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Background: MTHFR gene polymorphisms has been shown to be associated with methotrexate (MTX) toxicity in adult hematological malignancies; however, the results remain inconclusive. Materials & methods: To examine the role of common MTHFR variants in MTX toxicity prediction, we performed a meta-analysis via identifying relevant studies for quantitative data pooling. Results: Our results showed a significant association between MTHFR C677T polymorphism and increased risk of MTX-induced all-grade (grade 1–4) and severe (grade 3–4) hepatic and gastrointestinal toxicities in Caucasian independent of MTX dosage. MTHFR 677T allele increased risk of severe mucositis and all-grade hematological toxicity. MTHFR A1298C polymorphism was not significantly associated with hepatic and hematological toxicity, whereas perhaps having a protective effect on mucositis and gastrointestinal toxicity. Conclusion: MTHFR C677T polymorphism may be a good predictor for MTX toxicity in adult hematological malignancies.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1β (IL-1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.

BACKGROUND AND PURPOSE Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. EXPERIMENTAL APPROACH mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. KEY RESULTS mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. CONCLUSIONS AND IMPLICATIONS These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Summary Objective To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E 2 (PGE 2 ) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). Methods SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N 6-cyclohexyladenosine (CHA, A 1 ), 2-[ p -(2-carboxyethyl)-phenetyl-amino]-5′- N -ethylcarboxamidoadenosine (CGS 21680, A 2A ), 5′- N -ethylcarboxamidoadenosine (NECA, non-selective), N 6-(3-iodobenzyl)2-chloroadenosine-5′- N -methyluronamide (Cl-IB-MECA, A 3 ). SFs were treated with TNF-α (10ng/ml) and lipopolysaccharide (LPS) (1μg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-α- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75Hz, 1.5mT) during the period in culture (24h). PGE 2 release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. Results TNF-α and LPS stimulated PGE 2 release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE 2 production. EMFs inhibited PGE 2 production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE 2 release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE 2 levels were associated to modification of COX-2 expression. Conclusions This study supports anti-inflammatory activities of A 1 and A 2A adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A 2A receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Background and Objectives Common methylenetetrahydrofolate reductase gene variants (MTHFR C677T and A1298C) have been described to have opposite effects on cancer patients. They may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g. methotrexate) are utilized. We analyzed 110 patients with high-grade non-Hodgkin’s lymphoma (NHL), 68 of whom were eligible for a chemotherapy combination containing methotrexate (MACOP-B) and 42 for chemotherapy without methotrexate (CHOP).Design and Methods Patients were genotyped by polymerase chain reaction and stratified by MTHFR variants. These data were related to the toxicity (WHO grade GO-4) that the patients suffered and their survival. Overall 64 cases (58.2%) developed some form of toxicity and 23 (20.9%) had grade 3/4 toxicity.Results When considering toxicity of any grade (grade 1–4), the 677TT genotype was significantly over-represented among cases with mucositis (OR=4.85; 95%CI, 1.47–15.97; p=0.009) and those with hepatic toxicity (OR=3.43; 95%CI, 0.99–11.86; p=0.052). Sub-analyses in the group treated with MACOP-B showed a slight increase in the risk of developing mucositis (OR=5.22; 95%CI, 1.20–27.27; p=0.03), and a strong increase in the risk of hepatic toxicity (OR=7.08; 95%CI, 1.38–36.2; p=0.019) and thrombocytopenia (OR=7.69, 95%CI 1.0–58.94; p=0.05). Interestingly, compared to the risk of developing toxicity of any grade, the risk of developing severe (grade 3/4) mucositis was almost doubled in the whole group of cases with 677TT (OR=8.13; 95%CI 1.61–41.04; p=0.011) and dramatically increased in the MACOP-B-treated cases with this gene variant (OR=24.6; 95%CI 2.49–87.41; p=0.001). There were significant results for 1298CC cases exclusively for mucositis (any grade, OR=5.33; 95%CI, 1.25–22.70; p=0.023 and OR=9.15; 95%CI, 1.14–73.41; p=0.037; for the whole group and the MACOP-B-treated group, respectively). Similarly, the risk of 1298CC patients developing severe mucositis increased (OR=9.24; 95%CI, 1.47–58.0; p=0.017 and OR=11.53; 0.93–143.18; p=0.057; in the whole group and in the MACOP-B-treated group, respectively). Event-free survival analysis revealed a lower probability of event-free survival at 5 years for 677T-carriers (log-ranks, p=0.05 and p=0.07 in the whole group and in the MACOP-B-treated group, respectively). More significant results were obtained when 1298CC cases were excluded from the reference group (log-ranks, p=0.03 and p=0.04, respectively). No significant associations were found in the CHOP-treated group.Interpretation and Conclusions Our data suggest that MTHFR gene variants play a critical role in NHL outcome, possibly by interfering with the action of methotrexate with significant effects on toxicity and survival. Genotyping of folate pathway gene variants might be useful to enable reduction of chemotherapy toxicity and/or to improve survival by indicating when dose adjustments or alternative treatments are necessary.

Summary Objective To investigate the single and combined effects of electromagnetic field (EMF) exposure and the insulin growth factor-I (IGF-I) on proteoglycan (PG) synthesis of bovine articular cartilage explants and chondrocytes cultured in monolayers. Design Bovine articular cartilage explants and chondrocyte monolayers were exposed to EMF (75Hz; 1.5mT) for 24h in the absence and in the presence of both 10% fetal bovine serum (FBS) and IGF-I (1–100ng/ml). PG synthesis was determined by Na 2 – 35 SO 4 incorporation. PG release into culture medium was determined by the dimethylmethylene blue (DMMB) assay. Results In cartilage explants, EMF significantly increased 35 S-sulfate incorporation both in the absence and in the presence of 10% FBS. Similarly, IGF-I increased 35 S-sulfate incorporation in a dose-dependent manner both in 0% and 10% FBS. At all doses of IGF-I, the combined effects of the two stimuli resulted additive. No effect was observed on medium PG release. Also in chondrocyte monolayers, IGF-I stimulated 35 S-sulfate incorporation in a dose-dependent manner, both in 0% and 10% FBS, however, this was not modified by EMF exposure. Conclusions The results of this study show that EMF can act in concert with IGF-I in stimulating PG synthesis in bovine articular cartilage explants. As this effect is not maintained in chondrocyte monolayers, the native cell–matrix interactions in the tissue may be fundamental in driving the EMF effects. These data suggest that in vivo the combination of both EMF and IGF may exert a more chondroprotective effect than either treatment alone on articular cartilage.

Item does not contain fulltext Methotrexate (MTX), one of the important pillars in the treatment of different forms of cancer, is associated with the development of hepatotoxicity. The 677C>T variant (rs1801133) in the methylenetetrahydrofolate reductase (MTHFR) gene might affect the development of hepatotoxicity. Results in literature are, however, contradictive. The aim of this study was to evaluate the role of the MTHFR 677C>T polymorphism in MTX-induced hepatotoxicity by analyzing a Dutch cohort of pediatric patients treated with high doses of MTX and subsequently performing a meta-analysis. Ninety-eight patients receiving 542 courses of high-dose MTX were genotyped for the MTHFR 677C>T variant. Hepatotoxicity was evaluated retrospectively according to common terminology criteria for adverse events-National Cancer Institute criteria. The influence of MTHFR 677C>T on hepatotoxicity was examined using a generalized estimating equation (GEE) analysis. A fixed-effect meta-analysis based on this and previous studies investigating the association between the MTHFR 677C>T polymorphism and uniformly coded hepatotoxicity was performed. The GEE analysis showed an increased risk of developing hepatotoxicity for T versus C allele (odds ratio (OR) 1.8; 95% confidence interval (CI) 1.0-3.2, P=0.04). This finding was not supported by the meta-analysis including seven studies and 1044 patients; the OR for the 677T versus C allele was 1.1 (95% CI 0.84-1.5, P=0.25). Heterogeneity between studies was observed, possibly related to differences in MTX dose and leucovorin rescue. In conclusion, in patients with cancer, the MTHFR 677T allele has only a minor role in the development of MTX-induced hepatotoxicity. Observed heterogeneity between studies warrants further study into (tailored) leucovorin rescue.

To evaluate the efficacy and toxicity of electrochemotherapy (ECT) in elderly metastatic breast cancer (BC) patients. Retrospective analysis of 55 patients with superficial metastases who underwent ECT according to the European Standard Operative Procedures of electrochemotherapy. Treatment schedule consisted of intravenous or intratumoral bleomycin followed by locally delivered electric pulses. Statistical comparisons were performed between two groups: the patients aged

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.

Background The antifolate agent methotrexate is an important component of maintenance therapy in acute lymphoblastic leukemia, although methotrexate-related toxicity is often a reason for interruption of chemotherapy. Prediction of toxicity is difficult because of inter-individual variability susceptibility to antileukemic agents. Methotrexate interferes with folate metabolism leading to depletion of reduced folates.Design and Methods The aim of this study was to investigate the influence of polymorphisms for folate metabolizing enzymes with respect to toxicity and survival in adult patients with acute lymphoblastic leukemia treated with methotrexate maintenance therapy. To this purpose, we evaluated possible associations between genotype and hematologic and non-hematologic toxicity and effects on survival at 2 years of follow-up in patients with acute lymphoblastic leukemia.Results Polymorphisms in the genes encoding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and in dihydrofolate reductase (DHFR 19 bp deletion) significantly increased the risk of hepatotoxicity in single (odds ratio 5.23, 95% confidence interval 1.13–21.95 and odds ratio 4.57, 95% confidence interval 1.01–20.77, respectively) and in combined analysis (odds ratio 6.82, 95% confidence interval 1.38–33.59). MTHFR 677C>T also increased the risk of leukopenia and gastrointestinal toxicity, whilst thymidylate synthase 28 bp repeat polymorphism increased the risk of anemia (odds ratio 8.48, 95% confidence interval 2.00–36.09). Finally, patients with MTHFR 677TT had a decreased overall survival rate (hazard ratio 2.37, 95% confidence interval 1.46–8.45).Conclusions Genotyping of folate polymorphisms might be useful in adult acute lymphoblastic leukemia to optimize methotrexate therapy, reducing the associated toxicity with possible effects on survival.

In this study we analyzed whether the polymorphisms 676TG in the tumor necrosis factor receptor (TNFR) II gene and -308GA in the TNFalpha promoter gene may influence the response grading to anti-TNFalpha therapy in rheumatoid arthritis. We enrolled and genotyped 105 RA patients treated with etanercept (n = 55), infliximab (n = 40) and adalimumab (n = 10) for 1 year. The clinical response was evaluated according to the ACR criteria every 3 months. Patients with TNFRII 676TG genotype was significantly associated with lower ACR response compared with 676TT genotype, at 3 (OR 3.78 95% CI 1.07-13.31) and 12 months (OR 4.30 95% CI 1.16-15.99) of treatment. No significant association between TNFalpha -308GA polymorphism and the clinical response was found. TNFRII 676TG genotype is associated with a lower response to anti-TNFalpha therapy, independently from the specific agent used. This polymorphism could become a useful genetic marker for predicting the different response grading to anti-TNFalpha therapy.

Low Factor XIII (FXIII) activity has been reported in the blood of patients with chronic venous leg ulcer (CVU). In vivo studies have described increased wound healing in CVU patients treated with FXIII concentrate, and in vitro studies have shown increased regenerative capacity in FXIII-treated fibroblasts. In addition, a common G-to-T polymorphism in the FXIIIA-subunit gene (V34L) significantly increases the activity and modifies the cross-linking properties of the FXIII molecule and this variant has been investigated as a protective factor against thrombosis, a recognized risk factor for CVU establishment. Therefore, the role of FXIII levels, FXIII V34L, FVR506Q, and FIIG20210A, common gene polymorphisms in the pathogenesis of CVU was investigated. Ninety-one patients with CVU and 195 healthy controls (91 of them sex- and age-matched) were PCR-genotyped for the FXIIIV34L, FVR506Q, and FIIG20210A substitutions and FXIIIA-subunit levels were determined by immuno-electrophoresis. The extent of the venous ulcer surface in patients was measured by computer software. The allele frequency and the genotype distribution of the FXIII polymorphism did not show significant differences between the whole group of cases and controls as well as prothrombin variants did. On the contrary, the FVR506Q variant (FV Leiden) allele was more frequent in patients, yielding a significant OR value of 5.93 (95 percent CI, 1.83-19.17; p= 0.003). Considering only CVU cases secondary to a post-thrombotic syndrome (n= 24), FV Leiden yielded a greater OR value of 16.08 (95 percent CI, 4.33-59.6; p < 0.0001). When the CVU cases were stratified by the three possible FXIII genotypes, a significant trend toward a lower mean value of the ulcerated area was clearly evident as the number of the polymorphic alleles (L34) increased in the genotype of patients (VV = 11.9 cm(2,)+/- 23.6; VL = 6.1 cm(2,)+/- 6.9; LL = 4.1 cm(2,)+/- 2.8; p= 0.01). On the other hand, FXIIIA antigen levels were similar between CVU cases and matched controls, but 11 percent of cases had FXIII deficiency (FXIIIA

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject – the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations – showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus’ uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus’ two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu. Am. J. Hematol. 67:183–188, 2001. © 2001 Wiley-Liss, Inc.