Anthony I. Mallet

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B4 and monohydroxy-eicosatetraenoic acid (HETE)-like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified: 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10(-4)M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.

Levels of arachidonic acid and its metabolites in the skin in human allergic and irritant contact dermatitis

Increased concentrations of arachidonic acid and prostaglandin E2, but not 12‐hydroxy‐eicosatetraenoic acid, were found in the skin in human contact dermatitis due to nickel and chromate allergens. Significant levels of neutrophil chemokinetic activity, with similar properties to leukotriene B4, were found in a high proportion of exudates from inflamed skin treated with allergen but not in exudates from untreated skin. Neither arachidonic acid nor its metabolites were increased in primary irritant dermatitis due to benzylalkonium chloride.

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.

Isolation and characterization of a diuretic peptide common to the house fly and stable fly

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

The analysis of arachidonic acid metabolites in normal, uninvolved and lesional psoriatic skin

Collection of exudate from suction bullae is a commonly used method for sampling human skin for mediator analysis. It is satisfactory on skin of normal structure but is unreliable on lesional psoriatic skin in which there are major structural changes and excessive scaling. Collection of exudates from abraded sites was found to be a suitable alternative method for psoriatic skin. Arachidonic acid and 12-HETE, but not PGE2, were significantly higher in exudate from abraded lesional psoriatic skin (494 +/- 88, 45.9 +/- 4.2 and 9.6 +/- 1.8 ng/ml respectively, mean +/- sem, n = 5) compared to uninvolved skin (154 + 38, 18.5 + 5.1 and 7.7 + 1.9 ng/ml) or skin of normal volunteers (119 +/- 37, 14.5 +/- 6.7 and 4.5 +/- 1.6 ng/ml, n = 7) which were similar. The coefficient of variation for exudate collection and mediator analysis was usually less than 55%. The analysis of lipoxygenase and cyclooxygenase products was simplified by the use of chlorobutane to extract preferentially arachidonic acid and HETEs from neutral aqueous solutions.

Generation by the phosphoramidon‐sensitive peptidases, endopeptidase‐24.11 and thermolysin, of endothelin‐1 and C‐terminal fragment from big endothelin‐1

1 Phosphoramidon, a potent inhibitor of endopeptidase‐24.11 (E‐24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin‐1 (big ET‐1) in rats. To examine whether E‐24.11 or thermolysin convert big ET‐1 to endothelin‐1 (ET‐1) and C‐terminal fragment (CTF), the effects on porcine and human big ET‐1 of each of the purified enzymes were compared in vitro. 2 For E‐24.11, the relative rates of hydrolysis were ET‐1 > CTF >> big ET‐1. The relative half‐lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET‐1 > 24 h; ET‐1, 37 min; CTF, 57 min. For comparison, the half‐life for hydrolysis of substance P under similar conditions was 2.1 min. 3 For thermolysin the relative rates of hydrolysis were found to be big ET‐1 > CTF > ET‐1. The relative half‐lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET‐1, 25 min; ET‐1, 56 min; CTF, 47 min. 4 Because the low rate of conversion of big ET‐1 to ET‐1 by E‐24.11 did not yield sufficient ET‐1 for h.p.l.c. quantification a RIA specific for ET‐1 (16–21) was used to study further the hydrolysis of big ET‐1 by E‐24.11. Incubation of big ET‐1 (0.2–2 nmol) with E‐24.11 (4–400 ng) generated ET‐1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET‐1 (2 nmol) with E‐24.11 (40 ng) for 8 h showed that steady state levels of ET‐1 were achieved after 4 h indicating that the rate of ET‐1 degradation was then equal to the formation of new ET‐1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET‐1 had been produced, but the yield was insufficient for verification by mass spectrometry. 5 Both ET‐1‐like and CTF‐like peaks were detected at 214 nm when the products of big ET‐1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET‐1 with amounts in the range 120–160 pmol. 6 The hydrolysis profile of ET‐1 by E‐24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue. 7 Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E‐24.11 and could be useful for the design of ECE inhibitors. Since E‐24.11 can both synthesize and hydrolyse ET‐1, the presence of E‐24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.

Longitudinal Profiles of 15 Serum Bile Acids in Patients With Intrahepatic Cholestasis of Pregnancy

OBJECTIVES:Increased maternal serum bile acids are implicated in intrahepatic cholestasis of pregnancy. Individual bile acid profiles and their relationship with disease progression, however, remain unknown. The purpose of this prospective study was to determine the temporal changes in bile acids in normal pregnancy and in pregnancies complicated with intrahepatic cholestasis of pregnancy and pruritus gravidarum.METHODS:A validated method for the evaluation of 15 bile acids (conjugated and unconjugated) in a single serum sample was developed using high-performance liquid chromatography/mass spectrometry (HPLC-MS) with an electrospray interface. Bile acid concentrations were assessed in samples (16 weeks of gestation to 4 weeks postpartum) from women with, or who later developed, intrahepatic cholestasis of pregnancy (n=63) and were compared with those from normal pregnant women (n=26) and from women with pruritus gravidarum (n=43).RESULTS:Intrahepatic cholestasis of pregnancy was associated with a predominant increase in cholic acid conjugated with taurine and glycine, from 24 weeks of pregnancy. Ursodeoxycholic acid (UDCA) treatment (≥21 days, n=15) significantly reduced serum taurocholic and taurodeoxycholic acid concentrations (P<0.01). Bile acid profiles were similar in normal pregnancy and pregnancy associated with pruritus gravidarum.CONCLUSIONS:The bile acid profiles and effects of treatment by UDCA implicate a role for taurine-conjugated bile acids in the syndrome of intracranial pressure. With regard to individual bile acid profiles, pruritus gravidarum is a disorder quite distinct from intrahepatic cholestasis of pregnancy.

Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection

Tuberculosis has been a scourge of humans over many millennia, but questions remain regarding its evolution and epidemiology. Fossil biomarkers, such as DNA and long-chain mycolic acids, can be detected in ancient skeletal and other materials. The phthiocerol dimycocerosate waxes are also robust biomarkers for tuberculosis and sensitive methods are available for the detection of their mycocerosic acid components. The presence of mycocerosic acids was investigated in 49 individuals from the 1837-1936 Coimbra Identified Skeletal Collection (Portugal), half with documentary data indicating tuberculosis as a cause of death. Samples were hydrolysed, acidic components converted to pentafluorobenzyl esters, the non-hydroxylated long-chain esters isolated, and this fraction separated into multimethyl-branched and other esters by normal phase high performance liquid chromatography. Negative ion chemical ionisation gas chromatography mass spectrometry was used to detect diagnostic C29, C30 and C32 mycocerosic acids. Mycocerosic acids were detected in archaeological material for the first time, illustrating that they are valuable biomarkers for the diagnosis of ancient tuberculosis. A 72% correlation with the Coimbra burial record supported TB as the major cause of death. In addition, 30% of the skeletons, positive for mycocerosates, showed the presence of related long-chain mycolipenic acids.

Cholesterol‐independent endothelial dysfunction in virgin and pregnant rats fed a diet high in saturated fat

1 Western diets high in saturated fat are associated with an increased incidence of cardiovascular diseases. In this study we have evaluated vascular endothelial function and oxidative stress in virgin rats fed a normal (VC) or high in saturated fat diet (VHF) (20% lard and corn oil w/w) from weaning until adulthood, and throughout subsequent pregnancy (PC and PHF, respectively). 2 The saturated fat diet was associated with enhanced noradrenaline sensitivity in small mesenteric arteries from VHF rats (VHF vs. VC, P < 0.05) and blunted endothelium‐dependent relaxation in VHF and PHF rats (VHF vs. VC, P < 0.001; PHF vs. PC, P < 0.05). Endothelial dysfunction was attributable to a reduced nitric oxide component of relaxation in VHF rats, and blunted prostacyclin and endothelium‐derived hyperpolarizing factor components in PHF rats. 3 Other than plasma cholesterol, which was reduced in VHF and PHF rats, plasma lipids were normal. Fasting plasma insulin and glucose concentrations were raised in VHF rats (P < 0.05) and the plasma marker of oxidative stress, 8‐iso PGF2α, was increased in PHF animals (P < 0.01). 4 These findings suggest that endothelial dysfunction induced by a saturated fat diet is cholesterol independent and likely to be of different mechanistic origin in virgin and pregnant rats.

The role of chemo-attractant lipoxygenase products in the pathogenesis of psoriasis.

The histopathology of chronic, stable plaque psoriasis is characterized by acanthosis with mitotic figures in the lower epidermis, abnormalities of differentiation, vascular dilatation and tortuosity, and leukocyte infiltrates. These infiltrates are composed of neutrophils and mononuclear cells, the former apparently migrating from the dermal papilla, through the Malpighian layer and into the parakeratotic stratum corneum. Mononuclear cells are evident in the superficial dermis and in the lower half of the epidermis (Ragaz & Ackerman, 1979; Soltani & Van Scott, 1972; Helwig, 1958; Pinkus & Mehregan, 1966). The nature of the earliest histopathological change in developing psoriatic lesions is the subject of controversy with, for example, one group (Chowaniec et al, 1981) reporting infiltration of polymorphonuclear leukocytes into the epidermis to be the earliest change, another (Ragaz & Ackerman, 1979) finding a superficial perivascular lymphocyte infiltrate, and yet another (Brody, 1984a, b) reporting mast cell degranulation as the primary event in acute guttate psoriasis. In addition, Braun-Falco and Schmoeckel (1977) found macrophages to be the predominant infiammatory cells in initial psoriatic lesions. In postulating a role for an endogenously released chemical mediator in the genesis of a pathological change, it is important (i) to recover elevated levels of the mediator from the involved tissue in vivo at an appropriate time, (2) to reproduce at least in part the pathological change by introduction ofthe mediator into the tissue in vivo, in appropriate concentrations, (3) to demonstrate mechanisms for the release and removal of the mediator in the involved tissue, (4) to modify the pathological process by administration of specific enzyme inhibitors or receptor antagonists in vivo, (5) to determine the relative amounts of other chemical mediators with similar modes of action in the involved tissue in vivo and (6) to determine whether production ofthe mediator occurs in other lesions, i.e. whether it is specific to the pathological process under study. It is noteworthy that most ofthe above, which are an extension ofthe criteria of Dale (1934), can only be investigated by in vivo studies. We have chosen to examine the role of acidic lipid neutrophil chemo-attractants in the pathogenesis ofthe infiammatory changes in chronic plaque psoriasis. The evidence obtained to date is presented here in six sections, according to the above criteria.