scholarly journals Tackling penicillin allergy delabeling with ultra-sensitive in vitro test and human-like specific IgE standards

2021 ◽  
Author(s):  
Sergi Morais ◽  
Pedro Quintero-Campos ◽  
Paula Segovia ◽  
Ethel Ibáñez-Echevarria ◽  
Dolores Hernández-Fernández de Rojas ◽  
...  
Keyword(s):  
Human Serum ◽  
Limit Of Detection ◽  
Specific Ige ◽  
Penicillin Allergy ◽  
Penicillin G ◽  
Diagnostic Strategy ◽  
In Vitro Test ◽  
Serum Samples

Background: Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the quantification of specific IgE is highly useful to confirm immediate allergy to penicillins. However, discrepant results associated to the low sensitivity of the in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to tackle a novel diagnostic strategy for specific IgE testing based on a homologous interpolation scheme, using recombinantly produced standards. Methods: Serum samples from a cohort of allergic patients and controls were analysed by a chemiluminescence-based immunoassay, using a bispecific binanobody as standard. The novel standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. Results: Testing a cohort of 65 human serum samples, the method achieved a good agreement and strong positive relationship, reaching a limit of detection below 0.1 IU/mL. The sensitivity of the in vitro test significantly increased (66 %), doubling that of the ImmunoCAP reference in vitro assay with an overall specificity of 100 %. Conclusions: The new diagnostic strategy compares favourably with the results obtained on the ImmunoCAP system, paving the way towards the standardization of penicillin allergy testing. The recombinant standards are potent calibrators, highly stable, easy and inexpensive to produce, and overcome the limitation associated with preparations derived from pooled human serum, expediting the production of next generation standards with different specificities to successfully tackle β-lactam allergy delabeling by in vitro tests.

Immediate Allergic Reactions to β-Lactams: Diagnosis and Therapy

2003 ◽  
Vol 16 (1) ◽  
pp. 19-23 ◽  
Author(s):  
A. Romano ◽  
C. Mondino ◽  
M. Viola ◽  
P. Montuschi
Keyword(s):  
Anaphylactic Shock ◽  
Specific Ige ◽  
Ring Structure ◽  
Skin Tests ◽  
In Vitro Tests ◽  
Allergic Reactions ◽  
In Vitro Test ◽  
Immunological Mechanisms

β-Lactams are the antibiotics which most frequently provoke adverse reactions mediated by specific immunological mechanisms. These reactions, classifiable as immediate or non-immediate, can be produced by the four classes of β-lactams (penicillins, cephalosporins, carbapenems and monobactams) currently available, which share a common β-lactam ring structure. Immediate reactions occur within the first hour after drug administration and are characterized by urticaria, angioedema, rhinitis, bronchospasm, and anaphylactic shock. Immediate reading skin tests are the quickest and most reliable method for demonstrating the presence of β-lactam specific IgE antibodies. It is crucial to use in diagnosis the suspected β-lactams themselves, particularly cephalosporins, in addition to penicillin determinants. Serum specific IgE assays can be used as complementary tests. Negative test results should be interpreted in light of the time elapsed from the last exposure to the responsible β-lactam. In fact, both in vivo and in vitro test sensitivity is known to decrease over time. In some diagnostic work-ups, patients with a positive history and negative skin and in vitro tests with classic reagents undergo a controlled administration of the suspected β-lactam. The management of immediate allergic reactions should take into consideration their severity and type. Adrenaline is the drug of choice in the treatment of anaphylactic shock. In addition to adrenaline, corticosteroids and antihistamines should be administered. Histamine H1 receptor antagonists are the mainstay of the treatment of immediate allergic reactions such as urticaria, rhinitis and conjunctivitis.

scholarly journals Identification of Antigenic Components of Staphylococcus epidermidis Expressed during Human Infection

2006 ◽  
Vol 74 (8) ◽  
pp. 4644-4654 ◽  
Author(s):  
Mohammad R. Pourmand ◽  
Simon R. Clarke ◽  
Richard F. Schuman ◽  
James J. Mond ◽  
Simon J. Foster
Keyword(s):  
Human Serum ◽  
Staphylococcus Epidermidis ◽  
Binding Capacity ◽  
Repeat Sequence ◽  
Human Infection ◽  
Surface Proteins ◽  
Serum Samples ◽  
Control Disease

ABSTRACT A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

In Vitro Toxicology Methods in Risk Assessment

1996 ◽  
Vol 24 (3) ◽  
pp. 325-331
Author(s):  
Iain F. H. Purchase
Keyword(s):  
Risk Assessment ◽  
Hazard Assessment ◽  
Human Health Risk ◽  
In Vitro Test ◽  
In Vitro Methods ◽  
New Concepts ◽  
Vitro Test

The title of this paper is challenging, because the question of how in vitro methods and results contribute to human health risk assessment is rarely considered. The process of risk assessment usually begins with hazard assessment, which provides a description of the inherent toxicological properties of the chemical. The next step is to assess the relevance of this to humans, i.e. the human hazard assessment. Finally, information on exposure is examined, and risk can then be assessed. In vitro methods have a limited, but important, role to play in risk assessment. The results can be used for classification and labelling; these are methods of controlling exposure, analogous to risk assessment, but without considering exposure. The Ames Salmonella test is the only in vitro method which is incorporated into regulations and used widely. Data from this test can, at best, lead to classification of a chemical with regard to genotoxicity, but cannot be used for classification and labelling on their own. Several in vitro test systems which assess the topical irritancy and corrosivity of chemicals have been reasonably well validated, and the results from these tests can be used for classification. The future development of in vitro methods is likely to be slow, as it depends on the development of new concepts and ideas. The in vivo methods which currently have reasonably developed in vitro alternatives will be the easiest to replace. The remaining in vivo methods, which provide toxicological information from repeated chronic dosing, with varied endpoints and by mechanisms which are not understood, will be more difficult to replace.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

scholarly journals Berberine-Coated Biomimetic Composite Microspheres for Simultaneously Hemostatic and Antibacterial Performance

Polymers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 360
Author(s):  
Xiaojian Zhang ◽  
Kaili Dai ◽  
Chenyu Liu ◽  
Haofeng Hu ◽  
Fulin Luo ◽  
...  
Keyword(s):  
In Vitro Test ◽  
Water Emulsion ◽  
Biomedical Material ◽  
Composite Microspheres ◽  
Antibacterial Performance ◽  
New Strategy ◽  
Oil In Water Emulsion ◽  
Hemostatic Powder

Biomimetic microspheres containing alginate/carboxymethylcellulose/gelatin and coated with 0%, 1%, 3%, and 6% berberine (BACG, BACG-1B, BACG-3B, BACG-6B) were prepared by the oil-in-water emulsion method combined with spray drying. Through a series of physicochemical parameters and determination of hemostatic properties in vitro and in vivo, the results indicated that BACG and BACG-Bs were effective in inducing platelet adhesion/aggregation and promoting the hemostatic potential due to their biomimetic structure and rough surface. In addition, BACG-6B with high berberine proportion presented better hemostatic performance compared with the commercial hemostatic agent compound microporous polysaccharide hemostatic powder (CMPHP). BACG-6B also showed strong antibacterial activity in the in vitro test. The hemolysis test and cytotoxicity evaluation further revealed that the novel composite biomaterials have good hemocompatibility and biocompatibility. Thus, BACG-6B provides a new strategy for developing a due-functional (hemostat/antibacterial) biomedical material, which may have broad and promising applications in the future.

scholarly journals Influence of ionic and non-ionic radiographic contrast media on leukocyte adhesion molecules

2003 ◽  
Vol 12 (5) ◽  
pp. 269-275 ◽  
Author(s):  
Guy L. J. Vermeiren ◽  
Roel Willems ◽  
Marc J. Claeys ◽  
Chris Vrints ◽  
Herman Slegers ◽  
...  
Keyword(s):  
Coronary Angiography ◽  
Contrast Media ◽  
Adhesion Molecules ◽  
Leukocyte Adhesion ◽  
Serum Samples ◽  
Cd11b Expression ◽  
Radiographic Contrast ◽  
Radiographic Contrast Media

Background:Many papers have focused on the importance of granulocytes in the process of reperfusion and ischemia. Most of the clinical studies measured several parameters of this process during and after coronary angiography, without taking into account the effect of the radiographic contrast media (RCM) used during this procedure.Materials and methods:We performed a randomized patient study(n=37)to evaluate the effect of ionic and non-ionic RCM on granulocyte adhesion during coronary angiography. We also evaluated the influence of the ionicity and osmolarity of the different substances on granulocyte adhesion molecules inin vitroexperiments.Results:The osmolarity of patient serum samples increased from 302±1 to 309±1 mOsm/kg(P<0.05)after infusion of RCM. The CD11b expression in the samples of the non-ionic RCM treated group increased from 221±21 MFI to 377±30 MFI(P<0.05)measured as the absolute mean fluorescence intensity (MFI), yet did not alter significantly in the ionic RCM group. In contrast, thein vitroexperiments showed a reduction of the CD11b expression from 360±70 MFI to 149±30 MFI(P<0.05)in the ionic RCM group.Conclusions:The upregulation of adhesion molecules was significantly reducedin vivowith ionic RCM, while ionic substances caused opposite effectsin vitro. This effect should be taken into account when performing leukocyte functional analysis of samples taken during angiography.

scholarly journals Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Christina D. Orrú ◽  
Jason M. Wilham ◽  
Lynne D. Raymond ◽  
Franziska Kuhn ◽  
Björn Schroeder ◽  
...  
Keyword(s):  
Real Time ◽  
Blood Test ◽  
Prion Diseases ◽  
Proteinase K ◽  
Transmissible Spongiform Encephalopathies ◽  
Serum Samples ◽  
Spongiform Encephalopathies ◽  
Jakob Disease

ABSTRACT A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

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