Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site,Serum Response and Disease and Identify Disease Specific Pathways

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.     

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Δnef and challenged with pathogenic SIVmac251

Background To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live‐attenuated SIVmac239Δnef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. Results Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post‐challenge. Concomitantly, viremia correlated inversely with SIV‐specific IgG, complement‐mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10⁶ PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). Conclusions As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.     

Host intrinsic determinants and potential consequences of parasite infection in free‐ranging red‐fronted lemurs (Eulemur fulvus rufus)

Parasites and infectious diseases represent ecological forces shaping animal social evolution. Although empirical studies supporting this link abound in various vertebrate orders, both the study of the dynamics and impact of parasite infections and infectious diseases in strepsirrhine primates have received little empirical attention. We conducted a longitudinal parasitological study on four groups of wild red‐fronted lemurs (Eulemur fulvus rufus) at Kirindy Forest, Madagascar, during two field seasons in consecutive years to investigate i) the degree of gastrointestinal parasite infection on population and individual levels and ii) factors potentially determining individual infection risk. Using a comprehensive dataset with multiple individually assignable parasite samples as well as information on age, sex, group size, social rank, and endocrine status (fecal androgen and glucocorticoid), we examined parasite infection patterns and host traits that may affect individual infection risk. In addition, we examined whether parasite infection affects mating and reproductive success. Our results indicated high variability in parasite infection on individual and population levels. Time of year and group size was important determinants of variability in parasite infection. Variation in hormone levels was also associated with parasite species richness and parasite infection intensity. Differences in parasite infection between years indicate a potential immune‐enhancing function of steroid hormones on nematode infections, which has not been reported before from other vertebrates studied under natural conditions. Male mating and reproductive success were not correlated to any measure of parasite infection, which suggests a nonfunctional role of the parasites we examined in primate sexual selection. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

GABAB receptor subunit 1 binds to proteins affected in 22q11 deletion syndrome

GABA_B receptors mediate slow inhibitory effects of the neurotransmitter γ-aminobutyric acid (GABA) on synaptic transmission in the central nervous system. They function as heterodimeric G-protein-coupled receptors composed of the seven-transmembrane domain proteins GABA_B1 and GABA_B2, which are linked through a coiled-coil interaction. The ligand-binding subunit GABA_B1 is at first retained in the endoplasmic reticulum and is transported to the cell surface only upon assembly with GABA_B2. Here, we report that GABA_B1, via the coiled-coil domain, can also bind to soluble proteins of unknown function, that are affected in 22q11 deletion/DiGeorge syndrome and are therefore referred to as DiGeorge critical region 6 (DGCR6). In transfected neurons the GABA_B1-DGCR6 association resulted in a redistribution of both proteins into intracellular clusters. Furthermore, the C-terminus of GABA_B2 interfered with the novel interaction, consistent with heterodimer formation overriding transient DGCR6-binding to GABA_B1. Thus, sequential coiled-coil interactions may direct GABA_B1 into functional receptors.     

Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca Greb. Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 µM cadmium chloride (CdCl₂) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.     

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.     

Studies of the silencing of baculovirus DNA binding protein

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.     

IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.