In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.     

Arginine deficiency in bile duct-ligated rats after surgery: the role of plasma arginase and gut endotoxin restriction

BACKGROUND & AIMS: Arginine deficiency may underlie the cellular immune depression after surgery in obstructive jaundice, which is associated with gut-derived endotoxemia. The aim of this study was to study arginine metabolism in the bile duct-ligated rat (BDL) after laparotomy. METHODS: Treatment with cholestyramine, a known endotoxin binder, was used to evaluate the role of gut-derived endotoxemia. RESULTS: In BDL rats, arginine levels were lower compared with those in sham-operated controls (P < 0.005), despite a three-fold increase in renal arginine release (P < 0.01). Liver and gut arginine handling also could not explain the reduced arginine levels. Higher plasma arginase activity (P < 0.0001) was measured in BDL rats, explaining both the lower arginine levels (r = 0.73, P < 0.01) and the increase in arginase product levels: ornithine (P < 0.005 and r = 0.72; P < 0.01) and urea (P < 0.01). Cholestyramine treatment prevented the decrease in postoperative arginine deficiency by reducing plasma arginase activity by 43% (P < 0.005). In addition, it significantly lowered plasma levels of the other liver enzymes (aspartate transaminase, alanine transaminase, gamma-glutamyl transpeptidase, and alkaline phosphatase; P < 0.05) in BDL rats. CONCLUSIONS: The study concluded that arginine deficiency in BDL rats after surgery is caused by high plasma liver arginase activity. Cholestyramine prevented the arginine deficiency by reducing plasma arginase activity through the inhibition of additional endotoxin-mediated hepatocellular damage after surgery in BDL rats.     

Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.     

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.     

A general analysis of bit error probability for reference-basedBPSK mobile data transmission

Unified analysis of the performance of binary phase shift keying (BPSK) under static and mobile operating conditions is presented for the case in which a separate reference tone is used for channel sounding and subsequent `coherent' data detection. It is shown that under both Rician and Rayleigh fading conditions, the use of a reference can eliminate the irreducible error rate phenomenon, with minimal sacrifice in bit error rate performance over an ideal BPSK system     

Simultaneous occurrence of a supra- and an infratentorial glioma in a patient with Ollier's disease: more evidence for non-mesodermal tumor predisposition in multiple enchondromatosis

A case is presented in which two neuro-ectodermal tumors, an infra- and a supratentorial glioma, developed in a young man with multiple enchondromatosis of Ollier's disease. This is the third such case of multifocal low-grade glioma in Ollier's disease, suggesting a predisposition for non-mesodermal tumors in Ollier's disease. The related condition of multiple enchondromatosis and hemangiomas (Maffucci's syndrome) is well known for its malignant potential, developing both mesodermal and non-mesodermal tumors. Along with other authors, we support the concept of two variants of the same disease with a predisposition to development of tumors from various germ layers.     

Botulinum toxin in the management of the lower limb in cerebral palsy

The role of intramuscular botulinum toxin A in the treatment of 26 children with cerebral palsy was evaluated. The indication for injection was the presence of a dynamic contracture of lower-limb muscles interfering with positioning or walking. Spastic target muscles were identified by clinical examination and, in ambulant children, by gait analysis. Between 50 and 320 units of botulinum toxin were injected into each muscle group to a total dose of 100 to 400 units per child. The effects of injection were monitored by repeated clinical examination and gait analysis. There were no clinically detectable systemic side-effects, and all but one patient had a reduction in tone, which occurred within three days and persisted for two to four months. There were significant improvements in ambulatory status and in sagittal-plane kinematics. In some cases these gains persisted after the tone-reducing effects of the toxin had worn off.