TADG-12 as an early marker in the development of pancreatic ductal adenocarcinoma (PDAC): Involvement of insulin containing cells

TADG-12 is a serine protease that was characterized as expressed in ovarian and gastric carcinomas. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and its late detection results in poor prognosis. Therefore, we decided to examine whether TADG-12 appears early in PDAC development. In normal pancreas, pale to moderate immunostaining is present in islets of Langerhans, while exocrine tissue and ducts are free from labeling. In contrast, in cancer patients, who still preserve the integrity of the exocrine and the endocrine tissues, a pronounced immunolabelling of TADG-12 was evident mainly located in the insulin containing β cells. In a more progressive stage of the disease TADG-12 was also evident in the deteriorated exocrine tissue. TADG-12 was also heavily labeled in islets of Langerhans, which were embedded in the stroma of the residual pancreatic tissue. Again, there was a considerable overlap between the labeling of insulin and TADG-12 in these islets. Close correlation between insulin and TADG-12 was also evident in islets of Langerhans surrounded by adipose cells. The TADG-12 labeled was confined to the cytoplasm and the membrane of the cells. In the progressive stage of PDAC, the cancerous ducts were clearly labeled with TADG-12 with no labeling of insulin. At high magnification the TADG-12 clearly labeled the cytoplasm and the cell wall membrane of duct cells, while the nuclei remained unstained upon incubation with antibodies to TADG-12. The present findings may assist in early detection of PDAC as well as targeting of TADG-12 in order to attenuate the rapid progression of the disease.     

Dynamic distribution of ERK,p38 and JNK during the development of pancreatic ductal adenocarcinoma

We recently discovered that oncogenic c-kit is highly expressed concomitantly with the development of pancreatic ductal adenocarcinoma (PDAC). Since oncogenic c-kit may activate major pathways of protein tyrosine phosphorylation, we decided to investigate this issue in the major protein phosphorylation cascades. In normal pancreas labeling with antiphosphorylated ERK1/2 (pERK1/2) antibody was mainly confined to islets of Langerhans in close overlapping with insulin containing cells. Phosphorylated p38 (pp38) showed a similar pattern of distribution, while only weak labeling was evident for pJNK and no labeling of pMEK was observed. As expected, general ERK1/2 (gERK1/2), general p38 (gp38), general JNK (gJNK) as well as general MEK (gMEK) were all evident in islets of Langerhans and in the exocrine tissue. In early development of PDAC, pERK1/2 and pp38 retained their localization in islets of Langerhans. Intensive staining of pERK1/2 was also evident in the cancerous ducts, while the labeling with antibodies to pp38 was more moderate. While pJNK staining in islets of Langerhans was weak, with no labeling in the cancerous ducts, antibodies to gJNK revealed intensive staining suggesting the weak staining of pJNK is not due to the lack of the enzyme. In a more advanced stage of PDAC the carcinomas were clearly stained with pERK1/2 and pp38, while moderate staining with pJNK was also evident. In liver metastases, the cancer cells were heavily labeled with all three phospho-MAPKs. It should be noted that the localization of all three kinases was mainly in the cell nuclei. In the more advanced stage of PDAC, heavy labeling was evident using antibodies to gERK1/2, gp38, gJNK and gMEK. However, no labeling to pMEK was evident in parallel sections. Our data suggest that both in normal and cancerous pancreas, most of the MAPK activities are located in islets of Langerhans and cancerous ducts. It is suggested that using inhibitors to protein kinases may attenuate the progression of the disease.     

Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

The stem cell factor-c-kit system and mast cells in human pancreatic cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF-c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF-c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.     

Combined ultrastructural and immunocytochemical studies of pancreatic endocrine tumors]

9 surgically removed endocrine tumours of pancreas of female patients aged from 19 to 71 years were studied. The material was stained with hematoxylin and eosin, by Grimelius and Fontana--Masson; PAP method with polyclonal antibodies to insulin, glucagon and somatostatin was also used. Immunoreactivity was weaker in tumours with clinically pronounced hormonal activity as compared to the normal endocrine cells in the Langerhans islets. This is confirmed ultrastructurally: tumour cells contain comparatively low number of neuroendocrine granules. Functionally inactive tumours show the opposite immunohistochemical results. The multipotential properties of cells forming pancreatic endocrine tumours are discussed.     

Microchip-based engineering of super-pancreatic islets supported by adipose-derived stem cells

Type 1 diabetes mellitus (T1DM) is a chronic disorder characterized by targeted autoimmune-mediated destruction of the β cells of Langerhans within pancreatic islets. Currently, islet transplantation is the only curative therapy; however, donor shortages and cellular damage during the isolation process critically limit the use of this approach. Here, we describe a method for creating viable and functionally potent islets for successful transplantation by co-culturing single primary islet cells with adipose-derived stem cells (ADSCs) in concave microwells. We observed that the ADSCs segregated from the islet cells, eventually yielding purified islet spheroids in the three-dimensional environment. Thereafter, the ADSC-exposed islet spheroids showed significantly different ultrastructural morphologies, higher viability, and enhanced insulin secretion compared to mono-cultured islet spheroids. This suggests that ADSCs may have a significant potential to protect islet cells from damage during culture, and may be employed to improve islet cell survival and function prior to transplantation. In vivo experiments involving xenotransplantation of microfiber-encapsulated spheroids into a mouse model of diabetes revealed that co-culture-transplanted mice maintained their blood glucose levels longer than mono-culture-transplanted mice, and required less islet mass to reverse diabetes. This method for culturing islet spheroids could potentially help overcome the cell shortages that have limited clinical applications and could possibly be developed into a bioartificial pancreas.     

Endocrine pancreas in cystic fibrosis: an immunohistochemical study

The pancreases of 17 patients who had cystic fibrosis with and without diabetes mellitus were evaluated at autopsy by routine staining and immunohistochemical methods for insulin, glucagon, somatostatin, and pancreatic polypeptide. Qualitative assessment of the number of islets of Langerhans and the degrees of exocrine pancreatic atrophy, fibrosis, and fat replacement was made for each pancreas. Quantitative assessment of islet composition was performed in 15 of the 17 based on the immunochemical reactivity of each cell type. Nondiabetic patients with cystic fibrosis in the latter part of the first decade of life have classic fibrocystic changes of the pancreas, with some persisting exocrine tissue, islets that appear normal, and prominent nesidioblastosis. The latter process may protect these patients from glucose intolerance. Young adult diabetic patients with cystic fibrosis have total loss of exocrine pancreas with fat replacement, lack of nesidioblastosis, a qualitative decrease in the number of islets, fibrosis of and amyloid deposits in islets, decreased numbers of insulin-containing cells in each islet, and atrophy of islet cells, probably resulting from progressive ischemia. Although the potential exists for an increasing incidence of diabetes mellitus in patients with cystic fibrosis as their life spans increase, individual variation occurs in this disease.     

New markers for pancreatic islets and islet cell tumors

Islets of Langerhans account for 2 g of endocrine tissue in the pancreas, comprising approximately one million islets, with each containing 1000 endocrine cells. The major hormone secreted from the islets is insulin, which regulates blood glucose, the main fuel of the body. Islets also secrete glucagon, somatostatin and pancreatic polypeptide and all are involved in the paracrine mechanism. Islet cells can be stained immunohistochemically for the general endocrine markers, chromogranin A, synaptophysin, neuron-specific enolase and Leu7. Beta islet cells are well equipped with glucose transporter 2, which binds to glucose and regulates diffusion of glucose through the beta cell membrane. As all four islet hormones are initially synthesized as prohormones, all islet cells are equipped with prohormone convertase 1/3 and 2. In addition, islet cells also contain zinc-containing matrix metalloproteinases and their inhibitors, metallothionein, cyclin-dependent kinases and insulin-like growth factors, and many more hormones, peptides and enzymes. Thus, islets not only secrete insulin and other pancreatic hormones but are a complex organ whose major function is glucose homeostasis.     

Migration path of stem cells involved in the repair of damaged pancreatic tissue caused by pancreatitis

Objective: To investigate the migratory path of stem cells in pancreatic tissues damaged by pancreatitis and to preliminarily identify stem cells that efficiently contribute to the repair of damaged pancreatic tissues. Methods: An animal model of acute pancreatitis was established, in which rats in the experimental group were given intraperitoneal (IP) injections of caerulein. Before the rats were sacrificed, 5-bromo-2’-deoxyuridine (BrdU) was administered by IP injection to label proliferating pancreatic cells. The localization and distribution of the stem cell-specific marker proteins nestin and c-kit in pancreatic tissues were examined using an immunohistochemical approach, and proliferation-specific BrdU incorporation was also analyzed. Results: (1) The nestin-positive cells first appeared in the pancreatic interlobar vessels, and then, were observed in the pancreatic acinar and islet tissues. (2) C-kit-positive cells were located only in the pancreatic islets. (3) BrdU-positive cells first appeared in the area surrounding the interlobular region, and then were diffusely distributed and filled the pancreatic lobules. Conclusions: (1) The stem cells, participated in the repair of damaged pancreatic tissue, appear firstly in the pancreatic interlobar vessels, then migrate toward the pancreatic lobules by using the interlobar vessels as channels and penetrate through the vascular endothelium into the pancreatic acinar tissues. A portion of the stem cells eventually penetrate into the islet tissue. (2) Exogenous stem cells, rather than the tissue-resident stem cells, efficiently contribute to the repair of damaged pancreatic tissues.     

Islet inflammation and CXCL10 in recent‐onset type 1 diabetes

Type 1 diabetes results from a T cell‐mediated destruction of insulin‐producing pancreatic β cells. Little is known on local factors contributing to migration of T cells to pancreatic tissue. We recently demonstrated evidence of viral infection in β cells in several recent‐onset type 1 diabetes patients. Islet inflammation was analysed in a series of new‐ or recent‐onset type 1 diabetic patients and non‐diabetic control subjects. Autoimmune T cell reactivity was studied in lymphocytes derived from pancreas‐draining lymph nodes of one recent‐onset type 1 diabetes patient in partial clinical remission. Insulitic lesions were characterized by presence of β cells, elevated levels of the chemokine CXCL10 and infiltration of lymphocytes expressing the corresponding chemokine receptor CXCR3 in all pancreatic lesions of type 1 diabetes patients, regardless of enterovirus infection of β cells. CXCR3 and CXCL10 were undetectable in pancreata of non‐diabetic control subjects. T cells isolated from draining lymph nodes of a recent‐onset patient with virally infected β cells and in clinical remission reacted with multiple islet autoantigens and displayed a mixed interferon (IFN)‐γ/interleukin (IL)‐10 cytokine pattern. Our data point to CXCL10 as an important cytokine in distressed islets that may contribute to inflammation leading to insulitis and β cell destruction, regardless of local viral infection. We demonstrate further pro‐ and anti‐inflammatory islet autoreactivity, indicating that different adaptive and innate immune responses may contribute to insulitis and β cell destruction.     

Serotonergic regulation of insulin secretion

The exact physiological role for the monoamine serotonin (5‐HT) in modulation of insulin secretion is yet to be fully understood. Although the presence of this monoamine in islets of Langerhans is well established, it is only with recent advances that the complex signalling network in islets involving 5‐HT is being unravelled. With more than fourteen different 5‐HT receptors expressed in human islets and receptor‐independent mechanisms in insulin‐producing β‐cells, our understanding of 5‐HT's regulation of insulin secretion is increasing. It is now widely accepted that failure of the pancreatic β‐cell to release sufficient amounts of insulin is the main cause of type 2 diabetes (T2D), an ongoing global epidemic. In this context, 5‐HT signalling may be of importance. In fact, 5‐HT may serve an essential role in regulating the release of insulin and glucagon, the two main hormones that control glucose and lipid homoeostasis. In this review, we will discuss past and current understanding of 5‐HT's role in the endocrine pancreas.     

胰岛干细胞研究进展

胰岛干细胞移植是治疗糖尿病的一条新途径，它可避免胰岛移植治疗糖尿病所面临的胰腺供体匮乏及长期使用免疫抑制剂而产生严重副作用的问题。本文将对有关胚胎干细胞及成体干细胞诱导分化为胰岛素分泌细胞，胰岛干细胞的分子标志，及胰岛干细胞的临床应用做一简要概述。     

Immunological perspective of self versus tumor antigens: insights from the RIP-gp model

Self-reactive T cells in the body are controlled by mechanisms of peripheral tolerance that limit their activation and induction of immune pathology. Our understanding of these mechanisms has been advanced by the use of tissue-specific promoters to express neo-self-antigens. Here, we present findings using the RIP-gp (rat insulin promoter-glycoprotein) transgenic mouse, which expresses the lymphocytic choriomeningitis virus glycoprotein (LCMV-gp) specifically in the pancreatic β islet cells. T cells responsive to this antigen remain ignorant of the LCMV-gp expressed by the islets, and breaking tolerance is dependent upon the maturation status of antigen-presenting cells, the avidity of the T-cell receptor ligation, and the level of major histocompatibility complex expression in the pancreas. Furthermore, decreased activity of Casitas B-lineage lymphoma b, a negative regulator of T-cell receptor signaling, can allow recognition and destruction of the pancreatic islets. This review discusses the roles of these factors in the context of anti-tissue responses, both in the setting of autoimmunity and in anti-tumor immunity.     

Localization of nucleic acid‐sensing toll‐like receptors in human and mouse pancreas

Nucleic acid‐sensing toll‐like receptors (TLRs) (3, 7, 8, 9) have a role both in antiviral innate immunity and in autoimmune disorders. We assessed the expression of TLR3, 7, 8 and 9 in human and mouse pancreas focusing on the subpopulations of cells in the Langerhans islets. We studied eight human samples with normal pancreatic islets and two samples from patients with type 1 diabetes. Additionally, 10 CD‐1 mouse pancreases were analysed. Immunohistochemical double‐stainings for the TLRs and insulin, glucagon or somatostatin, respectively, were performed along with appropriate controls. In human pancreas, strong immunoreaction of TLR7 and TLR8 was observed in the insulin‐positive beta cells, whereas glucagon‐ or somatostatin‐expressing cells of the islets were weakly stained or negative. In type 1 diabetes, the expression in islets was weak or lost (TLR7: p = 0.014, TLR8: p = 0.053), correlating with loss of beta cells. TLR3 and 9 were expressed only weakly with no correlation with specific cell types. In mouse pancreas, only TLR9 was detected. Intra‐pancreatic nerve ganglia strongly expressed TLR7. The strong expression of TLR7 and TLR8 in the beta cells of normal human islets could be an important piece in the puzzle of type 1 diabetes pathogenesis, and be linked with destruction of this particular subpopulation of the islet cells. In normal mice, only TLR9 can be constantly detected in the islets, highlighting differences between the species.     

链脲佐菌素小鼠糖尿病胰岛结构改变的定量病理学测试及分析

目的:定量揭示链脲佐菌素（Streptozotocin, STZ）诱发的小鼠糖尿病胰岛结构改变的特点,为分析胰岛功能改变补充定量病理学基础。方法:腹腔注射STZ,诱发Balb/c小鼠糖尿病;过量CO2处死小鼠并取胰腺,常规病理制样、胰岛素免疫组化染色、图像测试及定量分析。结果:造模第8周,糖尿病组（DM组）小鼠血糖显著高于正常对照组（NC组）小鼠,DM组小鼠血糖均值为19.3 mmol/L;相比NC组小鼠,DM组小鼠胰岛数目、β细胞数目及面积显著减少（P<0.05）,计算得出β细胞数量丧失约62.7％,胰岛β细胞面积丧失约27.0%,胰岛素阳性单位（PU值）表达减少约53.8％。DM组小鼠胰岛β细胞面积密度（AAβ,PI）、面数密度（NAβ,PI）、数量百分比（βPer）均显著降低（P<0.05）,其中PU值、AAβ,PI、βPer与血糖升高呈负相关,皮尔森相关系数（R值）分别为-0.653、-0.736和-0.899（显著性均<0.05）;建立造模时间（t）与胰岛β细胞数量百分比改变的函数:[βPer(%)=79.68-9.74t+0.38t2+0.06t3-4.66×10-3t4+2.86×10-5t5+1.68×10-5t6]。结论:通过定量病理学技术测量糖尿病小鼠模型胰岛和β细胞形态学改变的相关指标,能够帮助准确评估胰岛的损伤情况,并建立血糖变化和所测量数值的相关数学模型,用以预测血糖升高导致胰岛损伤的具体情况。 【关键词】糖尿病;胰岛;定量病理学;阳性单位;链脲佐菌素     

大鼠胰组织块的三维重建及形态学测量

目的:观察大鼠胰组织块内部各结构的空间位置关系,并描述胰岛的立体形态学特征.方法:经过长序列连续切片、H-E染色、切片图像数字化及配准等步骤,建立正常大鼠胰组织块的数字化图像数据集,对其内部的动脉、静脉、导管及胰岛进行分割及三维重建,并对重建结果进行测量及分析.结果:所得数字化图像数据集分辨率高,配准准确,再现了原组织的结构,重建结果可在三维空间内自由观察.根据重建结果测量多种形态学指标.胰腺泡及结缔组织占胰总体积的绝大部分,胰岛占1.125%.结论:胰内动脉、静脉分支自主干垂直发出后,互相伴行,先于胰表面,随后向小叶深部发出分支.胰导管走行独立,分支间存在吻合.某些部位动脉、静脉与导管并行,形成三联管结构.胰岛与导管系统关系密切.     

Selective lack of the C16:0 fatty acid isoform of sulfatide in pancreas of type II diabetic animal models

Sulfatide (3'-sulfogalactosyl-ceramide) is a glycosphingolipid mainly located in the nervous system, but has also been found in the islets of Langerhans. Previous studies have suggested that sulfatide is involved in insulin processing and secretion. In this study, sulfatide expression and metabolism in pancreas and isolated islets of the type II diabetes models, ob/ob- and db/db mouse, was investigated using TLC-ELISA, metabolic labelling and electron microscopy. As in non-diabetic Lewis rat and human pancreas, sulfatide was located in secretory granules of the beta cells. However, the type II diabetic animal models and their background strains had an altered sulfatide expression, involving the lack of the C16:0 sulfatide fatty acid isoform, compared to non-diabetic Lewis rat, BALB/c mouse and human pancreatic tissue, in which the two dominating pancreatic sulfatide isoforms C16:0 and C24:0 are expressed. Correspondingly, in isolated ob/ob islets, sulfatide synthesis excluded the production of C16:0 sulfatide. Insulin administration to ob/ob mouse, which lowers beta cell activity, resulted in significantly increased sulfatide expression in pancreas (p=0.0003), but still no expression of the C16:0 sulfatide isoform. In vitro, the C16:0 sulfatide was shown to be the isomer involved in the preservation of insulin crystals. Thus, it is hypothesized that the selection of sulfatide isomers in pancreas might be a genetic factor contributing to disease development in type II diabetic animal models.     

Analysis of pancreatic islet cells and hormone content in the spontaneously diabetic KKAy mouse by morphometry,immunocytochemistry and radioimmunoassay

Summary The splenic pancreas of 165 day old diabetic KKAy and age-matched nondiabetic C57BL/ 6 mice was examined by morphometry and immunocytochemistry at the light microscopic level and by radioimmunoassay to evaluate the morphology, surface area, endocrine cell composition and hormone content of the pancreatic islets. The insulin cells of the diabetic mice were severely degranulated and many of the glucagon, somatostatin and pancreatic polypeptide cells were displaced from the mantle to the core of the islet tissue where the non-insulin cells appeared to lose their continuity. The topography of some of the islets of KKAy mice was further deranged by acinar cells among the endocrine tissue. Morphometric analysis revealed that the surface area of the islets of KKAy mice was significantly expanded in comparison with that of C57BL/6 mice. The volume and numerical percents of the insulin cells were significantly increased whereas those of the glucagon and somatostatin cells were decreased in the KKAy mice. Since only the mean absolute number of insulin cells was elevated in the diabetic mice, the alteration in the relative proportions of the noninsulin cells and hypertrophy of the islets seemed to be a manifestation of insulin cell hyperplasia. Pancreatic insulin and somatostatin contents were markedly diminished in the islets of KKAy compared with those of C57BL/6 mice. These results demonstrate that the microscopic anatomy, endocrine cell populations and hormone content of the pancreatic islets are deranged in the KKAy mouse with severe hyperinsulinemia and hyperglycemia.     

Ontogenetic appearance of immunoreactive endocrine cells in rat pancreatic islets

Summary Chronological development of immunoreactive, pancreatic endocrine cells was immunohistochemically studied in rats. The first immunoreaction occurs for glucagon on day 11.5 and for insulin on day 12.5 of gestation, respectively, in the cells located within the cap-like or tubular pancreatic primordium derived from the gut wall. Immunoreactive somatostatin cells appear first at the periphery of primitive islets on day 15.5. On day 18.5, the cells of the primitive islets obtain their definitive arrangement and the islets are now separated from the tissue of the exocrine pancreas. Decapitation or encephalectomy performed on day 16.5 embryos fails to influence the ensuing further development of endocrine pancreas. This suggests that the hypothalamus or pituitary does not play an essential role in the histogenesis of the pancreatic islets.