Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

<p>Abstract</p> <p>Background</p> <p>Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.</p> <p>Results</p> <p>We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.</p> <p>Conclusion</p> <p>In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.</p

Natural history of non-lethal raine syndrome during childhood

Background: Raine syndrome (RS) is a rare autosomal recessive disorder caused by biallelic loss-of-function mutations of FAM20C. The most common clinical features are microcephaly, exophthalmos, hypoplastic nose and severe midface hypoplasia, leading to choanal atresia. The radiological findings include generalized osteosclerosis and brain calcifications. RS is usually lethal during the neonatal period due to severe respiratory distress. However, there exists a non-lethal RS form, the phenotype of which is extremely heterogeneous. There is paucity of data about clinical course and life expectancy of these patients.Results: This is the first description of follow-up features of non-lethal RS patients. Moreover, we present three unpublished cases. There are five Asian and two Arab patients. All were born to consanguineous parents. The most common neonatal comorbidity was respiratory distress secondary to choanal atresia. A variable degree of neurodevelopmental delay was seen in the majority of our cases and seizures and hearing or vision involvement were also frequent. Neurological and orthopedic issues were the most frequent complications seen at follow-up in our group. Persistent hypophosphatemic rickets was the most striking endocrinological manifestation, which was scarcely responsive to therapy with phosphate salts and alfacalcidol. Life expectancy of our patients goes beyond childhood, with the oldest of those described being 18 years old at present.Conclusions: Manifestations of RS in those surviving the neonatal period are being increasingly recognized. Our study supports previous findings and provides clinical and biochemical observations and data from longer follow up. Finally, we propose multidisciplinary follow up for patients with non-lethal RS

VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology

Structural Homeostasis: Compensatory Adjustments of Dendritic Arbor Geometry in Response to Variations of Synaptic Input

As the nervous system develops, there is an inherent variability in the connections formed between differentiating neurons. Despite this variability, neural circuits form that are functional and remarkably robust. One way in which neurons deal with variability in their inputs is through compensatory, homeostatic changes in their electrical properties. Here, we show that neurons also make compensatory adjustments to their structure. We analysed the development of dendrites on an identified central neuron (aCC) in the late Drosophila embryo at the stage when it receives its first connections and first becomes electrically active. At the same time, we charted the distribution of presynaptic sites on the developing postsynaptic arbor. Genetic manipulations of the presynaptic partners demonstrate that the postsynaptic dendritic arbor adjusts its growth to compensate for changes in the activity and density of synaptic sites. Blocking the synthesis or evoked release of presynaptic neurotransmitter results in greater dendritic extension. Conversely, an increase in the density of presynaptic release sites induces a reduction in the extent of the dendritic arbor. These growth adjustments occur locally in the arbor and are the result of the promotion or inhibition of growth of neurites in the proximity of presynaptic sites. We provide evidence that suggest a role for the postsynaptic activity state of protein kinase A in mediating this structural adjustment, which modifies dendritic growth in response to synaptic activity. These findings suggest that the dendritic arbor, at least during early stages of connectivity, behaves as a homeostatic device that adjusts its size and geometry to the level and the distribution of input received. The growing arbor thus counterbalances naturally occurring variations in synaptic density and activity so as to ensure that an appropriate level of input is achieved

Efficient Detection of Proteins Retro-Translocated from the ER to the Cytosol by In Vivo Biotinylation

Retro-translocation from the ER to the cytosol of proteins within the secretory pathway takes place on misfolded molecules that are targeted for degradation by the cytosolically located 26S proteasome complex. Retro-translocation occurs also for other proteins (such as calreticulin) that, despite being synthesized and transported to the ER, are in part dislocated to the cytosol. We have taken advantage of the E. coli derived biotin-ligase (BirA) expressed in the cytosol of mammalian cells to specifically biotin-label in vivo proteins within the secretory pathway that undergo retro-translocation. We validated the method using four different proteins that are known to undergo retro-translocation upon different conditions: the human trans-membrane protein MHC class-I a chain (MHC-Ia), the Null Hong Kong mutant of the secretory a1 anti-trypsin (NHKa1AT), the immunoglobulin heavy chain (HC) and the ER chaperone calreticulin (Crt). We observed specific monobiotinylation of cytosolically dislocated molecules, resulting in a novel, reliable way of determining the extent of retrotranslocation

New tags for recombinant protein detection and O-glycosylation reporters.

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation

SOME REMARKS ON THE PRESCRIBED MEAN CURVATURE EQUATION ON COMPLETE MANIFOLDS

We obtain conditions on the behavior at infinity of the mean curvature of a graphunder volume growthassumptions. An L q comparison result is also given. 0. Introduction. Let (M,〈, 〉) be a complete (noncompact), m-dimensional, m ≥ 2, Riemannian manifold and, for a fixed reference point o ∈ M, set r(x) = dist (M,〈,〉)(o, x). Thus BR and ∂BR denote, respectively, the geodesic ball and sphere of radius R centered at o. In what follows we shall always assum