Accumulation of small hyaluronan oligosaccharides in tumour interstitial fluid correlates with lymphatic invasion and lymph node metastasis.

BACKGROUND: Association studies have implicated the glycosaminoglycan hyaluronan (hyaluronic acid, HA) and its degrading enzymes the hyaluronidases in tumour progression and metastasis. Oligosaccharides of degraded HA have been ascribed a number of biological functions that are not exerted by high-molecular-weight HA (HMW-HA). However, whether these small HA oligosaccharides (sHA) have a role in tumour progression currently remains uncertain due to an inability to analyse their concentration in tumours. METHODS: We report a novel method to determine the concentration of sHA ranging from 6 to 25 disaccharides in tumour interstitial fluid (TIF). Levels of sHA were measured in TIF from experimental rat tumours and human colorectal tumours. RESULTS: While the majority of HA in TIF is HMW-HA, concentrations of sHA up to 6 µg ml(-1) were detected in a subset of tumours, but not in interstitial fluid from healthy tissues. In a cohort of 72 colorectal cancer patients we found that increased sHA concentrations in TIF are associated with lymphatic vessel invasion by tumour cells and the formation of lymph node metastasis. CONCLUSIONS: These data document for the first time the pathophysiological concentration of sHA in tumours, and provide evidence of a role for sHA in tumour progression.

Flow cytometry-based isolation of dermal lymphatic endothelial cells from newborn rats.

The lymphatic system plays a key role in tissue homeostasis, fatty acid transport, and immune surveillance. Pathologically, dysfunction of the lymphatic system results in edema, and increased lymphangiogenesis can contribute to tumor metastasis. Lymphatic vessels are composed of lymphatic endothelial cells (LECs) that can be identified by distinct marker molecules such as Prox-1, podoplanin, VEGFR-3 and LYVE-1. Primary LECs represent a valuable tool for the study of basic functions of the lymphatic system. However, their isolation remains a challenge, particularly if rodent tissues are used as a source. We developed a method for the isolation of rat dermal LECs from the skin of newborn rats based on sequential enzymatic digestion with trypsin and Liberase followed by flow cytometric sorting using LYVE-1 specific antibodies. Cells isolated according to this protocol expressed the lymphatic markers Prox-1, podoplanin, LYVE-1 and VEGFR-3, and displayed an endothelial-like morphology when taken into culture. These primary cells can be used for studying lymphatic biology in rat models, and the protocol we describe here therefore represents an important extension of the experimental repertoire available for rats and for modeling the human lymphatic system.

PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice.

BACKGROUND: PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. METHODS: Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. RESULTS: Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCγ1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. CONCLUSIONS: PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

ASAP1 promotes tumor cell motility and invasiveness, stimulates metastasis formation in vivo, and correlates with poor survival in colorectal cancer patients.

We have previously performed an unbiased screen to identify genes whose expression is associated with the metastatic phenotype. Secondary screening of these genes using custom microarray chips identified ASAP1, a multi-domain adaptor protein with ADP-ribosylation factor-GAP activity, as being potentially involved in tumor progression. Here, we show that at least three different splice forms of ASAP1 are upregulated in rodent tumor models in a manner that correlates with metastatic potential. In human cancers, we found that ASAP1 expression is strongly upregulated in a variety of tumors in comparison with normal tissue and that this expression correlates with poor metastasis-free survival and prognosis in colorectal cancer patients. Using loss and gain of function approaches, we were able to show that ASAP1 promotes metastasis formation in vivo and stimulates tumor cell motility, invasiveness, and adhesiveness in vitro. Furthermore, we show that ASAP1 interacts with the metastasis-promoting protein h-prune and stimulates its phosphodiesterase activity. In addition, ASAP1 binds to the SH3 domains of several proteins, including SLK with which it co-immunoprecipitates. These data support the notion that ASAP1 can contribute to the dissemination of a variety of tumor types and represent a potential target for cancer therapy.

Stereotactic radiotherapy of targets in the lung and liver.

BACKGROUND: Stereotactic irradiation of extracranial targets offers a non-invasive treatment modality for patients with localized tumors, which are not amenable for surgery or other invasive approaches because of age or impaired medical condition. The purpose of the study was the evaluation of the method to achieve local control of irradiated targets in relation to treatment toxicity. PATIENTS AND METHODS: Irradiation was performed as hypofractionated treatment in three fractions of 10 Gy each, normalized to the PTV enclosing 65% isodose with patient fixation in a stereotactic body frame. The isocenter was localized by stereotactic coordinates. Targets were circumscribed tumors in the lung (n = 27) and liver (n = 24) not amenable for other treatment modalities: primary lung cancer (n = 12), local recurrences of lung cancer (n = 4), lung metastases (n = 11), liver metastases (n = 23) and one cholangiocellular carcinoma. Median CTV/PTV for targets in the lung was 57/113 cm3 (min/max 5-277 cm3/17-343 cm3) and for targets in the liver 50/102 cm3 (min/max 9-516 cm3/42-772 cm3). Median follow-up for targets in the lung was 8 months (2-33) and 9 months (2-28) for liver targets. Local control was defined as complete or partial remission and stable disease, measured by repeated CT scans after 6 weeks and in 3 months intervals. Treatment toxicity was evaluated according to the WHO score. RESULTS: Crude local control was 85% for pulmonary targets and 83% for hepatic targets. Actuarial local control after 1 and 2 years was 76% and 76% for lung tumors and 76% and 61% for liver tumors. Actuarial overall patient survival was 48% after 1 year and 21% after 2 years for targets in the lung and 71% and 43% for targets in the liver. No acute grade 3-5 side effects were observed. Serious late toxicity occurred in two patients: a chronic ulceration of the esophagus at a target close to the mediastinum after 3 months (grade 3) and fatal bleeding from the pulmonary artery after 9 months (grade 5) in a previously irradiated patient. It remained unclear, whether the bleeding was a side effect of irradiation or due to tumor infiltration. CONCLUSION: Hypofractionated stereotactic irradiation of targets in the lung and liver is a locally effective treatment with actuarial local control rates of 76% after 1 year and 61-76% after 2 years without relevant acute toxicity. Severe late toxicity did not occur, if targets close to the mediastinum were avoided.

Restoring vascular nitric oxide formation by L-arginine improves the symptoms of intermittent claudication in patients with peripheral arterial occlusive disease.

BACKGROUND: Administration of L-arginine improves nitric oxide (NO) formation and endothelium-dependent vasodilation in atherosclerotic patients. OBJECTIVES: We investigated in this double-blind, controlled study whether prolonged intermittent infusion therapy with L-arginine improves the clinical symptoms of patients with intermittent claudication, as compared with the endothelium-independent vasodilator prostaglandin E1, and control patients. METHODS: Thirty-nine patients with intermittent claudication were randomly assigned to receive 2 x 8 g L-arginine/day, or 2 x 40 microg prostaglandin E1 (PGE1)/day or no hemodynamically active treatment, for 3 weeks. The pain-free and absolute walking distances were assessed on a walking treadmill at 3 km/h, 12% slope, and NO-mediated, flow-induced vasodilation of the femoral artery was assessed by ultrasonography at baseline, at 1, 2 and 3 weeks of therapy and 6 weeks after the end of treatment. Urinary nitrate and cyclic guanosine-3', 5'-monophosphate (GMP) were assessed as indices of endogenous NO production. RESULTS: L-Arginine improved the pain-free walking distance by 230+/-63% and the absolute walking distance by 155+/-48% (each p < 0.05). Prostaglandin E1 improved both parameters by 209+/-63% and 144+/-28%, respectively (each p < 0.05), whereas control patients experienced no significant change. L-Arginine therapy also improved endothelium-dependent vasodilation in the femoral artery, whereas PGE1 had no such effect. There was a significant linear correlation between the L-arginine/asymmetric dimethylarginine (ADMA) ratio and the pain-free walking distance at baseline (r=0.359, p < 0.03). L-Arginine treatment elevated the plasma L-arginine/ADMA ratio and increased urinary nitrate and cyclic GMP excretion rates, indicating normalized endogenous NO formation. Prostaglandin E1 therapy had no significant effect on any of these parameters. Symptom scores assessed on a visual analog scale increased from 3.51+/-0.18 to 83+/-0.4 (L-arginine) and 7.0+/-0.5 (PGE1; each p < 0.05), but did not significantly change in the control group (4.3+/-0.4). CONCLUSIONS: Restoring NO formation and endothelium-dependent vasodilation by L-arginine improves the clinical symptoms of intermittent claudication in patients with peripheral arterial occlusive disease.

Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease.

BACKGROUND: We studied urinary nitrate and cGMP excretion rates, indices of systemic NO formation, and plasma concentrations of L-arginine and the endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and its inactive stereoisomer, symmetrical dimethylarginine, in 77 patients with peripheral arterial occlusive disease (PAOD) in Fontaine stages IIb through IV and in 47 young and 37 elderly healthy control subjects. METHODS AND RESULTS: Urinary nitrate excretion was 182.0+/-11.4 micromol/mmol creatinine and cGMP excretion was 186.2+/-13.0 nmol/mmol creatinine in young healthy control subjects. In elderly control subjects, both excretion rates were slightly lower (nitrate, 156.0+/-7.8 micromol/mmol creatinine; cGMP, 150.0+/-8.3 nmol/mmol creatinine; P=NS). In PAOD patients, there was a significant, progressive reduction of urinary nitrate (IIb, 138.4+/-11.9; III, 128.6+/-11.3; and IV, 91.9+/-8.0 micromol/mmol creatinine; P<.05) and cGMP (IIb, 139.9+/-25.2; III, 115.6+/-13.1; and IV, 76.9+/-7.9 nmol/mmol creatinine; P<.05) excretion rates related to the Fontaine stage of PAOD. These changes were independent of changes in renal excretory function. Plasma L-arginine concentrations were not significantly different between the groups, but ADMA concentrations were elevated in PAOD patients (young control subjects, 1.25+/-0.11; elderly control subjects, 1.01+/-0.05 micromol/L; IIb, 2.62+/-0.24; III, 3.06+/-0.48; and IV, 3.49+/-0.26 micromol/L; P<.05 for PAOD versus control subjects). There was a significant linear correlation between urinary nitrate and cGMP excretion rates and a significant negative linear correlation between plasma ADMA concentrations and urinary nitrate excretion. CONCLUSIONS: In PAOD patients, there is a progressive reduction in urinary nitrate and cGMP excretion rates, which may be caused in part by accumulation of ADMA, an endogenous inhibitor of NO synthase.

[Evaluating guidelines for "selection and training of staff for odor analysis"--a 7-year longitudinal study]. / Uberprüfung der Richtlinie zur "Auswahl und Schulung von Personen für die Geruchsanalyse"--eine 7 Jahres-Längsschnittuntersuchung.

During a 7 years longitudinal study on a representative group the guideline "selection and schooling of personal for odour analysis" was reexamined. It could be shown that the selection in two steps is well suited to recognize anosmia and hyposmia, respectively and that normal osmia is well to be characterized by smell scores of 30-42. A statistical significant training effect could be shown with the qualitative test for training of the odour memory as well as the articulation in describing of smell impressions. The result of the rank order test demonstrate the practicable subdivision of the odour intensity into 5 degrees.

[Gas chromatography studies of exhaust air from an animal body waste salvage industry]. / Gaschromatographische Untersuchungen der Abluft eines Tierkörperverwertungsbetriebes (TKVB).

The proceedings of animal body waste salvage plants are-as you know-connected with intense smell importunities of the immediate environment. Suited desodorization plants are required to limit this problem of the environmental hygiene. The presupposition for a high effectiveness of these plants is to know the qualitative composition of the exhaust air, the analysis of which is the subject of the article in hand.

[Atrial arrhythmias after surgical repair of an atrial septal defect (secundum-type). Six years follow-up in adults (author's transl)]. / Atriale Arrhythmien nach operativem Verschluss eines Vorhofseptumdefektes (Secundum-Typ). Postoperative Langzeitbeobachtungen im Erwachsenenalter.

Atrial arrhythmias before and after repair of an atrial septal defect (secundum-type) were analyzed in 100 patients. The mean postoperative follow-up was 5.8 years, the mean age at the last check-up was 33.4 years. Preoperatively, 10 patients showed atrial arrhythmias (6 pts atrial flutter or fibrillation [AF], 3 pts sinus node [SN] dysfunction, 1 pt atrial tachycardia). About 6 years after operation, 29 patients showed atrial arrhythmias (16 pts AF, 10 pts SN dysfunction, 3 pts atrial tachycardias). The 26 patients with AF and SN dysfunction were 10 years older and had a 3 years longer follow-up period postoperatively than those with sinus rhythm. In addition, they had a higher pulmonary artery pressure preoperatively. Especially the occurrence of AF after operation was strongly related to the age and the pulmonary artery pressure. All other factors had no significance. The data show that closure of an atrial septal defect cannot prevent the occurrence of AF in the following period. In addition, SN dysfunction is found postoperatively in a significant number of patients probably due to the intraoperative injury to the sinus node. However, it cannot be excluded that the unfavorable results may in part be due to the selection of the patients.

[Treatment of choledocholithiasis in the elderly]. / Zur Behandlung der Choledocholithiasis im höheren Alter.

Treatment of cholelithiasis and its complications in aged patients is a very difficult problem. Early cholecystectomy is regarded as riskless in patients less than 60 years old. In case of optimal indications we perform choledochoduodenostomy following cholecystectomy. To assess late results 68 patients aged 60-90 years were examined 20 to 7 years after primary operation. The results were good in 86,7 per cent.

The transfer of J blood-group activity from J-containing bovine serum to J-negative erythrocytes.

The J blood-group activity of bovine serum is contained both in a lipid and in a nonlipid fraction. This is also true for calf serum. It demonstrated that the J determinant is transferred from a serum protein onto the erythrocyte membrane by incubation in vitro. Even though the donor of J activity is a lipid-free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (probably a carbohydrate unit) must have been detached from a serum glycoprotein and transferred to a lipidic receptor (probably a glycosphingolipid) at the erythrocyte membrane. It is suggested than an enzyme system located in or at the erythrocyte membrane is responsible for the transfer of J substance. The transfer of J substance is inhibited by a polar lipid present in bovine serum.