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.

Teriflunomide for multiple sclerosis in real-world setting.

OBJECTIVES: Teriflunomide 14 mg is a once-daily oral disease-modifying treatment for relapsing-remitting multiple sclerosis. We examined adverse event (AE) profile and efficacy in real life. MATERIALS AND METHODS: In this observational cohort study, we retrospectively examined 1521 blood samples and data of 102 patients followed for up to 28 months. RESULTS: The number of female patients starting teriflunomide peaked in the fifth decade, 10 years later compared to male patients (P<.001), reflecting pregnancy concerns. Seventy-six percentages of patients shifted to teriflunomide from treatment with interferon-beta. Expanded disability status scale improved in 11% of patients (18.2±3.6 months follow-up) and remained constant in 67.5% (15±5.3 months follow-up). Of ten relapses, three occurred within 6 months after starting treatment. Seventeen patients (16.5%) discontinued teriflunomide: 53% because of AEs and 29% because of relapse. Levels of alanine aminotransferase (ALT) remained normal in 95.3% of the blood samples and remained below 1.5 times the upper limit of normal in 91% of the 4.7% abnormal samples. One-third of the patients had abnormal ALT values at least once. Haematological abnormalities were found in <4% of the blood samples, but at least one abnormal value was observed in up to 21% of the patients. CONCLUSIONS: Efficacy and safety of teriflunomide in real-life setting support data obtained by the pivotal trials. Laboratory abnormalities are rare among the large number of samples, but patients may commonly have a single mild, abnormal value if frequently tested.

Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells.

Microtubules are critically involved in membrane traffic and maintenance of epithelial cell polarity. In this study we examined the effect of microtubule disruption by colchicine on 1) the subcellular organization of the apical endocytic apparatus, 2) apical endocytosis, and 3) subcellular distribution of gp330 and Aquaporin-1 water channels in renal proximal tubule cells. Rats were treated in vivo with colchicine for 5 h before fixation of the kidneys, and sections of proximal tubules were studied using electron microscopy, morphometry and immunocytochemistry. In proximal tubule cells from colchicine-treated animals, virtually no endocytic invaginations are present, indicating that colchicine blocks the formation of endocytic invaginations. Furthermore, no large endocytic vacuoles are present, also consistent with a block of endocytosis. This was confirmed by functional studies revealing a colchicine-induced arrest in apical endocytosis of peroxidase. There is a marked reduction in dense apical tubules (the exocytic vehicle for membrane recycling) but an extensive accumulation of small vesicles, suggesting a disruption in membrane recycling. This disruption may be primary or secondary to the block of endocytosis. Immunofluorescence and immunoelectron microscopy reveal extensive colchicine-induced changes in the subcellular distribution of gp330 and Aquaporin-1. The localization of gp330 is normally restricted to the apical endocytic apparatus. However, after colchicine treatment gp330 is localized to numerous vesicles distributed throughout the entire cytoplasm, as previously shown (Gutmann et al., Am. J. Physiol. 257, C397-C407 (1989). Also Aquaporin-1 water channels, which are normally almost exclusively present in the plasma membranes, are redistributed to small vesicles in addition to the plasma membrane localization after colchicine treatment. In summary, the present study suggests that cytoplasmic microtubules are critically involved in 1) formation and stabilization of endocytic invaginations, 2) formation of large endocytic vacuoles, 3) apical endocytosis, 4) maintaining the polarized distribution of vesicles in the apical part of the cell, and 5) maintaining the polarized organization of gp330 in the apical endocytic apparatus, and maintaining Aquaporin-1 water channels in the plasma membranes.

Detection of Duffy antigen in the plasma membranes and caveolae of vascular endothelial and epithelial cells of nonerythroid organs.

The nonerythroid expression of the Duffy blood group protein (gp-Fy) was confined to certain cell types. Immunocytochemistry studies of the kidney showed gp-Fy in the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts. Gp-Fy was also produced in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In the thyroid, only the endothelial cells of capillaries produced gp-Fy. In the spleen, the endothelial cells of capillaries, high endothelial venule, and sinusoids produced abundant gp-Fy. Ultrastructural studies showed that apical and basolateral plasma membrane domains, including caveolae, had gp-Fy. Immunoblot analysis showed substantially less gp-Fy in nonerythroid cells than in erythrocytes. Moreover, the analyzed nonerythroid organs of Duffy-negative individuals did not produce more gp-Fy to compensate for the lack of this protein in their erythrocytes. The nucleotide sequence and the size of kidney mRNA from a Duffy-positive individual were the same as that of bone marrow. It is assumed, therefore, that nonerythroid Duffy protein is the product of the same gene as that of bone marrow. This notion is reinforced by the fact that nonerythroid and erythroid gp-Fy have the same antigenic domains.

Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways.

The purpose of this study was to determine the cellular and subcellular localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the presence of AQP8 mRNA in all kidney zones. LLC-PK(1) cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified antibodies, whereas controls using cells transfected with the vector, but without the insert, were negative. The labeling was almost exclusively associated with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26-28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequential ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry revealed modest labeling of proximal tubules and weak labeling of collecting ducts in cortex and medulla of rat kidney. The labeling was confined to cytoplasmic areas with no labeling of the brush border. Immunoblotting and RT-PCR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trachea. Consistent with this, immunohistochemistry revealed AQP8 labeling in the hepatocytes and spermatogenic cells in testis and in the basal cells in ductus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and tracheal glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. In conclusion, AQP8 is expressed at low levels in rat kidney proximal tubules and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronchi as well as in multiple glands, including salivary glands.