scholarly journals Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

1994 ◽  
Vol 180 (3) ◽  
pp. 885-895 ◽  
Author(s):  
Y Ichiyoshi ◽  
P Casali
Keyword(s):  
Defense Mechanisms ◽  
Expression System ◽  
Gene Segment ◽  
Immunoglobulin M ◽  
Class Switch ◽  
Natural Defense ◽  
Kappa Chain ◽  
Polyreactive Antibodies ◽  
Complementarity Determining Region

Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interaction of antibody-bearing small unilamellar liposomes with target free antigen in vitro and in vivo. Some influencing factors

1981 ◽  
Vol 200 (2) ◽  
pp. 203-210 ◽  
Author(s):  
Gregory Gregoriadis ◽  
Anne Meehan ◽  
Mon Moey Mah
Keyword(s):  
Affinity Chromatography ◽  
Specific Binding ◽  
Immunoglobulin M ◽  
Mouse Blood ◽  
Igg Binding ◽  
Rabbit Igg ◽  
Kappa Chain ◽  
Free Antigen

Affinity chromatography-purified and non-purified rabbit immunoglobulin G (IgG) raised against human immunoglobulin M (IgM) or kappa chain was incorporated into carboxyfluorescein-containing small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1). IgG incorporation was carried out by co-sonicating the immunoglobulin with the lipids (30% incorporated) (method A) or by interacting it with preformed liposomes bearing goat anti-(rabbit IgG) IgG (63 and 70% incorporated) (method B). (1) Judging from liposomal carboxyfluorescein-latency values, incorporation of IgG by either method did not affect liposomal stability. Furthermore, treatment of liposomes with papain released 75.1% (method A) and 93.3% and 95.1% (method B) of the IgG, suggesting that most of its antigen-recognizing Fab regions were available on the liposomal surface. This was strongly supported by the immunoelectrophoretic detection of Fab in papain-released products. (2) Liposomes bearing purified anti-IgM IgG bound 30%, (method A) and 45% (method B) of IgM in buffer. These values wee about 6-fold greater (both methods) than those obtained with corresponding liposomes bearing non-purified IgG. Binding of liposomes bearing anti-(kappa chain) IgG to kappa chain in buffer was 37% of that added. In the presence of mouse blood or serum, binding of IgM to liposomes bearing purified anti-IgM IgG was decreased slightly (24 and 30% for methods A and B). However, because of the nearly complete abolition of IgM binding to liposomes bearing non-purified IgG, these values were now 20–25-fold greater than those obtained with liposomes bearing non-purified IgG. (3) In mice pre-injected with IgM, at least 36.1% and 37.7% of the antigen was bound to subsequently injected liposomes bearing anti-IgM IgG incorporated by methods A and B respectively. No binding occurred with liposomes bearing the non-purified IgG. (4) Cholesterol-rich small unilamellar liposomes bearing affinity chromatography-purified antibodies may prove useful for the specific binding of free antigens in vivo.

Induction of Metalloproteinase 9 Secretion from Human Keratinocytes by Pleuran (β-Glucan from Pleurotus ostreatus)

2009 ◽  
Vol 64 (7-8) ◽  
pp. 597-600 ◽  
Author(s):  
Juraj Majtán ◽  
Pawan Kumar ◽  
Ján Koller ◽  
Jana Dragúńová ◽  
Ján Gabriž
Keyword(s):  
Wound Healing ◽  
Matrix Metalloproteinase ◽  
Defense Mechanisms ◽  
Primary Cultures ◽  
Human Keratinocytes ◽  
Water Soluble ◽  
Natural Defense ◽  
Human Keratinocyte ◽  
Metalloproteinase 9

Glucan preparations, primarily modifi ed water-soluble glucans, are involved in the activation of the body’s natural defense mechanisms and in the acceleration of the skin’s wound-healing processes. Pleuran, an insoluble β-D-glucan in hydrogel form, offers a natural alternative to more common chemically derivated soluble β-D-glucans. Pleuran was applied to human keratinocyte primary cultures, and after 24 h of incubation the release of matrix metalloproteinase 9 (MMP-9) and metalloproteinase 2 (MMP-2) by stimulated keratinocytes was detected using gelatine zymography. There was a concentration-dependent increase in pro-MMP-9 release after treatment with pleuran over the concentration range of 2 to 200 μg/ml, but pro-MMP-2 was detected at a constant level. Moreover, the active forms of both MMPs were not detectable, indicating that in vitro autoactivation of these zymogens did not occur. The results indicate that pleuran is a potent keratinocyte stimulator of pro- MMP-9 release, which implies its application in dermatological therapies

scholarly journals FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant

2021 ◽  
Author(s):  
Jianhua Yu ◽  
Wenjuan Dong ◽  
Jing Wang ◽  
Lei Tian ◽  
Jianying Zhang ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Defense Mechanisms ◽  
Defense Mechanism ◽  
Spike Protein ◽  
Natural Defense ◽  
Host Defense Mechanism ◽  
Direct Inhibitors

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID 19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.

Antifungal Proteins from Plant Latex

2020 ◽  
Vol 21 (5) ◽  
pp. 497-506
Author(s):  
Mayck Silva Barbosa ◽  
Bruna da Silva Souza ◽  
Ana Clara Silva Sales ◽  
Jhoana D’arc Lopes de Sousa ◽  
Francisca Dayane Soares da Silva ◽  
...  
Keyword(s):  
Cell Walls ◽  
Defense Mechanisms ◽  
Hydrolytic Enzymes ◽  
Fungal Species ◽  
Biologically Active ◽  
Protein Classification ◽  
Fungal Cell ◽  
Antifungal Proteins ◽  
Plant Latex

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants’ defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Seroprevalence of Anti-SARS-CoV-2 IgG and IgM among Adults over 65 Years Old in the South of Italy

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 483
Author(s):  
Immacolata Polvere ◽  
Alfredina Parrella ◽  
Giovanna Casamassa ◽  
Silvia D’Andrea ◽  
Annamaria Tizzano ◽  
...  
Keyword(s):  
Antibody Response ◽  
Immunoglobulin M ◽  
Molecular Testing ◽  
Elisa Assay ◽  
The South ◽  
Serum Samples ◽  
Serological Screening ◽  
Rna Detection ◽  
In Vitro Diagnostic

SARS-CoV-2 is a zoonotic betacoronavirus associated with worldwide transmission of COVID-19 disease. By the beginning of March, WHO reported about 113,820,000 confirmed cases including more than 2,527,000 deaths all over the world. However, the true extent of virus circulation or its real infection/fatality ratio is not well-estimated due to the huge portion of asymptomatic infections. In this observational study, we have estimated the prevalence of specific immunoglobulin M and G directed towards SARS-CoV-2 antigen in a cohort of 1383 adult volunteers aged over 65 years old, living in the district of Benevento, in the South of Italy. Serological screening was carried out on capillary blood in September 2020, seven months after pandemic outbreak in Italy, to evaluate virus circulation and antibody response among elderly adults, in which severe symptoms due to viral infection are more common. The overall seroprevalence of anti-SARS-CoV-2 antibodies was 4.70% (CI 3.70%–5.95%) with no statistically significant differences between sexes. Among these, 69.69% (CI 55.61%–77.80%) tested positive to IgM, 23.08% (CI 14.51%–34.64%) to IgG and 9.23% (CI 4.30%–18.71%) was positive for both. All patients that were positive to IgM underwent molecular testing through RT-qPCR on oral-rhino pharyngeal swabs and only one specimen was positive for SARS-CoV-2 RNA detection. Instead, the presence of IgG from screened volunteers was confirmed by re-testing serum samples using both an ELISA assay validated for in vitro diagnostic use (IVD) and a recently published synthetic peptide-based ELISA assay. In conclusion, our report suggests that (1) early restrictions were successful in limiting COVID-19 diffusion in the district of Benevento; (2) rapid serological analysis is an ideal testing for both determining real seroprevalence and massive screening, whereas detection of viral RNA remains a gold standard for identification of infected patients; (3) even among people without COVID-19 related symptoms, the antibody response against SARS-CoV-2 antigens has individual features.

scholarly journals Ophthalmic Drug Dosage Forms: Characterisation and Research Methods

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Przemysław Baranowski ◽  
Bożena Karolewicz ◽  
Maciej Gajda ◽  
Janusz Pluta
Keyword(s):  
Defense Mechanisms ◽  
Drug Application ◽  
Drug Dosage ◽  
Eye Drops ◽  
Eye Tissues ◽  
Ophthalmic Drug ◽  
Application Frequency

This paper describes hitherto developed drug forms for topical ocular administration, that is, eye drops, ointments,in situgels, inserts, multicompartment drug delivery systems, and ophthalmic drug forms with bioadhesive properties. Heretofore, many studies have demonstrated that new and more complex ophthalmic drug forms exhibit advantage over traditional ones and are able to increase the bioavailability of the active substance by, among others, reducing the susceptibility of drug forms to defense mechanisms of the human eye, extending contact time of drug with the cornea, increasing the penetration through the complex anatomical structure of the eye, and providing controlled release of drugs into the eye tissues, which allows reducing the drug application frequency. The rest of the paper describes recommendedin vitroandin vivostudies to be performed for various ophthalmic drugs forms in order to assess whether the form is acceptable from the perspective of desired properties and patient’s compliance.

scholarly journals In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

2005 ◽  
Vol 71 (3) ◽  
pp. 1522-1530 ◽  
Author(s):  
Amy M. Grunden ◽  
Francis E. Jenney ◽  
Kesen Ma ◽  
Mikyoung Ji ◽  
Michael V. Weinberg ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pyrococcus Furiosus ◽  
Expression System ◽  
Anaerobic Bacteria ◽  
Flavin Mononucleotide ◽  
Superoxide Reductase ◽  
Recombinant Enzymes ◽  
Significant Similarity ◽  
Midpoint Potential

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

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