Neonatal Fc receptor blockade by Fc engineering ameliorates arthritis in a murine model

Multiple autoimmune diseases are characterized by the involvement of autoreactive Abs in pathogenesis. Problems associated with existing therapeutics such as the delivery of intravenous immunoglobulin have led to interest in developing alternative approaches using recombinant or synthetic methods. Toward this aim, in the current study, we demonstrate that the use of Fc-engineered Abs (Abs that enhance IgG degradation [Abdegs]) to block neonatal FcR (FcRn) through high-affinity, Fc region binding is an effective strategy for the treatment of Ab-mediated disease. Specifically, Abdegs can be used at low, single doses to treat disease in the K/B×N serum transfer model of arthritis using BALB/c mice as recipients. Similar therapeutic effects are induced by 25- to 50-fold higher doses of i.v. Ig. Importantly, we show that FcRn blockade is a primary contributing factor toward the observed reduction in disease severity. The levels of albumin, which is also recycled by FcRn, are not affected by Abdeg delivery. Consequently, Abdegs do not alter FcRn expression levels or subcellular trafficking behavior. The engineering of Ab Fc regions to generate potent FcRn blockers therefore holds promise for the therapy of Ab-mediated autoimmunity.     

Glutaric acid as a spacer facilitates improved intracellular uptake of LHRH-SPION into human breast cancer cells

Superparamagnetic iron oxide nanoparticles (SPIONs) bound directly to luteinizing hormone releasing hormone (LHRH) have shown high efficiency for intracellular uptake to breast cancer cells, MDA-MB-435S.luc. We demonstrate in this communication that inclusion of a small spacer molecule such as glutaric acid (Glu) in between SPION and LHRH increases further receptor mediated intracellular uptake. LHRH-bound SPIONs with and without the spacer molecule were nontoxic.     

Genome-Wide Linkage Scan for Primary Open Angle Glaucoma: Influences of Ancestry and Age at Diagnosis

Primary open-angle glaucoma (POAG) is the most common form of glaucoma and one of the leading causes of vision loss worldwide. The genetic etiology of POAG is complex and poorly understood. The purpose of this work is to identify genomic regions of interest linked to POAG. This study is the largest genetic linkage study of POAG performed to date: genomic DNA samples from 786 subjects (538 Caucasian ancestry, 248 African ancestry) were genotyped using either the Illumina GoldenGate Linkage 4 Panel or the Illumina Infinium Human Linkage-12 Panel. A total of 5233 SNPs was analyzed in 134 multiplex POAG families (89 Caucasian ancestry, 45 African ancestry). Parametric and non-parametric linkage analyses were performed on the overall dataset and within race-specific datasets (Caucasian ancestry and African ancestry). Ordered subset analysis was used to stratify the data on the basis of age of glaucoma diagnosis. Novel linkage regions were identified on chromosomes 1 and 20, and two previously described loci—GLC1D on chromosome 8 and GLC1I on chromosome 15—were replicated. These data will prove valuable in the context of interpreting results from genome-wide association studies for POAG.     

A non-additive interaction in a single locus causes a very short root phenotype in wheat

Non-additive allelic interactions underlie over dominant and under dominant inheritance, which explain positive and negative heterosis. These heteroses are often observed in the aboveground traits, but rarely reported in root. We identified a very short root (VSR) phenotype in the F₁ hybrid between the common wheat (Triticum aestivum L.) landrace Chinese Spring and synthetic wheat accession TA4152-71. When germinated in tap water, primary roots of the parental lines reached ~15 cm 10 days after germination, but those of the F₁ hybrid were ~3 cm long. Selfing populations segregated at a 1 (long-root) to 1 (short-root) ratio, indicating that VSR is controlled by a non-additive interaction between two alleles in a single gene locus, designated as Vsr1. Genome mapping localized the Vsr1 locus in a 3.8-cM interval delimited by markers XWL954 and XWL2506 on chromosome arm 5DL. When planted in vermiculite with supplemental fertilizer, the F₁ hybrid had normal root growth, virtually identical to the parental lines, but the advanced backcrossing populations segregated for VSR, indicating that the F₁ VSR expression was suppressed by interactions between other genes in the parental background and the vermiculite conditions. Preliminary physiological analyses showed that the VSR suppression is independent of light status but related to potassium homeostasis. Phenotyping additional hybrids between common wheat and synthetics revealed a high VSR frequency and their segregation data suggested more Vsr loci involved. Because the VSR plants can be regularly maintained and readily phenotyped at the early developmental stage, it provides a model for studies of non-additive interactions in wheat.     

Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage

On induction of DNA damage with 405-nm laser light, proteins involved in base excision repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by latrunculin B, cytochalasin B, or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin but also of tubulin, linking cytoskeletal integrity to BER.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.     

Partial role of nitric oxide in infarct size limiting effect of quercetin and rutin against ischemia-reperfusion injury in normal and diabetic rats

Reperfusion injury is remarkable clinical issue that needs to be resolved as ischemia-reperfusion is a common phenomenon encountered in numerous clinical situations. The present communication report the involvement of nitric oxide (NO) in cardioprotection offered by flavonoids (rutin and quercetin) against myocardial ischemia reperfusion. Rutin produced better cardioprotection than quercetin in normal and diabetic rats. The observed cardioprotection offered with quercetin and rutin was partially abolished by prior administration of nitric oxide synthase inhibitor, L-NAME (N-nitro-L-arginine methyl ester) in both normal and diabetic rats. L-NAME abolished the cardioprotective actions of rutin more strongly than the cardioprotective actions of quercetin. However, mechanistic study with NOS inhibitor implied the possible partial role of nitric oxide in infarct size limiting effect of quercetin and rutin