Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography.

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by beta-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC(50) values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s.     

The use of recombinant-activated factor VII in von Willebrand disease: a case series.

Spontaneous and surgery-associated bleeding in patients with von Willebrand disease (vWD) cannot always be controlled with desmopressin or replacement therapy. This paper presents results on the use of recombinant-activated factor VII (rFVIIa) in patients with vWD included in the internet registry Haemostasis.com. Twenty-eight reports on the use of rFVIIa in vWD were identified from the database and included in this analysis. The bleeding episodes were classified as mild (n = 7), moderate (n = 16), or severe (n = 2), and were unspecified in three cases. The median dose of rFVIIa administered was 94 microg/kg body weight (40-127.3 microg/kg). Bleeding stopped in 23 of 27 evaluable patients (85%) and markedly decreased in three patients; the total response rate was 96% (26/27 patients). Response did not correlate with the type of vWD, the site or severity of the initial bleed, or the rFVIIa dose. Other replacement therapies were infrequently used, and their use was similar in the 24 h before and after rFVIIa administration. Eighteen patients also received antifibrinolytic treatment, but its impact on response was not recorded. Only one adverse event (mild fever) was observed. These cases suggest a role for rFVIIa as a safe and effective therapy for vWD.     

Standardized quick en bloc technique for procurement of cadaveric liver grafts for pediatric liver transplantation

This paper describes a quick procedure for cadaveric liver graft retrieval during multiple organ harvesting. The technique is based on minimal preliminary dissection, absence of in situ direct portal perfusion, and en bloc removal of the liver and pancreas, with an aortic patch encompassing the coeliac trunk and superior mesenteric artery. The results of 110 pediatric liver transplantations with 109 organs harvested using this technique are reported. There were no graft harvesting injuries. The liver graft primary nonfunction rate was 4.5% (5/110). The 3-month retransplantation rate was 10%. The actual patient survival rates were 93% at 3 months and 90% at 1 year; actual graft survival rates were 85.5% and 78%, respectively. The technique described was at least as safe as conventional procedures. A major advantage of the procedure is its flexibility, which allows for the easily combined procurement of other organs (whole pancreas and intestine).     

Localizing and photosensitizing mechanism by tetra(3-hydroxyphenyl)porphin in vivo on human malignant melanoma xenografts in athymic nude mice

Observations on time-dependent localization of tetra(3-hydroxyphenyl)porphin (3-THPP) in human malignant melanoma transplanted to athymic nude mice from 1 to 120 h after intraperitoneal (i.p.) 10 mg kg^–1 b.w. administration were made by means of fluorescence microscopy. Fluorescence was found on the membrane of the melanoma cells and in the cytoplasm with a peak fluorescence intensity at 24 h post-injection of 3-THPP. The growth of the tumour cells was obviously inhibited at an early stage after PCT. Morphological changes of the tumour at various intervals after treatment by PCT with 3-THPP were also observed. Diffuse degeneration of the tumour cells with swelling of mitochondria and endoplasmic reticulum, heterochromatin aggregation and margination, etc., and subsequently diffuse necrosis with little or no the background of tumorous vascular response were found at an early stage after PCT. On the other hand, it was also observed that the necrosis of the melanoma areas was caused as a consequence of tumorous vascular injury at a later stage after PCT. Thus, two tumoricidal processes caused by PCT with 3-THPP were seen: a direct phototoxic action on tumour cells at an early stage after PCT and an indirect effect secondary to tumorous vascular injury at a later period after PCT.     

Verapamil-digoxin interaction in chronic hemodialysis patients

Verapamil increases the serum-digoxin concentration (SDC) in digoxin treated normals due to a compromised renal and extrarenal clearance. In chronic hemodialysis patients (CHD-patients) treated with digoxin where the renal elimination is diminished, verapamil has been shown to cause substantial increases of SDC with risk of digoxin intoxication. The effect of verapamil treatment on SDC in 8 nearly anephric (Uvol less than 1 l/d) CHD-patients on digoxin treatment was assessed. The patients were continuously treated with verapamil for two periods of two weeks at two dosage levels, 120 mg/d and 240 mg/d, whereafter verapamil was withdrawn. SDC and serum-verapamil were measured weekly. The SDC increased from 1.1 mmol/l to 1.7 mmol/l (p less than 0.05, N = 7) during the first two weeks. Increasing the dose of verapamil to 240 mg/d did not cause a further increment in SDC; on the contrary, the mean SDC decreased. The SDC increments varied between 0 and 200% of baseline values. We conclude that verapamil treatment decreases digoxin clearance in CHD-patients and that the influence of verapamil on SDC in CHD-patients shows great interindividual variation with no close dose dependency and decreases to pretreatment level in 2-3 weeks.     

Hypophyseal Non-Hodgkin's Lymphoma presenting with clinical panhypopituitarism successfully treated with chemotherapy

Summary A patient is described with a testicular Non-Hodgkin's Lymphoma (NHL) presenting with panhypopituitarism caused by a hypophyseal localization. A⁶⁷Gallium scintigraphy showed avid uptake in the hypophyseal region. Obviously⁶⁷Gallium could reach the tumor, by the intravenous route, which was the reason to treat the patient with intravenous chemotherapy. A complete remission was induced, which seems to be lasting (+ 25 months). As far as we know this is the first report of panhypopituitarism caused by a hypophyseal NHL in the hypophysis and successfully treated by intravenous chemotherapy.     

Polymerization of lactams, 88. Copolymers poly(ϵ-caprolactam)-block-polybutadiene prepared by anionic polymerization,part III. Model polymerizations initiated with potassium salt of ϵ-caprolactam and accelerated with isocyanates and their derivatives

Anionic polymerization of ?-caprolactam was initiated with the potassium salt of ?-caprolactam and accelerated with phenyl isocyanate, toluylene diisocyanate, 4,4′-diphenylmethane diisocyanate, some derivatives of these isocyanates (urethanes, ureas, and allophanates), or combinations of phenyl isocyanate with its derivatives at 150°C. The effect of individual structures on the polymerization kinetics and their contribution to the preparation of block copolymers of ?-caprolactam with hydroxy-terminated prepolymers, in-situ functionalized with diisocyanates, are discussed on the basis of a detailed analysis of time functions of polymer yield and degree of polymerization.     

Fas receptor expression on B-lineage cells

Mice homozygous for the lpr mutation have B and T cell defects and develop autoantibodies, suggesting that lpr plays a role in their genesis. The lpr defect has been identified as a mutation in the apoptosis-associated Fas receptor (FasR) gene. To begin to define the role of FasR in B cells, we have surveyed FasR expression on B-lineage cells from early progenitors in the bone marrow through their maturation in the periphery. Contrary to some reports, we found that FasR is expressed on B cells at all stages of their development and is highest on germinal center B cells. FasR is not expressed on lpr/lpr-derived cells. These data are consistent with the idea that lpr/lpr mice have an intrinsic B cell defect that may be manifested in developing as well as peripheral B cells. An unexpected finding is that B-1 (CD5) B cells do not constitutively express FasR: FasR becomes detectable on B-1 B cells only after activation.