Cladribine modifies functional properties of microglia.

Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1-1 µM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory interleukin (IL)-4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL-4 up-regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1-1 µM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 µM increased the IL-4-induced gene expression of arginase 1 (Arg1) and LPS-induced expression of IL-1ß, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 µM potentiated the increased expression of anti-inflammatory TNF receptor 2 (TNF-R2) but not TNF-R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1-1 µM CdA that putatively overlaps human CSF concentrations. This may be related to the up-regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

Prevalence of variants in methylenetetrahydrofolate reductase and the severity of pulmonary vascular disease.

Pulmonary hypertension is a serious disease associated with constriction, cellular proliferation, inflammation, and in situ thrombosis of the small vessels of the lung. Some studies suggest that homozygous 677TT variants and compound heterozygous 677CT/1298AC variants in methylenetetrahydrofolate reductase may increase the risk for systemic vascular disease. We sought to determine the prevalence of variants in methylenetetrahydrofolate reductase in patients with pulmonary hypertension, and whether homozygous or compound heterozygous variants are associated with an increased severity of disease. The medical records of patients with pulmonary hypertension were retrospectively reviewed to identify 105 patients who were evaluated for variants in methylenetetrahydrofolate reductase. The frequency of the minor allele 677C > T was 0.352 and the frequency of the minor allele 1298A > C was 0.295. The number of patients who were homozygous 677TT, homozygous 1298CC or compound heterozygous 677CT/1298AC was similar to the number of control patients with corresponding variants in a meta-analysis of studies. Patients with homozygous or compound heterozygous variants had a significantly higher ratio of pulmonary to systemic vascular resistance (0.75 ± 0.07 vs. 0.56 ± 0.04, p = 0.019) during baseline heart catheterization. Twenty-five of 61 patients without, and 28 of 44 patients with, homozygous or compound heterozygous variants had moderate to severe disease (p = 0.030). Variants in methylenetetrahydrofolate reductase are common in the general population and in patients with pulmonary hypertension. It is unlikely that these variants cause pulmonary vascular disease; however, they may influence the progression or severity of disease.