Does daily folic acid supplementation reduce methotrexate efficacy?

Methotrexate is a mainstay treatment for autoimmune and inflammatory conditions in the field of Dermatology. However, in some patients, its use is associated with significant side effects and toxicity. Folate supplementation with either folic acid or folinic acid often mitigates side effects and reduces the incidence of systemic toxicity related to methotrexate. Although the value of methotrexate is clear, debate remains about folate supplementation. There is little agreement about the proper dosing or frequency of folate supplementation as many believe that daily folate supplementation can reduce methotrexate efficacy. Although daily use of folic acid does not appear to affect methotrexate efficacy, dosing of folinic acid close to methotrexate administration may hinder methotrexate efficacy. Therefore, folic acid should be used daily with methotrexate to ameliorate side effects, whereas folinic acid should only be used for methotrexate toxicity.

Cytokine regulation by MAPK activated kinase 2 in keratinocytes exposed to sulfur mustard.

Uncontrolled inflammation contributes to cutaneous damage following exposure to the warfare agent bis(2-chloroethyl) sulfide (sulfur mustard, SM). Activation of the p38 mitogen activated protein kinase (MAPK) precedes SM-induced cytokine secretion in normal human epidermal keratinocytes (NHEKs). This study examined the role of p38-regulated MAPK activated kinase 2 (MK2) during this process. Time course analysis studies using NHEK cells exposed to 200µM SM demonstrated rapid MK2 activation via phosphorylation that occurred within 15 min. p38 activation was necessary for MK2 phosphorylation as determined by studies using the p38 inhibitor SB203580. To compare the role of p38 and MK2 during SM-induced cytokine secretion, small interfering RNA (siRNA) targeting these proteins was utilized. TNF-α, IL-1ß, IL-6 and IL-8 secretion was evaluated 24h postexposure, while mRNA changes were quantified after 8h. TNF-α, IL-6 and IL-8 up regulation at the protein and mRNA level was observed following SM exposure. IL-1ß secretion was also elevated despite unchanged mRNA levels. p38 knockdown reduced SM-induced secretion of all the cytokines examined, whereas significant reduction in SM-induced cytokine secretion was only observed with TNF-α and IL-6 following MK2 knockdown. Our observations demonstrate potential activation of other p38 targets in addition to MK2 during SM-induced cytokine secretion.

A bioassay for the detection of neutralizing antibodies against the α-melanocyte stimulating hormone analog afamelanotide in patients with erythropoietic protoporphyria.

The tridecapeptide afamelanotide (Scenesse®) is a congener of α-melanocyte stimulating hormone (α-MSH). Upon binding to the melanocortin 1 receptor (MC1R) on the surface of pigment cells of the skin, the melanocytes, α-MSH or afamelanotide trigger the synthesis of cAMP, which stimulates the synthesis of melanin and therefore induces skin tanning. In a recent trial, afamelanotide administered as controlled release implants protected erythropoietic protoporphyria (EPP) patients from sunlight induced phototoxic skin reactions. Administration of biological therapeutic peptides may elicit unwanted immunogenic responses in recipients of these products. Although in a previous study using ELISA technique we excluded any newly developed immunogenicity during prolonged exposure to afamelanotide, we confirmed the previously published existence of low titers of antibodies against α-MSH in drug-naïve individuals that cross-reacted with afamelanotide. In order to investigate whether such antibodies are neutralizing, i.e. could block the biological effect of afamelanotide, we developed a cell culture-based bioassay. The basis of our assay was the measurement of afamelanotide-induced cAMP formation in a strain of the B16 mouse melanoma cell line, G4F-7, expressing the transfected human MC1R. Average half-effective concentrations of the natural hormone α-MSH and its congener afamelanotide were 38.8 ± 10.6 and 10.9 ± 7.17 nM (n=5), respectively. Neutralizing antibodies would reduce the cAMP formation. Two neutralizing anti-α-MSH antibodies served as positive controls. cAMP formation in the G4F-7 cells after addition of sera of drug-naïve (n=6) and of drug-exposed EPP patients (n=17) was significantly lower than after that from healthy volunteers (n=13). There was no difference between drug-naïve and drug-exposed patients. Using forskolin as a hormone-independent stimulator of cAMP formation, we excluded an unspecific interference of EPP sera with cAMP formation. We conclude that afamelanotide even after prolonged application to EPP patients did not elicit neutralizing antibodies. Further, the low titer immunoreactivity observed in sera of some drug-naïve individuals had no effect on the biological activity of afamelanotide.

Fatal influenza A(H1N1) respiratory tract infection in a patient having psoriasis treated with infliximab.

BACKGROUND: The use of biologic agents represents a remarkable advance for patients with psoriasis and psoriatic arthritis who have experienced an incomplete response to other therapeutic modalities. Decreased mortality and improved quality of life have been reported in patients undergoing treatment with these agents. Increased risk of bacterial, viral, granulomatous, and opportunistic infections also has been associated with the use of these medications. Enhanced patient education, watchful monitoring to promote early detection of infections, discontinuation of the medication when clinical symptoms are identified, and immediate availability of supportive care are advised to balance the benefit of treatment with biologic agents against the potential risk of infection. Herein, we discuss the risk of infection and the monitoring and vaccination guidelines in patients having psoriasis treated with biologic agents. OBSERVATIONS: A woman with obesity and psoriasis that had previously been successfully treated with efalizumab (Raptiva) for 3 years was started on a regimen of infliximab (Remicade) to treat a flare. She died 1 week after her first infusion of infliximab and was found to have had influenza A(H1N1). CONCLUSIONS: We report the first case to date of a patient with psoriasis who died of influenza A(H1N1) respiratory tract infection while undergoing treatment with infliximab. Further observations are needed to make a causal association.

Folic acid supplementation during treatment of psoriasis with methotrexate: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The value of folate supplementation in methotrexate (MTX)-treated patients remains controversial. OBJECTIVES: To determine the effect of folic acid (FA) on the efficacy of MTX and the frequency of side-effects associated with MTX therapy. METHODS: A 12-week double-blind clinical trial was conducted in patients with psoriasis stable on their long-term MTX doses but not receiving FA. They were randomized into two arms of either FA 5 mg or placebo daily. MTX doses were not changed throughout the study. Patients were monitored every 3 weeks by the same observer. Assessments included Psoriasis Area and Severity Index (PASI), a visual analogue scale (VAS) of patients' perception of their psoriasis severity and the Dermatology Life Quality Index (DLQI). Adverse events were systematically recorded. Haematological and biochemical monitoring was performed. RESULTS: Twenty-two patients with psoriasis were recruited. Age, sex and weekly MTX doses were similar in both groups. All 22 patients completed the study. The mean PASI in the FA group increased from 6.4 at baseline to 10.8 at 12 weeks. In the placebo group the mean PASI fell from 9.8 at baseline to 9.2 at 12 weeks. The mean change from baseline in the FA group was 4.4 vs. -0.6 in the placebo group (P < 0.05). Similar trends were observed in the changes in VAS and in the DLQI and differences between the groups were significant for both these parameters (P < 0.05). Few adverse effects were reported. CONCLUSIONS: This study suggests that supplementation with FA during long-term MTX treatment reduces the efficacy of MTX in the control of psoriasis. Due to the relatively small sample size and short duration of this study, no conclusions can be drawn regarding the possibility that FA may reduce the side-effects of MTX.

Prophylactic efficacy of amifostine and its analogues against sulphur mustard toxicity.

The successful implication of the chemical weapons convention stimulated research with a new vigour on the destruction of the stockpiled sulphur mustard (SM). A prophylactic agent for SM will be very useful for personnel engaged in the destruction of SM and during inspections by the Organisation for the Prohibition of Chemical Weapons. Due to simple method of preparation, SM can be used clandestinely during war or by terrorist groups. Inspite of research over several decades no satisfactory prophylactic or treatment regimen has evolved for SM. Amifostine an organophosphorothioate, originally developed as a radioprotector, and its analogues were evaluated as a prophylactic agent for SM. Three analogues by varying the chain length and substitution at the sulphur atom were synthesised and coded as DRDE-06, DRDE-07 and DRDE-08. LD(50) of amifostine and its analogues were estimated through intraperitoneal (i.p.) route. For the protection studies, amifostine and its analogues were administered i.p. in mice, 30 min before dermal (percutaneous) application of SM. The dose of the prophylactic agent was 0.2 LD(50) (i.p.) and that of SM was 152 mg/kg (undiluted) equal to 19-fold LD(50) of SM. Amifostine and one of its analogues, DRDE-07 gave significant protection. Further studies were carried out using amifostine and DRDE-07, and both of them significantly protected mice against SM (155 mg/kg, in PEG 300, equal to 19 LD(50)) when they were administered i.p. either 30 min before or simultaneously. LD(50) of amifostine and DRDE-07 were also estimated through the oral route (1049 or 1248 mg/kg, respectively). Prophylactically administered amifostine and DRDE-07 (0.2 LD(50), p.o.) significantly protected the mice against dermally applied SM (155 mg/kg, in PEG 300, equal to 19 LD(50)). The protection offered by DRDE-07 was better than that of amifostine by the oral route. DRDE-07 (0.2 LD(50), p.o.) also protected significantly with respect to the decrease in body weight and the depletion of GSH induced by SM. DNA damage induced by SM was also significantly reduced by amifostine and DRDE-07 (0.2 LD(50), p.o.). Further studies are in progress on the various pharmacological and toxicological properties of DRDE-07.

Assessment of sulfur mustard interaction with basement membrane components.

Bis-2-chloroethyl sulfide (sulfur mustard, HD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant HD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether HD directly modifies basement membrane components (BMCs), and to evaluate the effect of HD on the cell adhesive activity of BMCs. EHS laminin was incubated with [14C]HD, and extracted by gel filtration. Analysis of the [14C]HD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by [14C]HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with [35S]cysteine. 35S-labeled laminin isoforms, Ae.B1e.B2e. laminin and K.B1e.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by immunoprecipitation from the cell culture medium, treated with HD or ethanol as control, and then analyzed by SDS-PAGE. On reduced SDS gels, these three BMCs not treated with HD showed the typical profile of dissociated subunits. However, HD treatment caused the appearance of higher molecular weight bands indicative of cross-linking of subunits within these BMCs. The HD scavengers sodium thiosulfate and cysteine prevented the cross-linking of BMC subunits by HD. Finally, tissue culture dishes coated with laminin or fibronectin were treated with HD or ethanol as a control, and human keratinocytes were plated on the BMC-coated surfaces. After 20 h of incubation, it was observed that cell adhesion was decreased significantly on the BMC-coated surfaces treated with HD. As expected, the preincubation of HD with cysteine diminished the HD inhibition of cell adhesion. Thus, HD alkylates adhesive macromolecules of the basement membrane zone and inhibits their cell adhesive activity. These findings support the hypothesis that the alkylation of basement membrane components by HD destabilizes the epidermal-dermal junction in the process of HD-induced vesication. The failure of the HD-alkylated BMCs to support the attachment of keratinocytes might also contribute to the slow reepithelialization of the wound site which is characteristic of HD-induced blistering.