Use of the Skin Cancer Quality of Life Impact Tool (SCQOLIT) - a feasibility study in non-melanoma skin cancer.

BACKGROUND: Incidence of non-melanoma skin cancers (NMSCs) is increasing and can significantly impact on quality of life (QOL), yet there are few studies evaluating patient-reported outcome measures (PROMs) in NMSC populations. We undertook a prospective feasibility study to evaluate a skin cancer-specific PROM, the Skin Cancer Quality of Life Impact Tool (SCQOLIT), in patients with a new diagnosis of NMSC. OBJECTIVES: (i) To establish acceptability of SCQOLIT in dermatology clinics, (ii) a descriptive analysis of SCQOLIT scores in NMSC. METHODS: Patients with histologically confirmed NMSC completed SCQOLIT, EQ-5D and a transition item. Questionnaires were completed at baseline and 3 months for group 1 ('low-risk' NMSC) and group 2 ('high-risk' NMSC) with additional questionnaires at 6-9 months for group 2. Patients participated in structured interviews. Clinician experience was captured through staff evaluation forms and a focus group. Acceptability and psychometric properties of SCQOLIT were assessed. RESULTS: Overall, 318 patients consented to participate. Mean SCQOLIT score at baseline was 5.33, with 2.6% of patients scoring ≥20. No ceiling effects were observed, whilst 13.9% scored 0. Validity was demonstrated against EQ-5D. Cronbach's alpha 0.84 demonstrated internal consistency. Thirteen patients were interviewed and thought SCQOLIT was comprehensive, captured impact on health-related QOL and helped express their needs to clinicians. Most clinicians found SCQOLIT 'very useful' or 'useful to some extent' in facilitating discussions. CONCLUSIONS: This feasibility study demonstrates that SCQOLIT is acceptable to patients and staff in dermatology skin cancer clinics. The psychometric properties of SCQOLIT confirm its utility in NMSC populations.

Investigation of the cutaneous response to recall antigen in humans in vivo.

In this paper we provide a detailed description of an experimental method for investigating the induction and resolution of recall immune response to antigen in humans in vivo. This involves the injection of tuberculin purified protein derivative (PPD) into the skin, followed by inducing suction blisters at the site of injection, from which leucocytes and cytokines that are involved in the response can be isolated and characterized. Using this technique we found that although the majority of CD4(+) T cells in the skin that are present early in the response express cutaneous lymphocyte antigen (CLA), the expression of this marker is reduced significantly in later phases. This may enable these cells to leave the skin during immune resolution. Furthermore, interleukin (IL)-2 production can be detected both in CD4(+) T cells and also in the blister fluid at the peak of the response at day 7, indicating that mediators found in the blister fluid are representative of the cytokine microenvironment in vivo. Finally, we found that older humans have defective ability to respond to cutaneous PPD challenge, but this does not reflect a global immune deficit as they have similar numbers of circulating functional PPD-specific CD4(+) T cells as young subjects. The use of the blister technology enables further characterization of the skin specific defect in older humans and also general mechanisms that govern immune regulation in vivo.

Relative impact of CD4+CD25+ regulatory T cells and tacrolimus on inhibition of T-cell proliferation in patients with atopic dermatitis.

BACKGROUND: It has been established recently that CD4+CD25+ regulatory T cells (Tregs) play an important role in controlling various immune responses. Immunosuppressive drugs are often used to treat immune dysregulation but are frequently associated with undesirable side-effects. OBJECTIVES: We examined the suppressive capacity of circulating Tregs in patients with atopic dermatitis (AD). Combined effects of Tregs and tacrolimus on the inhibition of T-cell proliferation in vitro were also assessed. METHODS: CD4+CD25+ and CD4+CD25- T cells were isolated from peripheral blood mononuclear cells using immunomagnetic beads. CD4+CD25- T cells were stimulated with purified protein derivative (PPD) or house dust mite allergen (Der p1) for 6 or 7 days, respectively. A dose range of tacrolimus and CD4+CD25+ T cells were added separately, or together. Proliferation was measured by (3)H-thymidine incorporation. RESULTS: CD4+CD25+ T cells from normal controls and patients with AD are anergic and inhibit the proliferation of CD4+CD25- T cells in response to PPD and Der p1 in vitro in a dose-dependent manner. Addition of tacrolimus and Tregs together showed significantly stronger inhibition of proliferation than either on their own. This was true for both antigens and both in normal controls and in patients with AD. CONCLUSIONS: CD4+CD25+ T cells in patients with AD have normal suppressive activity compared with healthy controls. Tregs and tacrolimus have additive effects on the inhibition of proliferation in response to PPD and Der p1.

The immunomodulatory effects of regulatory T cells: implications for immune regulation in the skin.

Regulatory T cells are thought to have a critical role in the suppression of immune responses. In addition to the prevention of the development of autoimmunity, they are also thought to have a role in the prevention of allergic responses to environmental allergens, immune responses to tumours and the development of memory responses to chronic infections. They have been isolated within the skin and have been shown to express surface markers that enable skin-specific migration, suggesting that regulatory T cells have a functional role in the skin. There is accumulating evidence to suggest that regulatory T cells may be involved in numerous skin disorders and may also be modified by various therapeutic agents used to treat these disorders. We review the evidence for the presence of this T-cell subset in humans, the suppressive effects of regulatory T cells, and their role in the skin.

Identification of novel metabolites of pioglitazone in rat and dog.

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [(3)H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.

Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver.

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.

Rat liver cytosol catalyzes a reaction involving activated N-nitrosodimethylamine and a carbohydrate from the pentose phosphate pathway.

N-Nitrosodimethylamine is a liver toxin and mutagen following activation by cytochrome P450. The role of the cytosol in N-nitrosodimethylamine metabolism is not well understood. The effect of cytosol on N-nitrosodimethylamine metabolism was investigated using microsomes and cytosol from rat liver in in vitro reactions with N-nitrosodimethylamine and an NADPH generating system. Studies in which [(14)C]-N-nitrosodimethylamine and calf thymus DNA were used indicated that the addition of cytosol to the microsomal reaction mixture resulted in >200% enhancement of the radioactivity associated with DNA after the DNA was isolated from the reaction mixture by phenol extraction followed by ethanol precipitation. This stimulatory effect was associated with a cytosolic protein and was found to be dependent on both the microsomes and the carbohydrate used in the glucose-6-phosphate dehydrogenase system for the generation of NADPH. The carbohydrate requirement was found to be specific for intermediates of the pentose phosphate pathway, and maximum stimulation occurred with ribulose 5-phosphate. Most of the counts from [(14)C]-N-nitrosodimethylamine which were isolated with DNA after the addition of cytosol to reaction mixtures were not covalently bound to the DNA. HPLC analysis identified four radiolabeled metabolites derived from [(14)C]-N-nitrosodimethylamine following the in vitro incubations. One of the four products was formed only when both cytosol and ribulose 5-phosphate were added to the enzymatic incubations. This product also formed from [(14)C]-alpha-acetoxy nitrosodimethylamine in the absence of microsomes, only when cytosol and ribulose 5-phosphate were added to the reaction mixtures. Thus, these data demonstrate that an enzyme in the cytosol catalyzes a reaction involving a metabolite of N-nitrosodimethylamine (which is formed following cytochrome P450-mediated activation) and a carbohydrate related to the pentose phosphate pathway. A similar reaction also occurs with N-diethylnitrosamine but not with N-dipropylnitrosamine or N-dibutylnitrosamine.

Proposed mechanism for the cytochrome P450-catalyzed conversion of aldehydes to hydrocarbons in the house fly, Musca domestica.

Experiments were performed to elucidate the mechanism of hydrocarbon formation in microsomal preparations from the house fly, Musca domestica. Antibody to both house fly cytochrome P450 reductase and a purified cytochrome P450 (CYP6A1) from the house fly inhibited (Z)-9-tricosene (Z9-23:Hy) formation from [15,16-3H]-(Z)-15-tetracosenal (24:1 aldehyde). Chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) analyses of the n-tricosane formed by microsomal preparations from [2,2-2H2,2-13C]- and [3,3-2H2,3-13C]tetracosanoyl-CoA demonstrated that the deuteriums on the 2,2- and 3,3-positions were retained in the conversion to the hydrocarbon product. Likewise, CI-GC-MS analysis of the Z9-23:Hy formed from [1-2H]tetracosenal by microsomal preparations demonstrated that the aldehydic proton on the 1-carbon was transferred to the hydrocarbon product. Hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were able to support hydrocarbon production from [3H]24:1 aldehyde in place of O2 and NADPH for short incubation times. From these data, a cytochrome P450 mechanism is proposed in which the perferryl iron-oxene, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the C=O double bond of the carbonyl group of the aldehyde. The reduced perferryl attacks the 1-carbon of the aldehyde to form a thiyl-iron-hemiacetal diradical. The latter intermediate can fragment to form an alkyl radical and a thiyl-iron-formyl radical. The alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2.

Regulation of enzymatic activity involved in sex pheromone production in the housefly, Musca domestica.

Ovarian produced ecdysteroids regulate sex pheromone production in the female housefly, inducing the synthesis of (Z)-9-tricosene (Z9-23:Hy), cis-9,10-epoxytricosane, (Z)-14-tricosen-10-one and methylalkanes. Experiments were performed to gain a detailed understanding of the processes affected by 20-hydroxyecdysone (20-HE) that result in sex pheromone production as the female becomes reproductively mature. A novel microsomal fatty acid synthetase (FAS) is present in the epidermal tissue and plays a role in producing the methyl-branched fatty acid precursors to the methylalkanes. This FAS is released from the microsomes in the presence of 3 M KCl. A major enzyme activity influenced by 20-HE is the fatty acyl-CoA elongation system. A shift in the chain length specificity of the products of the elongation system causes the change in the chain lengths of the alkenes produced to switch from C27 and longer in the previtellogenic female to C23 in the mature female. Data is presented indicating that it is the condensation activity of the elongation system that is affected. Z9-23:Hy arises from a 24 carbon acyl group which is reduced to an aldehyde, and then converted to the hydrocarbon. Data is presented demonstrating that it is the fatty acyl-CoA derivative and not the free fatty acid that is the substrate. There does not appear to be a chain length specificity which regulates the conversion of fatty acyl-CoAs to hydrocarbons as both 24 and 28 carbon fatty acyl-CoAs are converted to hydrocarbon by both males and females of all ages.

Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2.

An unusual mechanism for hydrocarbon biosynthesis is proposed from work examining the formation of (Z)-9-tricosene (Z9-23:Hy), the major sex pheromone component of the female housefly, Musca domestica. Incubation of (Z)-15-[1-14C]- and (Z)-15-[15,16-3H2]tetracosenoic acid (24:1 fatty acid) with microsomes from houseflies gave equal amounts of [3H]Z9-23:Hy and 14CO2. The formation of CO2 and not CO, as reported for hydrocarbon formation in plants, animals, and microorganisms [Dennis, M. & Kolattukudy, P. E. (1992) Proc. Natl. Acad. Sci. USA 89, 5306-5310], was verified by trapping agents and by radio-GLC analysis. Incubation of (Z)-15-[15,16-3H2]tetracosenoyl-CoA with microsomal preparations in the presence of NADPH and O2 gave almost equal amounts of (Z)-15-3H2]tetrasosenal (24:1 aldehyde) and Z9-23:Hy. Addition of increasing amounts of hydroxylamine (aldehyde trapping agent) caused a decrease in hydrocarbon formation with a concomitant increase in oxime (aldehyde derivative) formation. The 24:1 aldehyde was efficiently converted to (Z)-9-tricosene only in the presence of both NADPH and O2. Bubbling carbon monoxide (20:80 CO/O2) or including an antibody against housefly cytochrome P450 reductase inhibited the formation Z9-23:Hy from 24:1 aldehyde. These data demonstrate an unusual mechanism for hydrocarbon formation in insects in which the acyl-CoA is reduced to the corresponding aldehyde and then carbon-1 is removed as CO2. The requirement for NADPH and O2 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.

Medullary sponge kidney presenting as polycystic renal disease.

We observed a 30-year-old woman in whom a large mass in the right lower quadrant and bilaterally enlarged cystic kidneys with markedly distorted collecting systems by excretory urography suggested a diagnosis of polycystic renal disease (PRD); but the linear and cystic dilatation of collecting tubules, many of which contained numerous calculi, was typical of medullary sponge kidney (MSK). Arteriography and CT scanning clearly showed that both the calcifications and the cysts were confined to the medullary portions. The cortical layer was entirely free of cysts except where large medullary cysts protruded to the surface. In patients in whom MSK imitates PRD, these conditions can be distinguished by CT scanning and arteriography.

Constrictive pericardial disease in the dog.

The clinicopathologic features of constrictive pericardial disease in 13 dogs were reviewed. The causes were infection (3 dogs), metallic foreign body (1 dog), and idiopathic (9 dogs). Owner complaints included abdominal enlargement, tachypnea, weakness or syncope, exertional fatigue, and weight loss. Ascites and jugular venous distention were consistently observed, whereas abnormalities of arterial pulses and heart sounds were variable and inconsistent. Diminished QRS voltages were common. Mild to moderate cardiomegaly, rounding of the cardiac silhouette, and variable and nonspecific angiographic findings were frequently observed. Cardiac catheterization consistently showed elevation and equilibration of atrial and ventricular diastolic pressures, but a prominent early diastolic (y) descent was uncommon. Fibrosis was confined to the parietal pericardium in 8 dogs, and included the epicardium in 5 dogs. Parietal pericardectomy was successful in relieving the syndrome in 6 of 10 dogs. Pulmonary thrombosis was the most common cause of early postoperative mortality.

Hemodynamics of progressive pneumopericardium in the dog.

The hemodynamics of progressive pneumopericardium were studied in 6 anesthetized healthy dogs (with intact thorax). Heart rate, cardiac output, and pericardial and intravascular pressures were recorded. Pressures were measured in the pericardial space by means of a percutaneously introduced, air-filled catheter, and in the right atrium, right ventricle, left atrium, left ventricle, pulmonary artery, and aorta by means of fluid-filled catheters. The gradual increase in mean pericardial pressure up to 11 +/- 2 mm of Hg was associated with increases in heart rate and mean right atrial, mean left atrial, right ventricular end-diastolic, and left ventricular end-diastolic pressures and with decreases in cardiac output and stroke volume, with no change in left ventricular systolic and mean aortic pressures. Critical cardiac tamponade occurred at a pericardial pressure of 12.2 +/- 2.8 mm of Hg, which was produced by 8.7 +/- 1.9 cm3 air/kg of body weight. This was characterized by a sudden marked decrease in heart rate, cardiac output, stroke volume, left ventricular systolic pressure, and mean aortic pressure, and equilibration of mean right atrial, mean left atrial, right ventricular end-diastolic, left ventricular end-diastolic, and pulmonary artery diastolic pressures. These hemodynamic changes were similar to those of experimental pericardial effusion.

The renal hemodynamic response to diatrizoate in normal and diabetic rats.

To study the renal hemodynamic response to a large intravenous bolus of a radiocontrast agent, 8 ml/kg body weight of 60% diatrizoate meglumine (D-60) was infused over 30 seconds in both normal rats and rats with streptozotocin-induced diabetes. The effect of equiosmolar mannitol (1350 mOsm/kg) was compared with the D-60 response in normal rats to examine the potential role of hypertonicity in mediating a response. A similar two-phase response was seen in all three groups. The effect of D-60 in normal rats was similar to that of mannitol, but all responses were reduced in diabetic rats. During Phase I in normal rats in response to D-60 there was a transient three-minute reduction in blood pressure (BP), and renal blood flow (RBF) fell by 62 +/- 7%. During Phase II blood pressure did not change from normal baseline values, but RBF fell by 29 +/- 5%; GFR fell by 42 +/- 3%, and filtration fraction (FF) diminished. In diabetic rats baseline FF was lower than normal and was not further reduced after infusion of D-60. It is suggested that D-60 reduces RBF and GFR by a nonspecific osmotic effect, perhaps related to tubuloglomerular feedback or to an acute increase in intratubular pressure. Responses to these mechanisms may be reduced in diabetic rats with a chronic glycosuric osmotic diuresis.

Chlordiazepoxide and dropping out of a detoxication service.

There were no significant differences in the age, sex or chlordiazepoxide therapy of patients completing and patients not completing a 7-day detoxication program.