APOL1 gene variants are associated with end-stage renal disease in African Americans. Here we investigate the impact of recipient APOL1 (apolipoprotein L-1) gene distributions on kidney allograft outcomes. We conducted a retrospective analysis of 119 African American kidney transplant recipients, and found that 58 (48.7%) carried two APOL1 kidney disease risk variants. Contrary to the association seen in native kidney disease, there is no difference in allograft survival at 5 years post-transplant for recipients with high-risk APOL1 genotypes. Thus, we were able to conclude that APOL1 genotypes do not increase risk of allograft loss after kidney transplantations, and carrying 2 APOL1 risk alleles should not be an impediment to transplantation.

Membrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F-actin: 1) small aggregates within the diffuse CNPase regions and 2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 micrograms/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F-actin is not altered under these conditions. In contrast, cytochalasin B disrupts F-actin, microtubules, and CNPase in the lacy networks, indicating that cross-linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for 1) direct association of CNPase with F-actin and tubulin in cytoskeletal structures and 2) organization of MBP into domains via association with microtubules in the lacy networks.

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Background and objectives: Nearly 30% of renal transplant recipients develops BK viremia, a prerequisite for BK nephropathy. Case reports have evaluated treatment options for BK virus, but no controlled studies have assessed prophylactic therapies. Fluoroquinolone antibiotics were studied for prevention of BK viremia after renal transplantation.Design, setting, participants, & measurements: This retrospective analysis evaluated adult renal transplant recipients with at least one BK viral load (blood) between 90 and 400 days after transplantation. Six to 12 months of co-trimoxazole was used for Pneumocystis prophylaxis. In sulfa-allergic/-intolerant patients, 6 to 12 months of atovaquone with 1 month of a fluoroquinolone was used. Fluoroquinolones can inhibit BK DNA topoisomerase. The two groups studied were those that received 30 days of levofloxacin or ciprofloxacin after transplantation and those that did not. The primary endpoint was BK viremia rates at 1 year. Of note, of the 160 patients not receiving fluoroquinolone prophylaxis, 40 received a fluoroquinolone for treatment of a bacterial infection within 3 months after transplantation. Subgroup analysis evaluating these 40 patients against the 120 who had no exposure to fluoroquinolones was completed.Results: A 1-month fluoroquinolone course after transplantation was associated with significantly lower rates of BK viremia at 1 year compared with those with no fluoroquinolone. In the subgroup analysis, exposure to fluoroquinolone for treatment of bacterial infections within 3 months after transplantation was associated with significantly lower 1-year rates of BK viremia.Conclusions: This analysis demonstrates that fluoroquinolones are effective at preventing BK viremia after renal transplantation.

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti-GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4-7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6-24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti-GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

Treatment of cultured oligodendrocytes with a monoclonal antibody to galactocerebroside (GalC) triggers a cascade of events including the redistribution of membrane surface GalC over internal domains of MBP and loss of microtubular structures within the sheets (Dyer and Benjamins: J Neurosci 8:4307-4318, 1988; Dyer and Benjamins: J Neurosci Res 24:212-221, 1989). In this report, wild type and myelin basic protein (MBP)-deficient shiverer oligodendrocytes were used to study the possible relationships between these events, and specifically to determine if MBP mediates signals which destabilize microtubular assemblies in cultured oligodendrocytes. We now show that MBP and GalC, which are both initially Triton X-100 soluble, become Triton X-100 insoluble following anti-GalC binding and anti-GalC:GalC complex redistribution, suggesting that the surface anti-GalC: GalC complexes become associated with cytoplasmic MBP. Mediation of the signaling event by MBP is further demonstrated by 1) a decreased phosphorylation of MBP in wild type oligodendrocytes after antibody binding, and 2) the absence of responses, such as GalC redistribution and microtubule loss, in MBP-deficient shiverer oligodendrocytes treated with anti-GalC. Continuous activation of the GalC/MBP pathway for 7 days in wild type oligodendrocytes results in enlarged cell bodies and production of numerous microprocesses, a morphology that is similar to MBP-deficient shiverer oligodendrocytes. A second signaling pathway which produces an opposite effect, i.e., the stabilization and apparent up-regulation of microtubular structures in cultured oligodendrocyte membrane sheets, remains functional in shiverer oligodendrocytes. Thus, MBP appears to be important for mediating extracellular signals that cause a loss of microtubular structures in oligodendrocyte membrane sheets and abnormal morphology.

Abstract. This is the first study to provide evidence that one function for the surface glycolipid galactocerebroside (GalC) is participation in the opening of Ca 2+ channels in oligodendroglia in culture. This glycolipid is a unique differentiation marker for myelin-producing cells; antibodies to GalC have been shown to markedly alter oligodendroglial morphology via disruption of microtubules (Dyer, C. A., and J. A. Benjamins. 1988. J. Neurosci. 8:4307--4318). This study demonstrates that extracellular EGTA blocks anti-GalC-induced disassembly of microtubules in oligodendroglial membrane sheets, demonstrating that an influx of extracellular Ca 2÷ mediates the cytoskeletal changes. The Ca 2÷ influx was examined directly by loading oligodendroglia with the fluorescent dye Indo-1 in defined medium, and measuring changes in Ca 2÷ in individual cells with a laser cytometer. Upon addition of anti-GalC IgG, a marked sustained increase in intracellular Ca 2÷ occurred in 80% of the oligodendroglia observed. EGTA blocked the increase, indicating the increase is due to an influx of extracellular Ca 2+, and not due to release from intracellular stores. The effect is specific, since Ca 2+ levels remain normal in oligodendroglia treated with nonimmune IgG; astrocytes do not respond to the anti-GalC. The Ca 2+ response in oligodendrocytes is dependent on concentration of antibody and GalC on the oligodendroglial membrane surface. The Ca 2+ influx is not mediated by voltage-sensitive Ca 2÷ channels: it is not blocked by cadmium, and depolarization with K ÷ does not mimic the response. The kinetics of the response suggest that second messenger-mediated opening of Ca 2÷ channels is involved. lvms have received increasing attention for their role in receptor-mediated signaling across cell membranes. In particular, glycolipids presumably associated with transmembrane proteins have been reported to participate in ligand binding and signaling; for example, cholera toxin binds to GMI ganglioside leading to activation of adenylate cyclase (see Fishman, 1982, for review). Glycolipids are involved in initiating such events as mitogenesis, morphogenesis, and cell recognition (Sharom and Grant, 1978;Grant and Peters, 1979;Hakomori, 1981;Spiegel and Wilchek, 1981;Thompson and Tillack, 1985;Facci et al., 1988;Curatolo, 1987;Bansal and Pfeiffer, 1989). However, little is known about the mechanisms underlying these effects.We have focused on the function of galactocerebroside (GalC), a glycolipid highly enriched on the surface of myelin-producing cells. Oligodendrocytes are the only cells in the central nervous system that express this lipid; although it is used widely as a unique marker for differentiation of oligodendrocytes, its role in the specialized membrane produced by these cells is not known. Several studies over the last decade indicate that antibodies to GalC can alter oligodendroglial morphology and myelination, providing indirect evidence that this glycolipid is involved in signal transduction (Diaz et al., 1978;Dorfman et al., ...

Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2-and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis. Keywords: axon, blood-brain barrier, dopamine, myelin, phenylketonuria, tyrosine. In the CNS, oligodendrocytes extend numerous processes, and from the distal tip of each process, a membrane sheet is assembled and wrapped around a segment of axon as an internode of myelin. Myelin is a highly metabolically active membrane that, under normal conditions, remains connected to and is supported by the oligodendrocyte cell body for the life span of the oligodendrocyte. Myelin is essential for the rapid conduction of action potentials and, therefore, there may be devastating consequences if oligodendrocytes fail to produce myelin (hypomyelination) or lose their myelin (demyelination) as a consequence of disease.One disease in which hypomyelination occurs in specific forebrain tracts, but neurons and their axons are spared, is the autosomal recessive disorder phenylketonuria (PKU) (Malamud 1966;Dyer et al. 1996). PKU is caused by a rise in blood phenylalanine (Phe) levels, due to a deficiency in the enzyme phenylalanine hydroxylase (PAH) (Jervis 1953). PAH is expressed primarily in liver and not in brain, and catalyzes the conversion of Phe to tyrosine (Hsieh and Berry 1979). Blood Phe levels normally are about 121 lM; however, in untreated individuals (and mice) ...