Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase.

AIMS: Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H(2)O(2) concentration that is low compared to current nonenzymatic formulations. METHODS AND RESULTS: Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l(-1) H(2)O(2) and 100 mmol l(-1) Br(-) at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme. CONCLUSIONS: The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.

Associations between genetic factors, tobacco smoking and autoantibodies in familial and sporadic rheumatoid arthritis.

OBJECTIVES: The objective of this study was to investigate the association between genes (HLA-DRB1 and PTPN22) and tobacco smoking, separately as well as combined, and serological markers of rheumatoid arthritis (RA) in a French population with RA. METHODS: 274 patients with RA with half of them belonging to RA multicase families, were genotyped for HLA-DRB1 allele and for PTPN22-1858 polymorphism. IgM rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) antibodies were determined by ELISA method. The search for association relied on chi(2) test and odds ratio with 95% confidence interval calculation. The interaction study relied on the departure-from-additivity-based method. RESULTS: The presence of at least one shared epitope (SE) allele was associated with anti-CCP antibodies presence (82.5% vs. 68.4%, p = 0.02), particularly with HLA-DRB1*0401 allele (28.0% vs. 16.4%, p = 0.01). Tobacco exposure was associated with anti-CCP antibodies, but only in presence of SE. A tendency toward an interaction was found between tobacco, the presence of at least one HLA-DRB1*0401 allele and anti-CCP antibodies (attributable proportion due to interaction = +0.24 (-0.21+0.76)). The cumulative dose of cigarette smoking was correlated with anti-CCP antibody titres (r = 0.19, p = 0.04). The presence of both SE and 1858T alleles was associated with a higher, but not significantly different, risk for anti-CCP antibodies presence than for each separately. No association was found between PTPN22-1858T allele and tobacco smoking for autoantibody positivity. CONCLUSIONS: Our findings suggest an association between SE alleles and tobacco smoking for anti-CCP positivity and a tendency toward an interaction between the HLA-DRB1*0401 allele and smoking for anti-CCP positivity in this sample of RA.

PADI4 gene in multiple sclerosis: a family-based association study.

In multiple sclerosis (MS) MBP is heavily citrullinated by peptidylarginine deiminase (PAD). This post-translational modification may be crucial for its pathogenesis. PADI4 is the isoform expressed in inflammatory infiltrates. The aim of this study was to analyse the role of PADI4 gene in conferring susceptibility to MS, by means of a family-based association study, testing three SNPs by RFLP. No association was found either with single SNPs or haplotypes. Similarly no significant association was detected partitioning the patients according to DRB1*15 positivity or disease severity. These results do not support a major role of the PADI4 gene, but further studies may contribute to clarify the genetic factors that regulate deimination.

Lack of association between the protein tyrosine phosphatase non-receptor 22 (PTPN22)*620W allele and systemic sclerosis in the French Caucasian population.

The minor allele of the R620W missense single-nucleotide polymorphism (SNP; rs2476601) in the PTPN22 (protein tyrosine phosphatase non-receptor 22) gene has been reported to be associated with multiple autoimmune diseases, including type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, autoimmune thyroiditis and vitiligo. Systemic sclerosis (SSc) is a connective tissue disease with some autoimmune abnormalities. The aim of our study was to test for association of the PTPN22*620W allele with SSc in a French Caucasian cohort with a case-control study of 121 patients with SSc and 103 controls. All patients and controls were genotyped for the PTPN22*R620W SNP. No association was found between the PTPN22*620W allele and SSc (7% v 9.2%, p = 0.39). The frequency of genotypes carrying at least one 620W allele was similar in both groups (13% v 17%, p = 0.38). The PTPN22*620W allele was also not associated with autoantibody patterns. Thus, the PTPN22*R620W polymorphism cannot be regarded as a genetic susceptibility factor for SSc in the French Caucasian population.

A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population.

BACKGROUND: Autoantibodies to citrullinated proteins (ACPA) are considered a specific marker for rheumatoid arthritis. Peptidylarginine deiminase (PAD) is the enzyme that converts arginyl into citrullyl residues; different isoforms of the enzyme are expressed in mammals. It has been suggested that the PADI4 gene may contribute to genetic susceptibility to rheumatoid arthritis, but conflicting results have been obtained in different populations. OBJECTIVE: To test the hypothesis that the PADI4 gene may confer susceptibility to rheumatoid arthritis in a white French population, using powerful and highly reliable family based association tests. METHODS: DNA samples were analysed from 100 families where one member was affected by rheumatoid arthritis and both parents were available for sampling. Five single nucleotide polymorphisms, located within the PADI4 gene and in its close proximity, were genotyped by restriction fragment length polymorphism, and haplotypes were constructed. The analysis involved use of the transmission disequilibrium test and genotype relative risk. ACPA were detected by ELISA on cyclic citrullinated peptides and on human deiminated fibrinogen. RESULTS: No single SNP or haplotype was associated with the disease, or was preferentially transmitted. No association was found when patients were partitioned according to ACPA positivity. CONCLUSIONS: No PADI4 haplotype is associated with rheumatoid arthritis in a white French population. The role of genes encoding the other PAD isoforms, or modulating tissue expression or enzyme activity, remains to be elucidated.

Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.

Ultraviolet A (UVA; 320-400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1, 4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.

Inactivation of intracellular and non-enveloped viruses by a non-ionic naphthalene endoperoxide.

Singlet oxygen (1O2, 1delta(g)) selectively oxidizes many biological targets, some of which, such as viruses, are located intracellularly under in vivo conditions. Considering the short lifetime of 1O2 in aqueous media, it is essential to generate this species in close proximity to the targets. Therefore, a water-soluble and non-ionic carrier of 1O2, DHPNO2, has been designed to convey 1O2 through lipid membranes. In contrast to the known anionic carrier NDPO2, which inactivates only extracellular enveloped viruses, the new compound exhibits virucidal activity on all types of viruses, enveloped (HIV) and non-enveloped (Poliovirus), extracellular and intracellular. HIV inactivation can also be achieved in the presence of red blood cells, suggesting the possible use of DHPNO2 in the decontamination of cellular blood products.

Virucidal activity of pure singlet oxygen generated by thermolysis of a water-soluble naphthalene endoperoxide.

Using the water-soluble naphthalene carrier of singlet oxygen NDPO2, we have shown that pure singlet oxygen is able to inactivate enveloped viruses (human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus, vesicular stomatitis virus), but has no effect on non-enveloped viruses (adenovirus and poliovirus 1). These results are related to the experiments on photoinactivation of viruses by hydrophobic photosensitizers (merocyanine 540, hypericin, phthalocyanines, hematoporphyrin and benzoporphyrin derivatives) and they strengthen the hypothesis that singlet oxygen plays a predominant role in this process.

Preparation of anti-HIV-low-density lipoprotein complexes for delivery of anti-HIV drugs via the low-density lipoprotein pathways.

Lipophilic prodrugs of 3'-azido-3'-deoxythymidine (AZT) and of 2',3'-didehydro-3'-deoxythymidine (D4T) have been synthesized. 3 beta-(2'-carboxymethoxy)-cholest-5-ene acid, palmitic acid, linolenic acid, linoleic acid, and cholanic acid have been covalently bound to AZT and D4T. In some experiments the fluorescent molecule NBD was simultaneously linked. These prodrugs were incorporated into LDL or acetylated LDL. The best incorporation was obtained with drugs presenting a steroid moiety (cholesterol derivative or cholanic acid) in their structure. The incorporation of prodrugs into LDL was estimated as approximately 200 molecules of prodrug per LDL particle. Cytofluorimetric studies clearly show that the NBD-steroid LDL or NBD-steroid acetylated LDL are bound and then internalized by the B-E receptor (U937) or the scavenger receptor (mouse peritoneal macrophage), respectively. The antiretroviral activity of palmitate-D4T, cholanic-AZT, and cholanic-AZT-LDL complex was similar to the activity of free D4T and free AZT, respectively. Development of lipid nucleoside-LDL complexes to attach specifically to cells involved in HIV infection might have a direct clinical relevance.

Synthesis and anti-HIV evaluation of D4T and D4T 5'-monophosphate prodrugs.

Several 5-monophosphate D4T derivatives and their analogues were synthesized as potential lipophilic prodrugs of D4T. Cholesteryl D4T phosphate diester and bis-5'-D4T phosphate inhibited HIV replication in CEM-Cl13 cells more efficiently than D4T itself as measured by the inhibition of cytopathic effect based on MTT assay or reverse transcriptase activity. The two compounds were devoid of toxicity on CEM-Cl13 cells at doses equal to 50 and 100 microM, respectively, which brought the selectivity index into the same range as AZT.