PI3Kγ Drives Priming and Survival of Autoreactive CD4+ T Cells during Experimental Autoimmune Encephalomyelitis

The class IB phosphoinositide 3-kinase gamma enzyme complex (PI3Kγ) functions in multiple signaling pathways involved in leukocyte activation and migration, making it an attractive target in complex human inflammatory diseases including MS. Here, using pik3cg ^−/− mice and a selective PI3Kγ inhibitor, we show that PI3Kγ promotes development of experimental autoimmune encephalomyelitis (EAE). In pik3cg^−/− mice, EAE is markedly suppressed and fewer leukocytes including CD4⁺ and CD8⁺ T cells, granulocytes and mononuclear phagocytes infiltrate the CNS. CD4⁺ T cell priming in secondary lymphoid organs is reduced in pik3cg^−/− mice following immunisation. This is attributable to defects in DC migration concomitant with a failure of full T cell activation following TCR ligation in the absence of p110γ. Together, this results in suppressed autoreactive T cell responses in pik3cg^−/− mice, with more CD4⁺ T cells undergoing apoptosis and fewer cytokine-producing Th1 and Th17 cells in lymphoid organs and the CNS. When administered from onset of EAE, the orally active PI3Kγ inhibitor AS605240 caused inhibition and reversal of clinical disease, and demyelination and cellular pathology in the CNS was reduced. These results strongly suggest that inhibitors of PI3Kγ may be useful therapeutics for MS.     

Muscular ETB receptors develop postnatally and are differentially distributed in specific segments of the rat vasculature.

The endothelin/endothelin-receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ETB receptor and investigated the distribution of ETB receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissue-specific and age-dependent presence of vasoconstrictor ETB receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ETB receptor immunoreactivity. The occurrence of ETB receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ETB receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ETB receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ETB receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ETB receptors, mainly on arterial smooth muscle cells in the vascular system of the rat.     

The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.     

General screening for celiac disease is advisable in children with type 1 diabetes.

The association between celiac disease (CD) and diabetes mellitus type 1 is well known. Only about one-third of all patients with CD are diagnosed in childhood as a result of typical gastrointestinal symptoms or growth retardation. To evaluate the feasibility of CD screening in diabetic children, we tested autoantibodies to tissue transglutaminase (tTGA) in all children with type 1 diabetes from our pediatric department during a 12-month period. In antibody-positive cases, we analyzed the clinical presentation and offered a duodenal biopsy to confirm the diagnosis and grade the severity of the inflammatory process. Of 205 children, 13 (6.3 %) were tTGA-positive. In seven of eight children who agreed to perform a biopsy, CD typical histological signs were detected (Marsh 1: n = 1, Marsh 3: n = 6). In three patients with confirmed disease, symptoms (iron deficiency, recurrent abdominal pain) remained undiscovered up to time of screening (latent form); in four, the disease was asymptomatic (silent form). Since clinical symptoms are mostly mild or absent in spite of severe signs of duodenal inflammation, we recommend tTGA screening in all diabetic children. This strategy may allow the identification of patients in an early stage in respect of prevention of long-term complications.     

Prevention of endotoxin-induced lethality, but not of liver apoptosis in poly(ADP-ribose) polymerase-deficient mice.

Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP-/-) to examine the differential sensitivity of wild-type (wt) and PARP-/- mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP-/- animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 microgram/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP-/- animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.     

Infinite zig-zag and cyclic-tetranuclear isomeric imidazolate-bridged polynuclear copper(II) complexes: Magnetic properties, catalytic activity and electrospray mass and tandem mass spectrometry characterization

Two mononuclear copper(II) complexes 1 and 2 with the unsymmetrical tridentate ligands 2- and 4-[((imidazol-2-ylmethylidene)amino)ethyl]pyridine have been prepared. In alkaline solution, deprotonation of the imidazole moiety in 1 and 2 promotes self-assembly, which yielded two structurally different species. Depending on the binding site in the imidazole ring, a polymeric complex with an infinite zig-zag-chain 3, or a cyclic-tetranuclear complex 4 is formed, as shown by spectroscopic and spectrometric analysis. Herein, structural characterization of these isomeric polynuclear complexes was performed by electrospray mass (ESI-MS) and tandem mass spectrometric experiments (ESI-MS/MS). Each isomer was shown to be stable in methanolic solutions and to display unique mass spectra with characteristic multiply charged molecular and fragment ions, corroborating previous data by EPR measurements. Magnetic data in the solid state fit a typical curve for an one-dimensional infinite regular chain system, with J = −(32.4 ± 1.2) cm⁻¹ and g = 2.03 for 3, and that of a cyclic-tetranuclear structure with J = −(55.5 ± 0.4) cm⁻¹ and g = 2.29 for 4. In the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) by molecular oxygen, both complexes were shown to act as efficient catalysts, exhibiting very similar ratios: k_cat/K_M = 9.12 × 10⁶ mol⁻¹ dm³ min⁻¹ for 3 and 8.73 × 10⁶ mol⁻¹ dm³ min⁻¹ for 4. These similar ratios indicate that interactions between the metal centres in 3 or 4 and the substrate in solution occur predominantly at the outside of the catalyst framework.     

Rate variation among nuclear genes and the age of polyploidy in Gossypium

Molecular evolutionary rate variation in Gossypium (cotton) was characterized using sequence data for 48 nuclear genes from both genomes of allotetraploid cotton, models of its diploid progenitors, and an outgroup. Substitution rates varied widely among the 48 genes, with silent and replacement substitution levels varying from 0.018 to 0.162 and from 0.000 to 0.073, respectively, in comparisons between orthologous Gossypium and outgroup sequences. However, about 90% of the genes had silent substitution rates spanning a more narrow threefold range. Because there was no evidence of rate heterogeneity among lineages for any gene and because rates were highly correlated in independent tests, evolutionary rate is inferred to be a property of each gene or its genetic milieu rather than the clade to which it belongs. Evidence from approximately 200,000 nucleotides (40,000 per genome) suggests that polyploidy in Gossypium led to a modest enhancement in rates of nucleotide substitution. Phylogenetic analysis for each gene yielded the topology expected from organismal history, indicating an absence of gene conversion or recombination among homoeologs subsequent to allopolyploid formation. Using the mean synonymous substitution rate calculated across the 48 genes, allopolyploid cotton is estimated to have formed circa 1.5 million years ago (MYA), after divergence of the diploid progenitors about 6.7 MYA.     

GENETIC DIVERSITY IN GOSSYPIUM HIRSUTUM AND THE ORIGIN OF UPLAND COTTON

Gossypium hirsutum has a large indigenous range encompassing most of Mesoamerica and the Caribbean, where it exhibits a diverse array of morphological forms spanning the wild-to-domesticated continuum. Modem, highly improved varieties (“Upland cotton”), which currently account for about 90% of world cotton commerce, are day-length neutral annuals derived from subtropical, perennial antecedents. To assess levels and patterns of genetic variation in the species and to elucidate the origin of Upland cotton, 538 accessions representing the full spectrum of morphological and geographical diversity were analyzed forallozyme variation at 50 loci. Levels of variation are modest overall but are low in Upland cotton. Relationships among accessions reflect pre-Columbian influences of aboriginal peoples and later European colonists superimposed on the preagricultural pattern. In contrast to expectations, two centers of diversity are evident, one in southern Mexico-Guatemala and the other in the Caribbean. Introgression of G. barbadense genes into G. hirsutum has been common in a broad area of sympatry in the Caribbean. The germplasm of present cultivars traces to Mexican highland stocks, which, in turn, were derived from material originally from southern Mexico and Guatemala. Despite the widespread belief that germplasm from several other species has been incorporated into modem Upland stocks through intentional breeding efforts, the 50 Upland cultivars examined contain no unique alleles, suggesting that retention of genes from transspecific sources has been minimal. The most recent infraspecific treatment, which recognizes seven races, does not adequately represent genetic relationships.     

Bidirectional cytoplasmic and nuclear introgression in the New World cottons Gossypium barbadense and G. Hirsutum (Malvaceae)

Patterns of interspecific cytoplasmic (plastid and mitochondrial) and nuclear introgression are typically asymmetrical: cytoplasmic gene flow or “capture” is frequently observed without evidence of nuclear introgression. In contrast, nuclear introgression without concomitant cytoplasmic introgression has rarely been demonstrated. Gossypium barbadense L. and G. hirsutum L. have large indigenous ranges in the New World semiarid tropics, with an extensive area of sympatry in the Caribbean and Central America. Numerous accessions of both species were surveyed for diagnostic cpDNA restriction sites. These data, in conjunction with previous information on nuclear markers, lead to several conclusions: 1) introgression between G. hirsutum and G. barbadense is bidirectional for both nuclear and cytoplasmic genes; 2) patterns of introgression between the two species are not symmetrical—in G. barbadense, introgression of G. hirsutum alleles is largely restricted to modem cultivars and is uncommon in areas of sympatry; in contrast, introgression of G. barbadense alleles into G. hirsutum is relatively common in areas of sympatry and is rare in modem cultivars; 3) nuclear introgression is geographically more widespread and more frequently detected than cytoplasmic introgression. Several mechanisms may underlie the differential patterns of cytoplasmic and nuclear gene flow observed, including differential fitness of infraspecific and interspecific cytonuclear combinations and selection against female function in interspecific backcrosses. Possible explanations for the observed asymmetrical patterns of introgression include differences in population sizes combined with phenological differences that bias interspecific pollen transfer.