Susana Martínez

Differential Th1/Th2 cytokine patterns in chronic arthritis: interferon gamma is highly expressed in synovium of rheumatoid arthritis compared with seronegative spondyloarthropathies.

OBJECTIVE To investigate possible differences in Th1 and Th2 cytokine mRNA expression in the synovial tissue (ST) of patients with rheumatoid arthritis (RA) and seronegative spondyloarthropathies (SpA) with diagnostic and/or pathogenic interest. METHODS Eleven RA patients and 14 SpA patients (10 with undifferentiated spondyloarthropathy (USpA), two with ankylosing spondylitis (AS) and two with psoriatic arthritis (PsA)) were included. Th1 (interferon γ, interleukin 2) and Th2 (interleukin 4, interleukin 5 and interleukin 10) cytokine mRNA levels from arthritic knee ST were quantified by using an optimised polymerase chain reaction method with a computerised analysis system. Protein levels of proinflammatory cytokines (interleukin 1, tumour necrosis factor α and interleukin 6) in synovial fluid were quantified with a specific ELISA test. RESULTS Th1 cytokines were detected in all of RA ST samples in contrast with 58% (interferon γ) and 71% (interleukin 2) of SpA samples. Th2 cytokines were expressed in 90% of RA ST samples, but the findings in SpA were interleukin 10 in 90%, interleukin 4 in 60% and interleukin 5 in 40% of ST samples. However, when the mRNA levels of each cytokine were quantified and corrected for T cell mRNA levels, only interferon γ levels were significantly higher in RA than in SpA (p<0.003). Thus, the Th1/Th2 cytokine ratio in RA was fivefold that of SpA. Synovial fluid interleukin 1β concentrations were higher in RA than in SpA (p<0.05); there were also higher synovial fluid levels of tumour necrosis factor α in RA than in SpA, but without statistical significance. CONCLUSION This study has detected both Th1 and Th2 cytokine gene expression in ST from RA and SpA patients. Synovium interferon γ mRNA levels and SF interleukin 1β protein levels were significantly higher in RA than in SpA, so reflecting the known proinflammatory activity of interferon γ through macrophage activation. Thus, the Th1 (interferon γ)/Th2 (interleukin 4) ratio is significantly higher in RA than in SpA ST. These data confirm previous studies on ST Th1/Th2 balance in RA and extend previous work in comparing ST RA with subgroups of SpA distinct of ReA.

ALCOHOL DEHYDROGENASE OF HUMAN AND RAT BLOOD VESSELS : ROLE IN ETHANOL METABOLISM

© 1997 Federation of European Biochemical Societies.

Assessment of exposure to drugs of abuse during pregnancy by hair analysis in a Mediterranean island

AIMS   This study aims to estimate the prevalence of drug use by pregnant women living in Ibiza, using structured interviews and biomarkers in maternal hair. In addition, the potentially detrimental effects of maternal drug abuse on their newborns were investigated. Ibiza has a large international night-life resort associated with clubs, music and use of recreational drugs. DESIGN, SETTING AND PARTICIPANTS   Hair samples were collected prospectively from January to March 2010 from a cohort of consecutive mothers after giving birth in the Hospital Can Misses in Ibiza. MEASUREMENTS   Opiates, cocaine, cannabis, methadone, amphetamines, 3,4-methylenedioxymethamphetamine (MDMA) and their metabolites were detected in a 3-cm-long proximal segment of maternal hair corresponding to the last trimester of pregnancy by gas chromatography coupled to mass spectrometry (n = 107). Data on socio-demographic characteristics and on tobacco, alcohol, drugs of prescription and drugs of abuse consumption during pregnancy were collected using a structured questionnaire. FINDINGS   Hair analysis showed an overall 16% positivity for drugs of abuse in the third trimester of pregnancy, with a specific prevalence of cannabis, cocaine, MDMA and opiates use of 10.3, 6.4, 0.9 and 0%, respectively. In the questionnaires, only 1.9% of mothers declared using drugs of abuse during pregnancy. Gestational drug of abuse consumption was associated with active tobacco smoking, a higher number of smoked cigarettes and the mother being Spanish. CONCLUSIONS   Illicit drug use is substantially under-reported among pregnant women living in Ibiza, particularly among Spanish nationals. Voluntary, routine objective biological toxicology screening should be considered as part of routine examinations in antenatal clinics on this Mediterranean island.

Structural and Enzymatic Properties of a Gastric NADP(H)- dependent and Retinal-active Alcohol Dehydrogenase*

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP+ rather than NAD+. Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP+ preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k cat/K m = 3.4·104 mm −1min−1). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58–60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

Molecular Basis for Differential Substrate Specificity in Class IV Alcohol Dehydrogenases A CONSERVED FUNCTION IN RETINOID METABOLISM BUT NOT IN ETHANOL OXIDATION

Mammalian class IV alcohol dehydrogenase enzymes are characteristic of epithelial tissues, exhibit moderate to high K m values for ethanol, and are very active in retinol oxidation. The human enzyme shows aK m value for ethanol which is 2 orders of magnitude lower than that of rat class IV. The uniquely significant difference in the substrate-binding pocket between the two enzymes appears to be at position 294, Val in the human enzyme and Ala in the rat enzyme. Moreover, a deletion at position 117 (Gly in class I) has been pointed out as probably responsible for class IV specificity toward retinoids. With the aim of establishing the role of these residues, we have studied the kinetics of the recombinant human and rat wild-type enzymes, the human G117ins and V294A mutants, and the rat A294V mutant toward aliphatic alcohols and retinoids. 9-cis-Retinol was the best retinoid substrate for both human and rat class IV, strongly supporting a role of class IV in the generation of 9-cis-retinoic acid. In contrast, 13-cisretinoids were not substrates. The G117ins mutant showed a decreased catalytic efficiency toward retinoids and toward three-carbon and longer primary aliphatic alcohols, a behavior that resembles that of the human class I enzyme, which has Gly117. TheK m values for ethanol dramatically changed in the 294 mutants, where the human V294A mutant showed a 280-fold increase, and the rat A294V mutant a 50-fold decrease, compared with those of the respective wild-type enzymes. This demonstrates that the Val/Ala exchange at position 294 is mostly responsible for the kinetic differences with ethanol between the human and rat class IV. In contrast, the kinetics toward retinoids was only slightly affected by the mutations at position 294, compatible with a more conserved function of mammalian class IV alcohol dehydrogenase in retinoid metabolism.

Amphibian Alcohol Dehydrogenase

Alcohol dehydrogenase (ADH) catalyses the reversible interconversion of a variety of alcohols and their corresponding aldehydes and ketones, and is widely distributed in organisms. It has been detected in all animals, in plants, and eukaryotic and prokaryotic microorganisms. In vertebrates the ADH system is complex, with at least seven different enzymatic classes (Jornvall and Hoog, 1995, Kedishvili et al., 1997). The study in sub- mammal species is of interest to understand the relationship between the classes found in mammals and the evolutionary origins of the present structures. Moreover, since the enzyme exhibits a wide substrate specificity, the comparative study of the enzymes from distant species may provide valuable information on the function of the enzyme, and how it may change among groups of vertebrates to adapt to different physiological needs.

Autosomal dominant hypercholesterolemia in Catalonia: Correspondence between clinical-biochemical and genetic diagnostics in 967 patients studied in a multicenter clinical setting

BACKGROUND Autosomal dominant hypercholesterolemia (ADH) is associated with mutations in the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes, and it is estimated to be greatly underdiagnosed. The most cost-effective strategy for increasing ADH diagnosis is a cascade screening from mutation-positive probands. OBJECTIVE The objective of this study was to evaluate the results from 2008 to 2016 of ADH genetic analysis performed in our clinical laboratory, serving most lipid units of Catalonia, a Spanish region with approximately 7.5 million inhabitants. METHODS After the application of the Dutch Lipid Clinic Network (DLCN) clinical diagnostic score for ADH, this information and blood or saliva from 23 different lipid clinic units were investigated in our laboratory. DNA was screened for mutations in LDLR, APOB, and PCSK9, using the DNA-array LIPOchip, the next-generation sequencing SEQPRO LIPO RS platform, and multiplex ligation-dependent probe amplification (MLPA). The Simon Broome Register Group (SBRG) criteria was calculated and analyzed for comparative purposes. RESULTS A total of 967 unrelated samples were analyzed. From this, 158 pathogenic variants were detected in 356 patients. The main components of the DLCN criteria associated with the presence of mutation were plasma LDL cholesterol (LDLc), age, and the presence of tendinous xanthomata. The contribution of family history to the diagnosis was lower than in other studies. DLCN and SBRG were similarly useful for predicting the presence of mutation. CONCLUSION In a real clinical practice, multicenter setting in Catalonia, the percentage of positive genetic diagnosis in patients potentially affected by ADH was 38.6%. The DLCN showed a relatively low capacity to predict mutation detection but a higher one for ruling out mutation.

109 – Histological Localization of Alcohol Dehydrogenase: Methods, Approaches and Applications

This chapter discusses the methods for histological localization of alcohol dehydrogenase. Tissue localization of multiple enzyme forms of human Alcohol Dehydrogenase (ADH) is of paramount importance to understand their physiological involvement in ethanol metabolism and organ susceptibility to alcohol damage. Histological techniques—such as immunohistochemistry and in situ hybridization, provide powerful and complementary approaches to identify definite cell layers or cell types that may contribute to ethanol elimination and, for that matter, be subjected to the local toxic effects of ethanol. Immunohistochemical methods rely on the availability of specific antibodies against different ADH forms. When polyclonal antiserum is the starting material, antibodies specific for a given ADH should be affinity purified and thoroughly checked for cross-reactivity against other ADH forms and proteins by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In situ hybridization methods, being highly sensitive, should use specific homologous probes, which have been previously characterized by Northern blot analysis. With either technique, it is essential to perform adequate control experiments by using pre-immune serum, pre-absorbed antiserum or sense RNA probes. Experimental conditions should always be carefully optimized and it is advisable that results be validated with appropriate functional tests or activity assays.