Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening

2020 ◽  
Vol 104 (6) ◽  
pp. 2501-2512
Author(s):  
Joana H. Ministro ◽  
Soraia S. Oliveira ◽  
Joana G. Oliveira ◽  
Miguel Cardoso ◽  
Frederico Aires-da-Silva ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Synthetic Antibody ◽  
Antibody Discovery

scholarly journals Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

2006 ◽  
Vol 80 (17) ◽  
pp. 8510-8520 ◽  
Author(s):  
Marina Kovaleva ◽  
Ingo Bussmeyer ◽  
Björn Rabe ◽  
Joachim Grötzinger ◽  
Enge Sudarman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phage Display ◽  
Cell Line ◽  
Primary Effusion Lymphoma ◽  
Recombinant Antibody ◽  
Cytokine Signaling ◽  
Hepatoma Cell Line ◽  
Single Chain ◽  
Synthetic Antibody ◽  
Stat3 Phosphorylation

ABSTRACT Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.

scholarly journals Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes

2021 ◽  
Author(s):  
Huimin Wang ◽  
Kai Yan ◽  
Ruixue Wang ◽  
Yi Yang ◽  
Yuelei Shen ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Immune Repertoire ◽  
Model Framework ◽  
Synthetic Antibody ◽  
Cdr3 Length ◽  
Germline Genes ◽  
Antibody Discovery ◽  
Human Immune ◽  
Antibody Heavy Chain

Abstract Background Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. In recent years, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Methods Using VH3–23 as a model framework, we analyzed the naturally and productively rearranged CDR-H3 diversity in human immune repertoire. With homology modeling, we further built VH3–23-based structural models to understand the spatial paratope and its influencing parameters. Results We observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Such CDR-H3 length dependent usage of human germline genes was not impacted by the choices of VH in the V(D)J recombination, ethnic background and health conditions. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Conclusions We quantitatively determined the CDR-H3 length-dependent usage of human germline genes, which makes it possible to design synthetic diversity fully mimicking that of natural immune repertoire. Our observations shed light on the design of next generation synthetic diversity with improved probability of success.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Ultrastructure of the Parotid Gland of the Nine-Banded Armadillo

1977 ◽  
Vol 35 ◽  
pp. 640-641
Author(s):  
J. R. Ruby
Keyword(s):  
Endoplasmic Reticulum ◽  
Parotid Gland ◽  
Periodic Acid ◽  
Acinar Cells ◽  
Duct System ◽  
Parotid Glands ◽  
Dasypus Novemcinctus ◽  
Anterior Portion ◽  
Periodic Acid Schiff ◽  
Thin Sections

Parotid glands were obtained from five adult (four male and one female) armadillos (Dasypus novemcinctus) which were perfusion-fixed. The glands were located in a position similar to that of most mammals. They extended interiorly to the anterior portion of the submandibular gland.In the light microscope, it was noted that the acini were relatively small and stained strongly positive with the periodic acid-Schiff (PAS) and alcian blue techniques, confirming the earlier results of Shackleford (1). Based on these qualities and other structural criteria, these cells have been classified as seromucous (2). The duct system was well developed. There were numerous intercalated ducts and intralobular striated ducts. The striated duct cells contained large amounts of PAS-positive substance.Thin sections revealed that the acinar cells were pyramidal in shape and contained a basally placed, slightly flattened nucleus (Fig. 1). The rough endoplasmic reticulum was also at the base of the cell.

Organisation in the Structure of the Cytoplasmic Ground Substance

1978 ◽  
Vol 36 (3) ◽  
pp. 627-639
Author(s):  
K.R. Porter
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Random Distribution ◽  
Ground Substance ◽  
The Matrix ◽  
Cell Processes ◽  
Cytoplasmic Ground Substance

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

Ultrastructural Changes Associated with Alcoholic Hyalin in Hepatocytes and Biliary Epithelial Cells

1971 ◽  
Vol 29 ◽  
pp. 572-573
Author(s):  
Odell T. Minick ◽  
Hidejiro Yokoo ◽  
Fawzia Batti
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Liver Biopsy ◽  
Alcoholic Hepatitis ◽  
Ultrastructural Changes ◽  
Main Mass ◽  
Biliary Epithelial Cells ◽  
Biopsy Specimens ◽  
Alcoholic Hyalin

To learn more of the nature and origin of alcoholic hyalin (AH), 15 liver biopsy specimens from patients with alcoholic hepatitis were studied in detail.AH was found not only in hepatocytes but also in ductular cells (Figs. 1 and 2), although in the latter location only rarely. The bulk of AH consisted of a randomly oriented network of closely packed filaments measuring about 150 Å in width. Bundles of filaments smaller in diameter (40-90 Å) were observed along the periphery of the main mass (Fig. 1), often surrounding it in a rim-like fashion. Fine filaments were also found close to the nucleus in both hepatocytes and biliary epithelial cells, the latter even though characteristic AH was not present (Figs. 3 and 4). Dispersed among the larger filaments were glycogen, RNA particles and profiles of endoplasmic reticulum. Dilated cisternae of endoplasmic reticulum were often conspicuous around the periphery of the AH mass. A limiting membrane was not observed.

Compound Symbiosis in Peridinium Balticum

1973 ◽  
Vol 31 ◽  
pp. 500-501
Author(s):  
R. N. Tomas
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
The Other ◽  
Exact Nature ◽  
Symbiotic Relationship

Peridinium balticum appears to be unusual among the dinoflagellates in that it possesses two DNA-containing structures as determined by histochemical techniques. Ultrastructurally, the two dissimilar nuclei are contained within different protoplasts; one of the nuclei is characteristically dinophycean in nature, while the other is characteristically eucaryotic. The chloroplasts observed within P. balticum are intrinsic to an eucaryotic photosynthetic endosymbiont and not to the dinoflagellate. These organelles are surrounded by outpocketings of endoplasmic reticulum which are continuous with the eucaryotic nuclear envelope and are characterized by thylakoids composed of three apposed lamellae. Girdle lamellae and membranebounded interlamellar pyrenoids are also present. Only the plasmalemma of the endosymbiont segregates its protoplast from that of the dinophycean cytoplasm. The exact nature of this symbiotic relationship is at present not known.

Golgi Apparatus and Melanogenesis: Ultrastructural Study of a Human Cellular Blue Nevus (Melanocytoma)

1973 ◽  
Vol 31 ◽  
pp. 380-381
Author(s):  
S.R. Allegra
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Ultrastructural Study ◽  
The Other ◽  
Blue Nevus ◽  
Normal Complement ◽  
Golgi Vesicles ◽  
Golgi System ◽  
The Subject ◽  
Cellular Blue Nevus

The respective roles of the ribo somes, endoplasmic reticulum, Golgi apparatus and perhaps nucleus in the synthesis and maturation of melanosomes is still the subject of some controversy. While the early melanosomes (premelanosomes) have been frequently demonstrated to originate as Golgi vesicles, it is undeniable that these structures can be formed in cells in which Golgi system is not found. This report was prompted by the findings in an essentially amelanotic human cellular blue nevus (melanocytoma) of two distinct lines of melanocytes one of which was devoid of any trace of Golgi apparatus while the other had normal complement of this organelle.

Modification of Pineal Ultrastructure by Exogenous Hormones

1967 ◽  
Vol 25 ◽  
pp. 170-171
Author(s):  
R. A. Turner ◽  
A. E. Rodin ◽  
D. K. Roberts
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Estradiol Benzoate ◽  
Female Rats ◽  
List Type ◽  
Type Number ◽  
Pregnant Rats ◽  
Gonadal Atrophy ◽  
Pineal Ultrastructure

There have been many reports which establish a relationship between the pineal and sexual structures, including gonadal hypertrophy after pinealectomy, and gonadal atrophy after injection of pineal homogenates or of melatonin. In order to further delineate this relationship the pineals from 5 groups of female rats were studied by electron microscopy:ControlsPregnant ratsAfter 4 weekly injections of 0.1 mg. estradiol benzoate.After 8 daily injections of 150 mcgm. melatonin (pineal hormone).After 8 daily injections of 3 mg. serotonin (melatonin precursor).No ultrastructural differences were evident between the control, and the pregnancy and melatonin groups. However, the estradiol injected animals exhibited a marked increase in the amount and size of rough endoplasmic reticulum within the pineal cells.

Hepatic Ultrastructure Changes Induced by Lithocholic Acid

1967 ◽  
Vol 25 ◽  
pp. 162-163 ◽  
Author(s):  
F. G. Zaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Lithocholic Acid ◽  
Smooth Endoplasmic Reticulum ◽  
Protein Diet ◽  
Low Protein Diet ◽  
Electron Microscopic ◽  
Hydropic Degeneration ◽  
Hepatic Cells ◽  
Low Protein ◽  
Microscopic Studies

Addition of lithocholic acid (LCA), a naturally occurring bile acid in mammals, to a low protein diet fed to rats induced marked inflammatory reaction in the hepatic cells followed by hydropic degeneration and ductular cell proliferation. These changes were accompanied by dilatation and hyperplasia of the common bile duct and formation of “gallstones”. All these changes were reversible when LCA was withdrawn from the low protein diet except for the hardened gallstones which persisted.Electron microscopic studies revealed marked alterations in the hepatic cells. Early changes included disorganization, fragmentation of the rough endoplasmic reticulum and detachment of its ribosomes. Free ribosomes, either singly or arranged in small clusters were frequently seen in most of the hepatic cells. Vesiculation of the smooth endoplasmic reticulum was often encountered as early as one week after the administration of LCA (Fig. 1).

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