Abstracts

Ohne Zusammenfassung

---

Abstracts

     

Morphometric analysis of murine skin wound healing: Standardization of experimental procedures and impact of an advanced multitissue array technique

Morphometric data based on skin wounding offer important information for the characterization of the phenotype of transgenic mouse models. The goal of this study was the comparison of technical procedures concerning wounding, processing, and evaluation of samples in different mouse strains. The multitissue array technique was used to estimate its adaptability for standardized analysis in wound healing. Skin wounds between days 1 and 14 after wounding were analyzed. The influence of mouse strain (C57BI/6 vs. FVB/N mice), sex, size of the punch biopsies, and preparation of the tissue sections was investigated on 94 mice. The parameters distance between the migration tongues (deltaMT) and surface not covered by epithelium were evaluated to describe the reepithelialization, and the distance between the adnexa was chosen to measure wound contraction. In addition, the techniques to measure the area of granulation tissue (GT) were evaluated. The data illustrate the requirement of standardized conditions for skin wound-healing experiments and demonstrate that histological preparation in serial sections is mandatory to detect slight differences in wound contraction. For the analysis of cellular composition in GT, multitissue arrays are useful tools in wound-healing studies.     

Adenine nucleotide levels in preserved and ischemically injured canine kidneys

This study explored the relationship between adenine nucleotide levels in canine renal cortex and renal function following: (a) graded periods of warm ischemia; (b) 48 hours' flush cooling with electrolyte solutions and ice storage; and (c) continuous hypothermic perfusion. Exposure to normothermic ischemia resulted in a rapid (within 15 minutes) degradation of ATP to ADP and AMP as well as a slow decline in total adenine nucleotide (TAN) to levels which were proportional to the duration of the ischemic injury. No functional impairment was evident after 15 minutes' ischemia, but with longer times, both the extent of decline in TAN and the degree of recovery following restoration of blood flow could be used to predict the quality of renal function.The relationship between TAN levels and function was of less predictive value following cold storage or continuous perfusion. The efficacy of intracellular flush solutions could not be attributed solely to conservation of TAN, nor did the well-maintained TAN levels during continuous perfusion necessarily lead to significantly better 48-hour storage than flush cooling with C2 solution.

---

Résumé Cette étude faite chez le chien examine la corrélation entre la fonction rénale et le taux d'adénine nucléotide dans le cortex rénal suite à: 1) différentes périodes d'ischémic chaude; 2) entreposage sur glace pendant 48 heures apres flushing avec des solutions électrolytiques; 3) hypotermie par perfusion continuelle. L'ischémie chaude a produit une dégradation rapide de l'ATD en ADP et AMP (en moins de 15 minutes) et une diminution progressive de la quantité totale d'adénine nucléotide (TAN) proportioneile à la durée de la période ischémique. La fonction rénale n'a pas été affectée par des périodes d'ischémie de 15 minutes ou moins. Suite à des périodes ischémiques plus longues cependant, la chutte des taux de TAN et leur niveau de rétablissement après réouverture de la circulation sanguine ont permis de prédire la qualité de la fonction rénale. Après storage au froid ou perfusion continue, la relation entre les taux de TAN et la fonction rénale est disparue. Le flushing à l'aide des solutions de type intracellulaire, malgré sa faible capacité à conserver les taux de TAN s'est avéré une méthode d'entreposage aussi efficace que la perfusion continuelle, laquelle a maintenu des taux normaux de TAN.

---

---

Supported by Veterans Administration Research Grant 1519-01.     

Risk factors for relapse in clinical stage I nonseminomatous testicular germ cell tumors: results of the German Testicular Cancer Study Group Trial.

PURPOSE: To prospectively assess potential risk factors for relapse in clinical stage I nonseminomatous germ cell tumors of the testis (CS I NSGCT). PATIENTS AND METHODS: From September 1996 to May 2002, 200 patients with CS I NSGCT were prospectively assigned to retroperitoneal lymph node dissection (RPLND), and risk factor assessment was performed within a multicenter protocol. One hundred sixty-five patients had an adequate minimum follow-up of 12 months (mean, 34.5 months) or had pathologic stage II. RESULTS: Pathologic stage II disease was found in 27.9% of patients. Only 0.6% of patients relapsed in the retroperitoneum after confirmation of pathologic stage I disease. With reference pathology, vascular invasion (VI) was most predictive of stage in multifactorial analysis (accuracy, 65.1%). However, the positive predictive value (PPV) of VI to predict patients who have metastatic disease or relapse during follow-up was only 52.7%. With absent VI, low-risk patients had a negative predictive value (NPV) of 76.9%. With a combination of several risk factors, the PPV increased to 63.6% and the negative predictive value increased to 86.5%. CONCLUSION: Even with an optimal combination of prognostic factors and reference pathology, more than one third of patients predicted to have pathologic stage II or relapse during follow-up will not harbor metastatic disease and, therefore, would be overtreated with adjuvant therapy. However, patients at low risk may be predicted at an 86.5% level, and thus, surveillance in highly compliant patients would be a valuable option. For high-risk patients, further reduction of adjuvant treatment is necessary.     

Serotonin and the 5‐HT7 receptor: The link between hepatocytes,IGF‐1 and small intestinal neuroendocrine tumors

Platelet‐derived serotonin (5‐HT) is involved in liver regeneration. The liver is also the metastatic site for malignant enterochromaffin (EC) cell “carcinoid” (neuroendocrine) neoplasms, the principal cellular source of 5‐HT. We hypothesized that 5‐HT produced by metastatic EC cells played a role in the hepatic tumor‐microenvironment principally via 5‐HT₇ receptor‐mediated activation of hepatocyte IGF‐1 synthesis and secretion. Using isolated rat hepatocytes, we evaluated 5‐HT₇ receptor expression (using PCR, sequencing and western blot). ELISA, cell transfection and western blots delineated 5‐HT‐mediated signaling pathways (pCREB, AKT and ERK). IGF‐1 synthesis/secretion was evaluated using QPCR and ELISA. IGF‐1 was tested on small intestinal neuroendocrine neoplasm proliferation, while IGF‐1 production and 5‐HT₇ expression were examined in an in vivo SCID metastasis model. Our results demonstrated evidence for a functional 5‐HT₇ receptor. 5‐HT activated cAMP/PKA activity, pCREB (130–205%, P < 0.05) and pERK/pAKT (1.2–1.75, P < 0.05). Signaling was reversed by the 5‐HT₇ receptor antagonist SB269970. IGF‐1 significantly stimulated proliferation of two small intestinal neuroendocrine neoplasm cell lines (EC₅₀: 7–70 pg/mL) and could be reversed by the small molecule inhibitor BMS‐754807. IGF‐1 and 5‐HT were elevated (40–300×) in peri‐tumoral hepatic tissue in nude mice, while 5‐HT₇ was increased fourfold compared to sham‐operated animals. We conclude that hepatocytes express a cAMP‐coupled 5‐HT₇ receptor, which, at elevated 5‐HT concentrations that occur in liver metastases, signals via CREB/AKT and is linked to IGF‐1 synthesis and secretion. Because IGF‐1 regulates NEN proliferation, identification of a role for 5‐HT₇ in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine‐producing mid‐gut tumors.     

Intravascular ultrasonic comparative analysis of degree of intimal hyperplasia produced by four different stents in the coronary arteries

Intravascular ultrasound studies were performed at angiographic follow-up on 121 native coronary lesions treated with 1 bare metal stent (n = 50), high-dose dexamethasone-eluting stents (n = 18), non-polymer-based paclitaxel-eluting stents (n = 18), or sirolimus-eluting stents (n = 35). Paclitaxel- and sirolimus-eluting stents reduced mean intimal hyperplasia thickness compared with bare metal stents by 49% and 90% (p = 0.048 and p <0.001), respectively, whereas mean intimal hyperplasia thickness treated with dexamethasone-eluting stents was similar to those lesions treated with bare metal stents.     

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.