Evidence for CCKB receptors in the guinea-pig kidney: localization and characterization by [125I]gastrin binding studies and by RT-PCR

Two types of receptors for gastrin and cholecystokinin (CCK) have been identified in the gastrointestinal tract and in the central nervous system: CCK_A and CCK_B receptors. Here we report evidence for the expression of CCK_B receptors in the guinea-pig kidney. Specific binding sites for [¹²⁵I]gastrin were detected in sections of the guinea-pig kidney: Binding was saturable, pH-, temperature- and time-dependent, and specific for gastrin-related peptides. The potencies for inhibition of binding of [¹²⁵I]gastrin were CCK-8 > gastrin 17-I > CCK_B receptor antagonist L-365,260 > des(SO₃)CCK-8 > CCK_A receptor antagonist L-364,718. Autoradiography demonstrated specific [¹²⁵I]gastrin binding to medullary collecting ducts and to a much lesser extent to glomeruli, but not over other structures. CCK_B receptor cDNA fragments were amplified by RT-PCR from total kidney, isolated tubuli and from tissues known to express CCK_B receptors such as stomach and brain. The kidney might therefore be a previously unidentified site of action for gastrin and cholecystokinin-related peptides. Received: 11 March 1998 / Accepted: 12 June 1998     

Combination of external-beam radiotherapy with intraoperative electron-beam therapy is effective in incompletely resected pediatric malignancies

PURPOSE: Intraoperative electron-beam radiotherapy (IOERT) has been applied for local dose escalation in over 1,400 patients in Heidelberg since 1991. Among these were 30 children, in 18 of whom IOERT was employed in radiation treatment with external-beam radiotherapy (EBRT) on account of incomplete resection. We address the question whether IOERT is able to compensate for microscopic or macroscopic tumor residue if employed in the overall radiation regimen. METHODS AND MATERIALS: The data of the aforementioned 18 children were analyzed with regard to local recurrence, overall survival, and complication rates. All children suffered from either sarcomas or neuroblastomas. In all children, IOERT was employed for local dose escalation after or before EBRT. RESULTS: After a median follow-up of 60.5 months, 15 of the treated children are alive. One local failure has been observed. Six children show clinically significant late morbidity, including the loss of a treated limb (Radiation Therapy Oncology Group Grade 4 [RTOG 4]), a severe nerve lesion (RTOG 3), an orthopedic complication (RTOG 2), a ureteral stenosis (not clinically significant), and a kidney hypotrophy (not clinically significant). In 1 child a fracture due to radionecrosis (RTOG 4) was diagnosed; however, in the follow-up, local tumor relapse was diagnosed as another possible reason for the fracture. CONCLUSIONS: Regarding the low incidence of local failure, IOERT seems to be able to compensate incomplete tumor resection in childhood sarcoma and neuroblastoma patients. The incidence of late morbidity is low enough to justify the employment of IOERT as part of the radiation treatment regimen for pediatric patients.     

Intraoperative electron boost radiation followed by moderate doses of external beam radiotherapy in limb-sparing treatment of patients with extremity soft-tissue sarcoma

PURPOSE: To analyze long-term prognosis and morbidity after limb-sparing treatment of patients with extremity soft-tissue sarcoma, with intraoperative electron boost radiotherapy (IOERT) followed by a moderate dose of external beam radiotherapy (EBRT). METHODS AND MATERIALS: A total of 153 patients who were treated in a single center from 1991 to 2004 were evaluated. Median IOERT dose was 15 Gy, mean EBRT dose 43 Gy (range, 40-50.4 Gy) in conventional fractionation (1.8-2 Gy). Median duration of follow-up was 33 months. Acute toxicity was assessed with Common Toxicity Criteria; late toxic effects were scored according to European Organization for Research and Treatment of Cancer/Radiation Therapy Oncology Group criteria. RESULTS: Five-year overall survival and 5-year local control rates were 77% and 78%, respectively. Whereas tumor size, patient age, and EBRT dose did not significantly affect outcome, resection status and grading were significant for survival; resection status and IOERT dose were significant for local control. Extremity salvage until death or time of follow-up was achieved in 90% of our patients, 86% of whom showed excellent limb function without impairment in activities of daily life. Acute toxicity Grade 2-4 was observed in 23% and late toxicity Grade 2-4 in 17% of patients. CONCLUSIONS: Treatment with IOERT combined with moderate doses of external beam irradiation yields high local control and extremity preservation rates in resected extremity soft-tissue sarcoma.     

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.     

In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen5,DTrp7,Dab8]urotensin II(4-11) (UFP-803)

The novel urotensin-II (U-II) receptor (UT) ligand, [Pen(5),DTrp(7),Dab(8)]U-II(4-11) (UFP-803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays. In the rat isolated aorta, UFP-803 was inactive alone but, concentration dependently, displaced the contractile response to U-II to the right, revealing a competitive type of antagonism and a pA(2) value of 7.46. In the FLIPR [Ca(2+)](i) assay, performed at room temperature in HEK293(hUT) and HEK293(rUT) cells, U-II increased [Ca(2+)](i) with pEC(50) values of 8.11 and 8.48. Urantide and UFP-803 were inactive as agonists, but antagonized the actions of U-II by reducing, in a concentration-dependent manner, the agonist maximal effects with apparent pK(B) values in the range of 8.45-9.05. In a separate series of experiments performed at 37 degrees C using a cuvette-based [Ca(2+)](i) assay and CHO(hUT) cells, urantide mimicked the [Ca(2+)](i) stimulatory effect of U-II with an intrinsic activity (alpha) of 0.80, while UFP-803 displayed a small (alpha=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22 degrees C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (alpha=0.11) and UFP-803 was completely inactive as an agonist. In vivo in mice, UFP-803 (10 nmol kg(-1)) antagonized U-II (1 nmol kg(-1))-induced increase in plasma extravasation in various vascular beds, while being inactive alone. In conclusion, UFP-803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP-803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.     

Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

We have compared the ability of three radioligands, [¹²⁵I]-cyanopindolol, [³H]-CGP 12,177 and [³H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [³H]-CGP 12,177 had very similar affinity for β₁- and β₂-adrenoceptors (about 40 pM), [¹²⁵I]-cyanopindolol and [³H]-dihydroalprenolol had 4- to 6-fold higher affinity for β₂- as compared to β₁-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [¹²⁵I]-cyanopindolol at β₃-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β₃-adrenoceptor affinity of [³H]-CGP 12,177 and [³H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [³H]-CGP 12,177 and [³H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β₃-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [¹²⁵I]-cyanopindolol appears the least unsuitable to label β₃-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β₃-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes.     

Treatment options for nausea and vomiting during pregnancy

Nausea and vomiting, common symptoms during pregnancy, often are regarded as an unpleasant but normal part of pregnancy during the first and early second trimesters. Nausea and vomiting of pregnancy (NVP) occurs in approximately 75-80% of pregnant women. The exact etiology and pathogenesis of NVP are poorly understood and are most likely multifactorial. Some theories for the etiology of NVP are psychological predisposition, evolutionary adaptation, hormonal stimuli, and Helicobacter pylori infection. Treatment ranges from dietary and lifestyle changes to vitamins, antiemetics, and hospitalization for intravenous therapy. Treatment generally begins with nonpharmacologic interventions; if symptoms do not improve, drug therapy is added. Although NVP has been associated with a positive pregnancy outcome, the symptoms can significantly affect a woman's life, both personally and professionally. Given the substantial health care costs, as well as indirect costs, and the potential decrease in quality of life due to NVP, providers need to acknowledge the impact of NVP and provide appropriate treatment.     

Detection of genetically modified DNA sequences in milk from the Italian market

The possible transfer and accumulation of novel DNA and/or proteins in food for human consumption derived from animals receiving genetically modified (GM) feed is at present the object of scientific dispute. A number of studies failed to identify GM DNA in milk, meat, or eggs derived from livestock receiving GM feed ingredients. The present study was performed in order to: (i) develop a valid protocol by PCR and multicomponent analysis for the detection of specific DNA sequences in milk, focused on GM maize and GM soybean; (ii) assess the stability of transgenic DNA after pasteurization treatment and (iii) determine the presence of GM DNA sequences in milk samples collected from the Italian market. Results from the screening of 60 samples of 12 different milk brands demonstrated the presence of GM maize sequences in 15 (25%) and of GM soybean sequences in 7 samples (11.7%). Our screening methodology shows a very high sensitivity and the use of an automatic identification of the amplified products increases its specificity and reliability. Moreover, we demonstrated that the pasteurization process is not able to degrade the DNA sequences in spiked milk samples. The detection of GM DNA in milk can be interpreted as an indicator of fecal or airborne contamination, respectively, with feed DNA or feed particles, although an alternative source of contamination, possibly recognizable in the natural environment can be suggested. Further studies, performed on a larger number of milk samples, are needed to understand the likely source of contamination of milk collected from the Italian market.     

Concentrations in plasma, urinary excretion and bactericidal activity of levofloxacin (500 mg) versus ciprofloxacin (500 mg) in healthy volunteers receiving a single oral dose

In a randomised crossover study, 14 volunteers received a single oral dose of 500 mg levofloxacin or 500 mg ciprofloxacin in order to assess plasma concentrations by high-pressure liquid chromatography (up to 24 h), urinary excretion and urinary bactericidal titres (UBTs) at intervals up to 120 h. The median maximum concentration of levofloxacin in plasma was 6.1 mg/L and that of ciprofloxacin was 2.3 mg/L. The median cumulative level of renal excretion of the administered dose of the parent drug was 81.2% for levofloxacin and 36.2% for ciprofloxacin. UBTs were determined for a reference strain and nine clinical uropathogens. The median UBTs of both quinolones measured within the first 12 h were between 0 and 1:≥1024, correlating with the minimum inhibitory concentrations (MICs) of the strains. For Gram-negative strains, the UBTs of both quinolones were comparable despite the lower MICs of ciprofloxacin. During further time courses, however, the UBTs of levofloxacin were significantly higher than those of ciprofloxacin. For Gram-positive strains, for which the MICs of levofloxacin were equal to or lower than those of ciprofloxacin, the UBTs of levofloxacin were already significantly higher from the beginning. It can be concluded that overall the doses of the two tested fluoroquinolones may be considered equivalent with regard to treatment of complicated urinary tract infections, although the recommended dosing is twice daily for ciprofloxacin and once daily for levofloxacin.     

Irinotecan (CPT-11) combined with bolus 5-fluorouracil/leucovorin (Saltz regimen) as first-line chemotherapy of patients with advanced colorectal cancer

Concerns about the safety of irinotecan (CPT-11) plus bolus 5-fluorouracil (5-FU)/leucovorin (LV) (the so-called Saltz regimen) have been previously reported. This prospective, multicenter, non-randomized study evaluated the anti-tumoral effect and toxicity of the Saltz regimen as first-line chemotherapy of 130 patients with advanced colorectal cancer (CRC). The median numbers of treatment cycles and infusions received per patient were 3 and 12, respectively. Eight (6.1) and 37 patients (28.5%) showed complete and partial responses, respectively [overall response rate=34.6% (95% confidence interval=20.7-48.5%)]. After a median follow up period of 9 months, 70 patients had died. The median progression-free survival and overall survival were 6.78 (0.3-33.8) and 8.26 months (range 0.3-33.8), respectively. The combined CPT-11/5-FU/LV treatment was well tolerated and no toxic deaths were reported. The most common grade 3/4 hematological toxicity was neutropenia (28% of patients and 3% of infusions), but no febrile neutropenia was reported. Delayed diarrhea was the most reported grade 3/4 non-hematological toxicity (21% of patients and 2% of infusions). Other non-hematological toxicities showed very low incidences. During the study five patients died due to factors not associated with disease progression. We conclude that the Saltz regimen administered on an outpatient basis was safe and well tolerated in patients with advanced CRC. Close monitoring of external patients together with an early treatment of toxicity was found to be essential to prevent severe and potentially fatal gastrointestinal or thromboembolic events previously reported with this CPT-11 combined regimen.