Horst Nizze

Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma

Pancreatic cancer can seldom be resected, and chemotherapy has only a limited effect on survival or tumour load. We did a phase I/II trial in 14 patients with pancreatic cancer to assess the safety of local activation of low-dose ifosfamide. We encapsulated genetically modified allogeneic cells, which expressed a cytochrome P450 enzyme, in cellulose sulphate and delivered them by supraselective angiography to the tumour vasculature. These cells locally activated systemically administered ifosfamide. The tumours of four patients regressed after treatment, and those of the other ten individuals who completed the study remained stable. Median survival was doubled in the treatment group by comparison with historic controls, and 1-year survival rate was three times better. Further studies of this cell-therapy-based treatment combined with chemotherapy for inoperable pancreatic cancer are warranted.

Tumour budding as prognostic factor in stage I/II colorectal carcinoma

Aims : The term tumour ‘budding’ has been coined for the detachment of tumour cells from the neoplastic glands of adenocarcinomas and is presumed to be an early step in the metastatic process. A limited number of studies have shown budding to be an adverse prognostic factor.

Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride

BACKGROUND & AIMS Regulatory mechanisms in chronic pancreatitis finally resulting in pancreatic fibrosis cannot be studied sufficiently in human pancreas. Results of a new pancreatitis model in rats suitable for investigation of the processes leading to pancreatic fibrosis are presented. METHODS Experimental pancreatitis was induced by intravenous application of 8 mg/kg body wt dibutyltin dichloride. Pancreatitis was characterized by histology, serum parameters, and immunohistochemistry, detecting inflammatory cells. Gene expression of collagen type I and transforming growth factor beta1 was shown by Northern blot analysis. RESULTS Dibutyltin dichloride induced an acute edematous pancreatitis within 24 hours. Extensive infiltration with mononuclear cells could be observed after day 7 followed by the development of fibrosis. Parallel to the cell infiltration, an upregulation of messenger RNA-encoding collagen type I and transforming growth factor beta1 could be shown. An active inflammatory process could be shown until the end of the observation period, i.e., 2 months. CONCLUSIONS The findings suggest that dibutyltin dichloride-induced pancreatitis in rats is suitable to study cellular interactions and mediators involved in the development of pancreatic fibrosis.

Targeted chemotherapy by intratumour injection of encapsulated cells engineered to produce CYP2B1, an ifosfamide activating cytochrome P450.

The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

Activation of the AKT/mTOR pathway in autosomal recessive polycystic kidney disease (ARPKD)

BACKGROUND Autosomal recessive polycystic kidney disease (ARPKD) [MIM 263200] belongs to a group of congenital hepatorenal fibrocystic syndromes and is caused by mutations in the PKHD1 gene encoding the multidomain protein fibrocystin/polyductin (FPC). The serine-threonine kinase mammalian target of rapamycin (mTOR) is one of the most important gate-keepers integrating numerous signals related to cell proliferation and growth. Whereas the direct activation of mTOR has been shown recently in autosomal-dominant PKD, no data are available on the role of mTOR signalling in proliferation and progression of ARPKD. METHODS Formalin-fixed and paraffin-embedded kidney specimens obtained during nephrectomy from children with ARPKD (n = 12) were used for immunohistochemical investigation of FPC expression (monoclonal antibody (mAb) 18, mAb 5a), proliferative activity (Ki-67) and activation of the mTOR pathway. Kidney specimens from children (n = 4) who died from causes not associated with kidney disease served as controls. For the detection of AKT, mTOR and S6K antibodies specifically recognizing the activated (phosphorylated) isoforms of these proteins were used. In all patients mutation analysis of the PKHD1 gene was performed. RESULTS In 10 out of 12 patients, we could confirm the diagnosis by the identification of PKHD1 mutations. The tubular cyst epithelium of all kidney specimens stained strongly positive with the FPC-specific monoclonal antibody (mAb) 18 but only very faint signals were obtained with mAb 5a. In contrast, healthy kidneys showed rather weak signals with both FPC-specific mAbs, indicating dysregulated expression of FPC in our patients. Phosphorylated AKT as well as activated mTOR and its down-stream effector S6K were strongly expressed in cystic epithelia of all kidney specimens but not in control tissues. No association between the activation of this pathway and the proliferative activity (Ki-67 expression) was observed. CONCLUSIONS Our results point to a central role of AKT/mTOR signalling in ARPKD and justify further investigations to evaluate the therapeutic potential of mTOR inhibitors in ARPKD patients.

Acute interstitial pancreatitis in rats induced by dibutyltin dichloride (DBTC): pathogenesis and natural course of lesions.

Dibutyltin dichloride (DBTC; 6 mg/kg body weight, i.v.) induced acute interstitial pancreatitis in rats. The course of the pancreatitis was examined within 28 days by light and electron microscopy as well as by pathobiochemistry (amylase, lipase, alkaline phosphatase, and bilirubin in serum; tin concentration in biliopancreatic juice, tissue, and concretions). The pathogenesis of the DBTC-induced pancreatitis in rats was studied by different experimental designs (in intact animals, after bile duct ligation, after surgical bypass of the bile duct). DBTC caused toxic necrosis of the biliopancreatic duct epithelium, which is then shed into the duct and forms obstructing plugs in the distal common bile duct. Interstitial pancreatitis occurred during the first 4 days, accompanied by significantly increased activities of serum α-amylase and lipase. After 7 days extensive infiltration of the pancreatic interstitium with mononuclear cells was observed. Twenty-eight days after administration of DBTC one-third of the rats showed periductal and interstitial fibrosis as well as an active inflammatory process in the pancreas. The findings suggest a twofold pathogenesis of the DBTC-induced pancreatitis: first, the cytotoxic effects on the biliopancreatic duct epithelium lead to epithelial necrosis with obstruction of the duct, subsequent cholestasis, and interstitial pancreatitis; and second, the hematogenic DBTC effects cause direct injury of pancreatic cells (mitochondrial damage, autophagy, cell necrosis) followed by interstitial edema and inflammation. Both processes lead to this special type of DBTC-induced acute pancreatitis with a tendency to a chronic course, when the obstruction of the duct and cholestasis persist.

Diagnostic value of urinary alanine aminopeptidase and N-acetyl-β-d-glucosaminidase in comparison to α1-microglobulin as a marker in evaluating tubular dysfunction in glomerulonephritis patients

To estimate the diagnostic value of tubular parameters, the urinary alanine aminopeptidase (AAP), N-acetyl-beta-D-glucosaminidase (NAG) and the alpha(1)-microglobulin (a1M) of 150 patients with histologically proven glomerulonephritis (GN) were determined. In addition, the reabsorption rate of the proximal tubule and the fractional excretion of sodium, the free water clearance and the renal function were assessed by inulin and p-aminohippurate (PAH) clearance. Compared to healthy controls, urinary AAP, NAG and a1M were found significantly elevated in GN patients. Morphological tubular changes were confirmed by significant differences in urinary laboratory parameters. In patients with tubular atrophy, the diagnostic sensitivity and specificity were calculated as follows: AAP (0.94/0.35), NAG (0.75/0.59) and a1M (0.73/0.52). In patients showing tubular protein droplets, the values were 0.90/0.17 for AAP, 0.78/0.76 for NAG and 0.84/0.74 for a1M and in patients with interstitial fibrosis, the values were AAP (0.95/0.35), NAG (0.75/0. 46) and a1M (68/0.38). Urinary AAP, NAG and a1M reflect histologically proven tubulus alteration in GN, although in most cases, the renal function is still intact. AAP indicates very early tubular impairment and, in some cases, AAP is elevated although NAG and a1M are still within normal ranges. We suggest that the enzyme activities are useful in the diagnostics of early stages of the disease.

Microvessel densities and microvascular architecture in colorectal carcinomas and their liver metastases: significant correlation of high microvessel densities with better survival.

Aims:  Microvessel densities in cancers have been shown to be a prognostic factor for some types of cancer. For colorectal cancer, however, the situation is far from clear.

Expression of Connexin26 in Islets of Langerhans Is Associated With Impaired Glucose Tolerance in Patients With Pancreatic Adenocarcinoma

Objectives: Impairment of glucose tolerance is one of the leading clinical presentations in patients with pancreatic carcinoma. The mechanism of disturbed glucose metabolism, however, is still under debate. Using microarray technology, key mechanisms of deregulated molecular functions of cancer cell–specific mRNAs and tumor-induced mRNAs in peritumorous tissue should be identified in pancreatic ductal adenocarcinoma (PDAC) by comparison to chronic pancreatitis and normal pancreas. Methods: Forty-three mRNAs were abundant in tissue specimens of patients operated due to pancreatic carcinoma but absent or of low abundance in chronic pancreatitis and normal pancreas. One of these mRNAs encodes the gap junction protein connexin26, known as a tumor suppressor, which was 10.8- and 6.9-fold more abundant in pancreatic carcinoma than in normal pancreas and chronic pancreatitis, respectively. Quantitative RT-PCR was performed for connexin26, with mRNA being expressed 26.7- and 2.9-fold more than in normal pancreas (n = 6), in pancreatic carcinoma (n = 7), and chronic pancreatitis (n = 8), respectively. Results: By immunohistochemistry, connexin26 was predominantly localized to the islets in the vicinity of the pancreatic carcinoma tissue. Control sections of tissue with chronic pancreatitis and normal pancreas show connexin26 expression in the islets as well. Interestingly, the level of mRNA abundance (fold over normal pancreas) in RT-PCR correlates (r = 0.62) with the 2h value of the pre-operative oral glucose tolerance test of these patients. Conclusion: Whether overexpressed connexin26 in pancreatic cancer is a cause of impaired glucose tolerance remains to be elucidated in further experimental studies.

Injection of Encapsulated Cells Producing an Ifosfamide‐Activating Cytochrome P450 for Targeted Chemotherapy to Pancreatic Tumors

Abstract: The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low‐dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.