepatic metastases showed hyper-enhancement during their arterial phase. Conelttsions Pancreatic islet cell tumor may display a wide spectrum of presentations in MSCT. Tumors with unusual appearances often present as diagnostic challenges. Non-contrast and post-contrast multiphase scans are recommended for the localization of functioning islet cell tumors.

CLINICOPATHOLOGIC FEATURES AND SURGICAL TREATMENT OF NONFUNCTIONING ISLET CELL TUMORS

Objective.To in vestigate clinicopathologic characteristics and surgical results of nonfunctiona l islet cell tumors.Methods.We performed retrospective analysis of50patient s with nonfunctional islet cell tumor treat-ed at Peking Union Medical College Hospital from July1968to July1999,and summarized clinical symptoms and signs,primary diagnosis before surgery,surgical treatments,pathologic and im munohisto-logical characteristics.Results.Of the50cases examined from July 1968to July1999,12were found during physical examinations. The most common symptoms were upper abdominal pain and upper abdominal dis-com fort,which appeared in20casesand17casesrespectively,an d the most common sign was abdominal masses.B-ultrasound and CT are the most commonly used means for preoperative examination,with posit ive rates of97.8%and100%respectively.Op-e rative mortality was2%.Five-year survival rate of radical surgery of maligna nt cases was75%.Immuno-histological examinations showed that tumors rich i n multi-peptide linked hormones,neuron-specific eno-lase and chromaffin were in65%cases,90.6%and73.3% respectively.The positive rates of insulin,glucagons,somatostatin,pancreat ic polypeptide,gastrin and vasoactive intestinal peptide were68.2% ,51.2%,42.9%,40% ,25%and15.8%respectively.Conclusion.Nonfuncti onal islet cell tumors lack diagnostic specificity.Ultrasound and CT are primar y examination methods.Immunohistological analysis indicates different hormones ,but lacks the presence of related clinical symptoms.Surgery is an effective treatment for nonfunctional islet cell tumors,and even for those with distal m etastasis,immediate surgical removal and treatment can improve prognosis.

Total pancreatectomy with islet cell transplantation vs intrathecal narcotic pump infusion for pain control in chronic pancreatitis

AIM: To evaluate pain control in chronic pancreatitis patients who underwent total pancreatectomy with islet cell transplantation or intrathecal narcotic pump infusion.METHODS: We recognized 13 patients who underwent intrathecal narcotic pump(ITNP) infusion and 57 patients who underwent total pancreatectomy with autologous islet cell transplantation(TP + ICT) for chronic pancreatitis(CP) pain control between 1998 and 2008 at Indiana University Hospital. All patients had already failed multiple other modalities for pain control and the decision to proceed with either intervention was made at the discretion of the patients and their treating physicians. All patients were evaluated retrospectively using a questionnaire inquiring about their pain control(using a 0-10 pain scale), daily narcotic dose usage, and hospital admission days for pain control before each intervention and during their last follow-up. RESULTS: All 13 ITNP patients and 30 available TP + ICT patients were evaluated. The mean age was approximately 40 years in both groups. The median duration of pain before intervention was 6 years and 7 years in the ITNP and TP + ICT groups, respectively. The median pain score dropped from 8 to 2.5(on a scale of 0-10) in both groups on their last follow up. The median daily dose of narcotics also decreased from 393 mg equivalent of morphine sulfate to 8 mg in the ITNP group and from 300 mg to 40 mg in the TP + ICT group. No patient had diabetes mellitus(DM) before either procedure whereas 85% of those who underwent pancreatectomy were insulin dependent on their last evaluation despite ICT. CONCLUSION: ITNP and TP + ICT are comparable for pain control in patients with CP however with high incidence of DM among those who underwent TP + ICT. Prospective comparative studies and longer follow up are needed to better define treatment outcomes.

Role of C-Peptide in Relation to Levels of Anti-GAD and Islet Cell Antibodies in Characterizing Types of Diabetes in the Young, in Eastern India

Background: Measuring fasting C-peptide (FCP) and antibodies against Glutamic acid decarboxylase (GADA) and Islet cell antibodies (ICA) are not so commonly explored in children and young adults. Objectives: To assess the levels of FCP, GADA and ICA in subjects below the age of 25 years with DM and compare their levels to differentiate between Autoimmune and Non-Autoimmune Type 1 DM. Methodology: Blood samples of 93 subjects diagnosed with DM, reporting to the tertiary care hospital, were analysed for ICA, GADA and FCP. Receiver operating characteristics (ROC) curves were analysed to check the ability of autoimmune markers, BMI and C-peptide to differentiate between Autoimmune (Ai) and Non-Autoimmune (NonAi) diabetes. Results: 30/93 (32.2%) were positive for anti-GAD ab and/or ICA and categorised as Autoimmune (Ai), the most common antibody being, anti-GAD ab (80%) in them. The level of FCP among Ai compared to NonAi, was significantly low (p 20.75 nmol/l) as a very dependable test for diagnosing Ai, Type 1 DM, in children and young adults. Its sensitivity and specificity are in the range of 86.2% and 96.8% respectively. Low level of C-peptide (Conclusion: This study revealed predominant positivity for anti-GAD ab (80%) among Ai+ patients. ROC analysis shows GADA above 20.75 nmol/l and Fasting C-peptide < 0.36 nmol/l as a good indicator for diagnosing Ai in children and young adults.

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells(ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as "guardians", controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and β-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation.

Imaging pancreatic islet cells by positron emission tomography

It was estimated that every year more than 30000 persons in the United States- approximately 80 people per day- are diagnosed with type 1 diabetes(T1D). T1 D is caused by autoimmune destruction of the pancreatic islet(β cells) cells. Islet transplantation has become a promising therapy option for T1 D patients, while the lack of suitable tools is difficult to directly evaluate of the viability of the grafted islet over time. Positron emission tomography(PET) as an important non-invasive methodology providing high sensitivity and good resolution, is able to accurate detection of the disturbed biochemical processes and physiological abnormality in living organism. The successful PET imaging of islets would be able to localize the specific site where transplanted islets engraft in the liver, and to quantify the level of islets remain alive and functional over time. This information would be vital to establishing and evaluating the efficiency of pancreatic islet transplantation. Many novel imaging agents have been developed to improve the sensitivity and specificity of PET islet imaging. In this article, we summarize the latest developments in carbon-11, fluorine-18, copper-64, and gallium-68 labeled radioligands for the PET imaging of pancreatic islet cells.

STUDIES OF EFFECTS OF PORTASYSTEMIC SHUNT ON THE FUNCTION OF HEPATIC AND PANCREATIC ISLET CELLS

The present study was aimed at dynamic observation of the ef fects of end to side portacaval shunt (PCS) and end to side mesocaval shunt (MCS) in dogs on the functions of the liver and pancreatic islet cells. According to correlation between the changes of plasma insulin level in the portal vein and hepatic flow and liver morphology after PCS and MCS, we conclude that the depletion of hepatic flow is the major factor in the deterioration of liver functions. The levels of insulin and glucagon in both the peripheral vein and the portal vein were decreased after PCS and MCS. There was also depletion of pancreatic islet A and B cells and vacuolar degeneration of the pancreas. These changes were more signifcant in PCS than in MCS, suggesting that portasystemic shunt, especially total portasystemie shunt, might damage pancreatic endocrine functions.

Bioengineered stem cells as an alternative for islet cell transplantation

Type 1 diabetes is an autoimmune and increasingly prevalent condition caused by immunological destruction of beta cells. Insulin remains the mainstay of therapy. Endeavours in islet transplantation have clearly demonstrated that type 1 diabetes is treatable by cellular replacement. Many challenges remain with this approach. The opportunity to use bioengineered embryonic or adult pluripotential stem cells, or islets derived from porcine xenograft sources could address future demands, but are still associated with considerable challenges. This detailed review outlines current progress in clinical islet transplantation, and places this in perspective for the remarkable scientific advances now occurring in stem cell and regenerative medicine approaches in the treatment of future curative treatment of diabetes.

Human pluripotent stem cell-derivedβcells:Truly immature isletβcells for type 1 diabetes therapy?

A century has passed since the Nobel Prize winning discovery of insulin,which still remains the mainstay treatment for type 1 diabetes mellitus(T1DM)to this day.True to the words of its discoverer Sir Frederick Banting,“insulin is not a cure for diabetes,it is a treatment”,millions of people with T1DM are dependent on daily insulin medications for life.Clinical donor islet transplantation has proven that T1DM is curable,however due to profound shortages of donor islets,it is not a mainstream treatment option for T1DM.Human pluripotent stem cell derived insulin-secreting cells,pervasively known as stem cell-derivedβcells(SC-βcells),are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy.Here we briefly review how isletβcells develop and mature in vivo and several types of reported SC-βcells produced using different ex vivo protocols in the last decade.Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown,the SC-βcells have not been directly compared to their in vivo counterparts,generally have limited glucose response,and are not yet fully matured.Due to the presence of extra-pancreatic insulin-expressing cells,and ethical and technological issues,further clarification of the true nature of these SC-βcells is required.

Pancreatic and peri-pancreatic lesions mimic pancreatic islet cell tumor in multidetector computed tomography

Objective This pictorial review aimed to summarize the most possible differential diagnosis of pancreatic islet cell tumor （PICT）. Data sources Data used in this review were mainly from Medline and Pubmed in English. And all clinical images in this review were from Department of Radiology, Peking Union Medical College Hospital, Beijing, China. Study selection Cases of pancreatic cystadenoma, solid pseudo-papillary tumor of the pancreas, pancreatic metastasis, pancreatic adenocarcinoma, para-pancreatic neuroendocrine tumors, Castleman disease, gastrointestinal stromal tumor, splenic artery aneurysm and accessory spleen were selected in this pictorial review for differential diagnosis of PICT.

Effects of human umbilical cord serum on proliferation and insulin content of human fetal islet-like cell clusters

BACKGROUND: Type 1 diabets is an autoimmune disease caused by the destruction of pancreatic β-cell with an in- creased incidence worldwide in the closing decades of the 20th century. This study was to investigate the effects of human umbilical cord serum (UCS) on the proliferation and function of human fetal islet-like cell clusters (ICCs) in vitro. METHODS: Eight fresh pancreatic glands obtained after in- duction of labor with water bag were mildly exposed to col- lagenase V, and the digested cells were cultured in a RPMI- 1640 medium plus 10% pooled UCS or fetal calf serum (FCS) to permit cells attachment and outgrowth of ICCs. RESULTS: In 8 consecutively explanted glands, develop- ment and proliferation of ICCs were observed. In the pre- sence of FCS, the outgrowth of ICC took place on the top of a flbroblast monocellular layer. UCS affected less growth of fibroblasts and increased the formation of ICCs about four-fold compared with explants from the same glands maintained in FCS. In both UCS and FCS, the insulin con- tent of the medium was variable to a certain extent and progressively declined from day 2 to day 6. Dithizone- stained ICCs in UCS suggested that most cell clusters were islet cells ( β-cells), and the purity of islets was estimated 80%-90%. The ultrastructure of the cultured cells showed a large number of granule-containing cells, most of which were identified as β-cells. CONCLUSION: We conclude that in comparison with ex- plants with FCS, the yield of ICCs and purification of islet cells are markedly increased by UCS and may facilitate the proliferation of pancreatic β-cells intended for islet trans- plantation.

Detection of Islet Cell Antibodies by Using Avidin-Biotin-Peroxidase Complex Technique

Autoimmunemechanisms may be involved in the pathogenesis of insulin dependent diabetes mellitus (IDDM).Multiple autoantibodies have been detected in patients with IDDM.Islet cell antibodies(ICA) complment-fixing islet cell antibodies(CF-ICA) and antibodies to an islet cell protein

EFFECTS OF GLUCAGON ON ISLET β CELL FUNCTION IN PATIENTS WITH DIABETES MELLITUS

Objective To evaluate islet β cell response to intravenous glucagon （ a non-glucose secretagogue） stimulation in diabetes mellitus. Methods Nineteen patients with type 1 diabetes （T1 D） and 131 patients with type 2 diabetes （T2D） were recruited in this study. T2D patients were divided into two groups according to therapy： 36 cases treated with insulin and 95 cases treated with diet or oral therapy. The serum C-peptide levels were determined at fasting and six minutes after intra- venous injection of 1 mg of ghicagon. Results Both fasting and 6-minute post-ghicagon-stimulated C-peptide levels in T1D patients were significantly lower than those of T2D patients （0. 76±0. 36 ng/mL vs. 1.81±0. 78 ng/mL, P 〈 0.05 ; 0.88±0.42 ng/mL vs. 3.68±0. 98 ng/mL, P 〈 0. 05 ）. In T1D patients, the C-peptide level after injection of ghicagon was similar to the fasting level. In T2D, patients treated with diet or oral drug had a significantly greater fasting and stimulated C-peptide level than those patients received insulin therapy （2.45±0. 93 ng/mL vs. 1.61±0. 68 ng/mL, P 〈 0.05 ; 5.26±1.24 ng/mL vs. 2.15±0.76 ng/mL, P 〈 0.05 ）. The serum C-peptide level after ghicagon stimulation was positively correlated with C-peptide levels at fasting in all three groups （ r = 0.76, P 〈 0.05 ）. Conclusions The 6-minute ghicagon test is valuable in assessing the function of islet β cell in patients with diabetes mellitus. It is helpful for diagnosis and treatment of diabetes mellitus.