Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25(high) cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25-Foxp3+ or CD25(low)Foxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25(high)Foxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25-Foxp3+ but not from the conversion of peripheral Foxp3-) cells. CD25(high)Foxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25-Foxp3+ cells and/or conversion of CD4+CD25-Foxp3- cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-beta is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.     

Codominance of TLR2-dependent and TLR2-independent modulation of MHC class II in Mycobacterium tuberculosis infection in vivo

Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010.     

Hierarchical patterns of paternity within crowns of Albizia julibrissin (Fabaceae)

The floral architecture and phenology of the tree species Albizia julibrissin (Fabaceae) offer the potential for flowers within inflorescences to share common pollen donors. Patterns of paternity within individual tree crowns may differ among isolated individuals and those in populations due to differences in pollinator foraging behavior. To determine how genetic diversity is partitioned within individual seed pools and whether these patterns differ among isolated and population trees, we obtained all fruits from three inflorescences from four clusters from three isolated trees and from three population trees in Athens, Georgia. We assayed 14 polymorphic allozymes to genotype all progeny within singly sired fruits to determine the multilocus genotype of each fruit's pollen donor. Inflorescences had multiple pollen donors, but simulation analyses revealed that redundancy of pollen donors tended to be more likely within inflorescences than randomly across the crown. Analysis of genetic and genotypic diversity indicated that individual maternal trees received pollen from many donors in uneven frequencies. Results suggest that isolated trees receive pollen from slightly fewer pollen donors and experience more within-plant pollinator movement than trees in populations. However, isolated trees receive qualitatively similar pollen from many sources, suggesting that these trees are not effectively isolated and that pollen moves long distances in this species.     

The identification of potent, selective, and bioavailable cathepsin S inhibitors

Highly potent, selective, and bioavailable inhibitors of human, mouse, or rat cathepsin S are described. The key structural features combine a sulfonyl moiety attached to a large group in P2 and a small substituent in P3.     

Computer simulation of feeding behaviour in the thylacine and dingo as a novel test for convergence and niche overlap

The extinct marsupial thylacine (Thylacinus cynocephalus) and placental grey wolf (Canis lupus) are commonly presented as an iconic example of convergence. However, various analyses suggest distinctly different behaviours and specialization towards either relatively small or large prey in the thylacine, bringing the degree of apparent convergence into question. Here we apply a powerful engineering tool, three-dimensional finite element analysis incorporating multiple material properties for bone, to examine mechanical similarity and niche overlap in the thylacine and the wolf subspecies implicated in its extinction from mainland Australia, Canis lupus dingo. Comparisons of stress distributions not only reveal considerable similarity, but also informative differences. The thylacine's mandible performs relatively poorly where only the actions of the jaw muscles are considered, although this must be considered in the light of relatively high bite forces. Stresses are high in the posterior of the thylacine's cranium under loads that simulate struggling prey. We conclude that relative prey size may have been comparable where both species acted as solitary predators, but that the dingo is better adapted to withstand the high extrinsic loads likely to accompany social hunting of relatively large prey. It is probable that there was considerable ecological overlap. As a large mammalian hypercarnivore adapted to taking small-medium sized prey, the thylacine may have been particularly vulnerable to disturbance.     

Degradation of matrix glycosaminoglycans by peroxynitrite/peroxynitrous acid: evidence for a hydroxyl-radical-like mechanism

The oxidant peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) is generated at sites of inflammation via reaction of O2.- with .NO. Previous studies have shown that these species can oxidize cellular targets, but few data are available on damage to extracellular matrix and its components, despite evidence for matrix modification in a number of pathologies. In the current study we show that reaction of ONOO-/ONOOH with glycosaminoglycans results in extensive polymer fragmentation. Bolus authentic ONOO-/ONOOH modifies hyaluronan, heparin, and chondroitin, dermatan, and heparan sulfates, in a concentration-dependent, but O2-independent, manner. The ONOO-/ONOOH generator 3-(4-morpholinyl)sydnoneimine produces similar time- and concentration-dependent damage. These reactions generate specific polymer fragments via cleavage at disaccharide intervals. Studies at different pH values, and in the presence of bicarbonate, are consistent with ONOOH, rather than the carbonate adduct, CO3.- or ONOO-, being the source of damage. EPR spin trapping experiments have provided evidence for the formation of carbon-centered radicals on glycosaminoglycans and related monosaccharides; the similarity of these spectra to those obtained with authentic HO. is consistent with fragmentation being induced by this oxidant. These data suggest that extracellular matrix fragmentation at sites of inflammation may be due, in part, to the formation and reactions of ONOOH.     

Gastric inflammatory markers and interleukins in patients with functional dyspepsia treated with astaxanthin

The chronic active inflammation caused by Helicobacter pylori is dominated by neutrophils, macrophages, lymphocytes and plasma cells. Several interleukins are involved in the inflammatory process. The aim of this study was to investigate the effect of astaxanthin on gastric inflammation in patients with functional dyspepsia. Forty-four consecutive patients were included, and biopsies were examined for IL-4, IL-6, IL-8, IL-10, interferon-gamma, CD4, CD8, CD14, CD19, CD25 and CD30. Patients were randomized: 21 patients were treated with 40 mg of astaxanthin daily, and 23 patients were treated with a placebo. There was a significant decrease in gastric inflammation in H. pylori-positive patients from both groups. There were no significant changes in the density of H. pylori or in any of the interleukins during or after treatment. There was a significant up-regulation of CD4 and down-regulation of CD8 in patients with H. pylori treated with astaxanthin. Astaxanthin had an effect on the inflammation and on the density of H. pylori in mice in a study where the diet could be standardized without antioxidants (Bennedsen et al., 1999). These dietary conditions are impossible in studies involving humans, and may be due to the minor effect when the host have access to antioxidants in their diet.     

Regulation of Th-1 T cell-dominated immunity to Neisseria meningitidis within the human mucosa

Neisseria meningitidis is commonly carried asymptomatically in the upper respiratory tract and only occasionally invades the bloodstream and meninges to cause disease. Naturally acquired immunity appears protective but the nature of the cellular immune response within the mucosa is uncertain. We show that following in vitro stimulation with N. meningitidis serogroup B (MenB) antigens, approximately 66% of the dividing mucosal CD4(+)CD45RO(+) memory population express the Th1-associated IL18-R while the remainder express CRTH2, a Th2-associated marker. The pro-inflammatory bias of this anti-MenB response is not evident in blood, demonstrating compartmentalization at the induction site; and occurs in the presence or absence of lipopolysacharide indicating that these responses are already fully committed. Depletion of CD25(+) cells reveals suppression of the effector CD4(+) T cell response restricted to the mucosa and most marked in children (i.e. those at greatest risk of disease). Mucosal T-regulatory cell (Treg) activity is partially overcome by blocking the human glucocorticoid-induced TNF receptor (GITR) and is not seen following stimulation with antigens from another mucosal pathogen, influenza virus. Pro-inflammatory, antimeningococcal T cell responses may limit invasive disease at the mucosa but Treg induction while reducing immunopathological damage, may also restrict the effectiveness of the protective response, particularly in children.     

Golgi Complex Organization in Skeletal Muscle: A Role for Golgi‐Mediated Glycosylation in Muscular Dystrophies?

The Golgi complex (GC) is the central organelle of the classical secretory pathway, and it receives, modifies and packages proteins and lipids en route to their intracellular or extracellular destinations. Recent studies of congenital muscular dystrophies in skeletal muscle suggest an exciting new role for an old and well-established function of the GC: glycosylation. Glycosylation is the exquisitely regulated enzymatic addition of nucleotide sugars to proteins and lipids mediated by glycosyltransferases (GTs). Mutations in putative Golgi-resident GTs, fukutin, fukutin-related protein and large1 cause these progressive muscle-wasting diseases. The appropriate localization of GTs to specific subcompartments of the Golgi is critical for the correct assembly line-like addition of glycan groups to proteins and lipids as they pass through the GC. Consequently, these studies of congenital muscular dystrophies have focused attention on the organization and function of the GC in skeletal muscle. In contrast to other cells and tissues, the GC in skeletal muscle has received relatively little attention; however, in recent years, several studies have shown that GC distribution in muscle is highly dynamic or plastic and adopts different distributions in muscle cells undergoing myogenesis, denervation, regeneration and maturation. Here, we review the current understanding of the dynamic regulation of GC organization in skeletal muscle and focus on the targeting of fukutin, fukutin-related protein and large1 to the GC in muscle cells.