Endosonography (ES) in the diagnosis of recurrent cancer of the rectum.

The ability of endosonography (ES) to detect local recurrence after "curative" surgery for rectal cancer was investigated in 65 patients. Fifteen patients developed local recurrence. All of these 15 patients had ES evidence of recurrence; in four cases recurrence was detected by ES alone. Ten of the 15 patients with local failure underwent reoperation. Four patients were candidates for reoperation for cure. Three patients with radical reoperation belonged to the group in which recurrence was detected by ES alone. ES is able to detect small tumors before there is evidence of recurrence in other available forms of examination.     

Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer

Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.     

Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.     

Über die sogenannten Myxome des Herzens

Zusammenfassung Die 15jährige Patientin litt wiederholt an Angina, die wohl über einer latenten Sepsis zu einer Endokardveränderung führte. Diese bedingte einen Thrombus des linken Vorhofes in der Gegend der Fossa ovalis. Aus der Zeit rühren embolische Narben der Milz und der linken Niere her. Unter eintretender Organisation des Thrombus wurde er durch den Blutstrom zu einem polypenförmigen Gebilde abgeglättet und ausgezogen. Er reichte nun durch das Ostium venosum sin. hindurch in die linke Herzkammer hinein und täuschte klinisch eine Insuffizienz der Klappe mit Stenose des Ostiums vor. Der organisierte Thrombus entartete in seinem größten Teile schleimig, während der neu angebildete Teil an der Basis auch jetzt noch wie ein typischer organisierter Thrombus aussah. Mikroskopisch konnte ich alle Bestandteile nachweisen, die die Autoren in den Myxomen beschrieben. Auch fand ich entzündliche Elemente, ebenso hyalines Fibrin, besonders in dem jüngeren, basalen Teile des Gebildes. Aus alledem komme ich zur Diagnose Thrombus myxomatodes und meine, daß auch alle die bisher beschriebenen primären Tumoren des endokards, außer den bösartigen Gewächsen, organisierte Thromben darstellen.Mit 5 Textabbildungen.     

Use of soluble recombinant TNF receptor to improve detection of TNF secretion in cultures of tumor infiltrating lymphocytes.

The accurate quantitation of picogram amounts of TNF is possible by ELISA and is useful in many areas of biomedical research, including studies of TNF release in vitro by stimulated lymphocytes and macrophages, and of serum levels in patients with cancer and sepsis. However, we show in this report that the detection of recombinant TNF standards by ELISA falls over time with incubation at 37 degrees C, and is further decreased when incubated with tumor infiltrating lymphocytes (TIL), making accurate quantitation difficult. We demonstrate that the soluble dimeric form of the TNF receptor can prevent this decrease, both in the presence and absence of TIL. In contrast, the soluble monomeric TNF receptor was much less effective in preventing this decrease. In addition, the dimeric but not the monomeric TNF receptor was found to inhibit bioactivity of TNF as measured by L929 cytotoxicity. The dimeric TNF receptor does not interfere with the detection of recombinant TNF standards by ELISA, and entirely stabilizes TNF levels incubated over 48 h at 37 degrees C in the presence and absence of TIL. This protection is specific, and the TNF receptor does not stabilize interferon-gamma. The dimeric form of the soluble TNF receptor has proven useful in detecting TNF released by TIL transduced with the TNF cDNA that are currently being used in studies of the gene therapy of cancer with TIL. The dimeric TNF receptor may also prove useful in the accurate quantitation of TNF released by stimulated lymphocytes and macrophages in vitro, and in the quantitation of serum TNF levels in patients.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Bestimmung der Bindung von Trijodthyronin an Serumproteine mittels Dextran-Gel-Filtration

Zusammenfassung 1. Es wird eine Methode zur gleichzeitigen Bestimmung des sog. freien und des proteingebundenen Anteils von in vitro zugesetztem L-Trijodthyronin-¹³¹Jod im Serum mittels Dextran-Gel-Filtration angegeben. In der beschriebenen Form ist diese Technik für die routinemäßige Anwendung in der Klinik zur Bestimmung der Bindungs- und Transportverhältnisse von Trijodthyronin geeignet.2. In sog. Verdrängungsversuchen wurde nichtmarkiertes Trijodthyronin dem Inkubationsgemisch von Serum und L-Trijodthyronin-¹³¹Jod zugesetzt. Die zugesetzten Trijodthyroninmengen erschöpfen die Gesamtbindungskapazität der Serumproteine in dem gewählten Konzentrationsbereich keineswegs. Im Gegensatz zum Verhalten der prozentualen Anteile des sog. freien und des proteingebundenen Trijodthyronins steigt die absolute Menge des proteingebundenen Trijodthyronins dabei steil an. Man findet eine Kurve, die nicht einer einfachen Sättigunskurve entspricht, sondern eine Resultante aus Sättigungskurven verschiedener Trijodthyronin-bindender Proteine und Verdrängungskurven kompetitiv gebundener Substanzen (z.B. Thyroxin) darstellt.3. Dextran-Gel wirkt nicht als einfaches Molekülsieb für Trijodthyronin. Es greift vielmehr durch Adsorptionsvorgänge kompetitiv in die Serumproteinbindungsverhältnisse des Trijodthyronins ein. Die physiologische Bedeutung des sog. freien Anteils an Trijodthyronin wird diskutiert.4. Die Methode zur Bestimmung des proteingebundenen Jods (PB¹²⁷I) mittels alkalischer offener Veraschung (Barker) wurde technisch vereinfacht und bezüglich ihrer Reproduzierbarkeit untersucht. Die¹³¹Jodausbeute aus zugesetztem L-Thyroxin-¹³¹Jod lag bei diesem Verfahren bei ca. 80%.

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Summary 1. A method allowing the simultaneous determination in serum of the socalled free and the protein bound part of 1-triiodothyronine-¹³¹I added in vitro, using dextran gel filtration, is presented. Assessment of serum protein binding and transport of triiodothyronine can be conveniently performed for clinical purposes by this procedure.2. In socalled discharge experiments non-labelled triiodothyronine is added to incubation mixtures of serum and l-triiodothyronine-¹³¹I. The amount of added triiodothyronine did not exhaust the total binding capacity of serum proteins for triiodothyronine. In contrast to the behaviour of the percentages of socalled free and protein bound triiodothyronine the absolute amount of protein bound triiodothyronine was rising linearly with rising concentrations of triiodothyronine added. The curve obtained was interpreted as resulting from saturation curves of different triiodothyronine binding proteins and discharge curves of competitively bound substances, e.g. thyroxine.3. Dextran gel is not acting merely as molecular sieve for triiodothyronine, but rather competing actively with serum proteins for triiodothyronine, adsorbing the latter. The physiological rôle of the socalled free triiodothyronine is discussed.4. With addition of l-thyroxine-¹³¹I 80% of¹³¹iodine was recovered, when the method ofBarker for determination of serum protein bound¹²⁷iodine (open alkaline ashing) was used.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.

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Herrn Prof. Dr. Dr. h. c.Carl Krauspe in Verehrung zum 70. Geburtstag gewidmet.