scholarly journals A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis

2020 ◽  
Vol 3 (3) ◽  
pp. 193-204
Author(s):  
Breno C B Beirão ◽  
Teresa P Raposo ◽  
Louise M Imamura ◽  
Max Ingberman ◽  
Ted Hupp ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Antibody Fragment ◽  
Cross Reactivity ◽  
Antibody Engineering ◽  
Model Systems ◽  
Native Form ◽  
Blocking Antibody ◽  
Macrophage Activity ◽  
Beneficial Effects ◽  
Variable Heavy

Abstract CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.

scholarly journals Generation and Evaluation of Chicken IgY-scFv for the Purposes of CPV Diagnosis and Therapy

2020 ◽  
Author(s):  
Shikun Ge ◽  
Long Xu ◽  
Ben Li ◽  
Fagang Zhong ◽  
XiaoYing Zhang
Keyword(s):  
Development Strategy ◽  
Antibody Fragment ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Antibody Engineering ◽  
Clinical Samples ◽  
Similar Reaction ◽  
Single Chain ◽  
Diagnosis And Therapy ◽  
T7 Phage Display

Abstract Background: Canine parvovirus (CPV) can cause acute and highly contagious enteritis in dog, antibodies have been used for diagnosis and therapy of CPV disease, generation of functional antibody fragments by using novel antibody engineering platforms is promising in veterinary practice. Results: The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system after immunizing hens with virus-like particles (VLP) of CPV-VP2. The titer of the primary scFv library reached to 1.5×10 6 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 hours. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. Conclusions: This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.

scholarly journals Selective Targeting of Epigenetic Readers and Histone Deacetylases in Autoimmune and Inflammatory Diseases: Recent Advances and Future Perspectives

2021 ◽  
Vol 11 (5) ◽  
pp. 336
Author(s):  
Mohammed Ghiboub ◽  
Ahmed M. I. Elfiky ◽  
Menno P. J. de Winther ◽  
Nicola R. Harker ◽  
David F. Tough ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
In Vivo Studies ◽  
Beneficial Effects ◽  
Domain Specific ◽  
Recent Advances ◽  
Autoimmune And Inflammatory Diseases

Histone deacetylases (HDACs) and bromodomain-containing proteins (BCPs) play a key role in chromatin remodeling. Based on their ability to regulate inducible gene expression in the context of inflammation and cancer, HDACs and BCPs have been the focus of drug discovery efforts, and numerous small-molecule inhibitors have been developed. However, dose-limiting toxicities of the first generation of inhibitors, which typically target multiple HDACs or BCPs, have limited translation to the clinic. Over the last decade, an increasing effort has been dedicated to designing class-, isoform-, or domain-specific HDAC or BCP inhibitors, as well as developing strategies for cell-specific targeted drug delivery. Selective inhibition of the epigenetic modulators is helping to elucidate the functions of individual epigenetic proteins and has the potential to yield better and safer therapeutic strategies. In accordance with this idea, several in vitro and in vivo studies have reported the ability of more selective HDAC/BCP inhibitors to recapitulate the beneficial effects of pan-inhibitors with less unwanted adverse events. In this review, we summarize the most recent advances with these strategies, discussing advantages and limitations of these approaches as well as some therapeutic perspectives, focusing on autoimmune and inflammatory diseases.

scholarly journals Isolation of the native form of chicken gizzard myosin light-chain kinase

1984 ◽  
Vol 218 (3) ◽  
pp. 863-870 ◽  
Author(s):  
P K Ngai ◽  
C A Carruthers ◽  
M P Walsh
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Native Form ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Chymotryptic Peptide ◽  
Dependent Protein

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.

scholarly journals Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
C. Gómez-Casado ◽  
M. Garrido-Arandia ◽  
P. Gamboa ◽  
N. Blanca-López ◽  
G. Canto ◽  
...  
Keyword(s):  
Food Allergy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Native Form ◽  
Ige Binding ◽  
Pru P 3

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named asPru p 3.01, Pru p 3.02, andPru p 3.03.Pru p 3.01showed very similar allergenic activity as the wild type byin vitroassays. However,Pru p 3.02andPru p 3.03presented reduced IgE binding with respect to the native form, byin vitro,ex vivo,and in vivo assays. In addition,Pru p 3.03had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, bothPru p 3.02andPru p 3.03maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus,Pru p 3.02andPru p 3.03could be good candidates for potential immunotherapy in peach-allergic patients.

scholarly journals The Function of the Histamine H4 Receptor in Inflammatory and Inflammation-Associated Diseases of the Gut

2021 ◽  
Vol 22 (11) ◽  
pp. 6116
Author(s):  
Bastian Schirmer ◽  
Detlef Neumann
Keyword(s):  
Inflammatory Diseases ◽  
Allergic Inflammation ◽  
Histamine H4 Receptor ◽  
Ongoing Research ◽  
Inflammatory Reactions ◽  
Beneficial Effects ◽  
Associated Diseases ◽  
Physiological Processes ◽  
H4 Receptor ◽  
Deficient Mice

Histamine is a pleiotropic mediator involved in a broad spectrum of (patho)-physiological processes, one of which is the regulation of inflammation. Compounds acting on three out of the four known histamine receptors are approved for clinical use. These approved compounds comprise histamine H1-receptor (H1R) antagonists, which are used to control allergic inflammation, antagonists at H2R, which therapeutically decrease gastric acid release, and an antagonist at H3R, which is indicated to treat narcolepsy. Ligands at H4R are still being tested pre-clinically and in clinical trials of inflammatory diseases, including rheumatoid arthritis, asthma, dermatitis, and psoriasis. These trials, however, documented only moderate beneficial effects of H4R ligands so far. Nevertheless, pre-clinically, H4R still is subject of ongoing research, analyzing various inflammatory, allergic, and autoimmune diseases. During inflammatory reactions in gut tissues, histamine concentrations rise in affected areas, indicating its possible biological effect. Indeed, in histamine-deficient mice experimentally induced inflammation of the gut is reduced in comparison to that in histamine-competent mice. However, antagonists at H1R, H2R, and H3R do not provide an effect on inflammation, supporting the idea that H4R is responsible for the histamine effects. In the present review, we discuss the involvement of histamine and H4R in inflammatory and inflammation-associated diseases of the gut.

scholarly journals Mesenchymal Stromal Cell Secretome for the Treatment of Immune-Mediated Inflammatory Diseases: Latest Trends in Isolation, Content Optimization and Delivery Avenues

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1802
Author(s):  
Elena Munoz-Perez ◽  
Ainhoa Gonzalez-Pujana ◽  
Manoli Igartua ◽  
Edorta Santos-Vizcaino ◽  
Rosa Maria Hernandez
Keyword(s):  
Stromal Cells ◽  
State Of The Art ◽  
Inflammatory Diseases ◽  
Therapeutic Approaches ◽  
Pharmacological Management ◽  
Beneficial Effects ◽  
New Therapeutic Approaches ◽  
Immune Mediated ◽  
High Prevalence ◽  
Inflammatory Processes

Considering the high prevalence and the complex pharmacological management of immune-mediated inflammatory diseases (IMIDs), the search for new therapeutic approaches for their treatment is vital. Although the immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSCs) have been extensively studied as a potential therapy in this field, direct MSC implantation presents some limitations that could slow down the clinical translation. Since the beneficial effects of MSCs have been mainly attributed to their ability to secrete a plethora of bioactive factors, their secretome has been proposed as a new and promising pathway for the treatment of IMIDs. Formed from soluble factors and extracellular vesicles (EVs), the MSC-derived secretome has been proven to elicit immunomodulatory effects that control the inflammatory processes that occur in IMIDs. This article aims to review the available knowledge on the MSC secretome, evaluating the advances in this field in terms of its composition, production and application, as well as analyzing the pending challenges in the field. Moreover, the latest research involving secretome administration in IMIDs is discussed to provide an updated state-of-the-art for this field. Finally, novel secretome delivery alternatives are reviewed, paying special attention to hydrogel encapsulation as one of the most convenient and promising strategies.

scholarly journals Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Thierry M. Work ◽  
Julie Dagenais ◽  
Anna Willimann ◽  
George Balazs ◽  
Kate Mansfield ◽  
...  
Keyword(s):  
Life Histories ◽  
Cross Reactivity ◽  
Tumor Formation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Native Form ◽  
Green Turtles ◽  
Tumor Viruses ◽  
The Caribbean ◽  
Lower Antibody Response

ABSTRACT Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation. IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

scholarly journals Dual Role of Metallic Trace Elements in Stress Biology—From Negative to Beneficial Impact on Plants

2019 ◽  
Vol 20 (13) ◽  
pp. 3117 ◽  
Author(s):  
Ewa Muszyńska ◽  
Mateusz Labudda
Keyword(s):  
Trace Elements ◽  
Molecular Mechanisms ◽  
Plant Protection ◽  
Model Systems ◽  
Elevated Concentration ◽  
Metallic Ions ◽  
Beneficial Effects ◽  
Metallic Trace Elements ◽  
Beneficial Impact ◽  
Evolutionary Consequences

Heavy metals are an interesting group of trace elements (TEs). Some of them are minutely required for normal plant growth and development, while others have unknown biological actions. They may cause injury when they are applied in an elevated concentration, regardless of the importance for the plant functioning. On the other hand, their application may help to alleviate various abiotic stresses. In this review, both the deleterious and beneficial effects of metallic trace elements from their uptake by roots and leaves, through toxicity, up to the regulation of physiological and molecular mechanisms that are associated with plant protection against stress conditions have been briefly discussed. We have highlighted the involvement of metallic ions in mitigating oxidative stress by the activation of various antioxidant enzymes and emphasized the phenomenon of low-dose stimulation that is caused by non-essential, potentially poisonous elements called hormesis, which is recently one of the most studied issues. Finally, we have described the evolutionary consequences of long-term exposure to metallic elements, resulting in the development of unique assemblages of vegetation, classified as metallophytes, which constitute excellent model systems for research on metal accumulation and tolerance. Taken together, the paper can provide a novel insight into the toxicity concept, since both dose- and genotype-dependent response to the presence of metallic trace elements has been comprehensively explained.

scholarly journals High doses of immunoglobulin G attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of C3b in C3bn- IgG complexes

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 184-193 ◽  
Author(s):  
HU Lutz ◽  
P Stammler ◽  
E Jelezarova ◽  
M Nater ◽  
PJ Spath
Keyword(s):  
Inflammatory Diseases ◽  
Factor H ◽  
Human Igg ◽  
Partial Protection ◽  
Beneficial Effects ◽  
High Concentrations ◽  
High Doses ◽  
Immune Aggregates

Abstract Intravenously applied human IgG has beneficial effects in treating inflammatory diseases, presumably because it has a complement attenuating role. This role of IgG was studied in vitro by following C3 activation and inactivation in sera that were supplemented with exogenous human IgG and incubated with immune aggregates. IgG added at 2 to 10 mg/mL stimulated the physiologic inactivation of C3b-containing complexes twofold to threefold in 20% sera. This, in turn, lowered the overall C3 activation by 28%, as new C3 convertases primarily assembled on C3b-containing complexes. Exogenous IgG (5 mg/mL) also stimulated inactivation of purified C3b2-IgG complexes, whereby their half-life dropped from 3–4 to 1.5 minutes in 20% serum. IgG appeared to act like a modulator of factor H and I because it did not stimulate inactivation of C3b-containing complexes in factor I-deficient serum. Thus, the known partial protection of C3bn-IgG complexes from inactivation by factor H and I was downregulated by high concentrations of IgG. The ability of high doses of IgG to stimulate complement inactivation is a novel regulatory role of IgG. This may be one of the molecular principles for its therapeutic efficacy in treating complement-mediated inflammations.

scholarly journals Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

2019 ◽  
Vol 01 (01) ◽  
pp. e33-e45 ◽  
Author(s):  
Xin Mei ◽  
Junsheng Chen ◽  
Jing Wang ◽  
Jianwei Zhu
Keyword(s):  
Clinical Trials ◽  
Cancer Therapy ◽  
Xenograft Model ◽  
Antibody Fragment ◽  
Model Systems ◽  
Specific Targeting ◽  
Protein Toxin ◽  
High Cytotoxicity ◽  
Hematological Tumors ◽  
Great Potency

AbstractImmunotoxins are proteins that consist of a protein toxin conjugated to a specific targeting moiety. The targeting moiety is usually an antibody or ligand, such as monoclonal antibody, antibody fragment, a cytokine, or a growth factor. The toxins usually come from plant toxins, bacterial toxins, or human-origin cytotoxic elements. Nearly all toxins work by enzymatically inhibiting protein synthesis. After binding to antigens or receptors on target cell surfaces, immunotoxins are internalized and translocated to the cytosol where they can kill the cells. Immunotoxins have demonstrated high cytotoxicity to cancer cells and to date two immunotoxins have been approved by U.S. Food and Drug Administration on the markets for the treatment of hematological tumors: Lumoxiti and Ontak. Many other molecules are under development or clinical trials for different forms of cancer. Although immunotoxins exhibit great potency in xenograft model systems and early clinical trials, there are obstacles that limit successful treatments, including immunogenicity, nonspecific toxicity, and poor penetration. However, efforts are underway to address these problems. In this review, we summarize immunotoxins currently in clinical trials for either hematological tumors or solid tumors, outline the design of immunotoxins utilizing variety of components, and discuss the prominent examples of redesigned immunotoxins with reduced immunogenicity and nonspecific toxicity, as well as the strategies in manufacturing immunotoxins. With further improvements, it is anticipated that immunotoxins will play an increasing role in cancer therapy.

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