Variation in the Measurement of Anti-Müllerian Hormone – What Are the Laboratory Issues?

Systematic Bias ◽

Dose Selection ◽

Anti-Müllerian Hormone (AMH) is a 140 kDa homodimeric glycoprotein consisting of two identical subunits linked by disulphide bonds and is synthesised by the testes and ovaries. Its clinical applications are prediction of ovarian response and gonadotropin dose selection upon in vitro fertilization. In males, AMH is used to investigate sexual developmental disorders and gonadal function. AMH is commonly assayed by enzyme-linked immunosorbent assay or automated immunoassay formats that show variation between methods. This review applies fundamental chemical pathology concepts to explain the observed analytical variation of AMH measurement. We examine the lack of standardisation between AMH assays, the impact of antibody design on variable measurements, consider the analytical detection of AMH isoforms, review analytical interference in AMH measurement, and briefly assess systematic bias between AMH assays. The improved attempt at standardising AMH measurement by the recent approval of a WHO Reference Reagent offers promise for harmonising immunoassay results and establishing consensus medical cut-off points for AMH in disease. Standardisation, however, will need to redress the issue of poor commutability of standard reference material and further assign a standard reference procedure to quantify AMH standard reference material. The improvement of the analytical phase of AMH testing will support harmonised method development and patient care.

The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-84117-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroaki Kanzaki ◽

Tetsuhiro Chiba ◽

Junjie Ao ◽

Keisuke Koroki ◽

Kengo Kanayama ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Erk Signaling ◽

Antitumor Effects ◽

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

Microfiltration results in the loss of analytes and affects the in vitro genotoxicity of a complex mixture of Alternaria toxins

Mycotoxin Research ◽

10.1007/s12550-020-00405-9 ◽

2020 ◽

Vol 36 (4) ◽

pp. 399-408

Author(s):

Georg Aichinger ◽

Natálie Živná ◽

Elisabeth Varga ◽

Francesco Crudo ◽

Benedikt Warth ◽

...

Keyword(s):

Membrane Filtration ◽

Oxidative Dna Damage ◽

Method Development ◽

Complex Mixture ◽

Monomethyl Ether ◽

Food Contaminants ◽

The Impact ◽

Ht 29 ◽

In Vitro Genotoxicity

Abstract Alternaria molds produce a variety of chemically diverse secondary metabolites with potentially adverse effects on human health. However, data on occurrence in food and human exposure is inconsistent for some of these mycotoxins. Membrane filtration is a frequent step in many sample preparation procedures for LC-MS-based methods analyzing food contaminants. Yet, little is known about the possibility of adsorptive phenomena that might result in analyte losses. Thus, we treated a complex extract of Alternaria toxins with several types of syringe filters and unraveled the impact on its chemical composition by LC-MS/MS. We observed significant, and in some cases complete, losses of compounds due to filtration. Particularly, two key Alternaria toxins, alternariol (AOH) and its monomethyl ether (AME), were heavily affected. As a comparison with published food surveys indicating a correlation of the type of filtration used with lower incidence reports in food, our results point at a possible underestimation of AME in past exposure assessment. Also, perylene quinones were greatly affected by filtration, underlining the importance to take this into consideration during analytical method development. Furthermore, we applied the comet assay in HT-29 cells to elucidate the impact of filtration on the genotoxicity of the extract. We observed strong coincidences with the loss of epoxide-carrying metabolites and also an intriguing induction of oxidative DNA damage by yet toxicologically uncharacterized Alternaria toxins. In conclusion, we highlight potential issues with sample filtration and call for a critical re-evaluation of previous food occurrence data in the light of the results at hand.

Biallelic mutations in the TOGARAM1 gene cause a novel primary ciliopathy

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-106833 ◽

2020 ◽

pp. jmedgenet-2020-106833

Author(s):

Valeria Morbidoni ◽

Emanuele Agolini ◽

Kevin C Slep ◽

Luca Pannone ◽

Daniela Zuccarello ◽

...

Keyword(s):

Sensory Neurons ◽

Developmental Disorders ◽

Cleft Lip ◽

Cleft Lip And Palate ◽

Missense Variant ◽

Causative Role ◽

Biallelic Mutations ◽

BackgroundDysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis.ObjectiveTo investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies.MethodsA trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in the TOGARAM1 gene. In silico, in vitro and in vivo (Caenorhabditis elegans) studies were carried out to explore the impact of mutations on protein structure and function, and relevant biological processes.ResultsTOGARAM1 encodes a member of the Crescerin1 family of proteins regulating microtubule dynamics. Its orthologue in C. elegans, che-12, is expressed in a subset of sensory neurons and localises in the dendritic cilium where it is required for chemosensation. Nematode lines harbouring the corresponding missense variant in TOGARAM1 were generated by CRISPR/Cas9 technology. Although chemotaxis ability on a NaCl gradient was not affected, che-12 point mutants displayed impaired lipophilic dye uptake, with shorter and altered cilia in sensory neurons. Finally, in vitro analysis of microtubule polymerisation in the presence of wild-type or mutant TOG2 domain revealed a faster polymerisation associated with the mutant protein, suggesting aberrant tubulin binding.ConclusionsOur data are in favour of a causative role of TOGARAM1 variants in the pathogenesis of this novel disorder, connecting this gene with primary ciliopathy.

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.5.586-592.2005 ◽

2005 ◽

Vol 12 (5) ◽

pp. 586-592 ◽

Cited By ~ 15

Author(s):

Peter C. Giardina ◽

Emma Longworth ◽

Renee E. Evans-Johnson ◽

Michaelene L. Bessette ◽

Hong Zhang ◽

...

Keyword(s):

Immunoglobulin G ◽

Capsular Polysaccharide ◽

Solid Phase ◽

Geometric Mean ◽

Serum Antibody ◽

Fluid Phase ◽

Antibody Titers ◽

ABSTRACT The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA+) and O-acetyl-negative (OA−) forms. This study investigates the impact of OA status (OA+ versus OA−) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA+ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA− antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA+ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA− antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA+ antigen than against the OA− antigen. These sera were also distinguished by the inability of fluid-phase OA− antigen to compete for antibody binding to OA+ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA+ and OA− target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA+ versus OA− W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA+ versus anti-OA− W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.

Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response

BioMed Research International ◽

10.1155/2016/4286528 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

N. Hojnik ◽

V. Vlaisavljević ◽

B. Kovačič

Keyword(s):

Implantation Rate ◽

Ovarian Response ◽

Time Lapse ◽

Poor Responder ◽

Poor Responders ◽

Poor Ovarian Response ◽

Developmental Potential ◽

The Impact ◽

Natural Cycles

Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations.

Borna Disease Virus Infection, a Human Mental-Health Risk

Clinical Microbiology Reviews ◽

10.1128/cmr.16.3.534-545.2003 ◽

2003 ◽

Vol 16 (3) ◽

pp. 534-545 ◽

Cited By ~ 76

Author(s):

Liv Bode ◽

Hans Ludwig

Keyword(s):

Disease Virus ◽

Diagnostic Systems ◽

Cross Sectional ◽

Borna Disease Virus ◽

Pros And Cons ◽

The Impact ◽

Borna Disease

SUMMARY This article focuses on human Borna disease virus (BDV) infections, most notably on the development of valid diagnostic systems, which have arisen as a major research issue in the past decade. The significance of a novel modular triple enzyme-linked immunosorbent assay that is capable of specifically measuring anti-BDV antibodies as well as major structural proteins N (p40) and P (p24) in the blood, either as free antigens in the plasma or as antibody-bound circulating immune complexes (CICs), is explained. The impact of CICs and plasma antigen, which indicate periods of antigenemia in the course of BDV infection, along with other infection markers that are still in use is discussed. The review further provides new insight into possible links of BDV to human diseases, summarizing cross-sectional and longitudinal data which correlate acute depression with the presence and amount of antigen and CICs. Moreover, BDV prevalence in healthy people is reevaluated, suggesting that this was previously underestimated. Antiviral efficacy of amantadine, in vivo and in vitro, is outlined as well, with emphasis on wild-type (human and equine) versus laboratory strains. Finally, the pros and cons of the association of BDV with human disease, as detailed in the literature, are critically discussed and related to our data and concepts. This article supports existing correlative evidence for a pathogenic role of BDV infection in particular human mental disorders, in analogy to what has been proven for a variety of animal species.

Characterization of the Plasmacytoid Dendritic Cell Response to Transmitted/Founder and Nontransmitted Variants of HIV-1

Journal of Virology ◽

10.1128/jvi.00157-18 ◽

2018 ◽

Vol 92 (19) ◽

Cited By ~ 4

Author(s):

Jordan Ari Schwartz ◽

Hongliang Zhang ◽

Zachary Ende ◽

Martin J. Deymier ◽

Terry Lee ◽

...

Keyword(s):

Dendritic Cell ◽

Plasmacytoid Dendritic Cell ◽

Female Genital Tract ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

The Impact ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1. IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.

Quercetin Improves the Endocrine Function of Rat Testicular Tissue Under in Vitro Conditions

Contemporary Agriculture ◽

10.2478/contagri-2021-0001 ◽

2021 ◽

Vol 70 (1-2) ◽

pp. 1-5

Author(s):

Filip Benko ◽

Patrik Hrnčiar ◽

Norbert Lukáč ◽

Róbert Kirchner ◽

Eva Tvrdá

Keyword(s):

Positive Impact ◽

Endocrine System ◽

Testicular Tissue ◽

Endocrine Function ◽

Beneficial Effects ◽

Wide Range ◽

Male Hormones ◽

Summary Compounds of natural origin are often used for their beneficial effects on the male endocrine system and the synthesis of steroid biomolecules in testicular tissue. One of such compounds is quercetin (QUE), which belongs to the flavonoid family and is found in a wide range of vegetables, fruits and plant products. The purpose of this study is to evaluate the impact of QUE on the endocrine function of rat testicular fragments under in vitro conditions. Testicular fragments from adult Wistar rats (n=9), cultured in the D-MEM medium with different concentrations of QUE (namely 1, 10 and 100 µmol/L) for 24 h at 37°C (5% CO2), were used in the experiment conducted. Following culture, the medium was separated and the levels of cholesterol (CHOL) and male hormones were measured. CHOL values were quantified spectrophotometrically, whereas the concentrations of androstenedione (ANDRO), dehydropeiandrosterone (DHEA) and testosterone (TEST) were quantified using the enzyme-linked immunosorbent assay (ELISA) commercial kit. The results obtained indicate that 10 µmol/L QUE significantly increased (P<0.001; P<0.05) the concentrations of all the steroid biomolecules considered (CHOL, ANDRO, DHEA and TEST) when compared to the control samples. Accordingly, our findings confirm the positive impact of QUE on the endocrine function and steroidogenesis of rat testicular tissue under in vitro conditions.

Effects of Saliva From Periodontally Healthy and Diseased Subjects on Barrier Function and the Inflammatory Response in in vitro Models of the Oral Epithelium

Frontiers in Oral Health ◽

10.3389/froh.2021.815728 ◽

2022 ◽

Vol 2 ◽

Author(s):

Antoine Roy ◽

Amel Ben Lagha ◽

Reginaldo Gonçalves ◽

Daniel Grenier

Keyword(s):

Inflammatory Response ◽

Matrix Metalloproteinase ◽

Barrier Function ◽

Chronic Inflammatory Disease ◽

In Vitro Models ◽

Oral Epithelium ◽

Chemokine Ligand ◽

Background: Periodontitis is a multifactorial, bacteria-mediated chronic inflammatory disease that results in the progressive destruction of the tooth-supporting tissues. It is well-known that saliva from subjects suffering from this disease generally contains higher levels of pro-inflammatory mediators, matrix metalloproteinases (MMP), and bacteria-derived toxic products. The aim of this study was to investigate and compare the effects of saliva from periodontally healthy and diseased subjects on the barrier function and inflammatory response in in vitro models of the oral epithelium.Methods: Unstimulated saliva samples from two groups of subjects, one with a healthy periodontium (n = 12) and one with severe generalized periodontitis (n = 11), were filter-sterilized. All the saliva samples were analyzed using an immunological multiplex assay to determine the levels of various cytokines and MMPs relevant to periodontitis. The impact of saliva on epithelial barrier integrity was assessed by monitoring transepithelial electrical resistance (TER) in an oral epithelium model using the B11 keratinocyte cell line. GMSM-K oral epithelial cells were treated with saliva from both groups to determine their ability to induce the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8), as determined by an enzyme-linked immunosorbent assay (ELISA).Results: Saliva from the periodontitis subjects contained significantly higher concentrations of matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), IL-8, and C-X-C motif chemokine ligand 1 (CXCL1) compared to saliva from the healthy subjects. Saliva from the healthy and periodontitis subjects affected cytokine secretion and TER in a similar manner. More specifically, saliva from both groups increased TER and induced IL-6 and IL-8 secretion in the in vitro oral epithelium models used.Conclusion: Independently of the presence or absence of periodontitis, saliva can increase the relative TER and the secretion of IL-6 and IL-8 in in vitro models of the oral epithelium.

Introduction of a Nist Instrument Sensitivity Standard Reference Material for X-Ray Powder Diffraction

Advances in X-ray Analysis ◽

10.1154/s0376030800009009 ◽

1991 ◽

Vol 35 (A) ◽

pp. 341-352 ◽

Cited By ~ 3

Author(s):

James P. Cline ◽

Susannah B. Schiller ◽

Ron Jenkins

Keyword(s):

Reference Material ◽

Powder Diffraction ◽

Standard Reference Material ◽

Systematic Bias ◽

Diffraction Intensity ◽

Preparation Methods ◽

X Ray ◽

Test Instrument ◽

Sensitivity Standard ◽

Instrument Sensitivity

AbstractImprovements in sample preparation methods have resulted in increased accuracy in x-ray powder diffraction intensity measurements. This improvement has focused scrutiny on the instrument as a potential source of error. NIST Standard Reference Material, SRM, 1976 consists of a sintered α alumina, corundum, plate certified with respect to 12 relative intensity values from 25 to 145 degrees 2θ. Its function is to allow for standardization of powder diffraction intensity as a function of 2θ angle (instrument sensitivity). An increase in the accuracy of interlaboratory comparisons of diffraction intensity and related determinations will result. Utilization of the SRM requires the user to collect intensity data from the test instrument in a manner which conforms to that used in the certification. Graphical interpretation of the ratio of these data to those on the certificate will allow for appropriate judgment as to the condition of the test instrument. Discontinuities in the data indicate a malfunctioning or misaligned instrument. Systematic bias, introduced by design characteristics of the test instrument, manifests itself in terms of a pattern in the data other than a horizontal straight line with value of one. This bias may be removed with the calculation and application of a correction curve.