Prognostic and predictive value of immunological parameters for chemoradioimmunotherapy in patients with pancreatic adenocarcinoma.

BACKGROUND: Chemoradioimmunotherapy of patients with pancreatic adenocarcinoma from the CapRI trial did not show any benefit of interferon-α in addition to a 5-fluorouracil (5FU)-based treatment. The aim of this study was to identify immunological parameters in patients from this trial to be used for predictive and/or prognostic purposes. METHODS: The following methods were used: tumour immunohistology, FACS analyses, cytokine measurement, as well as cytotoxicity and ELIspot. Immunological parameters were correlated with patients' survival using the Kaplan-Meier method. RESULTS: Irrespective of therapy type, high lymphocyte accumulation in tumours and frequencies of NK cells and effector (eff) CD8(+) T cells in peripheral blood of the patients were associated with patients' survival. Amount of CD3(+) and effector-memory CD8(+) blood lymphocytes, expression of CD152 and interleukin (IL)-2 serum level showed a predictive value for chemoradioimmunotherapy. Tumoural accumulation of CD3(+) and CD8(+) cells was predictive for outcome of chemotherapy alone. Besides, we identified the frequencies of CD3(+) lymphocytes, effCD8(+) T cells and NK cells in the peripheral blood of the patients, and IL-10 amount in serum, to be predictive values for 5FU-based chemotherapy. CONCLUSIONS: Immunological parameters, identified in this trial as possible markers, may be of interest in personalized medicine towards the improvement of the treatment and prognosis of pancreatic carcinoma patients.

Chronic alcohol intake increases the severity of pancreatitis induced by acute alcohol administration, hyperlipidemia and pancreatic duct obstruction in rats.

BACKGROUND: The mechanism of alcoholic pancreatitis is still unknown. It is of special interest why only about 5% of all alcoholics develop an episode of pancreatitis. We evaluated the role of long-term alcohol intake in the pathogenesis of alcoholic pancreatitis in rats. METHODS: To evaluate the effect of long-term alcohol intake, rats were fed either a Lieber-DeCarli control diet (CD) or a Lieber-DeCarli alcohol diet (AD) for 6 weeks. Then, rats were infused over 2 h with either Ringer's solution (CO) or ethanol (E). In additional animals, alcoholic pancreatitis was induced by ethanol combined with hyperlipidemia and temporary pancreatic duct obstruction (EFO). Controls received Ringer's solution combined with hyperlipidemia and temporary pancreatic duct obstruction (RFO). Intravital microscopy (pancreatic perfusion and leukocyte adhesion), alcohol concentrations, amylase, lipase, cholesterine and triglyceride levels in plasma, myeloperoxidase activity and histology were evaluated at different time intervals. RESULTS: In those animals which received the Lieber-DeCarli control diet, capillary perfusion was reduced in the E group and further reduced in the EFO group as compared to the controls (CO, RFO; p < 0.01). Leukocyte adhesion was significantly increased in rats receiving E (p < 0.01), and was further increased in the combination group EFO (p < 0.01). EFO induced histologically evident acute pancreatitis. The additional administration of a long-term alcohol diet further increased microcirculatory disturbances and pancreatic injury significantly (EFO-AD > EFO-CD). CONCLUSIONS: This study shows that alcoholic pancreatitis is induced by the combination of ethanol and individual cofactors. Chronic alcohol abuse intensifies these changes. Therefore, long-term alcohol intake seems to be a major factor in the pathogenesis of alcoholic pancreatitis.

Intracapillary leucocyte accumulation as a novel antihaemorrhagic mechanism in acute pancreatitis in mice.

BACKGROUND: Pancreatic infiltration by leucocytes represents a hallmark in acute pancreatitis. Although leucocytes play an active role in the pathophysiology of this disease, the relation between leucocyte activation, microvascular injury and haemorrhage has not been adequately addressed. METHODS: We investigated intrapancreatic leucocyte migration, leucocyte extravasation and pancreatic microperfusion in different models of oedematous and necrotising acute pancreatitis in lys-EGFP-ki mice using fluorescent imaging and time-lapse intravital microscopy. RESULTS: In contrast to the current paradigm of leucocyte recruitment, the initial event of leucocyte activation in acute pancreatitis was represented through a dose- and time-dependent occlusion of pancreatic capillaries by intraluminally migrating leucocytes. Intracapillary leucocyte accumulation (ILA) resulted in dense filling of almost all capillaries close to the area of inflammation and preceded transvenular leucocyte extravasation. ILA was also initiated by isolated exposure of the pancreas to interleukin 8 or fMLP, demonstrating the causal role of chemotactic stimuli in the induction of ILA. The onset of intracapillary leucocyte accumulation was strongly inhibited in LFA-1(-/-) and ICAM-1(-/-) mice, but not in Mac-1(-/-) mice. Moreover, prevention of intracapillary leucocyte accumulation led to the development of massive capillary haemorrhages and transformed mild pancreatitis into lethal haemorrhagic disease. CONCLUSIONS: ILA represents a novel protective and potentially lifesaving mechanism of haemostasis in acute pancreatitis. This process depends on expression of LFA-1 and ICAM-1 and precedes the classical steps of the leucocyte recruitment cascade.

Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

BACKGROUND: Near-infrared (NIR) imaging of liver segments provides substantial information for surgeons performing liver resection. It was hypothesized that ramucirumab, an endothelium-specific antibody approved by the Food and Drug Administration, could be used for liver segment imaging using the endothelium capture principle. METHODS: The capture efficacy of anti-vascular endothelial growth factor receptor (VEGFR) 2 monoclonal antibodies (mAbs) and segment imaging were studied in a mouse model. Binding of ramucirumab in human and porcine tissues was studied using immunofluorescence staining. Isolated porcine liver perfusion was used to analyse the labelling and NIR imaging of selected liver segments. RESULTS: VEGFR2 is well expressed on the endothelium of the smallest microvascular blood vessels in mouse, porcine and human liver tissues, as well as in human liver tumours. Perfusion of selected segments in the isolated liver model showed high capture of the anti-VEGFR2 (clone 522302) mAb and ramucirumab in mice and pigs respectively. NIR imaging of selected segments was achieved using isolated porcine liver perfusion with IRDye® 800CW-conjugated ramucirumab. CONCLUSION: VEGFR2 is well expressed on the smallest microvascular blood vessels and can capture antibodies during single intravascular passages with high efficacy. The ex vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this antibody for intraoperative navigation in liver surgery. Surgical relevance Imaging of liver segments provides substantial information for surgeons when performing liver resection. The antivascular endothelial growth factor receptor (VEGFR) 2 antibody ramucirumab conjugated with near-infrared dye could visualize selected liver segments using an endothelial capture-based approach in an isolated perfusion liver model. The ex vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this anti-VEGFR2 antibody for intraoperative navigation in liver surgery.

ANTECEDENTES: La obtención de imágenes quasi infrarrojas (near-infrared, NIR) de los segmentos hepáticos proporciona información importante a los cirujanos que realizan resecciones hepáticas. Se estableció la hipótesis de que el ramucirumab, un anticuerpo específico para el endotelio, aprobado por la FDA, podría ser útil para obtener imágenes de los segmentos hepáticos utilizando el principio de captura del endotelio. MÉTODOS: Se estudió la eficacia en la captura de anticuerpos monoclonales (monoclonal antibodies, mABs) contra el receptor del factor de crecimiento endotelial vascular 2 (anti-VEGFR2) y de su capacidad para obtener imágenes de segmentos hepáticos en un modelo de ratón. Se estudió la incorporación del ramucirumab en tejidos humanos y porcinos mediante tinción por inmunofluorescencia. Para analizar la expresión y las imágenes NIR de los segmentos hepáticos, se utilizó un sistema de perfusión hepática aislada en cerdos. RESULTADOS: El VEGFR2 se expresa bien en el endotelio de los territorios microvasculares de calibre más pequeño en el hígado de ratón y de cerdo, así como en tejidos hepáticos y tumores humanos. En el modelo de hígado aislado, la perfusión segmentaria mostró una elevada captura del mAb anti-VEGFR2 (clon 522302) y del ramucirumab en ratones y cerdos, respectivamente. La captura endotelial del ramucirumab conjugado con IRDye 800CW permitió obtener imágenes selectivas de los segmento usando NIR en hígado porcino aislado. CONCLUSIÓN: El VEGFR2 se expresa bien en los territorios microvasculares más pequeños y puede captar anticuerpos durante el paso intravascular de una dosis con alta eficacia. La imagen ex vivo de un determinado segmento usando endocapt de ramucirumab demuestra el potencial de este anticuerpo para la navegación intraoperatoria en cirugía hepática.

Transfection with CD40L induces tumour suppression by dendritic cell activation in an orthotopic mouse model of pancreatic adenocarcinoma.

OBJECTIVE: Patients with adenocarcinoma of the pancreas have only limited promising therapy options. Therefore, immunotherapeutic approaches might be considered promising and have gained importance over the last few years. In this study, CD40L gene transfer was tested as potent immunotherapy. METHODS: The efficacy of CD40L gene transfer in initiating anti-tumour immune response was investigated in a pancreatic ductal adenocarcinoma orthotopic syngeneic mouse model. In addition, the role of dendritic cells was determined. RESULTS: A significantly slower tumour growth rate and less metastasis were observed following administration of the CD40L plasmid. Such an effect of the plasmid was not observed in immunodeficient mice. Tumours of treated mice were found to be infiltrated with T cells and dendritic cells. The latter were mature and of myeloid origin. Tumour-infiltrating lymphocytes were tumour-specific as shown in IFN-gamma ELISPot assays. Using intravital microscopy it was possible to show a significant induction of leukocytes sticking to the tumour endothelium after CD40L treatment. Adoptive cell transfer experiments have revealed that tumour-derived dendritic cells and CD8 cells from CD40L-treated donor mice either harbour anti-tumour activity or induce it in the recipients. Distinctly, CD8 cells from donor spleens were found to migrate directly into the recipient's tumour. CONCLUSIONS: The induction of anti-tumour activity initiated after treating mice with the CD40L plasmid was achieved. Further investigations showed that this is mediated by mature myeloid dendritic cells which activate CD8 cells. Clinical trials investigating CD40L-based therapies should be extended.

Taurine protects from liver injury after warm ischemia in rats: the role of kupffer cells.

BACKGROUND/AIMS: Warm ischemia to liver with subsequent Kupffer cell-dependent pathology is associated with many clinical conditions. Taurine prevents Kupffer cell activation and improves graft survival after experimental cold ischemia and liver transplantation. Thus this study was designed to assess its effects after warm hepatic ischemia. METHODS: The left liver lobe of female Sprague-Dawley rats (170-210 g) underwent 60 min of warm ischemia. Animals were given either intravenous taurine or Ringer's solution 10 min prior to warm ischemia. Transaminases, histology, in vivo microscopy, intercellular adhesion molecules-1 (ICAM-1) expression, TNF-alpha and tissue hydroperoxide were compared between groups using analysis of variance (ANOVA) or ANOVA on ranks as appropriate. RESULTS: Taurine significantly decreased transaminases and improved histologic outcome. Phagocytosis of latex beads, serum TNF-alpha levels and tissue hydroperoxide concentrations were also significantly reduced. Stickers in sinusoids and post-sinusoidal venules significantly decreased. In parallel, both leukocyte infiltration and ICAM-1 expression decreased (p < 0.05), while flow velocity of red blood cells as well as sinusoidal perfusion rate were improved (p < 0.05). CONCLUSION: This study demonstrates that taurine blunts Kupffer cell-dependent hepatic pathology after warm ischemia in vivo via mechanisms including leukocyte-endothelial interaction, microcirculation disturbances and protection against lipid peroxidation.

Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas.

Transferrin receptor (TFRC) is a membrane-bound protein expressed in larger amounts in proliferating, e.g., malignant, cells than in quiescent cells. The specific expression of TFRC can represent a diagnostic tool or a therapeutic target in solid tumours expressing this antigen. Whether TFRC is expressed in human pancreatic tumours is unknown. The aim of this study was the investigation of the expression of TFRC and transferrin in human pancreatic cancer and in neuroendocrine tumours of the pancreas. Fifty one specimens of human pancreatic cancer and 14 samples of pancreatic neuroendocrine tumours were obtained after surgery. The expression of TFRC, transferrin and cytokeratin was studied by standard immunohistochemistry. Flow cytometry was used for the investigation of TFRC expression in nine cell lines of ductal pancreatic cancer in vitro. In contrast to normal tissue, 93% of pancreatic tumour cells showed positive (82%) or heterogeneous (11%) expression of TFRC. It was strongly expressed by malignant epithelial cells; normal stromal and endothelial cells were not stained by anti-TFRC antibodies. Primary tumours and metastases showed a similar frequency of TFRC expression. Three neuroendocrine carcinomas showed positive expression of TFRC by malignant tumour cells. The expression of TFRC was negative in benign neuroendocrine tumours of the pancreas. The cell lines of pancreatic cancer were characterised by a low expression of TFRC in vitro. In contrast to normal pancreatic tissue and benign neuroendocrine tumours of the pancreas, pancreatic cancer and neuroendocrine carcinoma are therefore characterised frequently by high expression of TFRC. Hence, TFRC represents a marker of malignant transformation in the pancreas that could be applied as potential diagnostic and therapeutic target.

Effect of different immunosuppressive agents on acute pancreatitis: a comparative study in an improved animal model.

BACKGROUND: Immunosuppressive drugs have been associated with the development and progression of acute pancreatitis after organ transplantation. Consequently, a reduction or a change in immunosuppressive therapy has been recommended once posttransplantation pancreatitis has been suspected. However, it is not known which of the available immunosuppressive agents is most harmful to the pancreas and which may be used safely in this situation. The objective of this study was to investigate the effect of different immunosuppressive drugs in various dosages on intrapancreatic protease activation, acinar cell necrosis, and mortality in an improved model of acute necrotizing pancreatitis in the rat. The rat model of acute necrotizing pancreatitis, like posttransplantation pancreatitis, is characterized by ischemia and microcirculatory disorders. METHOD: Acute pancreatitis was induced in rats by using a combination of low-dose controlled intraductal glycodeoxycholic acid superimposed on intravenous cerulein hyperstimulation. Six hours thereafter, animals were randomized to intravenous therapy with 2, 10, or 50 mg/kg/day prednisolone (PRED); 3, 15, or 60 mg/kg/day cyclosporine A (CsA); 10 mg/kg/day azathioprine (AZA); 0.6 mg/kg/day orthoclone OKT3 (OKT3); or saline. After 36 hr, surviving animals were killed to determine acinar cell necrosis and trypsinogen activation peptides levels (TAP) in blood and ascites. RESULTS: Compared with saline-treated control rats, animals treated with 60 mg/kg/day CsA developed significantly more acinar cell necrosis and had increased amounts of TAP in ascites. Likewise, there was more extensive acinar cell necrosis in animals subjected to AZA therapy. However, this was not associated with incremental TAP. Animals treated with 3 or 15 mg/kg/day CsA, OKT3, or PRED showed no significant changes in these target parameters. Animals given 10 or 50 mg/kg/day PRED even had decreased hematocrit values and produced significantly less ascites than animals in the other groups. CONCLUSION: The present results suggest that AZA and high doses of CsA aggravate acute pancreatitis and should, therefore, be avoided once posttransplantation pancreatitis has been suspected, whereas lower doses of CsA, OKT3, and PRED may be used safely. PRED can even be used at higher doses as may be required when graft rejection is suspected.

Cigarette smoke enhances ethanol-induced pancreatic injury.

Alcohol induces pancreatic ischemia, but the mechanisms promoting pancreatic inflammation are unclear. We investigated whether cigarette smoke inhalation is a cofactor in the development of ethanol-induced pancreatic injury. Cigarette smoke was administered to anesthetized rats alone or in combination with intravenous ethanol infusion. Control animals received either saline or ethanol alone. Pancreatic capillary blood flow and leukocyte-endothelium interaction in postcapillary venules were evaluated by intravital microscopy. Leukocyte sequestration was assessed by measurement of myeloperoxidase activity in pancreatic tissue, and pancreatic injury evaluated by histology. Ethanol decreased pancreatic blood flow progressively over 90 minutes (p < 0.001 vs. baseline), but neither leukocyte-endothelium interaction nor leukocyte sequestration was altered. Cigarette smoke alone reduced pancreatic blood flow temporarily (p < 0.01 vs. baseline) and increased leukocyte-endothelium interaction (roller p < 0.001, sticker p < 0.01 vs. baseline). Cigarette smoke potentiated the impairment of pancreatic capillary perfusion caused by ethanol, and both the number of rolling leukocytes and myeloperoxidase activity levels were increased compared to ethanol or nicotine administration alone (p < or = 0.05 and p < or = 0.01, respectively). This study demonstrates that ethanol induces pancreatic ischemia and that cigarette smoke leads to both temporary pancreatic ischemia and minimal leukocyte sequestration. Cigarette smoke potentiates the amount of pancreatic injury generated by ethanol alone. Smoking therefore seems to be a contributing factor in the development of alcohol-induced pancreatitis in the rat model.

Characterization and reduction of ischemia/reperfusion injury after experimental pancreas transplantation.

Reperfusion injury after pancreas transplantation is a cause of early graft pancreatitis. The aim of this study was to quantify pancreatic microcirculation after pancreas transplantation in correlation with cold ischemia time. In a second step the effect of N-acetylcysteine on reperfusion damage was tested. Pancreas transplantation was performed in three different groups of male Lewis rats. Groups 1 and 2 received no special treatment. Cold ischemia time was 1.5 hours in group 1 and 16 hours in groups 2 and 3. In group 3 donor and recipient were both treated with N-acetylcysteine (300 mg/kg) 1.5 hours after reperfusion graft microcirculation was quantified by means of intravital microscopy. Rhodamine-labeled leukocytes, fluoroscein isothiocyanate-labeled erythrocytes, and fluoroscein isothiocyanate-albumin were used as fluorochromes. After a cold ischemia time of 16 hours, functional capillary density, erythrocyte velocity, and leukocyte-endothelium interaction were reduced significantly compared to a cold ischemia time of 1.5 hours (P<0.05). After 16 hours of cold ischemia, treatment with N-acetylcysteine improved all of these parameters (P

Isovolemic hemodilution with dextran prevents contrast medium induced impairment of pancreatic microcirculation in necrotizing pancreatitis of the rat.

BACKGROUND: Previous studies demonstrated that intravenous contrast medium (CM), as used in contrast enhanced computed tomography, aggravates the impairment of pancreatic microcirculation (PM) characteristic of severe pancreatitis and increases necrosis and mortality in necrotizing pancreatitis (NP) in rats. This study evaluates the use of isovolemic hemodilution, which can enhance the microcirculation in severe pancreatitis, for preventing CM-induced injury. METHODS: NP was induced in 30 dextran-tolerant Wistar rats by intraductal glycodeoxycholic acid and intravenous cerulein for 6 hours. PM was quantified by intravital microscopy using fluorescein isothiocyanate labeled erythrocytes. Based on previous results, areas with low blood flow (< 1.6 nL/min/cap) were identified and baseline recordings of capillary blood flow taken. A reduction of hematocrit to 75% of baseline was achieved by replacement of 5 mL/kg of blood with 25 mL/kg Ringer's lactate (RL) or by exchange of 8 mL/kg of blood for the same amount of dextran 70.6%. Thereafter, the nonionic CM iopamidol (Solutrast, Byk Gulden, Konstanz, Germany) was injected during 1 minute and PM measurements repeated after 30 and 60 minutes. RESULTS: Despite hemodilution with RL, pancreatic capillary perfusion was significantly decreased to 87% of baseline (0.83 +/- 0.04 mL/min/cap; n = 216) 60 minutes after CM infusion (P < 0.05). In contrast, capillary blood flow was significantly increased to 161% (1.56 +/- 0.05 nL/min/cap; n = 278) in the group treated with dextran. Moreover, the percentage of capillaries developing complete stasis was significantly lower in the dextran group (2.3 +/- 1.2%) compared to animals diluted with RL (22.3 +/- 4.8%) (P < 0.002). CONCLUSION: Isovolemic hemodilution with dextran prevents the additional impairment of pancreatic microcirculation induced by CM in NP.

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer.

Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mum h(-1) was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.

Effect of Flt3 ligand gene transfer in experimental pancreatic cancer.

BACKGROUND: Fms-like tyrosine kinase 3 receptor (Flt3) is an important receptor expressed on the cell membrane of immature antigen-presenting cells. The binding of Flt3 to its ligand (FL) activates the proliferation of dendritic cells (DCs). This mechanism is currently being evaluated in the therapy of malignant tumors. The aim of the present study was to study the effect of FL gene transfer on the immune response and tumor growth in experimental pancreatic cancer. MATERIALS AND METHODS: The rat FL was sequenced and cloned from total mRNA extract of the spleen. Transfection efficiency of subcutaneously growing rat duct-like pancreatic cancer (DSL6A) with DOTAP-/cholesterol-based liposomes was tested using a pcDNA3.1-lacZ construct. Flt3 ligand production of in vitro transfected tumor cells and in vivo transfected tumors was measured by enzyme-linked immunosorbent assay. Tumor induction was achieved in Lewis rats by a subcutaneous inoculation of syngeneic pancreatic tumor cells (DSL6A). The animals were allocated into three groups: control, mock treatment, and treatment with FL plasmid. The plasmid was injected intratumorally three times per week for 2 weeks. The total observation time was 6 weeks. RESULTS: The tumor volume was significantly lower in the FL-transfected group during the first 3 weeks. The number of responders was significantly higher in the FL group compared with control and mock treatment. The number of CD80+ DCs in the spleen was significantly higher after FL gene transfer. The responders showed a significantly higher number of splenic natural killer (NK) cells. There were no differences of infiltrating lymphocytes, proliferation, and tumor blood vessels between the groups. CONCLUSION: Intratumoral gene transfer of FL in rats activated proliferation of DCs and NK cells, which causes a moderate reduction of tumor growth. This improvement of local tumor control during the first weeks could be explained by an improved antigen presentation.

Angiogenesis inhibition with TNP-470, 2-methoxyestradiol, and paclitaxel in experimental pancreatic carcinoma.

INTRODUCTION: Inhibition of tumor angiogenesis is a novel therapeutic modality for various malignancies. AIM: To investigate the effect of different antiangiogenic agents (TNP-470, 2-methoxyestradiol, and paclitaxel) on growth and neovascularization of experimental pancreatic cancer. METHODOLOGY: In 25 male Lewis rats, tumor induction was achieved by orthotopic and subcutaneous tumor fragment implantation of ductlike pancreatic cancer DSL6A. Four weeks after tumor implantation, the animals were randomly treated with TNP-470, 2-methoxyestradiol, or paclitaxel. After 2 weeks of antiangiogenic therapy, total tumor volume, vital tumor surface, vascular density, and apoptosis were measured. RESULTS: Total tumor volume and vital tumor surface were not significantly different in any of the treatment groups. Similarly, vascular density and apoptosis were not altered by treatment with the various angiogenesis inhibitors at the specific doses used. CONCLUSION: We conclude that in contrast to many earlier studies, angiogenesis inhibition by a single-drug application and by the doses used in the present model did not reveal a favorable therapeutic effect on pancreatic cancer DSL6A. The combination of different angiogenesis inhibitors or higher doses might be more effective.

Expression of HLA class I/II antigens and T cell immune response in human neuroendocrine tumors of the pancreas.

Peptide presentation by HLA class I and II antigens regulates specific antigen recognition by T cells. The present study aimed to investigate T cell infiltration and its relation to HLA antigen expression in pancreatic neuroendocrine tumors. Fresh tissue samples were collected from five insulinomas and six other neuroendocrine tumors (one gastrinoma, one glucagonoma, two carcinoid, and two neuroendocrine carcinomas). Normal pancreatic and splenic tissue samples were used as controls. Investigation of infiltrating lymphocyte populations, as well as staining of HLA class I and II antigens, were performed by standard immunohistochemistry. The majority of investigated tumors demonstrated an intratumoral infiltration by CD3+, CD4+ and CD8+ T cells that was significantly higher than in normal pancreatic islets. Only a minority of tumor-infiltrating T cells showed the CD45RO+ phenotype. The expression of HLA class I antigen was altered in 10 of 11 tumors. A loss of beta-2microglobulin represented the most frequent type of alteration to HLA class I expression, although the total loss of HLA class I was found in only one case of neuroendocrine carcinoma. HLA class II molecules were expressed by endothelial and lymphoid cells and not by tumor cells. In conclusion most neuroendocrine pancreatic tumors induce a T cell mediated immune response resulting in an intratumoral infiltration with CD3+, CD4+ and CD8+ T cells. Loss of beta-2microglobulin is a frequent alteration in these tumors, which may influence the normal function of the HLA class I antigen complex. In contrast to malignant tumors of the exocrine pancreas, expression of HLA class II was absent in neuendocrine pancreatic tumor cells.

[Lack of stimulation of CD11b/CD18 induced leukocyte adhesioin by platelet activating factor (PAF) and f-MLP in malignant tumor endothelium in experimental pancreas cancer of the rat]. / Fehlende Stimulierbarkeit der CD11b/CD18-vermittelten Leukozytenadhäsion durch Platelet-Activating-Faktor (PAF) und f-MLP im malignen Tumorendothel beim experimentellen Pankreaskarzinom der Ratte.

UNLABELLED: The interaction between immunocompetent cells and tumor-endothelium is essential for effective immunologic recognition. In the present study we evaluated resting and CD11b/CD18-mediated leukocyte adhesion on tumor-endothelium of experimental pancreatic carcinoma and in healthy pancreatic venules. METHODS: 22 male Lewis rats (120-140 g) were anesthetized. Duct-like pancreatic carcinoma (DSL6A, Am. J. Pathol. 1993; 143:292) was induced by intrapancreatic implantation of tumor fragments between inert polymethylmetacrylat plates. After 4 wks the tumor-bearing pancreas was exposed and the microcirculation studied. Parameters in tumor vessels (15-40 microns) and healthy pancreatic collecting venules (20-40 microns) included: Erythrocyte velocity, Leukocyte adhesion, Vessel diameter and wall shear rate. Measurements were obtained before and 5 min after adding f-MLP (100 mM) or PAF (50 mM), two CD11b/CD18 agonists of different potency to the immersion chamber. RESULTS: [table: see text] CONCLUSION: In experimental pancreatic carcinoma leukocyte adhesion of low affinity is reduced despite comparable wall shear rates. The CD11b/Cd18-mediated adhesion of high affinity, which is inducible by f-MLP and PAF in healthy pancreatic venules, is absent in tumor vessels. This may be a mechanism by which malignant tumors escape immune control.

Trypsinogen activation peptides (TAP) in peritoneal fluid as predictors of late histopathologic injury in necrotizing pancreatitis of the rat.

The levels of trypsinogen activation peptides (TAP) were quantified by ELISA immunoassay in acute pancreatitis of the rat and compared to the degree of late histopathological sequelae and exocrine functional impairment 4 and 12 weeks after the acute phase of the disease. For this purpose acute pancreatitis of different severity was induced using a suitable rat model recently described. Forty five surviving animals were studied. The level of TAP in peritoneal exudate measured 3 and 6 hr after pancreatitis induction correlated well with the amount of the late histopathological injury at the end of the corresponding observation period (at 4 weeks after 3 hr: r = 0.75, P = 0.003, after 6 hr: r = 0.72, P = 0.005, Pearson; and at 12 weeks after 3 hr: r = 0.86, P = 0.0001, after 6 hr: r = 0.84, P = 0.0001, Pearson). A negative correlation of TAP with the impairment of exocrine function was found only at 4 weeks for the secretion of total protein (r = -0.76 after 3 hr; r = -0.62 after 6 hr) and for exocrine function (r = -0.67 after 3 hr, r = -0.57 after 6 hr), but not at 12 weeks after acute pancreatitis. No correlation with plasma amylase and lipase was found. We conclude that quantitation of TAP in ascites provides an accurate prediction of late histopathologic sequelae. Pancreatic exocrine function could be predicted by TAP assay only in the early stage after pancreatitis induction (eg, four weeks). In later stages of the disease (eg, 12 weeks) remaining pancreatic tissue seems to compensate for any exocrine deficits that have occurred.

Influence of gastrointestinal hormones on tumor microcirculation of experimental pancreatic cancer in the rat.

BACKGROUND/AIMS: Gastrointestinal hormones influence the microcirculation in the normal pancreas. In the present study, we studied the effect of cerulein and somatostatin on pancreatic cancer microcirculation after orthotopic and nonorthotopic tumor implantation. METHODS: In 36 male Lewis rats (150-180 g) induction of a ductlike pancreatic cancer was achieved by intrapancreatic or intraperitoneal tumor fragment interposition between two inert transparent polymethyl methacrylate plates. After 4 weeks, intravital microscopy of the tumor microcirculation was performed in a temperature-controlled immersion chamber. The animals received 5 microg/kg cerulein or 3 mg/kg somatostatin for 1 h intravenously. The erythrocyte velocity in normal pancreatic capillaries or in tumor vessels was measured. RESULTS: The erythrocyte velocity in the capillaries of the normal pancreas was 1.01 +/- 0.11 mm/s at baseline and increased to 1.64 +/- 0.09 mm/s after cerulein stimulation (p = 0.007). Pancreatic cancer vessels demonstrated no increase in erythrocyte velocity after orthotopic (baseline 0.95 +/- 0.14 mm/s, after 1 h 0.86 +/- 0.13 mm/s; n.s.) and nonorthotopic tumor implantation (baseline 0.91 +/- 0.12 mm/s, after 1 h 0.95 +/- 0.14 mm/s; n.s.) after cerulein stimulation. Somatostatin decreased the erythrocyte velocity both in normal pancreas (baseline 0.87 +/- 0.02 mm/s, after 1 h 0.60 +/- 0.07 mm/s; p = 0.01) and in pancreatic cancer (baseline 0.85 +/- 0.20 mm/s, after 1 h 0.63 +/- 0.18 mm/s; p = 0.02) after orthotopic tumor implantation. There was no effect of somatostatin after nonorthotopic tumor implantation (baseline 0.90 +/- 0.10 mm/s, after 1 h 0.88 +/- 0.14 mm/s; n.s.). CONCLUSION: These data suggest that pancreatic cancer microcirculation lacks physiological blood flow control by stimulatory hormones, in contrast to the normal pancreas.

In vivo assessment of angioarchitecture and microcirculation in experimental liver cancer: a new model in rats.

The rising incidence of unresectable hepatocellular malignancies remains a therapeutic challenge. Little is known about the mechanisms of angiogenesis and immunological aspects of liver tumor vessels. The aim of this study was to investigate hepatic and tumor microcirculation and leukocyte-endothelium interaction by means of intravital microscopy in a rat model of hepatocellular carcinoma. Off-line analysis showed that the angioarchitecture as well as blood flow velocity in liver cancer is heterogeneous. The leukocyte-endothelium interaction is significantly reduced compared to normal liver tissue. The data suggest that the main mechanism is a reduced expression of adhesion molecules demonstrating an effective immune escape mechanism of this tumor. The model represents a useful experimental tool to explore angiogenesis inhibition or immunological therapeutic strategies in experimental liver cancer.

Measurement of blood flow in the main arteriole of the villi in rat small intestine with FITC-labeled erythrocytes.

Changes of blood flow in the intestine occur under various pathological conditions. The mucosa of the intestine is especially sensitive to tissue damage resulting in swelling, loss of tissue integrity, and ulceration. Changes of blood supply to the mucosa may contribute to local tissue damage. Therefore, the quantification of the perfusion of the intestinal mucosa in an animal model may help to elucidate the involved pathophysiological mechanisms. In our study, autologous erythrocytes were labeled with fluorescein-isothiocyanate and used for the evaluation of erythrocyte velocity in the main arteriole of the villi in the distal part of the ileum using intravital microscopy. In addition, the arteriolar diameter was determined, and the arteriolar blood flow was calculated. Under stable cardiovascular and respiratory conditions, blood flow ranged between 6.6 +/- 0.3 and 5.9 +/- 0.3 nl/min (means +/- SEM) during the observation period of 120 min. Our results suggest that this approach is a feasible method to quantify blood flow in the main arteriole of the villi and is therefore a suitable method for further investigating changes of mucosal blood flow in acute and chronic states of bowel disease.