Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

Effect of retroviral endostatin gene transfer on subcutaneous and intraperitoneal growth of murine tumors.

BACKGROUND: Inhibiting tumor angiogenesis is a promising new strategy for treating cancer. Difficulties with the stability, manufacture, and long-term administration of recombinant antiangiogenic proteins have prompted investigators to use gene therapy to generate these proteins in vivo. We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the murine liver cell line NMuLi could inhibit tumor growth in vivo. METHODS: NMuLi cells were transduced with retroviral vectors containing the murine endostatin gene. The presence and function of endostatin in transduced cell supernatants were confirmed by competitive enzyme immunoassay and endothelial cell proliferation assays. Nude mice were given a subcutaneous or intraperitoneal injection with NMuLi cells, control transduced cells (NEF-null), or endostatin-transduced clones (NEF-Endo1 to 4) and were monitored for tumor growth. All statistical tests were two-sided. RESULTS: Supernatants from the clone secreting the lowest amount of endostatin (NEF-Endo4, 28 ng/mL) inhibited endothelial cell proliferation by 6% (95% confidence interval [CI] = 0% to 12%), and those from the clone secreting the highest amount (NEF-Endo1, 223 ng/mL) inhibited endothelial cell proliferation by 20% (95% CI = 13% to 27%). Increased levels of endostatin were detected in tumor lysates, but not serum, of mice given a subcutaneous injection of NEF-Endo1 cells. After 63 days, mice given a subcutaneous injection of parental NMuLi or NEF-null cells had tumor volumes of 2400 mm(3) (95% CI = 1478 mm(3) to 3300 mm(3)) and 2700 mm(3) (95% CI = 2241 mm(3) to 3144 mm(3)), respectively, compared with mean tumor volumes of less than 30 mm(3) in mice given an injection of NEF-Endo clones, a statistically significant difference (P<.001). After 123 days, all 16 mice given an intraperitoneal injection of parental NMuLi or NEF-null cells had died, compared with only three (9%) of 32 mice given an injection of NEF-Endo clones. CONCLUSIONS: Retroviral endostatin gene transfer leads to secretion of functional endostatin that is sufficiently active to inhibit tumor growth. Further studies of retroviral endostatin gene transfer for the treatment of cancer are warranted.

Role of tumor necrosis factor on toxicity and cytokine production after isolated hepatic perfusion.

PURPOSE: Isolated limb or liver perfusion with tumor necrosis factor (TNF) and melphalan results in regression of advanced cancers in the majority of treated patients. However, the contribution of TNF to the efficacy of isolation perfusion with melphalan has not been demonstrated conclusively in random assignment trials. Furthermore, TNF is an inflammatory cytokine and may be associated with significant systemic and regional toxicity. This study was conducted to characterize the toxicity and secondary cytokine production attributable to TNF by comparing these parameters in patients undergoing isolated hepatic perfusion (IHP) using melphalan with or without TNF. EXPERIMENTAL DESIGN: Thirty-two patients with unresectable colorectal cancer confined to the liver underwent a 60-min hyperthermic IHP using 1.5 mg/kg melphalan alone (n = 17) or with 1.0 mg of TNF (n = 15) with inflow via the gastroduodenal artery and outflow via an isolated segment of inferior vena cava. Complete vascular isolation was confirmed using the I-131 radiolabeled albumin-monitoring technique. Post-IHP parameters of hepatic and systemic toxicity and cytokine levels [TNF, interleukin (IL)-6 and IL-8] in perfusate and serum were measured. RESULTS: Levels of IL-6 and IL-8 in perfusate at the end of the 60-min IHP were significantly higher in TNF-treated patients (P < or = 0.001). Peak systemic IL-6 and IL-8 levels post-IHP were also significantly higher in TNF-treated compared with non-TNF-treated patients (P < 0.0001) by 28- and 268-fold, respectively. The peak levels of these cytokines were associated with significantly lower systolic blood pressure and higher heart rate and mean pulmonary artery blood pressure in TNF-treated patients during the first 48 h post-IHP (P < or = 0.03). Serum bilirubin levels were significantly higher (P = 0.017) and platelets lower (P = 0.03) in TNF-treated compared with non-TNF-treated patients. However, elevations in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were not significantly different between groups and returned toward baseline within 1 week after IHP. CONCLUSIONS: Addition of TNF to melphalan during IHP results in significant differences in post-IHP production of IL-6 and IL-8 with associated changes in mean arterial blood pressure and greater regional toxicity, as reflected in higher levels of serum bilirubin. However, these measurable differences were transient and did not appear to be of major clinical consequence. Prior to its routine use, the benefit of TNF in isolation perfusion should be demonstrated in random assignment trials.

Effects of hyperthermia and tumour necrosis factor on inflammatory cytokine secretion and procoagulant activity in endothelial cells.

The application of hyperthermia (HT) and tumour necrosis factor alpha (TNF) in isolation perfusion of the limb or liver results in regression of advanced cancers confined to these regions of the body in most patients and are thought to exert anti-tumour effects primarily on tumour neovasculature. However, the individual contribution of either treatment factor on endothelial cells (EC) are not known. In this study, we investigated the in vitro effects of moderate and severe HT on human umbilical vein EC (HUVEC) with and without TNF in clinically relevant doses. HUVEC were exposed to normothermia (37 degrees C) or moderate (39 degrees C) and severe (41 degrees C) HT for 90 or 180 min with or without TNF (1 microg/ml). Cell viability, cytokine secretion (IL-6, IL-8, VEGF, ICAM-1, VCAM-1, RANTES, E-selectin, P-selectin, L-selectin, and PECAM-1), and induction of procoagulant activity as reflected in tissue factor (TF) production were assessed at the end of the treatment period and at several time points thereafter. Neither HT nor TNF exerted significant cytotoxic effects on EC at the doses and temperatures used. HT resulted in increased production of PECAM-1 with little or no additional effect when combined with TNF. TNF caused increased secretion of IL-6, IL-8, ICAM-1, and VCAM-1 with little or no additional effect from HT. Increased E-selectin and RANTES levels were observed with TNF and HT only at 24 h after treatment. HT and TNF had mainly antagonistic effects on VEGF secretion with HT causing primarily decreased production and TNF causing increased VEGF secretion under all temperatures. Most notably, there was a rapid, prolonged and synergistic peak increase in procoagulant activity when TNF and HT were used in combination compared to TNF or HT treatment alone. These results indicate that TNF and HT exert primarily independent effects on inflammatory cytokine production in EC but synergistically increase procoagulant activity as reflected in TF production. These data provide a possible mechanism for the thrombotic effects in tumour neovasculature seen following isolation perfusion with these agents and provide a rationale for their combined use in this treatment setting.

Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage IV clear cell renal cancer.

Clear cell renal carcinoma (CCRC) is a highly angiogenic tumor known to secrete vascular endothelial cell growth factor (VEGF). Endostatin is an endogenous antiangiogenic agent with antitumor activity in mice. The purpose of this study was to evaluate serum levels of endostatin in normal subjects and in patients with CCRC and to examine the relationship of these levels to circulating VEGF levels. Fifteen patients (mean age, 48 years) on a clinical protocol for stage IV CCRC at the National Cancer Institute were included in the study. Archived prenephrectomy serum samples were analyzed for endostatin and VEGF concentrations. Endostatin and VEGF levels were compared with those of an age-matched group of volunteer blood donors (n = 18) using a competitive enzyme immunoassay. Data were analyzed using the Mann-Whitney U test and the Spearman rank correlation. Median serum endostatin levels were 24.6 ng/ml (range, 15.1-54.0 ng/ml) in CCRC patients versus 14.1 ng/ml (range, 1.0-19.3 ng/ml) in healthy controls (P < 0.0001). Median VEGF levels were 3.4 ng/ml (range, 0.1-11.2 ng/ml) and 2.5 ng/ml (range, 0.1-4.2 ng/ml), respectively (P = 0.065). A highly significant correlation was observed between endostatin and VEGF levels among the CCRC patients (r = 0.81, P = 0.0003) but not among controls (r = -0.22, P = 0.37). Endostatin levels are detectable in serum from healthy subjects as well as from CCRC patients. Levels are significantly elevated and correlate with VEGF levels in CCRC patients. Elucidating the nature of this correlation may lend insight into the regulation of tumor angiogenesis in patients with renal cancer.

Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia.

Several inflammatory cytokines, most notably tumor necrosis factor (TNF) and IL-1, induce anorexia and loss of lean body mass, common manifestations of acute and chronic inflammatory conditions. In C57BL/6 female mice, the administration of TNF, IL-1, and, to a lesser extent, leukemia inhibitory factor (LIF), produced a prompt and dose-dependent increase in serum leptin levels and leptin mRNA expression in fat. IL-10, IL-4, ciliary neurotrophic factor, and IL-2, cytokines not known to induce anorexia or decrease food intake, had no effect on leptin gene expression or serum leptin levels. After administration of Escherichia coli lipopolysaccharide (LPS), leptin gene expression and leptin levels were increased. These findings suggest that leptin levels may be one mechanism by which anorexia is induced during acute inflammatory conditions.

Comparison of five commercial kits for the determination of glycosylated haemoglobin.

Five commercial chromatography kits for the measurement of glycosylated haemoglobin have been compared using blood specimens obtained from diabetic patients and from non-diabetic controls. Assessment was based on accuracy, general impressions of practicality, and overall performance. The precision of each technique was assessed by selecting five diabetic and five non-diabetic specimens and testing each one by each technique. All systems tested were of a high standard and the advantages and disadvantages of each system are discussed.

Production of ethylene and other volatiles and changes in cellulase and laccase activities during the life cycle of the cultivated mushroom, Agaricus bisporus.

Nine volatile hydrocarbons, as well as methyl chloride, carbonyl sulphide and carbon disulphide, have been identified by mass spectrometry as products of Agaricus bisporus in the compost used in comerical mushroom beds. Of these, only ethylene showed a pattern of production that could be correlated with developmental phases of the crop, high levels being produced whenever fruit bodies were rapidly enlarging. In laboratory flask cultures, under controlled conditions, high levels of ethylene occurred whenever young fruit bodies entered the expansion phase. The enhanced rate of ethylene production continued over several days, irrespective of whether fruit bodies were removed. Production occurred within the colonized compost; no ethylene was evolved by the fruit body itself. When the first fruit bodies expanded, either in beds or culture flasks, laccase levels in the compost fell and those of a beta-1,4-glucanase (cellulase) rose. The enzyme switch occurred once only, during maturation of the first fruit bodies, whereas an elevated ethylene production was associated with each occasion when fruit body maturation took place. The low level of laccase and high of cellulase characterized the whole of the reproductive stage of A. bisporus, whereas the phasic periods of high ethylene production distinguished between periods of fruit body maturation and intervening resting periods.

Rapid fermentation confirmation of Neisseria gonorrhoeae.

A rapid sugar fermentation procedure has been developed for the confirmation of Neisseria gonorrhoeae from either primary isolation media or purification media. A lightly buffered salt solution with pH indicator and sugars was heavily inoculated with presumptively positive growth. Proper fermentation patterns are obtained in 1 to 4 h with no interference from inhibited contaminants or variation in results due to differing growth requirements of gonococcal strains.

Particulate Fractions of Neisseria gonorrhoeae.

Passage of gonococcal cells through a Ribi cell fractionator produced a mixture of protoplasm and cell particulates, the latter being separable in sucrose solutions. Separation of these particulates resulted in the detection of objects identified as cell walls and "plasts." The latter objects had an average diameter of 1.00 +/- 0.05 mum and consisted of spatially oriented granules surrounded by a membrane-like structure only. Cell wall structures were not observed with these plasts. Plasts were morphologically stable in distilled water and at low pH values. Although 50% more pressure was required to rupture plasts than whole cells, plast morphology was destroyed by organic solvents. Whole cell and plast morphology was examined by electron and phase microscopy, and certain correlations were made between structure and morphological stability. Cell wall antiserum reactivities were quantitatively different for plasts and cell walls. Serological reactivities of plasts were separable by sonic oscillation and differential centrifugation of the sonically treated material.