Reduced-intensity unrelated cord blood transplantation for patients with advanced malignant lymphoma.

We report the results of reduced-intensity unrelated cord blood transplantation (RI-UCBT) in patients with advanced malignant lymphoma. Twenty patients (median age, 46.5 years; range, 27-66 years) underwent RI-UCBT with a preparative regimen consisting of fludarabine 125 mg/m2 , melphalan 80 mg/m 2 , and 4 Gy of total body irradiation. The median infused total cell dose was 2.75 x 10(7)/kg (range, 2.3-3.4 x 10(7)/kg). Graft-versus-host disease (GVHD) prophylaxis was composed of cyclosporine or tacrolimus alone. Fifteen patients achieved primary neutrophil engraftment after a median of 20 days. Eight patients developed grade II to IV acute GVHD, and 2 developed chronic GVHD. Of the 16 patients with evaluable disease, 10 achieved a complete response. Primary disease recurred in 1 patient, and transplant-related mortality within 100 days occurred in 8 of 20 patients. The estimated 1-year probability of progression-free survival was 50%. These data suggest that RI-UCBT is a feasible option for patients with refractory lymphoma who lack an HLA-matched donor.     

Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia

We report here that loss of the Sprouty2 gene (also known as Spry2) in mice resulted in enteric nerve hyperplasia, which led to esophageal achalasia and intestinal pseudo-obstruction. Glial cell line-derived neurotrophic factor (GDNF) induced hyperactivation of ERK and Akt in enteric nerve cells. Anti-GDNF antibody administration corrected nerve hyperplasia in Sprouty2-deficient mice. We show Sprouty2 to be a negative regulator of GDNF for the neonatal development or survival of enteric nerve cells.     

Committing embryonic stem cells to early endocrine pancreas in vitro

A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothio-glycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin+ or C-peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of beta-cell specification and differentiation factors activin beta B, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.     

Advance in animal experiments with the undulation pump total artificial heart: 50 and 54 day survival periods with 1/R control

The undulation pump total artificial heart (UPTAH) is a unique, implantable, total artificial heart (TAH) that uses undulation pumps. To achieve long-term survival in animals with physiologic hemodynamic conditions, a control method based on conductance and arterial pressure was applied to UPTAH. With this control method, called 1/R control, survival periods of 50 days (No. 0016, 49.6 kg) and 54 days (No. 0030, 42.5 kg) were obtained in adult female goats. In No. 0016, 1/R control was applied to the left pump, whereas in No. 0030, it was applied to the right pump. Another pump was used for left-right balance control. The control stability was better in No. 0030 than in No. 0016. The sucking effect of the left atrium was remarkable in No. 0016, possibly because of a time delay when left-right balance control was performed with the right pump. In No. 0016, the cause of death was probably a thrombus flown from a panus in the left atrium. It is possible that the left atrial suction effect influenced the thrombus and panus formation in the left atrium. In No. 0030, the cause of death was a small rupture of the membrane in the right pump. The rupture may have been caused by excessive negative pressure inside the pump. This pressure resulted from suction of the right atrium because of an unexpected control excursion, which was probably caused by a software bug. It will be necessary to redesign the undulation pump and improve the software to achieve longer survival periods for animals with physiologic hemodynamic conditions.     

Cationic polymerization of vinyl monomers with latent initiators, 3. 1-(p-methoxybenzyl)tetrahydrothiophenium hexafluoroantimonate as a new potent cationic initiator

Cationic polymerization of styrene in the presence of 1-(p-methoxybenzyl)tetrahydrothiophenium hexafluoroantimonate ( 3 ) as an efficient cationic initiator was investigated. 3 was synthesized in an excellent yield by the reaction of p-methoxybenzyl bromide with tetrahydrothiophene and the subsequent exchange of bromide ions for hexafluoroantimonate ions. In the polymerization of styrene, 3 acted as a potent thermally latent initiator which initiates the polymerization at a slightly higher temperature than room temperature, although no polymerization occurs at room temperature for 30 min. 3 is a much more active initiator than the previously reported benzylsulfonium salt 1 . The enhanced activity of 3 was also confirmed in the polymerization of glycidyl^b phenyl ether.     

Prospective clinical evaluation of the serologic tuberculous glycolipid test in combination with the nucleic acid amplification test

We have conducted a prospective controlled multicenter study to evaluate differences in the levels of clinical utility of the tuberculous glycolipid (TBGL) serodiagnostic test and the nucleic acid amplification test in patients with smear-negative active pulmonary tuberculosis (TB). The TBGL test and the PCR test were individually not so useful for the rapid diagnosis of smear-negative active pulmonary TB. However, clinical utility was considerably improved by using the TBGL test and the PCR test in combination, especially in patients with smear-negative and culture-negative active pulmonary TB and in patients with minimally advanced lesions.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation

Accumulation of unfolded and malfolded proteins causes endoplasmic reticulum (ER) stress, stimulating unfolded protein response (UPR) and c-Jun N-terminal kinase (JNK) activation and activating caspase-12 located on the ER. Little is known about the relationship between the ER stress and polyglutamine [poly(Q)] aggregates. Poly(Q)72 repeats [poly(Q)(72)] induced the stimulation of ER stress signals such as JNK activation, upregulation of Grp78/Bip and caspase-12 activation in C2C5 cells. We prepared antiserum against the cleavage site of mouse caspase-12 at D(318) (anti-m12D318), and showed that poly(Q)(72) with perinuclear aggregates, cytoplasmic inclusions and nuclear inclusions stimulated JNK activation and anti-m12D318 immunoreactivity, but poly(Q)(72) with dispersed aggregates and small nuclear aggregates showed a significantly less effect. Poly(Q)(72) and poly(Q)(11) dispersed in cytoplasm did not. Anti-m12D318-positive cells showed apoptotic features. Unlike anti-m8D387 immunoreactivity, the anti-m12D318 immunoreactivity was not coaggregated with poly(Q). Ac-IETD-fmk (caspase-8 inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) did not prevent the anti-m12D318 immunoreactivity induced by poly(Q)(72) aggregates. Anti-m12D318 immunoreactivity was detected in caspase-8(-/-) and caspase-3(-/-) mouse embryonic fibroblasts expressing poly(Q)(72) aggregates. Thus, caspase-12 was activated by poly(Q)(72) aggregates via a pathway independent of caspase-8 and caspase-3 activation, and caspase-12 activation was closely associated with poly(Q) aggregate-mediated cell death. Stimulation of ER stress signals may be involved in the pathogenesis of neurodegenerative disorders with poly(Q) expansion.     

ARK5 expression in colorectal cancer and its implications for tumor progression

A novel member of the human AMPK family, ARK5, was recently discovered to be a key molecule in mediating cancer cell migration activity in human pancreas cancer cell line PANC-1, and its activation was found to be induced by Akt-dependent phosphorylation at Ser 600. DNA array analysis with 241 paired cDNAs from 13 different types of tumors and corresponding normal tissues derived from cancer patients revealed ARK5 overexpression in the samples of colorectal cancer. ARK5 expression was measured and an in vitro invasion assay was performed in six human colorectal cancer cell lines, WiDr, HCT-15, DLD-1, SW620, LoVo, and SW480, and since high invasion activity was concordant with higher ARK5 expression, ARK5 expression was examined in relation to tumor progression and metastatic activity in clinical samples. In 56 clinical samples of primary colorectal cancers and their liver metastases, higher ARK5 expression was observed in the samples from more advanced cases, and much higher expression was observed in the liver metastases. In situ hybridization analysis showed ARK5 overexpression in tumor cells. Based on these findings, we propose that ARK5 overexpression is involved in tumor progression of colon cancer clinically.