Immunomodulatory effects of vitamin D: implications for GVHD.

GVHD remains a major source of morbidity and mortality after allogeneic BMT. GVHD is mediated by alloreactive T cells derived from the hematopoietic graft that target host tissues. Pre-clinical models have shown that presentation of alloantigens by host DCs results in the activation of donor-derived T cells that mediate GVHD. Strategies that interfere with the Ag-presenting capacity of DCs after allogeneic transplantation may decrease the risk of developing GVHD. Vitamin D is a hormone essential for calcium metabolism that shows immunomodulatory properties. We showed that correction of vitamin D deficiency appeared to mitigate manifestations of GVHD. In pre-clinical studies, we have shown that vitamin D inhibits DC maturation, polarizes T-cell populations toward the expression of Th2 as compared with Th1 cytokines, and blunts allogeneic T-cell proliferation in response to DC stimulation. Exposure to vitamin D resulted in increased expression of IDO, an enzyme responsible for tryptophan metabolism that is upregulated in tolerizing DCs. These data suggest that exposure to vitamin D results in immature DC populations that bias toward tolerizing rather than stimulatory T-cell populations. Vitamin D may therefore have a role in the prevention of GVHD.

Validation of a gas chromatography/thermal conductivity detection method for the determination of the water content of oxygenated solvents.

A gas chromatographic (GC) method for the determination of the water content of acetone solutions containing methyl isobutyl ketone (MIBK) and other by-products of MIBK synthesis was developed and validated. Linearity is demonstrated (R2=0.9999) over a broad range of water concentrations in acetone ranging from 0.04 to 15.2% (w/w). The quantitation limit (QL) of this technique was found to be 0.346% (w/w) and the detection limit (DL) was estimated to be 13ppm. Validation of this analytical method's accuracy against a coulometric Karl Fischer titration (KFT) method showed excellent agreement between these two techniques. The GC/TCD method is accurate to within a relative error of 3.49% with respect to the KFT method for water concentration above the QL. However, the analysis of six samples below the QL showed an average relative error of 10.96%. Consequently, the KFT method is recommended for the moisture analysis of samples for water concentrations below 0.35% (w/w). Excellent precision is realized for water concentration above the QL (RSD<<0.8%). Good precision (RSD=1.1%) was obtained for the sample with the lowest water concentration investigated (0.04%, w/w). Repeatability, robustness and excellent specificity of this technique are demonstrated.

Surgical oncology in university departments of surgery in the United States.

Data on surgical oncology and multidisciplinary cancer program activity were obtained from 124 of 126 university surgery departments in the United States. Most of these institutions have American College of Surgeons-approved cancer programs (84%) as well as divisions of medical (95%), radiation (94%), pediatric (76%), and gynecologic (79%) oncology. Only 47 departments (38%) have formal divisions of surgical oncology. There are no major staffing or activity differences in surgical departments with or without such divisions, but multidisciplinary cancer program activity is greater in those institutions with a surgical oncology focus. Peer-reviewed cancer research grants are more frequent in departments of surgery with a surgical oncology division (68% vs 47%). The activities of the existing 47 divisions of surgical oncology are mainly operative, with breast cancer, melanoma, and soft-tissue sarcomas being the major clinical responsibilities. Chemotherapy is also frequent (81%). Cancer education for undergraduate and postgraduate surgical trainees is a major responsibility of most divisions, but only a small proportion (28%) have postresidency surgical oncology training programs. In contrast to the growth of some oncologic specialties, the establishment of surgical oncology within university departments has been slow, and the manpower needs appear modest.

Differentiation-associated changes in in-vitro mRNA translation in the HL60 cell line.

In order to characterize certain aspects of gene expression during the granulocytic differentiation of the HL60 cell line, we have analysed changes in the population of mRNA available for translation in vitro. RNA extracts of DMSO-induced and control cells were translated in vitro in a wheat germ cell-free protein synthesizing system. Translation products were analysed by two-dimensional electrophoresis followed by autoradiography. Autoradiograms were analysed by a computer-assisted method utilizing a drum-scanning microdensitometer. Spots were identified by their relative positions on the films and their relative intensity was estimated. One hundred and eighty-one peptides were identified in both the DMSO-induced and untreated control HL60 cells, 31 of which showed differentiation-associated changes in synthesis in vitro. The 11 peptides which decreased in synthesis did so early in the differentiation process, whereas most of the 20 peptides which increased did so at a later time. Three peptides were shown to increase more than 8-fold by day 4 of induction. A comparison with normal granule proteins from human leukocytes suggests that at least two of these may correspond to functional granule proteins. The changes in peptide patterns which we describe demonstrates that the program of gene expression during HL60 differentiation includes changes in the relative abundance of specific mRNA transcripts. The data described here also provides a standard for comparison of other proteins, such as oncogene products, as they are identified.

Proliferation-associated changes in in vitro mRNA translation in the HL60 cell line.

In order to characterize patterns of gene expression during the proliferation cycle of HL60 cells, we have analysed changes in the population of mRNA available for translation in vitro. HL60 cells were separated into cell cycle phases by centrifugal elutriation, monitoring the separation with flow cytometry. RNA was extracted from cell fractions highly enriched in G1, S or G2+M phases and translated in vitro. Translation products were analysed by two-dimensional electrophoresis followed by autoradiography. Autoradiograms were analysed by a computer-assisted method utilizing a drum-scanning microdensitometer. Spots were identified by their relative positions on the films and their relative intensity was estimated. Of the 159 peptides studied for cell cycle-associated changes in synthesis, nine showed phase-associated changes. The most significant changes were the accumulation of four peptides that showed maximal synthesis only in G2+M phases. An additional four peptides were synthesized maximally in both S and G2+M phases. One peptide showed maximal synthesis in S phase. These changes in gene expression suggest that these relatively abundant transcripts are regulated primarily at a quantitative level during proliferation and may be related to the doubling of structural proteins prior to mitosis.

Characterization of proteins in flagellates and growing amebae of Naegleria fowleri.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.

Intracellular crystalline deposits by bacteria grown in urine from a stone former.

Several species of bacteria were found to form an intracellular crystalline material when grown in urine obtained from a subject with a history of infrequent renal calculi formation. The following species: Proteus mirabilis, Proteus rettgeri, Providencia stuartii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, and Candida albicans formed crystals of hydroxyapatite. Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris produced crystals of calcite -II. Several of these bacteria have been isolated from the kidneys of patients with renal caculi indicationg that microorganisms may be involved in the nucleation process during calculogenesis.

Formation of crystalline deposits by several genera of the family Enterobacteriaceae.

Several species of bacteria from the family Enterobacteriaceae formed crystalline materials containing calcium when grown in a defined culture medium. Enterobacter aerogenes, Proteus vulgaris, Citrobacter freundii, and C. intermedius produced calcium pyrophosphate crystals. Edwardsiella tarda and Escherichia coli formed calcite III crystals, whereas Proteus mirabilis, Klebsiella pneumoniae, Providencia stuartii, and Serratia marcescens produced hydroxyapatite crystals. Several of these bacteria have been isolated from the kidneys of patients with kidney stones, indicating that microorganisms may be involved in the enucleation process of kidney stone formation.