Impact of a donor source on adult Philadelphia chromosome-negative acute lymphoblastic leukemia: a retrospective analysis from the Adult Acute Lymphoblastic Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation.

BACKGROUND: We aimed to clarify the impact of the donor source of allogeneic stem cell transplantation (allo-SCT) on Philadelphia chromosome-negative acute lymphoblastic leukemia [Ph(-) ALL] with focus on cord blood (CB). PATIENTS AND METHODS: We retrospectively analyzed data of 1726 patients who underwent myeloablative allo-SCT for adult Ph(-) ALL. The sources of the allo-SCT were related donors (RD; N = 684), unrelated donors (URD; N = 809), and CB (N = 233). RESULTS: Overall survival (OS) in patients after CB allo-SCT in first complete remission (CR1) was comparable with that after RD or URD allo-SCT (RD: 65%, URD: 64% and CB: 57% at 4 years, P = 0.11). CB was not a significant risk factor for relapse or non-relapse mortality as well as for OS in multivariate analyses. Similarly, the donor source was not a significant risk factor for OS in subsequent CR or non-CR (RD: 47%, URD: 39% and CB: 48% in subsequent CR, P = 0.33; RD: 15%, URD: 21% and CB: 18% in non-CR, P = 0.20 at 4 years). CONCLUSION: Allo-SCT using CB led to OS similar to those of RD or URD in any disease status. To avoid missing the appropriate timing, CB is a favorable alternative source for adult Ph(-) ALL patients without a suitable RD or URD.

A prospective dose-finding trial using a modified continual reassessment method for optimization of fludarabine plus melphalan conditioning for marrow transplantation from unrelated donors in patients with hematopoietic malignancies.

BACKGROUND: Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. PATIENTS AND METHODS: The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. RESULTS: Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m(2) (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. CONCLUSIONS: mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

"Transmural air leak": a computed tomographic finding following endoscopic submucosal dissection of gastric tumors.

BACKGROUND AND STUDY AIMS: A small amount of free air, visible on CT but not on plain chest radiography, which appeared following endoscopic submucosal dissection (ESD) of a gastric neoplasm without endoscopically visible perforation, was defined as a "transmural air leak", and a prospective, consecutive entry study was performed to determine its incidence and clinical significance. PATIENTS AND METHODS: Between January 2006 and September 2008, ESD was performed for 246 gastric lesions in 246 consecutive patients. Abdominal CT scan was performed 1 day after ESD. In addition, chest radiography and blood biochemistry tests were performed at different time points before and after ESD. RESULTS: Two hundred and nineteen lesions (89 %) were curatively removed by ESD. Among the total of 246 patients, we encountered endoscopically visible perforation during ESD in 2 patients (0.8 %), and clinically suspected perforation diagnosed by the presence of free air on chest radiography but invisible during ESD in 3 patients (1 %), while transmural air leak was observed in another 33 (13 %). Air leak occurred in cases where resection size was larger, procedure time longer, and the muscularis propria on the ulcer base was exposed at the end of ESD. Patients with air leaks developed pyrexia at a higher rate than those without (36 % vs. 16 %, P = 0.018). These patients recovered with antibiotics and required no endoscopic or surgical intervention. The presence of an air leak did not affect the duration of hospital stay. CONCLUSIONS: A transmural air leak was observed in 13 % of the patients undergoing ESD. Larger resection size, prolonged procedure time, and exposure of the muscularis propria on the ulcer base were risk factors for transmural air leak, but the outcome of patients with this complication was good.

The Schizosaccharomyces pombe mei4+ gene encodes a meiosis-specific transcription factor containing a forkhead DNA-binding domain.

The mei4+ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation. The mei4 disruptant failed to complete meiosis-I but could proliferate normally. mei4+ was transcribed only in meiosis-proficient diploid cells after premeiotic DNA replication. The mei4+ open reading frame encodes a 57-kDa serine-rich protein comprised of 517 amino acids with a forkhead/HNF3 DNA-binding domain in the amino-terminal region. Transcription of spo6+, a gene required for sporulation, was dependent on the mei4+ function. Two copies of the GTAAAYA consensus sequence, proposed as the binding site for human forkhead proteins, were found in the promoter region of spo6+. A gel mobility shift assay demonstrated the sequence-dependent binding of the GST-Mei4 forkhead domain fusion protein to DNA fragments with one of the consensus elements. Deletion of this consensus element from the spo6 promoter abolished the transcription of spo6+ and resulted in a sporulation deficiency. One-hybrid assay of Mei4 which was fused to the Gal4 DNA-binding domain localized the transcriptional activation domain in the C-terminal 140 amino acids of Mei4. These results indicate that Mei4 functions as a meiosis-specific transcription factor of S. pombe.

Increase of bone marrow macrophages and CD8+ T lymphocytes predict graft failure after allogeneic bone marrow or cord blood transplantation.

Graft failure (GF) remains an obstacle to survival after allogeneic hematopoietic stem cell transplantation. However, differentiating GF from delayed engraftment (DE) can be difficult. Host CD8+ lymphocytes have been reported to mediate graft rejection, but the impact of macrophages on DE or GF is yet to be clarified. Peri-engraftment bone marrow (BM) specimens of 32 adult patients with normal engraftment, DE or GF were retrospectively evaluated to identify the potential associations of CD163+ macrophage and CD8+ lymphocyte infiltration into BM. The macrophage or CD8+ lymphocyte number/total nucleated cell number was defined as the Mac ratio and CD8 ratio, respectively. Both DE and GF groups had significantly higher Mac ratios at day 14 than the normal group (P<0.0001), but no significant difference was observed between the DE and GF groups (P=1.000). The CD8 ratio at day 14 was significantly higher in the GF than in the normal group (P=0.005), whereas the CD8 ratios of the DE and normal groups were similar (P=0.07). A high Mac ratio at day 14 was associated with a risk of DE or subsequent GF. Patients with increased CD8 ratio at day 14 had a further risk of GF. The Mac ratio and the CD8 ratio appear to be well suited for predicting engraftment status.

Elevation of 4 beta-galactosyltransferase activity for paragloboside synthesis in sera of patients with cancer.

Galactosyltransferase activities in sera of cancer patients were determined by assaying the formation of paragloboside from UDP-galactose and lactotriaosylceramide immobilized on microtiter plates by means of the enzyme-linked immunosorbent assay using a monoclonal antibody, H-11, directed to paragloboside. Enzyme properties were as follows. Optimum pH was 6.8 in cacodylate buffer, and Km values were 2 microM for lactotriaosylceramide and 29 microM for UDP-galactose. The enzyme activity was inhibited by the addition of alpha-lactalbumin. Glucose (20 mM) inhibited the enzyme activity in the presence of alpha-lactalbumin (0.1 mg/ml) but not in its absence. These enzyme properties are similar to those of bovine milk galactosyltransferase, indicating that the enzyme in the sera might be lactose synthetase. The enzyme activities in sera from patients with cancer, patients with benign disease, or a reference sample group were assayed. The activity was below the limit of detection (5.5 pmol/25 microliters serum/2 h) in the reference sample group. Remarkable elevations of the enzyme activity were observed with high incidence in patients with cancer, especially those with blood cancer (100%). A high incidence was observed in the progressive stage, and the enzyme activity was detected at stage 1 in lung, esophagus, stomach, colorectal, and testis cancer. The enzyme activity in sera from patients with benign disease was elevated in 22% of the patients. After effective therapies, the enzyme activity decreased to below the limit of detection. Release of the galactosyltransferase into culture medium of cancer cells could be demonstrated. These observations suggest that the galactosyltransferase is released from cancer tissue into the circulation. The present method for the assay of galactosyltransferase may be useful for the detection of patients with cancer and for monitoring neoplastic recurrence after therapy.

Impact of MRD and TKI on allogeneic hematopoietic cell transplantation for Ph+ALL: a study from the adult ALL WG of the JSHCT.

To assess the impact of minimal residual disease (MRD) and tyrosine kinase inhibitor (TKI) administration on allogeneic hematopoietic cell transplantation (allo-HCT) for Ph-positive ALL (Ph+ALL), we retrospectively analyzed data from a registry database for 432 adult Ph+ALL patients in first CR (CR1) who received pre-transplant TKI administration. Negative MRD (MRD(-)) at allo-HCT was achieved in 277 patients. OS in patients transplanted in MRD(-) was significantly better than that in patients transplanted in MRD(+) (MRD(-): 67% vs MRD(+): 55% at 4 years; P=0.001). MRD(-) at allo-HCT was a significant risk factor for survival along with age at allo-HCT in multivariate analyses. Incidence of relapse in patients transplanted in MRD(-) was significantly lower than that in patients transplanted in MRD(+) (MRD(-): 19% vs MRD(+): 29% at 4 years; P=0.006). In multivariate analyses, MRD(+) at allo-HCT was a significant risk factor for relapse. A post-transplant TKI was administered to 103 patients. In subanalyses regarding the effect of post-transplant TKI administration, post-transplant TKI administration was a significant risk factor for relapse in multivariate analyses (P<0.0001). MRD status at allo-HCT is one of the most important predictive factors for Ph+ALL patients transplanted in CR1.

Purification and partial characterization of a novel cellular retinol-binding protein, type three, from the piscine eyes.

A novel cellular retinol-binding protein, termed type three (CRBP III), was isolated from eyes of the bigeye of tuna. CRBP III showed a molecular weight of 15,400, an isoelectric point of 4.80, alpha 1-mobility in electrophoresis, and a lambda max of 350 nm. All-trans-retinol, the endogenous ligand, could be competitively displaced by retinoic acid but not by retinal. CRBP III was differentiated from purified piscine and rat cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins (CRABP) by its amino-acid composition, electrophoretic mobility, fluorescence spectra and ligand-binding specificity.

Thyrotropin, prolactin, and growth hormone responses to thyrotropin-releasing hormone in anorexia nervosa and bulimia.

Serum thyrotropin (TSH), prolactin (PRL), and growth hormone (GH) levels were measured before and after stimulation with thyrotropin-releasing hormone (TRH) in 10 patients with bulimia, 7 with features of the restricting subtype of anorexia nervosa, and 6 with bulimic subtype of anorexia nervosa. The mean basal levels of TSH, PRL, and GH did not differ among the three groups. A delayed TSH response was found in 86% of the restricting anorectics, 80% of the bulimic anorectics, and 22% of the bulimics. The PRL response was normal in all patients, with no significant difference among the three groups. Elevated basal GH levels were found in 29% of the restricting anorectics, 33% of the bulimic anorectics, and 33% of the bulimics. An abnormal GH increase after TRH stimulation was observed in 50% of the restricting anorectics, 20% of the bulimic anorectics, and 13% of the bulimics. These results suggest that some patients with bulimia, and some with anorexia nervosa, have a hypothalamic dysfunction. These neuroendocrine abnormalities do not appear to be due solely to low weight or to metabolic changes resulting from binge eating and are not associated with depressive symptoms.

TRH test and DST in schizoaffective mania, mania, and schizophrenia.

The thyrotropin-releasing hormone (TRH) test and the Dexamethasone Suppression Test (DST) were given to 10 patients who met Research Diagnostic Criteria (RDC) for schizoaffective disorder, manic type, 9 who met the criteria for mania, and 27 who met the criteria for schizophrenia. A blunted thyrotropin (TSH) response to TRH was observed in 3 of the 10 schizoaffective manics, 4 of the 9 manics, and 3 of the 27 schizophrenics. Nonsuppression on the DST was observed in 5 of the 10 schizoaffective manics, 2 of the 9 manics, and 2 of 22 schizophrenics. The schizoaffective manic and the manic patients had similar rates of TSH blunting and DST nonsuppression, and these were significantly higher than the rates in the schizophrenic patients. This difference was not attributable to baseline TSH and cortisol levels or to neuroleptic treatment. It is suggested that patients with RDC schizoaffective mania and mania have more disturbance in the hypothalamic-pituitary adrenal and thyroid axes than patients with schizophrenia.

Rapid purification of protein phosphatase 2A from mouse brain by microcystin-affinity chromatography.

Microcystin LR, which is a monocyclic heptapeptide containing two L-amino acids, leucine and arginine, is a new inhibitor of protein phosphatases 1 and 2A. Microcystin LR-affinity chromatography was used to purify protein phosphatase 2A as a holoenzyme. Five mg of microcystin LR were immobilized to ECH Sepharose 4B by the carbodiimide coupling reaction. Following DEAE-cellulose column chromatography, microcystin-affinity chromatography, as the second step in the procedure, resulted in purification of protein phosphatase 2A in a pure form. The enzyme isolated from mouse brain consisted of two regulatory subunits of 67 kDa and 58 kDa and a catalytic subunit of 41 kDa. Microcystin-affinity chromatography is useful for isolation of protein phosphatase 2A.

Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma.

A polyclonal antibody was raised against a recombinant ligand binding domain construct of the human retinoid X receptor (RXR) alpha. This antibody reacted with an endogenous 54 kDa nuclear protein from human hepatoma-derived HuH7 cells in immunoblot analyses. Immunoblotting of nuclear proteins from human hepatocellular carcinomas (HCCs) and their surrounding tissues revealed the presence of a 44 kDa RXR distinct from the 54 kDa RXR and a dramatic decrease in the relative amounts of 44 kDa RXR to 54 kDa RXR in all HCCs compared with normal tissue. In vitro shift and intracellular conversion from 54 kDa RXR to 44 kDa species were observed with the nuclear extracts of HuH7 cells. Furthermore, transfection of hRXR alpha cDNA into HuH7 cells resulted in the increase of 54 kDa RXR, whereas transfected mouse hepatocytes accumulated 44 kDa RXR. These results strongly indicated that 44 kDa RXR was a physiological proteolytic fragment of 54 kDa RXR and that post-translational metabolism of RXR was impaired in HCC and the HuH7 hepatoma-derived cell line.

Mechanisms of action of okadaic acid class tumor promoters on mouse skin.

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are the okadaic acid class of non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, which do not bind to the phorbol ester receptors in cell membranes or activate protein kinase C in vitro. They have potent tumor-promoting activities on mouse skin, as strong as TPA-type tumor promoters, such as TPA, teleocidin, and aplysiatoxin. DNA samples isolated from tumors induced by dimethylbenz[alpha]anthracene and each of the okadaic acid class tumor promoters had the same mutation at the second nucleotide of codon 61 (CAA to CTA) in the c-H-ras gene. Okadaic acid receptors, protein phosphatases 1 and 2A, are present in the particulate as well as cytosolic fractions of various mouse tissues. The apparent "activation" of protein kinases by the okadaic acid class tumor promoters, after their incubation with 32P-ATP, protein kinases, and protein phosphatases, was observed. This activation was caused by inhibition of protein phosphatases 1 and 2A by the okadaic acid class tumor promoters. Treatment of primary human fibroblasts and human keratinocytes with the okadaic acid class tumor promoters induced the hyperphosphorylation of a 60-kDa protein in nuclear and cytosolic fractions, due to the inhibition of protein phosphatases. The 60-kDa protein is a proteolytic fragment of nucleolin, a major nonhistone protein and is designated as "N-60." The mechanisms of action of the okadaic acid class tumor promoters are discussed with emphasis on the inhibition of protein phosphatase activity.

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR.

Certain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the two L-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathione S-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Extrarenal Wilms' tumor in the adult patient. A case report and review of the world literature.

A case of an extrarenal Wilms' tumor arising in the retroperitoneal region of a 49-year-old male is reported. A review of the world literature indicates that the incidence of the tumor arising in the extrarenal region is extremely rare. A total of 14 cases have previously been reported, but the number of cases that occurred in adult patients is only 2. The clinical and pathologic features are briefly discussed.

A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.