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Purpose: Experimental studies indicate that perioperative hypoperfusion impairs anastomotic healing. In bowel surgery, the part of bowel that will be anastomosed is often pedicled, leaving the blood supply dependent on the marginal artery only. Little is known about the blood supply in such a segment, and whether anastomotic strength is affected when flow would be reduced. This study describes oxygenation and blood flow in pedicled bowel segments in the rat and investigates whether early anastomotic strength changes with variations in blood flow., Methods: In rats, pedicled segments were created in ileum and colon by successive ligation of the feeding arteries. Oxygenation and blood flow were measured in the distal part of this segment by use of near-infrared spectroscopy with indocyanine green as an intravascular tracer. In a second experiment, a short pedicled colonic segment was created and, after flow measurements, an anastomosis was constructed. Wound strength and hydroxyproline content were analyzed 2 and 5 days after operation., Results: After creation of a pedicled segment, the concentration of oxygenated hemoglobin decreased significantly. Blood flow also significantly decreased to even less than 10% of baseline. A very large variation was observed between animals, in particular, after ligation of the first arteries. The strength of colonic anastomoses was not significantly correlated with the blood flow in the pedicled segment before anastomotic construction., Conclusions: The creation of a pedicled bowel segment greatly reduces tissue oxygenation and blood flow to its distal part. Such impaired perioperative flow does not significantly affect early wound strength after anastomotic construction.

Background: Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series., Methods: Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing., Findings: SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively., Interpretation: Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome., Funding: The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

Pheochromocytomas (PCC) are catecholamine-producing tumors arising from the adrenal medulla that occur either sporadically or in the context of hereditary cancer syndromes, such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease (VHL), neurofibromatosis type 1, and the PCC-paraganglioma syndrome. Conventional comparative genomic hybridization studies have shown loss of 1p and 3q in the majority of sporadic and MEN2-related PCC, and 3p and 11p loss in VHL-related PCC. The development of a submegabase tiling resolution array enabled us to perform a genome-wide high-resolution analysis of 36 sporadic benign PCC. The results show that there are two distinct patterns of abnormalities in these sporadic PCC, one consisting of loss of 1p with or without concomitant 3q loss in 20/36 cases (56%), the other characterized by loss of 3p with or without concomitant 11p loss in 11/36 (31%). In addition, we found loss of chromosome 22q at high frequency (35%), as well as the novel finding of high frequency chromosome 21q loss (21%). We conclude that there appear to be two subgroups of benign sporadic PCC, one of which has a pattern of chromosomal abnormalities that is comparable with PCC from patients with MEN2 and the other that is comparable with the PCC that arise in patients with VHL disease. In addition, genes on 21q and 22q might play a more important role in PCC pathogenesis than had been assumed thus far.

The treatment of choice for non-metastatic pheochromocytoma is surgical resection. Its goals are to abolish catecholamine hypersecretion, normalize blood pressure, and prevent further tumor growth or progression to metastatic disease. Data on long-term mortality and morbidity after pheochromocytoma surgery are limited. We here report a retrospective study on the long-term outcome after surgery for apparently benign pheochromocytoma at the Radboud University Nijmegen Medical Centre. Data on clinical presentation, treatment, post-surgical blood pressure and recurrence, metastasis and death were collected of 69 consecutive patients (January 1966-December 2000; follow-up: until death or January 2006). Survival was compared with survival of a matched reference population. Two patients died of surgical complications. All ten patients with metastatic disease (including three diagnosed at first surgery) died. At follow-up, 40 patients were alive and recurrence free and three patients were lost to follow up. Two patients experienced a benign recurrence. Mean+/-s.d. follow-up was 10.2+/-7.5 (median 9, range 1-38) years. Kaplan-Meier estimates for 5- and 10-year survival since surgery were 85.8% (95% CI: 77.2-94.4%) and 74.2% (95% CI: 62.0-86.4%) for patients versus 95.5 and 89.4% in the reference population (P<0.05). Sixty-four percent of all patients with hypertension prior to surgery showed a significant decrease in blood pressure, but remained hypertensive after surgery. In conclusion, compared with the general population patients have a reduced life expectancy following pheochromocytoma surgery, due to their risk of developing metastatic disease. Only one-third becomes normotensive without antihypertensive medication. Therefore, lifelong follow-up is warranted.

The clinical behavior of endocrine pancreatic tumors (EPTs) is difficult to predict in the absence of metastases or invasion to adjacent organs. Several markers have been indicated as potential predictors of metastatic disease, such as tumor size > or =2 cm, Ki67 proliferative index > or =2%, cytokeratin (CK) 19 status, and recently in insulinomas, chromosomal instability (CIN). The goal of this study was to evaluate the value of these markers, and in particular of the CIN, to predict tumor recurrence or progression and tumor-specific death, using a series of 47 insulinomas and 24 non-insulinoma EPTs. From these EPT cases, a genomic profile has been generated and follow-up data have been obtained. The proliferative index has been determined in 68 tumors and a CK19 expression pattern in 50 tumors. Results are statistically analyzed using Kaplan-Meier plots and the log-rank statistic. General CIN, as well as specific chromosomal alterations such as 3p and 6q loss and 12q gain, turned out to be the most powerful indicators for poor tumor-free survival (P< or =0.0004) and tumor-specific death (P< or =0.0113) in insulinomas. The CIN, chromosome 7q gain, and a proliferative index > or =2% were reliable in predicting a poor tumor-free survival in non-insulinoma EPTs (P< or =0.0181, whereas CK19 expression was the most optimal predictor of tumor-specific death in these tumors. In conclusion, DNA copy number status is the most sensitive and efficient marker of adverse clinical outcome in insulinomas and of potential interest in non-insulinoma EPTs. As a consequence, this marker should be considered as a prognosticator to improve clinical diagnosis, most practically as a simple multi-target test.

Pheochromocytomas (PCCs) are rare tumors that arise from chromaffin tissue in the adrenal medulla, but can also occur in the abdomen outside the adrenals and are then called sympathetic paragangliomas (sPGLs). According to the literature, between 15 and 25% of apparently sporadic adrenal PCC and sPGL are caused by germline mutations in RET, von Hippel-Lindau disease (VHL), succinate dehydrogenase subunit B (SDHB), or subunit D SDHD. However, few studies have addressed the mutationfrequency of these candidate genes in selected subgroups of PCC andsPGL, such as bilateral adrenal PCC or extra-adrenal sPGL, and none have looked at somatic mutations by analyzing tumor tissue. Therefore, we have investigated the occurrence of germline and somatic mutations in RET, VHL, SDHB, and SDHD in comparatively large series of bilateral adrenal PCC (n = 33 patients) and sPGL (n = 26 patients), with the aim of determining the mutation frequency of each of these genes and to establish a genetic testing algorithm. Twenty-one RET, two VHL germline, and one SDHD mutations were found in the patients with bilateral adrenal PCC. In sPGL, one novel SDHB germline and one novel SDHB somatic mutation were observed. In addition, two SDHD germline mutations were found. We conclude that germline RET mutations are predominantly found in bilateral PCC, and that somatic and germline SDHB and SDHD mutations usually occur in sPGL, which has practical consequences for genetic testing algorithms. We suggest that sequential mutation analysis should be directed first at RET, followed by VHL and SDHD for patients with bilateral adrenal PCC at diagnosis, and at SDHB and SDHD for patients with sPGL.

Both polyamine metabolism and DNA methylation play an important role in normal and malignant growth. Specific enzyme inhibitors or drugs that interfere with these metabolic pathways have proven to be potential anticancer agents. Since DNA methylation and polyamine metabolism depend on a common substrate, i.e. S-adenosylmethionine, interaction between both pathways can be expected. Little is known about the relationship between these pathways but studies are available indicating that polyamines and DNA methylation are directly or indirectly interconnected, metabolically as well as physiologically with respect to the regulation of cell growth, differentiation and cancer development. These considerations give rise to the possibility that, by targeting both pathways, a more profound and effective inhibitory effect on the growth of malignant cells can be achieved. In previous studies we showed that 6-MP (6-mercaptopurine) as well as MTX (methotrexate), well-known drugs in the treatment of acute lymphoblastic leukaemia, inhibit DNA methylation and induce apoptosis in malignant blood cells. Our recent results show that combined treatment with 6-MP, MTX and drugs interfering with polyamine metabolism has additive/synergistic effects on the growth, cell viability and/or apoptotic death of leukaemic cells. Such a combination therapy could have great clinical value for patients in which therapy using inhibitors of thiopurines/purine metabolism has failed.

Objective: Pheochromocytomas are uncommon tumours arising from chromaffin cells of the adrenal medulla and related paraganglia. So far, one of the few reported markers to discriminate malignant from benign tumours is the betaB-subunit of inhibin and activin, members of the transforming growth factor (TGF)-beta superfamily of growth and differentiation factors., Design: We investigated the expression of the mRNAs coding for activin and inhibin subunits, their receptors and binding proteins by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and studied the presence of the inhibin betaB-subunit in human pheochromocytomas by immunohistochemistry., Patients: Samples from resected pheochromocytomas of patients operated between 1973 and 2003 were used for experiments., Results: The immunohistochemical investigations revealed that staining of the inhibin betaB-subunit was positive in 12 of 36 (33%) benign and 5 of 34 (15%) malignant pheochromocytomas (P > 0.05). Therefore, it was not possible to discriminate between benign and malignant tumours solely on the basis of inhibin betaB-subunit immunohistochemistry. Quantitative real-time RT-PCR in nine benign and four malignant tumours showed expression of inhibin alpha-, betaA- and betaB-subunits, the activin receptors Alk-4, ActRIIA, and ActRIIB, and the inhibin- and activin-binding proteins betaglycan and follistatin in all samples. No correlations were detected between individually coupled expression of mRNAs of these activin- and inhibin-related genes in the 13 pheochromocytomas. Only inhibin betaA-subunit expression was different in malignant compared to benign pheochromocytomas (P = 0.020)., Conclusions: No clear role for activin and inhibin was found in discriminating between benign and malignant pheochromocytomas.

Introduction: Gemcitabine has been shown to exert a radiosensitizing effect in various epithelial cancers. The aim of the present studies was to investigate whether the efficacy of radioimmunotherapy (RIT) using the (131)I-labeled anti-CEA monoclonal antibody (MAb) MN-14 could be enhanced by coadministration of gemcitabine in nude mice with small (1-3 mm) peritoneal metastases of colonic origin., Materials and Methods: Firstly, the maximum tolerated dose (MTD) of gemcitabine was determined, when administered intraperitoneally at two different dosing schedules (0.11-3.0 mg/mouse/administration on days 0, 3, 6, and 9, or 0.022-0.60 mg/mouse/administration on days 0, 1, 2, 3, and 4). In two separate therapy studies in which these two administration regimens were applied, the efficacy of gemcitabine monotherapy was compared to that of RIT alone (125 muCi (131)I-MN-14/mouse) or RIT combined with gemcitabine., Results: When administered every 3rd day for a total of 4 administrations, or daily for 5 consecutive days, the gemcitabine was considered safe at 0.33 mg/mouse/administration and 0.066 mg/mouse/administration, respectively. In the first therapy study, median survival of the control mice was 39 days. Gemcitabine monotherapy at 0.11 mg or 0.33 mg/mouse/administration every 3rd day (total, 4 administrations) resulted in a median survival of 52 and 57 days, respectively (p = 0.0003, compared to controls). RIT alone resulted in a median survival of 66 days (p < 0.0001, compared to controls). The combination of RIT and gemcitabine coadministration resulted in a median survival of 73 and 94 days, respectively (p = 0.12, for trend). In the second therapy study, median survival of the control mice was 48 days, which was similar to the median survival of the mice treated with daily administrations of gemcitabine monotherapy at 0.022 mg/mouse/administration on 5 consecutive days (49 days; p = 0.17). RIT alone resulted in a significantly improved median survival of 66 days (p= = 0.0010, compared to controls). Combination therapy using RIT and gemcitabine resulted in a median survival of 64 days, which did not differ significantly from the survival of the mice treated with RIT alone (p = 0.43)., Conclusions: At the dose regimens employed, gemcitabine did not enhance the efficacy of RIT of experimental small-volume peritoneal carcinomatosis of colonic origin.

Pheochromocytomas (PCCs) are neuroendocrine tumors of chromaffin tissue that produce catecholamines. They are usually located in the adrenal medulla, although in about 10% the tumors arise from extra-adrenal chromaffin tissue. The majority of PCCs arise sporadically, but PCCs occur also in the context of hereditary cancer syndromes. Familial PCC is inherited as an autosomal dominant trait alone or as a component of the multiple endocrine neoplasia Type 2 (MEN2) syndrome (RET gene), Von Hippel-Lindau (VHL) disease (VHL gene), neurofibromatosis Type 1 (NF1 gene), or familial pheochromocytoma-paraganglioma (PCC-PGL) syndrome (SDHD/B and C genes). It has been reported that 24% of apparently sporadic PCCs patients harbor germline mutations in these PCC-causing genes. We investigated the contribution of the inherited PCC-causing genes in a partly retrospectively and partly prospectively obtained series of 213 apparently sporadic PCCs. Mutation analysis was performed for RET (56 cases), VHL (136 cases), and SDHD (126 cases) and SDHB (47 cases). No germline RET mutations, six (4.4%) germline VHL mutations, two (1.5%) germline SDHD mutations, and one germline (1.6%) SDHB mutation were found. In total we found germline mutations in about 7.5% of the investigated apparently sporadic PCCs. Although 7.5% germline mutations in a series of apparently sporadic PCCs are far less than the more than 20% reported in the literature, the figure is significant enough to consider germline mutation testing for each patient with PCC.

Background: Multiple factors contribute to the process of prosthetic graft failure. Some of them are specifically related to the biological behavior of the used materials. To pursue the ideal substitute for the autologous vein graft, many materials have been taken into consideration. Of these, polyester (Dacron) and human umbilical vein (HUV, Dardik) bypass grafts have gained much attention in vascular surgical practice over the years. This study compares the results of both in vivo and in vitro investigations on graft thrombogenicity and neo-intimal formation in collagen-coated heparin bonded Dacron and in HUV bypass grafts. It is an adjunct to our clinical comparison of graft materials in infrainguinal arterial reconstruction., Methods: In 12 adult Beagle dogs, a patch was sewn onto the abdominal aorta (Dacron, n = 6; HUV, n = 6). At defined interval times, thrombocyte aggregation was measured with nuclear imaging of 99mTechnetium labeled platelets. Post-mortem histological analysis of the interface between the native vessel wall and the patch was performed in all animals., Results: At 4 h (2.67, SD = 0.77) and after 2 weeks (2.21, SD = 0.28) after implantation, significantly higher thrombogenicity was measured in the HUV grafts compared to Dacron grafts (1.98, SD = 0.10 and 1.98, SD = 0.11, P = 0.02 and 0.025, respectively). At 4 weeks, no significant difference could be found (HUV, 2.26; SD = 0.29; Dacron, 2.11; SD = 0.16; P = 0.23). Measurement of 'neo-intimal' thickness after explantation of the patch at 28 days after the initial procedure showed a significant difference: in HUV grafts the mean thickness of the inner lining was 0.76 mm (SD = 0.50), compared to 0.16 mm (SD = 0.10) in the Dacron grafts (P = 0.013)., Conclusion: HUV grafts showed a higher thrombogenicity at 4 h and 2 weeks after insertion of the graft compared to Dacron grafts. At 4 weeks this difference is not present. After 28 days the inner ('neo-intimal') lining is significantly more pronounced in HUV grafts than in Dacron grafts.

Pheochromocytomas (PCC) are relatively rare neuroendocrine tumors, mainly of the adrenal medulla. They arise sporadically or occur secondary to inherited cancer syndromes, such as multiple endocrine neoplasia type II (MEN2), von Hippel-Lindau disease (VHL), or neurofibromatosis type I (NF1). Loss of 1p is the most frequently encountered genetic alteration, especially in MEN2-related and sporadic PCC. Previous studies have revealed three regions of common somatic loss on chromosome arm 1p, using chromosome-based comparative genomic hybridization (CGH) and LOH analysis. To investigate these chromosomal aberrations with a higher resolution and sensitivity, we performed microarray-based CGH with 13 sporadic and 11 syndrome-related (10 MEN2A-related and 1 NF1-related) tumors. The array consisted of 642 overlapping bacterial artificial chromosome (BAC) clones mapped to 1p11.2-p36.33. Chromosomal deletions on 1p were detected in 18 of 24 cases (75%). Among 9 tumors with partial 1p loss, the deleted region was restricted to 1cen-1p32.3 in six cases (25%), indicating a region of genetic instability. The consensus regions of deletion in this study involved 1cen-1p21.1, 1p21.3-1p31.3, and 1p34.3-1p36.33. In conclusion, these data strongly suggest that chromosome arm 1p is the site for multiple tumor suppressor genes, although the potential candidate genes CDKN2C and PTPRF/LAR are not included in these regions., (Copyright 2005 Wiley-Liss, Inc)

Background: Inhibition of the COX-2 enzyme has been shown to have a radiosensitizing effect in epithelial cancers. The aim of this study was to investigate whether the efficacy of radioimmunotherapy (RIT) using 131I-labeled anti-CEA monoclonal antibody MN-14 could be enhanced by co-administration of the selective COX-2 inhibitor Parecoxib in mice with small volume (1-3 mm) peritoneal carcinomatosis of colonic origin., Methods: First, the efficacy of 14 daily injections of Parecoxib monotherapy (0-0.2-1.0-5.0-25.0 mg/kg) was determined in mice with intraperitoneal LS174T xenografts. Second, the influence of Parecoxib (1.0 or 5.0 mg/kg) on the biodistribution of 125I-MN-14 was assessed. Finally, the efficacy of RIT alone [125 microCi 131I-MN-14/mouse approximately 1/4 of the maximal tolerated dose (MTD)] was compared with that of Parecoxib monotherapy and RIT combined with daily injections of Parecoxib (1.0 or 5.0 mg/kg)., Results: Parecoxib had no measurable antitumor effect up to the highest dose level (25 mg/kg). Parecoxib had no effect on the uptake of 125I-MN-14 in the intraperitoneal tumor xenografts or on normal tissue distribution. Median survival of the control mice and the mice treated with Parecoxib monotherapy (1.0 or 5.0 mg/kg) was 48.5 days, 52 days and 52 days (P=0.47). RIT alone significantly delayed the growth of the intraperitoneal xenografts resulting in a median survival of 87 days (P<0.0001). Mice treated with RIT + Parecoxib at 1.0 or 5.0 mg/kg had a median survival of 73.5 days and 76 days, respectively, which was not statistically different from survival after RIT alone (P=0.15)., Conclusion: The COX-2 inhibitor Parecoxib does not enhance the therapeutic efficacy of RIT of experimental small volume peritoneal carcinomatosis of colonic origin.

Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic makeup. Insulinomas represent the predominant syndromic subtype of EPTs. The metastatic potential of insulinomas can frequently not be predicted using histopathological criteria, and also molecular markers indicating malignant progression are unreliable because of the small number of cases per subtype studied so far. For the identification of reliable indicators of metastatic disease, we investigated 62 sporadic insulinomas (44 benign and 18 tumors with metastases) by means of comparative genomic hybridization (CGH). In addition, the role of MEN1 (multiple endocrine neoplasia type 1) gene mutations was determined to assess specific chromosomal alterations associated with dysfunction of this endocrine tumor-related tumor suppressor gene. Only one case with a somatic MEN1 mutation was identified (1527del7bp), indicating that the MEN1 gene plays a minor pathogenic role in sporadic insulinomas. CGH analysis revealed that the total number of aberrations per tumor differs strongly between the benign and the malignant group (4.2 vs 14.1; P<0.0001). Furthermore, chromosome 9q gain was found to be the most frequent aberration in both benign and malignant insulinomas, whereas chromosome 6q losses and 12q, 14q and 17pq gains are strongly associated with metastatic disease. Our study shows that chromosomal instability, as defined by > or =5 gains together with > or =5 losses, or total number of gains and losses > or =8, rather than parameters such as tumor size and proliferation index, is the most powerful indicator for the development of metastatic disease in patients with sporadic insulinoma.

Purpose: We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas., Patients and Methods: This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies., Results: Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function., (Copyright The Histochemical Society, Inc.)

Matrix metalloproteinases (MMPs) have been implicated as mediators of tissue damage in several inflammatory diseases. Since the multiple organ dysfunction syndrome (MODS) is thought to result from systemic inflammation, overactivation of MMPs could contribute to the organ damage observed. The expression and activity of several MMPs were studied in a murine model for MODS. Sixty mice were given an aseptic intraperitoneal injection of lipopolysaccharide, followed, after 6 days, by zymosan. At days 2, 5, 8, 12, and 16 after the injection of zymosan, the liver, lungs, spleen, and kidneys were collected from groups of mice for either RNA extraction, gelatinase zymography and collagenase (MMP-1 and -13) assays (six mice per time point), or immunohistochemistry (three mice per time point). A group of nine mice did not receive zymosan and acted as controls. The expression of MMP-2 mRNA in zymosan-treated mice was strongly up-regulated in liver tissue only. For MMP-9, this was the case in all organs examined. Quantitative gelatin zymography demonstrated the near complete absence of any gelatinase activity in tissues from control mice. However, in the liver, lungs, and especially the spleen of zymosan-treated animals, significantly increased activity of proform and active MMP-2 and -9 was observed with time. Overall, MMP-1 and -13 activities were very low in all samples from the liver and lungs. In the spleen, however, high levels of MMP-1 and -13 were observed in zymosan-treated animals. Immunohistochemical staining for MMP-2 was detected in the liver and spleen, but not in lung and kidney tissue of zymosan-treated animals. Staining for MMP-9 could be detected in liver, lung, and spleen tissues of zymosan-treated mice. For both MMPs, staining appeared to be limited to phagocytes. In conclusion, the data suggest a role for MMPs, especially MMP-9, in the pathogenesis of MODS., (Copyright 2004 Pathological Society of Great Britain and Ireland)

Unlabelled: Therapeutic efficacy in radioimmunotherapy depends, among other things, on the choice of the radionuclide. The aim of the present study was to determine the most suitable radionuclide for radioimmunotherapy with monoclonal antibody MN-14 to carcinoembryonic antigen in an experimental model of small peritoneal metastases of colorectal origin., Methods: In nude mice with intraperitoneal LS174T tumors (diameter, 1-3 mm), the biodistributions of MN-14 labeled with (131)I ((131)I-MN-14), (186)Re-mercaptoacetyltriglycine ((186)Re-MN-14), and (88)Y-diethylenetriaminepentaacetic acid (DTPA) ((88)Y-MN-14) after intravenous and intraperitoneal administration were determined. Subsequently, the therapeutic efficacies of equally toxic activity doses of (131)I-MN-14 (9.25 MBq per mouse), (186)Re-MN-14 (9.25 MBq per mouse), (90)Y-MN-14 (3.15 MBq per mouse), and MN-14 labeled with (177)Lu-DTPA ((177)Lu-MN-14) (8.33 MBq per mouse) after intraperitoneal administration were determined., Results: Each of the radioimmunoconjugates preferentially accumulated in tumor nodules, both after intravenous administration and after intraperitoneal administration. Values for clearance from blood were similar for all radioimmunoconjugates. The uptake of (88)Y-MN-14 in the liver and spleen was significantly higher than the uptake of (131)I-MN-14 or (186)Re-MN-14. Maximal uptake values (mean +/- SD) in tumors were 58 +/- 7 percentage injected dose per gram of tissue (%ID/g) for (131)I-MN-14 (24 h after administration), 83 +/- 19 %ID/g for (186)Re-MN-14 (72 h after administration), and 148 +/- 89 %ID/g for (88)Y-MN-14 (192 h after administration). Dosimetric analysis of the biodistribution data estimated that the radiation doses guided to the tumor by intraperitoneally administered (131)I-MN-14, (186)Re-MN-14, (90)Y-MN-14, and (177)Lu-MN-14 were 150, 100, 45, and 200 Gy, respectively. The median survival time of control mice, treated with unlabeled MN-14, was 42 d, whereas the median survival times of mice treated with (131)I-MN-14, (186)Re-MN-14, (90)Y-MN-14, and (177)Lu-MN-14 were 100 d (range, 58-142; P < 0.0001), 72 d (range, 46-84; P = 0.0002), 82 d (range, 46-142; P < 0.0001), and 136 d (range, 56-142; P < 0.0001), respectively. At the completion of the experiment (142 d after tumor cell inoculation), no residual disease was found in 8 of 9 long-term survivors ((131)I, n = 3; (90)Y, n = 1; and (177)Lu, n = 4)., Conclusion: The uptake of (88)Y-MN-14 in small peritoneal LS174T xenografts was higher than the uptake of (131)I-MN-14 or (186)Re-MN-14. The present study indicates that (131)I and (177)Lu are the most suitable radionuclides for the radioimmunotherapy of small peritoneal metastases.

A hallmark of Type 2 diabetes mellitus (T2DM) is chronic hyperglycemia, which is thought to play a role in pancreatic beta-cell failure. Here we investigated whether treatment of Zucker diabetic fatty (ZDF) rats, an animal model for T2DM, with the renal glucose transport inhibitor phlorizin could prevent alterations in the pancreatic islets. ZDF rats were treated with phlorizin or vehicle for 13 weeks starting with 6-week-old rats and before the onset of hyperglycemia. During the treatment, blood glucose levels in sham-treated ZDF rats increased rapidly from 7.7 +/- 0.3 to 24.8 +/- 0.6 mmol/l, whereas those in phlorizin-treated ZDF rats increased only slightly, but significantly, from 7.0 +/- 0.2 to 8.9 +/- 0.6 mmol/l. Phlorizin prevented the decrease in plasma insulin levels and caused a higher increase in body weight of the ZDF rats. Compared to 6-week-old untreated ZDF rats, in 19-week-old sham- and phlorizin-treated ZDF rats similar changes were found in islet architecture (more irregular boundaries and a disrupted mantle of peripheral islet cells) and in the mitochondria at the ultrastructural level (swelling of the matrix and disruption of the cristae). Using reverse transcriptase-polymerase chain reaction, no differences in mRNA expression levels were found for insulin, islet amyloid polypeptide (IAPP), and the prohormone convertase (PC) 1 and PC2 between 6-week-old untreated ZDF rats and 19-week-old sham- and phlorizin-treated ZDF rats. However, immunohistochemistry revealed similar decreases in insulin and IAPP protein expression in 19-week-old sham- and phlorizin-treated ZDF rats compared to those in 6-week-old untreated ZDF rats. These observations indicate that during aging of ZDF rats phlorizin treatment does not prevent the decreases in insulin and IAPP protein expression and the progressive histopathological changes in the pancreatic islets. Therefore, it is highly unlikely that these changes are caused by chronic hyperglycemia.

The importance of polyamines in prostatic growth and differentiation has prompted studies to evaluate the clinical relevance of the ornithine decarboxylase/polyamine system in prostatic cancer. These studies show that differences in biological behaviour of prostatic (cancer) cells are associated with changes in polyamine levels and/or the activity of their metabolic enzymes. Faulty antizyme regulation of polyamine homoeostasis may play an important role in the growth and progression of prostatic carcinoma. Treatment of human prostate carcinoma cells with inhibitors of polyamine metabolic enzymes or polyamine analogues induces cell growth arrest or (apoptotic) cell death. Our recent in vitro studies using conformationally restricted polyamine analogues show that these compounds inhibit cell growth, probably by inducing antizyme-mediated degradation of ornithine decarboxylase. Sensitivity of human prostate cancer cells for these compounds was increased in the absence of androgens. These results suggest that these analogues might have chemotherapeutic potential in case prostatic cancer has become androgen-independent. Pilot data in an in vivo model show that these analogues have effects on tumour cell proliferation, vascularity, blood perfusion and tissue hypoxia. Overall, these studies show that polyamines may serve as important biomarkers of prostatic malignancy and provide a promising target for chemotherapy of prostatic cancer.

Background: The strength of intestinal anastomoses is relatively low in the first days after operation, possibly as a result of localized degradation of the supporting matrix by enzymes from the matrix metalloproteinase (MMP) family. The aim of this study was to examine whether doxycycline, a drug known to inhibit MMP activity, could enhance anastomotic strength., Methods: Male Wistar rats received anastomoses in both ileum and colon. From the day before operation onwards, animals were treated daily with doxycycline (orally or subcutaneously) in a dose of 10 mg/day or with saline only. Rats were killed 1, 3, or 5 days after operation, and anastomotic bursting pressure and breaking strength were measured. At day 3, anastomotic hydroxyproline levels were measured, MMP (gelatinase) activity was analyzed by gelatin zymography, and anastomotic histology was examined., Results: Doxycycline enhanced wound strength, but only at day 3, when it was at its lowest. Subcutaneous administration of 10 mg/day increased median colonic and ileal breaking strength by 27% (P =.0019) and 104% (P =.0376), respectively. Colonic bursting pressure was increased by 93% (P =.0002). Wound histology was similar in experimental and control groups., Conclusions: Administration of doxycycline enhances anastomotic strength and should be investigated further as a means to preserve anastomotic integrity.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Increased polyamine levels are required for growth, differentiation, and transformation of cells. In situ detection of ODC in cells and tissues has been performed with biochemical, enzyme cytochemical, immunocytochemical, and in situ hybridization techniques. Different localization patterns at the cellular level have been described, depending on the type of cells or tissues studied. These patterns varied from exclusively cytoplasmic to both cytoplasmic and nuclear. These discrepancies can be partially explained by the (lack of) sensitivity and/or specificity of the methods used, but it is more likely that (sub)cellular localization of ODC is cell type-specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Intracellular translocation of ODC may be a prerequisite for its regulation and function.

The purpose of the study was to investigate the course of the zymosan-induced multiple organ dysfunction syndrome (MODS) in the absence of tumor necrosis factor (TNF) in a murine model. Tumor Necrosis Factor-alpha-lymphotoxin-a knockout (TNF/LT-/-) mice (n = 36) and wild-type (TNF/LT+/+) mice (n = 36) received 40 microg of lipopolysaccharide (LPS) intraperitoneally followed by zymosan at a dose of 1 mg/g body weight 6 days later (day 0). Animals were monitored daily for body weight and temperature and clinical symptoms. At day 22, most of the surviving mice were killed to examine organ weight and histology. A small number of animals were followed until day 48. In all animals, zymosan induced an acute sterile peritonitis phase followed by an apparent recovery. From day 8 onwards the TNF/LT+/+ mice entered a third-MODS-like-phase, characterized by loss of body weight, decreased body temperature, and significant mortality. At day 22, survival in the TNF/LT-/- mice (92%) was significantly (P = 0.01) higher than in the TNF/LT+/+ mice (60%). In addition, average body temperature and average relative (vs. weight at day 0) body weight were higher in the TNF/LT-/- mice than in the TNF/LT+/+ mice (35.9 degrees C and 100% vs. 33.3 degrees C and 84%, respectively). However, at this time point, surviving animals from both groups showed similar and significant organ damage, indicated by an increase in absolute and relative (vs body weight) weight of lung, spleen, and liver (liver only in the TNF/LT-/- mice). Moreover, histopathological examination of organs from the surviving animals showed a similar degree of microscopic damage in both groups. Interestingly, besides mononuclear cells, inflammatory infiltrates in lungs and livers of TNF/LT+/+ but not of TNF-/- mice contained neutrophils. In conclusion, TNF-deficient mice exhibit significantly improved morbidity and mortality during zymosan-induced MODS. However, the absence of TNF does not completely protect against MODS in this murine model.

Purpose: The mechanisms that cause diabetes to impair the development of anastomotic strength in the intestine are poorly understood. We investigated whether short-term uncontrolled diabetes causes alterations in microscopic aspects of anastomoses from the ileum and colon., Methods: Eighteen Wistar rats were rendered diabetic one week before operation by intravenous streptozotocin injection (50 mg/kg), resulting in nonfasting blood glucose levels of approximately 20 mmol/l. Another 18 age-matched rats were used as controls with a normal blood glucose range of 5 to 7 mmol/l. All rats underwent resection and anastomosis of both the ileum and colon. Animals were killed at one, three, or seven days after operation. Cellular and architectural parameters of anastomotic healing were scored in hematoxylin and eosin-stained sections. Anastomotic collagen content was analyzed by image analysis in picrosirius red-stained sections., Results: Anastomotic necrosis, edema, and epithelial recovery were not affected by diabetes. In diabetic rats, the number of polymorphonuclear cells and macrophages was significantly (P = 0.025 and 0.0002, respectively) increased in ileal anastomoses one and three days after operation. In colonic anastomoses, the number of polymorphonuclear cells was increased at one (P = 0.001) and seven (P = 0.014) days after operation. Repair of the submucosal-muscular layer in colonic anastomoses from diabetic rats was impaired seven days after surgery (P = 0.0071), but in ileal anastomoses no difference was found. In the anastomotic area, collagen deposition at postoperative Days 1, 3, and 7 remained unaffected by diabetes., Conclusion: Experimental diabetes leads to alterations in cellular components involved in the early phase of repair of intestinal anastomoses but not to a reduced accumulation of wound collagen.

Thus far, histopathological changes in the pancreatic islets of Zucker Diabetic Fatty (ZDF) rats, an animal model of type 2 diabetes mellitus (or non-insulin-dependent diabetes mellitus), have only been studied in male rats and in 18-weeks old rats or younger. In this study, we have examined in both male and female ZDF rats the histopathological changes longitudinally, from 6 to 32 weeks of age. We studied islet architecture and cellular distribution of the various islet hormones both in ZDF and control rats. In the ZDF rats, aging was initially associated with an enlargement of the islets. From 18 weeks onwards, no further enlargement was noted but islet boundaries became increasingly irregular, leading to the appearance of projections of endocrine cells into the surrounding exocrine tissue. At the islet boundaries as well as within the islets progressive fibrosis was observed with increasing amounts of collagen and reticular fibers. In the islets, staining intensity of both insulin and islet amyloid polypeptide (IAPP) increased slightly till 10 weeks of age and thereafter decreased rapidly. In contrast, the staining intensities of glucagon, somatostatin, and pancreatic polypeptide (PP) did not change. Even at the age of 32 weeks, just the beta-cells and not the other endocrine islet cells appear to be affected. In control rats, aging evoked only minor changes. Thus, we observed that during prolonged development of diabetes mellitus in both male and female ZDF rats histopathological changes in the pancreatic islets became progressively more severe, eventually leading to disintegration of the islets.

Background: Recent studies indicate that N-terminally bis-ethylated-polyamine analogs have significant antitumor activity in several human solid-tumor models. In this study, the in vitro and in vivo antitumor potential of the polyamine analog N(1), N(11)-diethylnorspermine (DENSpm) in human prostate carcinoma cells was examined., Methods: The antiproliferative and biochemical effects of DENSpm were tested in four human prostate cancer cell lines, i.e., PC-3, TSU-pr1, DU-145, and JCA-1. The in vivo antitumor potential was explored in two groups of nude mice bearing small or more developed xenografts of the DU-145 cell line. The mice were treated with 40 mg/kg DENSpm, three times per day for two cycles of 6 days, on days 1-6 and 8-13., Results: In vitro studies showed that all four tested human prostate carcinoma cell lines were sensitive to DENSpm in micromolar concentrations. In tumor-bearing mice, DENSpm clearly prevented tumor growth in both size groups, which became significant after day 17. Treatment with DENSpm evoked intracellular accumulation of the analog and various regulatory responses, e.g., downregulation of the polyamine biosynthesis, the induction of the catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT), and the depletion or decrease of natural polyamines. The cellular sensitivity to growth inhibition by DENSpm only correlated with the degree of ODC inhibition and SSAT induction., Conclusions: DENSpm has sustained inhibitory effects on the growth of human prostate carcinoma cells in vitro as well in vivo. This polyamine analog may provide a new tool in the chemotherapy of prostate cancers with various phenotypes., (Copyright 2000 Wiley-Liss, Inc.)

To investigate whether polyamines may be valuable diagnostic and prognostic markers in prostate cancer, the presence of polyamines was studied in various human prostatic tissues using both proton magnetic resonance (MR) spectroscopy and high-pressure liquid chromatography (HPLC). The HPLC results showed that normal and benign hyperplastic prostatic tissues were characterized by a high content of spermine. Spermine levels were reduced in tumor tissue, especially in prostatic carcinoma with metastases, and in xenografts of human prostatic carcinoma cells. These preliminary results indicate that spermine may be used as a biomarker for malignant behavior. The MR spectroscopy study showed that it is possible to detect spermine resonances in prostatic biopsy material by one-dimensional and two-dimensional J-resolved MR spectroscopy at high field (600 MHz). Localized one-dimensional in vitro MR spectra obtained at the clinical field strength of 1.5 T showed spermine signals in the region between 3.0 and 3.3 ppm. In in vivo MR spectra of the human prostate, however, these signals were obscured by resonances of choline (3.2 ppm) and creatine (3.0 ppm).

Purpose: After acute arterial obstruction of the lower extremity, muscle damage is the critical determinant for clinical outcome. The extent of muscle damage and limb viability are currently assessed by clinical examination, which is inaccurate. Tc-99m-pyrophosphate (PYP) has been applied for imaging ischaemia-reperfusion damage. More recently, a new imaging agent, Tc-99m-glucarate (GLUC), was introduced for delineating early myocardial infarction after ischaemia-reperfusion. The aim of this study was to determine if GLUC could delineate early skeletal muscle damage after ischaemia-reperfusion. Both tracers were used in a novel murine model of hindlimb ischaemia-reperfusion., Methods: In anaesthetised mice, ischaemia of one hindlimb was maintained for 2, 3 and 4h using a tourniquet, followed by a reperfusion period of 1h. Additionally, reperfusion periods of 3, 24 and 96h were studied after 3h of ischaemia. PYP or GLUC was injected 45min before end of reperfusion. Concentrations of both agents were determined in blood, reperfused and contralateral muscle. Reperfused-to-contralateral muscle ratios were calculated. In separate experiments, muscle biopsies were obtained for histologic examination., Results: Ischaemia and reperfusion damage was demonstrated histologically. Using scintigraphy GLUC depicted reperfusion significantly better than PYP. After 2, 3 and 4h of ischaemia, the reperfused-to-contralateral ratios for GLUC were 10.7+/-0.9, 8.9+/-0.9 and 8.6+/-1.1, as compared to 4.5+/-0.7, 4.9+/-0.4 and 4.5+/-0.4 for PYP (P<0.05 at all points). For longer periods of reperfusion, the ratios for GLUC decreased to similar levels as observed for PYP., Conclusion: The present study indicates that GLUC is a specific early marker of myocyte necrosis after ischaemia-reperfusion. GLUC may become an useful agent for clinical, early, non-invasive monitoring of skeletal muscle damage after ischaemia-reperfusion.

The natural polyamines (putrescine, spermidine and spermine) are ubiquitous low-molecular aliphatic amines that play multifunctional roles in cell growth and differentiation. Recently, evidence has merging that polyamines are actively involved in cell death. Changes in polyamine homeostasis have been reported during cell death of nerve cells, in programmed cell death of embryonic cells and in various in vitro models of apoptosis. Polyamines and many of their structural analogs exert cytotoxic effects in vitro as well in vivo. Furthermore, polyamine analogs and inhibitors of the polyamine anabolic/catabolic pathways modulate processes of cell death in a cell-type specific way. Much ambiguity exists in the working mechanisms by which polyamines mediate apoptosis since they have been shown to act as promoting, modulating or protective agents in apoptosis. Nevertheless, from the studies reviewed here it can be concluded that polyamines are critically involved in cellular survival which makes them suitable targets for therapeutic intervention that is specifically directed to cell death pathways.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)

Background: Intraarterial infusion of the free radical donor tert. -butyl-hydroperoxide (tert.-BuOOH) into one extremity of the rat induces vascular permeability and considerable skeletal muscle damage. However, it remains unclear what the role of polymorphonuclear neutrophils (PMNs) is in oxidative stress-related processes. Therefore, we investigated possible differences between neutropenic and normal animals in this model., Methods: Neutropenia was induced in male rats by intraperitoneal administration of cyclophosphamide. tert.-BuOOH was continuously infused intraarterially into one hindlimb of normal or neutropenic nonanesthetized rats for 24 h. The control neutropenic rats were infused with the same volume of saline. After the infusion, 99mTc-IgG was administered intravenously followed by scintigraphic imaging analysis of the left/right uptake ratio of the hindlimbs and by gamma counting of the tissue samples of the gastrocnemius and gluteus maximus muscles. Samples of these muscles were analyzed by light microscopy., Results: The uptake ratios were significantly increased in the normal and neutropenic tert.-BuOOH-infused animals as compared with the saline-infused neutropenic rats (P < 0.05). The uptake ratios were significantly higher in normal than in neutropenic tert.-BuOOH-infused rats (P < 0.05). Histological analysis of the saline infused skeletal muscles showed unaffected skeletal muscles with intact arterioles and arteries. In the gastrocnemius and gluteus maximus muscles of the normal tert. -BuOOH-infused and neutropenic rats, similar morphological damage was observed., Conclusions: PMNs can increase, to some extent, the vascular permeability of the free radical damaged small arteries and arterioles of a tert.-BuOOH-infused hindlimb. However, in the present animal model, tert.-BuOOH alone can induce oxidative stress-related abnormalities with skeletal muscle tissue damage that is mainly independent of the presence of PMNs., (Copyright 1999 Academic Press.)

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

A 25-year-old woman developed Nelson's syndrome, 3 years after successful bilateral adrenalectomy for Cushing's disease. Despite pituitary surgery and radiotherapy the tumour showed invasive growth, leading to visual disturbance, paresis of the oculomotor nerve and, 34 years after adrenalectomy, to death by widespread purulent leptomeningitis. Autopsy revealed a large adenohypophyseal carcinoma with a metastasis attached to the dura, both tumours showing immunocytochemical staining for ACTH and TSH. We review the literature on metastatic adenohypophyseal carcinoma in Cushing's disease and Nelson's syndrome and discuss the role of proliferation markers as indicators of malignant progression.

Purpose: to determine whether intraoperative radiotherapy causes long-term negative effects on the healing of colonic anastomoses in the rat., Methods and Materials: 175 rats were divided into seven equal groups. One group served as sham-irradiated control group. In the others, following a colonic resection, 1 or 2 cm of the distal bowel limb was irradiated with a single dose of 10, 15, or 20 Gy (groups 10/1, 15/1, 20/1, 10/2, 15/2, and 20/2, respectively). Subsequently, an anastomosis was constructed. The animals were killed after 6 (n = 10 in each group) or 12 (n = 15) months. The abdomen was inspected for abnormalities and the colonic diameter was measured. The anastomotic segment was analyzed biochemically (hydroxyproline) and histologically., Results: During the experimental period, 1 rat (group 15/1) died because of anastomotic leakage and 3 others died from unknown causes. There was no difference in colonic diameter between groups. Altogether 17 rats developed an adenocarcinoma in the irradiated area: 11 of these had received a dose of 20 Gy. Histological observation indicated that fibrosis was present only in a limited number of animals, mostly after irradiation with a dose of 15 or 20 Gy. All anastomoses were functional and showed normal histology. The hydroxyproline content of the anastomotic segment was increased--with respect to the control group--only in the 20/2 group after 6 months. After 12 months, the hydroxyproline concentration in the (irradiated) segment distal to the anastomosis proper was higher in the 10/1 and 15/1 groups than in the control group. Otherwise, there were no differences between groups., Conclusion: Intraoperative irradiation with a single dose of 10-20 Gy, delivered to the distal limb used for anastomotic construction, does not appear to constitute a threat to anastomotic integrity. Dose-related changes included formation of adenocarcinomas and fibrosis, but function and histology of the anastomosis proper remained unaffected.

Rat/mouse T cell hybridoma-derived PC60 R55/R75 cells were used as a model to study tumour necrosis factor (TNF)-induced apoptosis. The role of ornithine decarboxylase (ODC) activity and polyamines in this process was investigated. In PC60 R55/R75 cells, TNF-induced ODC activity was completely suppressed by externally added spermine (Spm). TNF decreased the intracellular levels of the three polyamines Spm, spermidine (Spd) and putrescine (Put). A reduction of the intracellular [Spm] with methylglyoxal bis(quanyl hydrasone) (MGBG), CGP48644a, or bis(ethyl)norspermine (BENSpm), clearly sensitized the cells towards the apoptotic effect of TNF. Conversely, an increase in intracellular [Spm] with DFMO or externally added Spm reduced cellular sensitivity. Similar results were obtained after TNF treatment of the human cell lines Kym 39A6 (rhabdomyosarcoma), HeLaH21 (cervix carcinoma) and U937 (histocytoma) and after alphaFas treatment of HeLaH21, U937 and CEM-CM3 (human T cell line). These results suggest that a decrease of intracellular Spm levels rather then ODC activity per se is involved in the sensitization towards apoptosis induced by TNF or alphaFas., (Copyright 1998 Academic Press Limited.)

Hyperthermia is a promising method for increasing the efficacy of radiation therapy of colorectal cancer. To study the histological aspects of healing of an anastomosis in the colon, after combined preoperative (sham) irradiation and (sham) hyperthermia treatment, 48 male Wistar rats were divided randomly into four groups. In each animal, a segment of the colon was treated successively by (sham) irradiation (single dose of 25 Gy X rays) and/or (sham) hyperthermia (44 degrees C, 30 min). After 5 days, a resection of the colon was performed by construction of an anastomosis: The distal limb consisted of (sham-) irradiated and/or (sham-) hyperthermia-treated bowel. Rats were killed 3 or 7 days after the surgical procedure. Evaluation of healing of the anastomosis was made by: (1) histological analysis of sections stained with hematoxylin and eosin, (2) semiquantitative measurement of collagen in the area of the anastomosis and (3) semiquantitative analysis of the number of macrophages by immunocytochemistry. Healing of the anastomoses in animals receiving irradiation or hyperthermia alone and in control animals was relatively uneventful. There were no differences between groups in formation of collagen or infiltration by macrophages in the area of the anastomosis. Animals treated with both radiation and hyperthermia showed marked necrosis, infiltration by polymorphonuclear leukocytes and rupture of the anastomosis. It is concluded that preoperative irradiation with a single dose of 25 Gy in combination with local hyperthermia at 44 degrees C for 30 min leads to disturbed repair of anastomoses.

A well defined animal model is a prerequisite to test intervention strategies aimed at preventing the development of the multiple organ dysfunction syndrome. This study compares changes in clinical parameters to histopathologic changes in tissues, observed over a 12 day period after a single intraperitoneal injection of zymosan in C57BL/6 mice. Administration of zymosan induced gradual and progressive changes in wet and dry organ weight of the liver, kidneys, and particularly, lungs and spleen that correlated with increasing histopathology. From 6 days after zymosan injection onwards, hemorrhagic spots were found in the lungs evolving into massive hemorrhages at 12 days, when thickened interstitial walls and loss of the honey comb-like structures were observed. The liver displayed progressive accumulation of macrophage-like and mononuclear cells. After 12 days, numerous granuloma-like structures were disseminated throughout the liver parenchyma. The spleen displayed great changes in red and white pulp with increasing numbers of megakaryocytes and plasma-like cells. In the kidneys, necrosis of the tubular epithelium adjacent to granulomas on the ventral (peritoneal) side was found. In mice, a single intraperitoneal challenge with zymosan leads to progressive multiple organ damage, which becomes apparent at some time after the insult. This animal model displays a number of features encountered in human multiple organ dysfunction syndrome and appears suitable to conduct intervention studies.

Oxygen-derived free radicals are suspected to play an important role in the pathogenesis of inflammation and ischemia/reperfusion of an extremity. In this study we investigated the repair capacity of a free radical-damaged hindlimb of the rat and the effect of the anti-oxidant N-acetyl-L-cysteine (NAC). In nonanesthetized rats (n = 39), the left hindlimb was continuously infused intra-arterially (1 ml/hr) for 24 hr with the free radical donor tert-butylhydroperoxide (tert-BuOOH, 25 mM). Subsequently the infusion system was disconnected and the repair of soft tissue damage was observed with special attention to various pain tests, vascular permeability ((99m)Tc-IgG scintigraphy), and histology for a maximum period of 6 weeks. In 12 of these tert-BuOOH-infused rats the antioxidant NAC was injected intraperitoneally. Six of the NAC-treated rats were killed after 24 hr of infusion, while the remaining 6 rats were disconnected, reinjected with NAC, and observed for 1 week. Tert-BuOOH infusion for 24 hr led to significantly increased pain sensations, vascular permeability, and histological damage. Treatment with NAC significantly reduced pain sensations and vascular permeability, though not to control levels. One week after disconnection, tissue damage was almost completely repaired in the NAC-treated rats. In the untreated rats, repair took longer but histology and vascular permeability were completely normalized within the observation period. Soft tissue damage, induced by 24-hr infusion of the free radical donor tert-BuOOH, showed spontaneous repair within 6 weeks. The antioxidant NAC significantly reduced the soft tissue damage and shortened the repair period.

We present a novel animal model which allows the continuous intra-arterial infusion in one hindlimb of non-anaesthetized rats, without inducing ischemia. Using this model the effect of continuous infusion (1 ml/h) for 24 h with tert-butylhydroperoxide (tert-BuOOH) at a concentration of 25 mM on soft tissue of the left hind limb was studied and compared to the effect of saline infusion (control group). The tert-BuOOH-infused foot showed increased skin temperature, increased circumference, redness of the plantar skin, impaired function and increased pain sensation, while in the contralateral foot and in rats only perfused with saline these signs of inflammation were absent (p < 0.01). Histological analysis of the left gastrocnemius muscle showed edema, muscle cell degeneration with a patchy distribution pattern and vascular damage. All these features increased in severity from 4 to 24 h tert-BuOOH infusion. After 24 h of tert-BuOOH infusion infiltration of neutrophils in the interstitium was observed. Vascular permeability, expressed as left to right gastrocnemius muscle 99mTc-IgG uptake ratio, was similarly increased after 4 h (2.09 +/- 0.26) and 12 h (2.04 +/- 0.08) of tert-BuOOH infusion compared to saline (1.05 +/- 0.08) (p < 0.001), and further increased after 24 h (3.84 +/- 0.13): (p < 0.001). In this animal model free radical-related soft tissue damage was induced, by continuous infusion of tert-BuOOH, followed by increasing necrosis and vascular permeability in skeletal muscle coinciding with neutrophilic infiltration.

An accurate and reliable quantitation of angiogenesis is an essential requirement for a detailed study of new blood vessel growth during healing of colon anastomoses. In the present study we applied a computer-based digital image processing system for quantitative analysis of the anastomotic vascularization and perfusion. Rats underwent colonic resection (1 cm) followed by construction of an end-to-end anastomosis. The animals were killed 3 or 7 days after operation. The vascularization and perfusion were analysed in cyrostat sections using an anti-collagen type IV antibody and the fluorescent marker bisbenzimide H 33342, respectively. If compared with 3-day-old anastomoses, a significant increase in the median vascular and perfusion areas was found 7 days after operation. Within the same anastomosis, the vessel density and perfusion were lower in the central region than in the peripheral region of the wound. The present computerized digital image analysis system is a reliable technique that is suited for quantitative measurements of microvascular changes occurring in colon anastomoses during the first postoperative week.

Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of xenobiotics and are divided into four subclasses. Alpha, Mu, Pi and Theta. Most human gastrointestinal tumors contain increased amounts of GST Pi. In order to compare data on the expression of GSTs obtained by biochemical as well as immunohistochemical methods, we characterized the presence of GST Alpha and Pi by Western blot analysis and immunohistochemistry in 22 samples of human gastric carcinoma and adjacent non-neoplastic mucosa. Biochemical analyses revealed the presence of GST Alpha and Pi in 95% and 91% of normal tissues and in 82% and 100% of tumor specimens, respectively. Immunohistochemically all cases of normal gastric tissue stained for both GST Alpha and Pi, whereas immunostaining for GST Alpha and Pi was seen in 36% and 100% of the gastric tumor specimens, respectively. No statistically significant correlation however was observed between biochemical and immunohistochemical determination of GST Alpha and Pi both in normal as well as in malignant tissue. The absence of a statistically significant correlation between biochemical and immunohistochemical determination of GST Alpha and Pi implies that a high degree of caution must be taken in interpretating data derived solely from biochemical or immunohistochemical assays.

Objective: To obtain data on the ontogeny of catecholamines and other chromaffin vesicle components, which could serve as a basis for the study of their role during fetal life in normal and pathologic conditions., Design: Epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A contents were measured in the porcine adrenal gland during various stages of gestation., Animals: 934 porcine fetuses representing 22 gestational ages between 43 and 108 days., Procedure: Total homogenates of adrenal glands were extracted and contents of different neurochemical markers were measured, using high-performance liquid chromatography, immunoassays, and western blotting. Immunohistochemical studies also were performed., Results: Epinephrine and norepinephrine contents as a function of gestational age can be represented by a sigmoidal curve. Norepinephrine content rises early in gestation, whereas epinephrine content increases later. Maximal increase was significantly higher for epinephrine content. A progressive appearance of separate epinephrine- and norepinephrine-storing cells was documented. Dopamine-beta-hydroxylase content as a function of gestational age can be adequately represented by a parabolic curve. No quantitative changes in chromogranin A concentration were observed, but western blotting revealed qualitative changes with progressing gestational age., Conclusions: Important changes occur in catecholamine formation around day 60 of gestation. The sharp increase in epinephrine/norepinephrine contents and the appearance of separate epinephrine- and norepinephrine-storing cells may be related to the progressive splanchnic innervation of the adrenal gland. The presence of chromogranin A early in gestation may indicate its necessity for catecholamine storage.

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells against cytotoxic and carcinogenic agents. The distribution and levels of GST Alpha and Pi in normal and malignant gastric tissue of 34 patients with gastric cancer were examined immunohistochemically. Expression of GST Alpha and Pi was observed in 47 and 100 percent of the tumors, respectively. In normal mucosa both enzyme classes were present in 100 percent of the specimens. Mucous cells showed staining for GST Alpha and Pi in 88 and 97 percent, parietal cells in 93 and 67 percent, and chief cells in 82 and 30 percent, respectively. No correlation was observed between the amount or pattern of GST Alpha or Pi in carcinomas and the clinical and pathological characteristics of the patients. So it can be concluded that both GST Alpha and Pi cannot be considered as prognostic factors for gastric cancer.

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

Indirect single- and double-staining immunofluorescence techniques were used to study the serotoninergic, peptidergic and GABAergic innervation of the ventrolateral (Onuf's nucleus) and dorsolateral (innervating intrinsic foot sole muscles) nuclei, located in the S1/S2 segments of the cat spinal cord. The relative density of 5-hydroxytryptamine-, thyrotropin-releasing hormone-, substance P- and gamma-aminobutyric acid-immunoreactive axonal varicosities was similar in both nuclei. The highest relative density was recorded for varicosities immunoreactive to gamma-aminobutyric acid, while those immunoreactive to 5-hydroxytryptamine or thyrotropin-releasing hormone yielded the lowest values. The density of enkephalin-immunoreactive varicosities was higher in the ventrolateral than in the dorsolateral nucleus. Calcitonin gene-related peptide-like immunoreactivity could be seen in neurons of the ventrolateral and dorsolateral nuclei. Occasionally, calcitonin gene-related peptide-immunoreactive axonal fibers were also encountered in these nuclei. Virtually all thyrotropin-releasing hormone-immunoreactive varicosities in the ventrolateral and dorsolateral nuclei also contained 5-hydroxytryptamine-like immunoreactivity, while a somewhat smaller number of them were co-localized with substance P. About 5-10% of the 5-hydroxytryptamine-immunoreactive varicosities were devoid of peptide-like immunoreactivity, and the number of 5-hydroxytryptamine-immunoreactive varicosities lacking thyrotropin-releasing hormone-like immunoreactivity was higher in the dorsolateral than in the ventrolateral nucleus. Finally, the free fraction of substance P-immunoreactive varicosities, i.e., those lacking both 5-hydroxytryptamine and thyrotropin-releasing hormone, was about 39% in the ventrolateral and 26% in the dorsolateral nucleus. Spinal cord transection at the lower thoracic level induced a depletion of 5-hydroxytryptamine and thyrotropin-releasing hormone-immunoreactive fibers from the ventrolateral and dorsolateral nuclei, indicating an exclusive supraspinal origin for these fibers. A reduction in substance P-like immunoreactivity following spinal cord transection alone or spinal cord transection combined with unilateral dorsal rhizotomy was also detected in both nuclei, suggesting a dual origin for substance P-immunoreactive fibers, i.e., both supra- and intraspinal. The decrease in number of substance P-immunoreactive fibers was however smaller than expected from the analysis of the fraction of substance P-immunoreactive fibers co-localized with 5-hydroxytryptamine, indicating thus that the experimental lesions may have triggered a sprouting of substance P-immunoreactive axons originating from spinal cord sources. The distribution of gamma-aminobutyric acid in the ventrolateral and dorsolateral nuclei was not affected by the different lesion paradigms. It is therefore assumed that these inputs are intrinsic to the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Dividing cells from the midline of the ventral rhombencephalon and medulla oblongata have been transduced with a modulatable oncogene, (ts)SV40-T, using retroviral gene transfer. At the permissive temperature of the oncogene (33 degrees C), cells replicated and were isolated as individual, homogeneous clones. The effects of simply raising the temperature to the oncogene's non-permissive value, namely 39 degrees C, were analyzed by immunohistochemical methods. In one clone in particular (921202-6), cells ceased replication and started to differentiate. Certain neuronal characteristics became apparent: neurone-specific enolase-like immunoreactivity developed, as did the ability to take up exogenously applied 5-hydroxytryptamine (5HT). In addition, the cells took up exogenous 5-hydroxytryptophan (5HTP), and subsequently decarboxylated it to 5HT. However, they were unable to synthesize immunohistochemically detectable amounts of 5HT using L-tryptophan as a precursor. No 5HT uptake was found either in mitotic cells of this clone held at 33 degrees C, or in several other neuronal clones differentiating at 39 degrees C. Neither the neuronal nor the serotoninergic characteristics of clone 921202-6 developed in the presence of retinoic acid. It is concluded that 921202-6 cells differentiate under basal conditions down a neuronal pathway typical of an APUD cell, and that the choice of this pathway is made prior to the end of cell cycling. Furthermore, predisposition of the precursor cells to the neuronal/APUD phenotype can be overridden by extraneous epigenetic factors.

We have applied non-isotopic in situ hybridization (ISH) to interphase cell nuclei of 23 phaeochromocytomas (18 primary and 5 metastatic tumours) within routine paraffin-embedded tissue sections. Each tumour was screened for numerical aberrations with a defined alphoid repetitive DNA probe set containing DNA probes specific for chromosomes 1, 7, 15, and Y. Normal adrenal medullas and other normal human cell types served as cytogenetic controls. Preservation of tissue morphology enabled targeted analysis of tumour cells. The presence of numerical chromosome changes in the tumour cells could easily be evaluated by comparing the ISH results of the DNA probes. Numerical abnormalities not previously reported in this neoplasm included overrepresentation of chromosomes 1 and 7, loss of chromosome 15, and both gain and loss of chromosome Y (P values < 0.01). The percentage of aneuploid cell nuclei in a tumour correlated well with the percentage of cells in the 4C peak of flow cytometric DNA histograms from these neoplasms. We conclude that interphase ISH can be used for the identification of new and reported cytogenetic changes in tumour cell nuclei within archival tissue sections. This novel procedure also allows for retrospective analysis of previously not karyotyped material.

The adrenal medulla of mammals has a heterogeneous population of cells. In adults most are epithelial cells containing a particular type of cytoplasmic granule. Based on a variety of cytochemical and ultrastructural studies it is now accepted that 2 different adrenal medullary chromaffin cell types can be distinguished, i.e. noradrenaline (NA) and adrenaline (A) synthesising and storing cells. Other cell types present in the adrenal medulla include neuronal elements comprising either cell bodies or nerve fibres entering from outside the gland (extrinsic innervation). It is assumed that adrenal medullary cells have a limited life span, i.e. they are replaced after a certain period. Data on this replacement process are scarce. Recently, we initiated an investigation into this question using cytochemical procedures that enable the detection of DNA duplication to measure mitotic activity in individual cells. Female Sprague-Dawley rats aged 22-36 wk received a single i.p. injection of BrdU or BrdU was administered continuously via an implanted mini-osmotic pump. Cell nuclei that had incorporated BrdU were demonstrated using an indirect immunoperoxidase staining technique. At 1 h after a single injection, 0.46 +/- 0.07% of the adrenal medullary (chromaffin) cells were labelled. This increased to 0.77 +/- 0.08% after 12 h with no further increase during the next 7-8 d. With continuous infusion of BrdU the fraction of labelled cells increased gradually to about 40% after 73 d (the longest period studied). These results show that in adult rats adrenal medullary cells are able to divide, although at a slow rate (renewal rate of about 1%/day).

The distribution of individual cytokeratin polypeptides in the adult rat lung parenchyma was investigated by immunohistochemistry with 44 monoclonal and 2 polyclonal antibodies. Simple epithelial cytokeratins 7, 8, 18 and 19 were found to be expressed differently in alveolar and bronchial epithelial cells. Three distinct types of alveolar cells were detected according to their pattern of immunoreactivity: type II cells strongly expressing cytokeratins 8 and 18 and weakly expressing cytokeratins 7 and 19 in the cell periphery; type I cells predominantly positive for cytokeratins 7 and 19 and weakly for cytokeratin 8; and a newly defined third cell type III (alveolar brush cell) with cytokeratin 18 abundantly expressed but organized in an unusual intracellular ("globular") structure. The latter cell type failed to bind the type II specific Maclura pomifera lectin, and contained no surfactant proteins. Bronchial epithelial cells exhibited a more or less uniform staining pattern for cytokeratins 8, 18 and 19 and focally for cytokeratins 4 and 7.