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Cell Host & Microbe Dec 2019H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to...
H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection in vivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.
Topics: Animals; Antibodies, Heterophile; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Birds; Epitopes; Humans; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Influenza in Birds; Influenza, Human; Mice; Neuraminidase; Orthomyxoviridae Infections; Pre-Exposure Prophylaxis; Vaccination; Vaccines, Inactivated; Viral Proteins; Virus Release
PubMed: 31757769
DOI: 10.1016/j.chom.2019.10.003 -
Medicina 2019We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal...
We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal TSH. After ruling out a thyrotropic adenoma and in the absence of clinical symptoms of hyperthyroidism, the possibility of analytical interference in the immunoassays used to measure hormones was investigated. Interferences caused by heterophile antibodies, macro TSH, anti-thyroid antibodies, biotin, and to a lesser extent anti-streptavidin and anti-ruthenium antibodies have been described. The analysis of the patient was carried out in a self-analyzer whose platform uses the streptavidin-biotin system that is very susceptible to several interferents. A proposed algorithm includes a series of simple tests to perform and interpret that allow detecting or ruling out the presence of interferents. Accordingly, a comparison was made with a different analytical platform (which does not use the streptavidin-biotin system), serial dilutions, precipitation with polyethylene glycol 6000 and treatment with microparticles coated with streptavidin. Results obtained confirmed the presence of anti-streptavidin antibodies in the patient's serum. In the case of disagreements between clinical manifestations and laboratory results, the possibility of methodological interferences should be investigated in order to avoid the potential iatrogenic risk involved in an erroneous biochemical interpretation.
Topics: Adenoma; Adult; Antibodies, Anti-Idiotypic; Diagnostic Errors; Female; Humans; Hyperthyroidism; Pituitary Neoplasms; Pregnancy; Streptavidin; Thyrotropin; Thyroxine; Triiodothyronine
PubMed: 31671397
DOI: No ID Found -
Xenotransplantation Jan 2020Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal...
BACKGROUND
Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation.
METHODS
Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro.
RESULTS
Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect.
CONCLUSION
We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.
Topics: Animals; Antibodies, Heterophile; CRISPR-Cas Systems; Cells, Cultured; Complement System Proteins; Galactosyltransferases; Graft Rejection; HLA Antigens; Heterografts; Histocompatibility Antigens Class I; Humans; Kidney Transplantation; Mixed Function Oxygenases; N-Acetylgalactosaminyltransferases; Swine; Transplantation, Heterologous
PubMed: 31591751
DOI: 10.1111/xen.12560 -
The American Journal of Case Reports Aug 2019BACKGROUND Few cases of falsely undetectable PSA due to the presence of an inhibitory serum factor have been reported in the world literature. We present a case of... (Review)
Review
BACKGROUND Few cases of falsely undetectable PSA due to the presence of an inhibitory serum factor have been reported in the world literature. We present a case of falsely low-to-undetectable PSA with data from a serum dilution series, the current literature on biochemical assay interference, and the implications for prostate cancer salvage treatment. CASE REPORT A 63-year-old man was treated with prostatectomy for high-risk prostate cancer and was found to have a rising PSA after approximately 3 years following surgery. He subsequently transferred his care to a different health system and was found to have an undetectable PSA. He was eventually found to have an elevated PSA once again after the particular assay at this institution was changed. He thus received salvage prostate radiotherapy and androgen deprivation therapy. CONCLUSIONS While falsely low PSA results cannot be explained by the presence of serum heterophile antibodies, competitive antibody interference against the immunoassay reagents or anti-PSA antibodies are possible explanations for the results of the dilution experiments performed in this case study. We suggest that unexpected PSA testing results should raise concern for assay interference and warrant further clinical workup.
Topics: False Negative Reactions; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Neoplasms; Salvage Therapy
PubMed: 31444319
DOI: 10.12659/AJCR.917137 -
Clinical Diabetes and Endocrinology 2019Pitfalls in hormonal assays are commonly seen in clinical practice and may lead to erroneous clinical impressions and treatments. In this article, we address common... (Review)
Review
Pitfalls in hormonal assays are commonly seen in clinical practice and may lead to erroneous clinical impressions and treatments. In this article, we address common laboratory pitfalls encountered during evaluation of patients with real or presumed endocrine disorders including high dose hook effect and falsely normal prolactin in cases of macroprolactinomas, macroprolactinemia and falsely elevated prolactin, macrothyrotropinemia and falsely elevated TSH, heterophile antibodies leading to false elevation of hormonal concentration, biotin interference with different hormonal assays, cross-reactivity of steroid hormones immunoassays, and others. We describe the mechanisms of such laboratory pitfalls, review clinical scenarios in which they might occur, and discuss the ways to resolve such conundrums. The aim of this article is to present a learning material for the endocrine trainees and practitioners.
PubMed: 31367466
DOI: 10.1186/s40842-019-0086-7 -
Scientific Reports Jul 2019To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from...
To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid μ-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP μ-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells (P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid μ-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo.
Topics: Animals; Antigens, Heterophile; Cells, Cultured; Diabetes Mellitus, Experimental; Humans; Insulin; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Nude; Microscopy, Electron; Recombinant Proteins; Secretory Vesicles
PubMed: 31341242
DOI: 10.1038/s41598-019-47257-7 -
Immunogenetics Jul 2019Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies...
Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.
Topics: Animals; Antibodies, Monoclonal; Antigens, Heterophile; Arginine; Epitopes; HEK293 Cells; Histocompatibility Antigens Class I; Humans; Mutagenesis, Site-Directed; Point Mutation; Swine
PubMed: 31270568
DOI: 10.1007/s00251-019-01123-y -
Journal of Virology Sep 2019Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others...
Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others previously showed that pseudotyping of oncolytic rhabdoviruses such as maraba virus and vesicular stomatitis virus (VSV) with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) results in only a weak induction of neutralizing antibodies. Moreover, LCMV-GP-pseudotyped VSV (VSV-GP) was significantly more stable in normal human serum (NHS) than VSV. Here, we demonstrate that depending on the cell line used for virus production, VSV-GP showed different complement sensitivities in nonimmune NHS. The NHS-mediated titer reduction of VSV-GP was dependent on activation of the classical complement pathway, mainly by natural IgM antibodies against xenoantigens such as galactose-α-(1,3)-galactose (α-Gal) or -glycolylneuraminic acid (Neu5Gc) expressed on nonhuman production cell lines. VSV-GP produced on human cell lines was stable in NHS. However, VSV-GP generated in transduced human cells expressing α-Gal became sensitive to NHS. Furthermore, GP-specific antibodies induced complement-mediated neutralization of VSV-GP independently of the producer cell line, suggesting that complement regulatory proteins potentially acquired by the virus during the budding process are not sufficient to rescue the virus from antibody-dependent complement-mediated lysis. Thus, our study points to the importance of a careful selection of cell lines for viral vector production for clinical use. Systemic application aims to deliver oncolytic viruses to tumors as well as to metastatic lesions. However, we found that xenoantigens incorporated onto the viral surface from nonhuman production cell lines are recognized by natural antibodies in human serum and that the virus is thereby inactivated by complement lysis. Hence, to maximize the effective dose, careful selection of cell lines for virus production is crucial.
Topics: A549 Cells; Animals; Antibodies, Neutralizing; Antigens, Heterophile; Cell Line; Chlorocebus aethiops; Complement System Proteins; Cricetinae; Genetic Vectors; Glycoproteins; Humans; Lymphocytic choriomeningitis virus; Mice; Oncolytic Virotherapy; Oncolytic Viruses; Vesicular Stomatitis; Vesicular stomatitis Indiana virus; Vesiculovirus
PubMed: 31243134
DOI: 10.1128/JVI.00567-19 -
Indian Journal of Clinical Biochemistry... Apr 2019The practice of medicine depends on the accuracy of biochemical assays. The high prevalence of incidental masses on imaging necessitates a correct biochemical diagnosis...
The practice of medicine depends on the accuracy of biochemical assays. The high prevalence of incidental masses on imaging necessitates a correct biochemical diagnosis before proceeding to radiological studies. Hormonal assays, tumour markers, and markers of cardiac injury are particularly susceptible to heterophile antibody interference which may lead to inaccurate and misleading results, inappropriate investigation and/or treatment, patient concern and potential harm. A case of heterophile antibody interference in the measurement of ACTH in a patient with Cushing's syndrome resulting in unnecessary invasive investigation is presented. Close collaboration and communication between laboratory and clinical staff is essential where laboratory results and the clinical picture are not congruent.
PubMed: 31093000
DOI: 10.1007/s12291-018-0770-x -
Xenotransplantation Jul 2019The role of complement in xenotransplantation is well-known and is a topic that has been reviewed previously. However, our understanding of the immense complexity of its... (Review)
Review
The role of complement in xenotransplantation is well-known and is a topic that has been reviewed previously. However, our understanding of the immense complexity of its interaction with other constituents of the innate immune response and of the coagulation, adaptive immune, and inflammatory responses to a xenograft is steadily increasing. In addition, the complement system plays a function in metabolism and homeostasis. New reviews at intervals are therefore clearly warranted. The pathways of complement activation, the function of the complement system, and the interaction between complement and coagulation, inflammation, and the adaptive immune system in relation to xenotransplantation are reviewed. Through several different mechanisms, complement activation is a major factor in contributing to xenograft failure. In the organ-source pig, the detrimental influence of the complement system is seen during organ harvest and preservation, for example, in ischemia-reperfusion injury. In the recipient, the effect of complement can be seen through its interaction with the immune, coagulation, and inflammatory responses. Genetic-engineering and other therapeutic methods by which the xenograft can be protected from the effects of complement activation are discussed. The review provides an updated source of reference to this increasingly complex subject.
Topics: Adaptive Immunity; Animals; Animals, Genetically Modified; Antibodies, Heterophile; Blood Coagulation; Blood Coagulation Factors; Blood Platelets; Complement Activation; Complement System Proteins; Endothelium, Vascular; Graft Rejection; Heterografts; Humans; Immunosuppressive Agents; Inflammasomes; Inflammation; Primates; Receptors, Complement; Swine; Tissue and Organ Harvesting; Transplantation Immunology; Transplantation, Heterologous
PubMed: 31033064
DOI: 10.1111/xen.12517