Factors influencing the collection of peripheral blood stem cells in patients with acute myeloblastic leukemia and non-myeloid malignancies

Factors influencing the collection of autologous peripheral blood stem cells (PBSCs) were studied in 182 mobilization procedures performed on 145 consecutive patients with acute myeloblastic leukemia (AML; n=67) and with various non-myeloid malignancies (NMM; n=78). PBSC were collected following mobilization with chemotherapy, treatment with granulocyte colony-stimulating factor (G-CSF) or chemotherapy plus G-CSF. Fewer colony-forming unit granulocyte-macrophages (CFU-GMs) were collected from patients with AML than from patients with NMM (P<0.0001), although there were no differences in the numbers of CD34+ cells collected between both groups. Multiple regression analysis showed that chemotherapy alone was predictive of a low CD34+ yield in patients with NMM (regression coefficient (RC)=-2.1; P=0.003). In addition, the interactions "diagnosis mutliple myeloma (MM)xmobilization with chemotherapy" (RC=2.9; P=0.004) and "diagnosis MMxmobilization with chemotherapy plus G-CSF" (RC=2.1; P=0.04) also remained in the model, both showing a favorable influence. In AML, mobilization with chemotherapy plus G-CSF was associated with higher CD34+ yields (P=0.003). In this subgroup of patients, multiple regression analysis identified the number of cycles of previous chemotherapy (< or =2 cycles; RC=1.3; P=0.03) and peripheral blood counts (WBC > or =1.5 x 10(9)/l and monocytes >20%; RC=0.8; P=0.02) as the factors most predictive of CD34+ cell yield. These findings emphasize the need to optimize harvesting technique to enhance safety and minimize morbidity and costs of this valuable procedure.     

Concomitant lower thoracic spine disc disease in lumbar spine MR imaging studies

Our objective was to study the coexistence of lower thoracic-spine disc changes in patients with low back pain using a large field of view (FOV) in lumbar spine MR imaging. One hundred fifty patients with low back pain were referred to an MR examination. All patients were studied with a large FOV (27 cm), covering from the coccyx to at least the body of T11. Discs were coded as normal, protrusion, and extrusion (either epiphyseal or intervertebral). The relationship between disc disease and level was established with the Pearson χ² test. The T11–12 was the most commonly affected level of the lower thoracic spine with 58 disc cases rated as abnormal. Abnormalities of T11–12 and T12–L1 discs were significantly related only to L1–L2 disease (p=0.001 and p=0.004, respectively) but unrelated to other disc disease, patient's gender, and age. No correlation was found between other discs. Magnetic resonance imaging of the lumbar spine can detect a great amount of lower thoracic disease, although its clinical significance remains unknown. A statistically significant relation was found within the thoracolumbar junctional region (T11–L2), reflecting common pathoanatomical changes. The absence of relation with lower lumbar spine discs is probably due to differences in their pathomechanisms. Electronic Publication     

Carotid endarterectomy in octogenarians with symptomatic high-grade internal carotid artery stenosis: long-term clinical and duplex follow-up

The objective of this study was to evaluate the clinical and duplex outcome after carotid endarterectomy (CEA) in recently symptomatic patients aged 80 years or older. Information was assembled from a prospective data collection of all CEAs performed from January 1986 to December 1999. Included were all patients with recently symptomatic carotid artery stenosis who were aged 80 years or older at time of operation. Thirty-two patients, with a mean age of 82 years, were included. Outcome events were stroke, death, and restenosis (more than 50% diameter reduction) during routine duplex scan follow-up. Conventional surgical technique was used regarding anesthesia and selective shunting or patching. None of the operated-on patients suffered a stroke at any time during follow-up. One patient (3.1%) died in the early postoperative phase (<30 days). An additional 8 patients died during follow-up. None of these deaths were of cerebrovascular origin. Survival at 3 years was 73% (life table analysis). Routine duplex scan follow-up showed 2 patients with a diameter reduction of more than 50%, both 3 months after CEA. Restenosis rate on duplex scan was 7.4% after 1 year. The authors conclude that there seems to be no reason to deny the very elderly the benefits of CEA. Stroke-free survival and survival rates show that carotid surgery is a safe procedure in patients aged 80 and over who are in apparently good health. These findings are supported by a low incidence of restenosis on duplex scan follow-up, indicating a durable repair.     

B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface. None of the B-ZIP proteins contain only aliphatic amino acids in the'a' and 'd' position. Only six of the Drosophila B-ZIP proteins contain a "canonical" hydrophobic interface like the yeast GCN4, and the mammalian JUN, ATF2, CREB, C/EBP, and PAR leucine zippers, characterized by asparagine in the second 'a' position. Twelve leucine zippers contain polar amino acids in the first, third, and fourth 'a' positions. Circular dichroism spectroscopy, used to monitor thermal denaturations of a heterodimerizing leucine zipper system containing either valine (V) or asparagine (N) in the 'a' position, indicates that the V-N interaction is 2.3 kcal/mole less stable than an N-N interaction and 5.3 kcal/mole less stable than a V-V interaction. Thus, we propose that the presence of polar amino acids in novel positions of the 'a' position of Drosophila B-ZIP proteins has led to leucine zippers that homodimerize rather than heterodimerize.     

P2Y receptor-mediated stimulation of Müller glial DNA synthesis

PURPOSE: To determine whether activation of P2Y receptors may increase the DNA synthesis rate of cultured Müller cells and to investigate whether adenosine 5'-triphosphate (ATP)-induced Müller cell proliferation is mediated by an intracellular calcium increase. METHODS: Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine immunoassay, and ATP-induced elevations of the intracellular calcium concentration were recorded by fura-2 imaging. RESULTS: ATP or uridine triphosphate (UTP) increased the DNA synthesis rate whereas alpha,beta-methylene-ATP, 2-methyl-thio-ATP, and adenosine were ineffective, indicating that the action of ATP was through P2Y receptors. The effect of ATP was dose dependent, with an EC(50) of 5.9 microM. The mitogenic effect of ATP required an elevation of the intracellular calcium and a calcium influx into Müller cells. Blockers of calcium-permeable channels (nickel ions) or of calcium-dependent potassium (BK) channels (iberiotoxin, charybdotoxin) inhibited the ATP-stimulated DNA synthesis. In calcium-imaging experiments, ATP-evoked intracellular calcium transients were significantly shortened in the presence of extracellular nickel ions or of iberiotoxin. A correlation was found between the duration of the ATP-evoked calcium transients and the basal proliferation rate of the cultures. CONCLUSIONS: The results indicate that the ATP-induced elevation of Müller glial DNA synthesis is dependent on an influx of calcium ions from the extracellular space and that the inhibiting effect of BK channel blockers on ATP-evoked DNA synthesis is caused by an inhibition of this influx. The amount of the calcium influx seems to be directly correlated to the strength of the ATP-evoked proliferation.     

Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin.

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by (1)H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenylisothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.     

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon

Indole-3-carbinol (I3C) inhibits the formation of colonic aberrant crypt foci and DNA adducts in rats given heterocyclic amine colon carcinogens, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Mechanism studies indicate that I3C induces cytochromes P4501A1 and 1A2 (CYP1A1 and CYP1A2), isozymes that respectively metabolize IQ via ring hydroxylation or activate the carcinogen by N-hydroxylation. The present study examined the dose-response for induction of CYP1A1 versus CYP1A2 by I3C, and compared the profiles of induction with the dose- response for inhibition of IQ-DNA adducts in the colon of the F344 rat. Dietary equivalent doses of I3C in the range 100-1000 p.p.m. increased in a dose-related manner both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in the liver and colonic mucosa, and Western blots showed a corresponding induction of CYP1A1 and CYP1A2 proteins. However, dietary equivalent doses of I3C in the range 10-25 p.p.m. (i) reduced hepatic EROD and MROD activities and CYP1A protein levels compared with controls, (ii) increased the ratio of CYP1A2 versus CYP1A1, and (iii) activated IQ to a more potent mutagen when liver microsomes from rats given I3C were used for metabolic activation in the Salmonella assay. Rats given a single oral dose of I3C shortly before administering IQ (5 mg/kg body wt, p.o.) exhibited dose-related inhibition of colonic IQ-DNA adducts in the range 25-100 p.p.m. I3C, reaching 95% inhibition at doses > or = 100 p.p.m. I3C, but IQ-DNA adducts were elevated slightly at the lowest I3C dose as compared with the controls. The possible significance of the low versus high dose effects of I3C are discussed in the context of human dietary exposures to I3C and the reported chemopreventive mechanisms of I3C in vivo.      

Schwann cell tumors express characteristic patterns of CD44 splice variants

Summary Members of the CD44 family of cell surface hyaluronate-binding proteins have been implicated in cell migration, cell-matrix interactions and tumor progression. To determine whether these proteins might play a role in the normal functions of Schwann cells and in their tumorigenesis, we examined the patterns of CD44 expression in Schwann cells from rat peripheral nerve, rat Schwann cell tumor lines, and human schwannomas. Normal rat spinal nerves and primary Schwann cell cultures expressed standard CD44 (CD44s) but not alternatively spliced variant isoforms. In contrast, rat Schwann cell tumor lines expressed both CD44s and a number of variants, including proteins containing sequences encoded by exon v6. Furthermore, we found that these cell lines bind hyaluronate, and that their cell surface hyaluronate binding correlates with CD44 expression. All of the human schwannomas also expressed CD44 variants, especially epitopes encoded by exon v5, the border between v7 and v8, and v9-10. These data indicate that Schwann cells normally express CD44s, that Schwann cell tumors express both CD44s and particular variants of CD44, and that CD44s and possibly variants of CD44 are involved in hyaluronate recognition by Schwann cell tumors.