A cutaneous lymphoma international prognostic index (CLIPi) for mycosis fungoides and Sezary syndrome.

BACKGROUND: There is no prognostic index for primary cutaneous T-cell lymphomas such as mycosis fungoides (MF) and Sezary syndrome (SS). METHOD: Two prognostic indices were developed for early (IA-IIA) and late stage (IIB-IVB) disease based on multivariate data from 1502 patients. End-points included overall survival (OS) and progression free survival (PFS). External validation included 1221 patients. FINDINGS: Significant adverse prognostic factors at diagnosis consisted of male gender, age >60, plaques, folliculotropic disease and stage N1/Nx for early stage, and male gender, age >60, stages B1/B2, N2/3 and visceral involvement for late stage disease. Using these variables we constructed two separate models each defined using 3 distinct groups for early and late stage patients: 0-1 (low risk), 2 (intermediate risk), and 3-5 factors (high risk). 10 year OS in the early stage model was 90.3% (low), 76.2% (intermediate) and 48.9% (high) and for the late stage model 53.2% (low), 19.8% (intermediate) and 15.0% (high). For the validation set significant differences in OS and PFS in early stage patients (both p<0.001) were also noted. In late stage patients, only OS differed between the groups (p=0.002). INTERPRETATION: This proposed cutaneous lymphoma prognostic index provides a model for prediction of OS in early and late stage MF/SS enabling rational therapeutic choices and patient stratification in clinical trials.

A novel technique for the examination of skin biopsies by laser capture microdissection.

BACKGROUND: Skin is an inherently heterogeneous tissue, thus the procurement of pure cell populations is critical for the accurate correlation of a molecular profile to a particular cell type or histological location. Laser Capture Microdissection (LCM) permits the efficient procurement of cells and mapping of genetic changes from histologically prepared samples. METHODS: This paper describes a robust LCM protocol established in our laboratory for the extraction of high quality DNA which sequenced from 100% of microdissected samples without the need for cloning. The unique properties of skin, in particular its strong intercellular adhesive forces, have dictated a significant modification to the normal procedure of tissue preparation to ensure reliable cell procurement. RESULTS: Using the methods outlined below we were able to precisely map the pattern of genomic mutations in our target gene of interest in normal skin, actinic keratosis and squamous cell carcinoma. CONCLUSIONS: The capability to select pure cell populations from the skin will revolutionise our ability to understand the processes involved in cutaneous tumourigenesis.

Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.

Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: the roles of glutathione and glutathione peroxidase.

Three different types of red blood cells (RBC) were used: (i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH and (iii) RBC from high-GSH sheep treated with CDNB to deplete GSH. Incubation of these RBC with t-butyl hydroperoxide (tBHP, 3 mM) for 10 min caused the formation of TBARS, oxidation of haemoglobin and degradation and aggregation of membrane proteins in RBC from low-GSH sheep and GSH-depleted RBC. By contrast, RBC from high-GSH sheep (normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreitol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TBARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted RBC or normal RBCs in the presence of 1.5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degradation and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymatic system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known antioxidants, Butylated hydroxytoluene, ascorbate, alpha-tocopherol and desferrioxamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insult.

Haemolysis of human and sheep red blood cells in glycerol media: the effect of pH and the role of band 3.

Haemolysis of red blood cells (RBC) in glycerol media may be measured spectrophotometrically. The haemolytic process in a rapid phase obeys a first order rate law. The rate constant expresses the rate of haemolysis. To gain a better understanding of the mechanism of haemolysis in glycerol media, the effects of pH and band 3 inhibitors on the rate of haemolysis in human and sheep RBC were observed. Over the pH range used (pH 5.8-10.0), the rate of haemolysis decreased with increase in pH in sheep RBC. By contrast, the rate of haemolysis increased from pH 5.8 to 6.4 and decreased above pH 6.4 in human RBC. The different effects of pH on the rate of haemolysis are due to inhibition of glycerol permeability by H(+) in human RBC but not in sheep RBC. This is supported by the different effects of temperature and Cu(2+) on the rate of haemolysis in human and sheep RBC. We did not observe complete inhibition of haemolysis by the classical band 3 inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Another band 3 inhibitor 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) showed only weak inhibition. Phenylgloxal (PG), another band 3 inhibitor, had no effect whatsoever on the rate of haemolysis. These results indicate that the anion pathway of band 3 is not the preferred route of transport of glycerol in mammalian RBC.

Glutathione reductase activity and flavin concentration in guinea-pig tissues.

Glutathione reductase (GR) activity and flavin concentration were studied in systemic tissues (brain, heart, lung, liver, spleen, stomach, pancreas, muscle, kidney, testis) and blood components (erythrocytes and plasma) from male guinea-pigs. GR activity and the flavin concentration were high in kidney and liver, and low in muscle. GR activity in erythrocytes was found in a range of tissues, but flavin concentration in erythrocytes was lower than in any tissues. GR was saturated with flavin adenine dinucleotide (FAD) in almost all tissues, but not in muscle or erythrocytes.

Lysine and glutamate transport in the erythrocytes of common brushtail possum, Tammar Wallaby and eastern grey, kangaroo.

It was recently coincidentally discovered, using 1H NMR spectroscopy, that the erythrocytes of two species of Australian marsupials, Tammar Wallaby (Macropus eugenii) and Bettong (Bettongia penicillata), contain relatively high concentrations of the essential amino acid lysine (Agar NS, Rae CD, Chapman BE, Kuchel PW. Comp Biochem Physiol 1991;99B:575-97). Hence, in the present work the rates of transport of lysine into the erythrocytes from the Common Brushtail Possum (Dactylopsilia trivirgata) and Eastern Grey Kangaroo (Macropus giganteus) (which both have low lysine concentrations), and Tammar Wallaby were studied, to explore the mechanistic basis of this finding. The concentration-dependence of the uptake was studied with lysine alone and in the presence of arginine, which may be a competitor of the transport in some species. In relation to GSH metabolism, glutamate uptake was determined in the presence and absence of Na+. The data was analysed to yield estimates of the maximal velocity (Vmax) and the Km in each of the species. Erythrocytes from Tammar Wallaby lacked saturable lysine transport in contrast to the other two species. The glutamate uptake was normal in all three animals for adequate GSH biosynthesis.

Phospholipid composition of erythrocyte membranes and plasma of mammalian blood including Australian marsupials; quantitative 31P NMR analysis using detergent.

The phospholipid classes of erythrocyte membranes and plasma from several domestic animals and marsupials were quantified by 31P NMR using detergents. Washed erythrocyte samples were thoroughly haemolysed by tip-sonication and dissolved in sodium cholate; plasma samples were dissolved in Triton X-100. The species studied were: common wombat (Vombatus ursinus), black-striped wallaby (Macropus dorsalis), bandicoot (Isoodon macrocarpus), Eastern grey kangaroo (Macropus giganteus), Tammar wallaby (Macropus eugenii), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus), horse (Equus caballus), dog (Canus familiaris) and rabbit (Orytolagus caniculus). There were considerable species variations in the relative abundance of erythrocyte and plasma phospholipid classes. The variations may be attributed to the habitats and diets of the animals as well as to their phylogenetic differences.

Glutathione removal reveals kinases as common targets for K-Cl cotransport stimulation in sheep erythrocytes.

K-Cl cotransport is activated by swelling, lowering of cellular free Mg (Mgi), and thiol modification of erythrocytes. Direct actions by thiol reagents on the K-Cl cotransport complex were separated from indirect effects through nonoxidative changes in cellular glutathione (GSH). We used 1-chloro-2,4-dinitrobenzene (CDNB), which, conjugated to GSH, is extruded from the erythrocyte as a thioether. CDNB caused a small biphasic effect (inhibition and stimulation) on K-Cl cotransport and, at 1 mM, abolished its stimulation by N-ethylmaleimide (NEM), diazenedicarboxylic acid bis[N,N-dimethylamide], methyl methanethiosulfonate, and staurosporine, a kinase inhibitor, independent of the order of treatment. Hence, NEM and other activating-thiol reagents, and perhaps GSH removal itself, target unidentified kinases involved in activation of K-Cl cotransport. CDNB also abrogated K-Cl cotransport stimulation by Mgi depletion independent of the order of treatment, indicating inhibition at a second site nearer to the transporter. Furthermore, CDNB treatment elevated and rendered K-Cl cotransport insensitive to osmotic shrinkage, suggesting uncoupling from the regulator.

Comparative erythrocyte metabolism in marsupials and monotremes.

Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

Comparative study of the antioxidant defence systems in the erythrocytes of Australian marsupials and monotremes.

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes "detoxify" H2O2, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC.

Antioxidant systems and erythrocyte life-span in mammals.

1. Erythrocyte antioxidant systems--superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR)--were discussed in relation to life-spans in some mammalian species. 2. The erythrocyte life-span of different mammals was found to be correlated with the levels of SOD, GSH-Px and GSH. 3. Data reviewed indicates that the erythrocyte life-span of each species is governed by both the oxygen radical formation and the efficiency of intrinsic antioxidant systems.

Hematology and erythrocyte biochemistry of spectacled hare-wallaby, Lagorchestes conspicillatus (Macropodidae Marsupialia).

Hematology and the red cell enzymes, metabolites, and rates of lactate production were measured in a small marsupial, the spectacled hare-wallaby Lagorchestes conspicillatus. In common with other marsupials of similar body weights, the red cells of this wallaby were found to have low levels of ATP and higher levels of 2,3-diphosphoglycerate (DPG) and produced no lactate when incubated with inosine. One of the five animals examined was suffering from anemia (hematocrit of 22.0% and hemoglobin concentration of 7.2 g/dl compared with the normal values of 46.6% and 15.8 g/dl, respectively); its red cells had almost twice the concentrations of ATP and DPG and had significantly higher activities of several enzymes of the glycolytic pathway.

Relation between reduced glutathione content and Heinz body formation in sheep erythrocytes.

To clarify the oxidant defense functions of reduced glutathione (GSH) in erythrocytes, the effect of GSH deficiency on in vitro oxidant defense was studied, using GSH-deficient sheep erythrocytes (low-GSH cells). The formation of Heinz bodies in low-GSH cells was higher than that in high-GSH cells when the cells were incubated with an oxidant drug, acetylphenylhydrazine (APH). Artificial depletion of GSH by 1-chloro-2,4-dinitrobenzene in high-GSH cells resulted in increased Heinz body formation in these cells incubated with APH. Furthermore, high negative correlation was observed between Heinz body formation and GSH content in sheep erythrocytes exposed to APH. These results clearly indicate that erythrocyte GSH is indispensable for erythrocyte defense against oxidative damage induced by APH, and support the previous observations that sheep with low-GSH erythrocytes were more susceptible to oxidative agents than were sheep with high-GSH erythrocytes.

Developmental changes in the erythrocyte metabolism in bandicoot Isoodon macrourus.

Activities of 13 different enzymes were measured in the erythrocytes of juvenile and adult bandicoots, Isoodon macrourus. Seven of these enzymes had significantly (p < 0.05 or less) greater activities in the juveniles compared to the adult animals. The activity of one enzyme--phosphofructokinase--was significantly (p < 0.01) lower in the juveniles. However, the activity of NADH-methaemoglobin reductase (MR) was similar in the two groups of animals. The rates of lactate production using four different substrates (glucose, galactose, inosine and adenosine) were also higher in the juveniles. These results indicate that the erythrocytes of juvenile bandicoots are metabolically more active than those of the adult animals and follow the general pattern of higher metabolic activity in the young red cells in eutherian mammals, with possible exception of NADH-MR activity.

Studies on the blood of fallow deer (Dama dama) and red deer (Cervus elaphus): haematology, red cell enzymes, metabolic intermediates and glycolytic rates.

1. Blood samples were obtained from fallow deer (Dama dama) and red deer (Cervus elaphus). Basic haematology, red cell enzymes, and metabolic intermediates and the glycolytic rate of the red cells incubated with different substrates were measured. 2. The major findings were (i) the activity of glucose phosphate isomerase was notably high in the red blood cells of the red deer; (ii) red deer cells also utilized adenosine more efficiently than those of fallow deer and (iii) red cells of both species utilized galactose more efficiently than other species of ruminants.

The relationship between reduced glutathione level and glutathione S-transferase activity in sheep erythrocytes.

The relationship between reduced glutathione (GSH) level and glutathione S-transferase (GST) activity in erythrocytes was examined, using sheep erythrocytes, which have varying GSH concentrations, and dog erythrocytes with an inherited high concentration of GSH. There was a positive correlation (r = 0.529, p < 0.001) between the GSH level and GST activity in sheep erythrocytes. In dog erythrocytes, the GST activity in high-GSH cells was significantly (p < 0.001) higher than that in normal-GSH cells. These results indicate that the activity of GST in erythrocytes is directly correlated with the intracellular GSH level.

Erythrocyte metabolism in two species of bats: common bent-wing bat (Miniopterus schreibersii) and red fruit bat (Pteropus scapulatus).

1. Metabolic intermediates, substrate utilization and enzyme activities were determined in the red blood cells of the common bent-wing bat and the red fruit bat. Standard haematological parameters and oxy-haemoglobin dissociation curves were also determined in both species. 2. The glycolytic rate as measured by lactate production was much higher for all substrates in the bent-wing bats. The activities of the glycolytic enzymes were also much higher in this species. 3. The standard haematological parameters were similar for the two species. The levels of 2,3-diphosphoglycerate (DPG) in the red cells of the fruit bat were nearly twice as high as those in the bent-wing bats. 4. The oxy-haemoglobin dissociation curve for the red fruit bat was located to the right of that for the bent-wing bat and both these curves were located to the right of that normally seen for human blood. 5. Both species of bat show blood characteristics well adapted to carrying the increased oxygen demands of flight.

Hemolytic and microbicidal actions of diethyldithiocarbamic acid.

Micromolar concentrations of diethyldithiocarbamic acid (DDC) kill fungi, bacteria and malaria. DDC forms chelates with copper and the microbicidal effectiveness of this drug is enhanced greatly by small amounts of copper. DDC, in the presence of at least 1 molar equivalent of copper, also causes lysis of human erythrocytes. To explore the cytocidal actions of DDC and copper, we have used human erythrocytes and Escherichia coli as models. We found that: (1) the combination of DDC and copper also lysed E. coli spheroplasts, suggesting a possible common mechanism of hemolytic and microbicidal action; (2) higher ratios of drug: metal (greater than 4:1) diminished hemolytic and, as observed earlier, microbicidal effects; (3) cobalt, known to suppress the microbicidal effects of DDC:Cu, also prevented red cell lysis; (4) despite the necessary involvement of copper in DDC-mediated hemolysis, there was no evidence of oxidative damage to erythrocytes, and both lysis of erythrocytes and killing of E. coli were undiminished in the absence of oxygen; (5) the DDC:Cu chelate preferentially located in organic solvents and in membranes of erythrocytes. The chelate was quite soluble in chloroform but much less so in a C-16 hydrocarbon (hexadecane) which resembled erythrocyte membrane lipid. In hexadecane and at greater than 10(-4) M DDC and 5 x 10(-5) copper, an amphipathic drug:metal complex accumulated at the organic:aqueous interface; and (6) this amphipathic complex may permeabilize the lipid bilayer, causing leakage of ions and cell water and eventuating in colloid osmotic lysis. Red cells and E. coli exposed to the chelate showed early loss of intracellular rubidium (86Rb+). Furthermore, lysis of erythrocytes and E. coli spheroplasts was suppressed by the inclusion of either dextran or sucrose. Thus, it appears that DDC:Cu chelates are cytocidal by virtue of concentrating in the lipid bilayer and, perhaps, forming amphipathic complexes which disrupt membrane integrity. Drugs with similar behavior hold promise for therapy of malaria because metals capable of forming such complexes may accumulate within parasitized red cells.

1H NMR spectroscopic survey of plasma and erythrocytes from selected marsupials and domestic animals of Australia.

1. 1H NMR spectra were acquired from whole plasma, intact erythrocytes, and ultrafiltrates of erythrocytes from nine native and eight introduced (domestic) Australian animals; single-pulse, spin-echo and 2-dimensional spectra were obtained. The aim was to detect and at least semi-quantify metabolites in the samples and compare the profiles amongst the species. 2. The Australian natives that were studied were all marsupials: greater brown bandicoot; bettong; eastern grey kangaroo; red kangaroo; koala; possum; red necked pademelon; Tammar wallaby; and wombat. The introduced mammals that were studied were: cat; cattle; dog; goat; horse; pig; rabbit; and sheep. 3. Because of the range of habitats and diets amongst the animals, it was postulated that the concentrations of the common metabolites in the blood would show marked differences and that there would also be some metabolites that were peculiar to a given animal. There were several major differences in the spectra: in the spectra of plasma, the glycoprotein and lipoprotein resonances showed the largest inter-species variation, whereas the most dramatic finding from the spectra of erythrocytes was a very high concentration of lysine in the cells from the Tammar wallaby.