The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

Identification of a flavobacterium strain virulent against Giardia lamblia cysts

We have isolated from a Kentucky stream a bacterial strain capable of killing the cyst form of Giardia lamblia. This bacterium, designated Sun4, is a Gram-negative, aerobic rod which produces a yellow pigment, but not of the flexirubin-type. Although true gliding motility has not been observed in Sun4, this strain does exhibit a spreading colony morphology when grown on R2A agar. Strain Sun4 has been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the genus Flavobacterium, and is most closely related to Cytophaga sp. strain Type 0092 and associated Flavobacterium columnare strains. Lipid analysis also identified fatty acids characteristic of the Cytophaga–Flavobacterium group of bacteria. In culture, Sun4 is able to degrade casein and cellulose, but not chitin, gelatin, starch, or agar. Degradation of Giardia cysts by Sun4 appears to require direct cellular contact as neither cell-free extracts nor cells separated from the cysts by dialysis membranes showed any activity against cysts. Activity against Giardia cysts is rapid, with Sun4 killing over 90% of cysts within 48 h. Strain Sun4 requires elevated levels of Ca²⁺ for optimal growth and degradative activity against Giardia cysts. We propose that bacterial strains such as Sun4 could be used as biological control agents against Giardia cysts in drinking water treatment systems.     

Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.     

The synthesis of ribosomal protein and ribosomal RNA in a rat-mouse hybrid cell line

A rat-mouse hybrid cell line was examined for the presence of ribosomal RNA and ribosomal proteins from both parents. As demonstrated by banding of centromeric heterochromatin, the hybrid cell line contained most of the mouse genome and at least 13 rat chromosomes. The ability of rat, but not mouse, ribosomes to dimerize was used to show that the hybrid contained both rat and mouse 28S ribosomal RNA. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicated the presence of both rat and mouse ribosomal proteins.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Intact amino acid uptake by northern hardwood and conifer trees

Empirical and modeling studies of the N cycle in temperate forests of eastern North America have focused on the mechanisms regulating the production of inorganic N, and assumed that only inorganic forms of N are available for plant growth. Recent isotope studies in field conditions suggest that amino acid capture is a widespread ecological phenomenon, although northern temperate forests have yet to be studied. We quantified fine root biomass and applied tracer-level quantities of U–¹³C₂–¹⁵N-glycine, ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in two stands, one dominated by sugar maple and white ash, the other dominated by red oak, beech, and hemlock, to assess the importance of amino acids to the N nutrition of northeastern US forests. Significant enrichment of ¹³C in fine roots 2 and 5 h following tracer application indicated intact glycine uptake in both stands. Glycine accounted for up to 77% of total N uptake in the oak–beech–hemlock stand, a stand that produces recalcitrant litter, cycles N slowly and has a thick, amino acid-rich organic horizon. By contrast, glycine accounted for only 20% of total N uptake in the sugar maple and white ash stand, a stand characterized by labile litter and rapid rates of amino acid production and turnover resulting in high rates of mineralization and nitrification. This study shows that amino acid uptake is an important process occurring in two widespread, northeastern US temperate forest types with widely differing rates of N cycling.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.