scholarly journals Blood functional assay for rapid clinical interpretation of germline TP53 variants

2020 ◽  
pp. jmedgenet-2020-107059 ◽  
Author(s):  
Sabine Raad ◽  
Marion Rolain ◽  
Sophie Coutant ◽  
Céline Derambure ◽  
Raphael Lanos ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Transcriptional Response ◽  
Functional Assay ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Patients With Cancer ◽  
Pathogenic Variants ◽  
Functional Variants ◽  
Multiplex Ligation Probe Amplification ◽  
P53 Mrna

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription–multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis.ResultsIn 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5–22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1–7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

scholarly journals An Autoimmune Disease-Associated Risk Variant in theTNFAIP3Gene Plays a Protective Role in Brucellosis That Is Mediated by the NF-κB Signaling Pathway

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Lixin Lou ◽  
Wanguo Bao ◽  
Xianjun Liu ◽  
Hongxiao Song ◽  
Yang Wang ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Mononuclear Cells ◽  
Protective Role ◽  
Negative Regulator ◽  
Functional Study ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
Tnfaip3 Gene ◽  
Functional Variants

ABSTRACTNaturally occurring functional variants (rs148314165 and rs200820567, collectively referred to as TT>A) reduce the expression of the tumor necrosis factor alpha-induced protein 3 (TNFAIP3) gene, a negative regulator of NF-κB signaling, and predispose individuals to autoimmune disease. In this analysis, we conducted a genetic association study of the TT>A variants in 1,209 controls and 150 patients with brucellosis, an infectious disease, and further assessed the role of the variants in brucellosis. Our data demonstrated that the TT>A variants were correlated with cases of brucellosis (P= 0.002; odds ratio [OR] = 0.34) and with individuals who had a positive serum agglutination test (SAT) result (titer of >1/160) (P= 4.2 × 10−6; OR = 0.23). A functional study demonstrated that brucellosis patients carrying the protective allele (A) showed significantly lower expression levels of the TNFAIP3 gene in their peripheral blood mononuclear cells and showed increased NF-κB signaling. Monocytes from individuals carrying the A allele that were stimulated withBrucella abortushad lower mRNA levels of TNFAIP3 and produced more interleukin-10 (IL-10), IL-6, and IL-1β than those from TT allele carriers. These data showed that autoimmune disease-associated risk variants, TT>A, of the TNFAIP3 locus play a protective role in the pathogenesis of brucellosis. Our findings suggest that a disruption of the normal function of the TNFAIP3 gene might serve as a therapeutic target for the treatment of brucellosis.

scholarly journals A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rachel Tanner ◽  
Andrew D. White ◽  
Charelle Boot ◽  
Claudia C. Sombroek ◽  
Matthew K. O’Shea ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mononuclear Cells ◽  
Functional Assay ◽  
Intermediate Precision ◽  
Peripheral Blood Mononuclear ◽  
Growth Inhibition Assay ◽  
Early Evaluation ◽  
Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

scholarly journals Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia

2019 ◽  
Vol 97 (6) ◽  
pp. 562-569 ◽  
Author(s):  
Anthony Cannavicci ◽  
Qiuwang Zhang ◽  
Si-Cheng Dai ◽  
Marie E. Faughnan ◽  
Michael J.B. Kutryk
Keyword(s):  
Growth Factor ◽  
Peripheral Blood ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Manifestations ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Peripheral Blood Mononuclear ◽  
Micro Rnas

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription–quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

Reduced Expression of Annexin A2 is Associated with Impaired Cell Surface Fibrinolysis and Venous Thromboembolism

Blood ◽  
2021 ◽  
Author(s):  
Hannah Fassel ◽  
Huigen Chen ◽  
Mary Ruisi ◽  
Neha Kumar ◽  
Maria T DeSancho ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thromboembolism ◽  
Cell Surface ◽  
Mononuclear Cells ◽  
Annexin A2 ◽  
Clot Lysis ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Intravascular Thrombosis

Reduced plasma fibrinolysis has been identified as a potential risk factor for venous thromboembolism (VTE), but the role of cell surface fibrinolysis in VTE is unknown. The annexin A2/S100A10 complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), augmenting plasmin generation by 60-fold on the endothelial cell surface. Several studies in both mice and humans support the concept that A2 regulates fibrin homeostasis and intravascular thrombosis in vivo. Here, we examined A2 protein expression and function in 115 adult subjects with venous thromboembolism (VTE) and 87 healthy controls. Using peripheral blood mononuclear cells (PBMCs) as a surrogate for endothelial cells, we found a 41% mean decrease in cell surface tPA-dependent fibrinolytic activity in subjects who had a positive personal and family history of VTE, but tested negative for known inherited thrombophilias. A2 protein was reduced on average by 70%, and mRNA levels by 30%, but neither decrease correlated with anticoagulant therapy. [Sentence omitted] Neither cell A2 protein nor cell surface plasmin generation correlated with plasma-based clot lysis times, suggesting that the plasma and cell surface fibrinolytic systems operate independently of one another. These data suggest that reduced expression of annexin A2 protein is associated with cell surface hypofibrinolysis and may represent a novel risk factor for inherited thrombophilia.

scholarly journals Next-Generation Sequencing Reveals a Controlled Immune Response to Zaire Ebola Virus Challenge in Cynomolgus Macaques Immunized with Vesicular Stomatitis Virus Expressing Zaire Ebola Virus Glycoprotein (VSVΔG/EBOVgp)

2015 ◽  
Vol 22 (3) ◽  
pp. 354-356 ◽  
Author(s):  
Fredrik Barrenas ◽  
Richard R. Green ◽  
Matthew J. Thomas ◽  
G. Lynn Law ◽  
Sean C. Proll ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Host Response ◽  
Ebola Virus ◽  
Mononuclear Cells ◽  
Transcriptional Response ◽  
Peripheral Blood Mononuclear ◽  
Virus Challenge ◽  
Cynomolgus Macaques ◽  
Vesicular Stomatitis ◽  
Generation Sequencing

ABSTRACTVesicular stomatitis virus expressing Zaire Ebola virus (EBOV) glycoprotein (VSVΔG/EBOVgp) could be used as a vaccine to meet the 2014 Ebola virus outbreak. To characterize the host response to this vaccine, we used mRNA sequencing to analyze peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques after VSVΔG/EBOVgp immunization and subsequent EBOV challenge. We found a controlled transcriptional response that transitioned to immune regulation as the EBOV was cleared. This observation supports the safety of the vaccine.

scholarly journals Gene Expression of Adhesion Molecules in Endothelial Cells from Patients with Peripheral Arterial Disease Is Reduced after Surgical Revascularization and Pharmacological Treatment

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Franca Marino ◽  
Luigina Guasti ◽  
Matteo Tozzi ◽  
Laura Schembri ◽  
Luana Castiglioni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Adhesion Molecules ◽  
Mononuclear Cells ◽  
Venous Blood ◽  
Statin Treatment ◽  
Early Event ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear

Atherosclerosis is an inflammatory disease characterized by immunological activity, in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage. We investigated gene expression of the adhesion molecules (AMs) ICAM-1, VCAM-1, andβ1-integrin in endothelial cells (ECs) isolated from venous blood (circulating EC, cEC) and purified from femoral plaques (pEC) obtained from 9 patients with peripheral artery disease (PAD) submitted to femoral artery thrombendarterectomy (FEA). In addition, in peripheral blood mononuclear cells (PBMCs) of the same subjects, we investigated gene expression of IFN-γ, IL-4, TGF-β, and IL-10. Patients were longitudinally evaluated 1 month before surgery, when statin treatment was established, at the time of surgery, and after 2 and 5 months. All AM mRNA levels, measured by means of real-time PCR, in cEC diminished during the study, up to 41–50% of initial levels at followup. AM mRNA expression was significantly higher in pEC than in cEC. During the study, in PBMCs, TGF-βand IL-10 mRNA levels remained unchanged while IFN-γand IL-4 levels increased; however, the ratio IFN-γ/IL-4 showed no significant modification. In PAD patients, FEA and statin treatment induce a profound reduction of AM expression in cEC and affect cytokine mRNA expression in PBMCs.

Data on cytokine mRNA expression in CSF and peripheral blood mononuclear cells from MS patients as detected by PCR

1998 ◽  
Vol 4 (3) ◽  
pp. 143-146 ◽  
Author(s):  
Philippe Monteyne ◽  
Christian JM Sindic
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Inflammatory Diseases ◽  
Clinical Activity ◽  
Immune Reactivity ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Csf Cells ◽  
Blood Mononuclear Cells

Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the mRNA coding for different cytokines in peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) cells from 18 multiple sclerosis (MS) patients as compared with 21 other neurological patients. mRNA levels were quantitated by radioactive hybridization of the PCR products. Expression of tumor necrosis factor (TNF)-a, interferon (IFN)-g, and interleukin (IL)-10 mRNA was elevated in CSF cells of MS patients. In many MS patients, both proinflammatory and immunoregulatory cytokine messages were detected in the CSF compartment. Such immune reactivity of CSF cells, as opposed to PBMC, was not associated with higher clinical activity of the disease. Expression of the B7.1 accessory molecule mRNA was similarly investigated. In the CSF, it was detected only in some clinically active MS cases and in other inflammatory diseases.

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis

2000 ◽  
Vol 6 (2) ◽  
pp. 61-65 ◽  
Author(s):  
Wen-Xin Huang ◽  
Ping Huang ◽  
Jan Hillert
Keyword(s):  
Multiple Sclerosis ◽  
Mononuclear Cells ◽  
Cd40 Ligand ◽  
Autoimmune Response ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Organ Specific

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT-PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P <0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.

Dasatinib (BMS-354825) pharmacokinetics correlate with pSRC pharmacodynamics in phase I studies of patients with cancer (CA180002, CA180003)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 3046-3046 ◽  
Author(s):  
F. R. Luo ◽  
F. R. Luo ◽  
Y. Barrett ◽  
P. Ji ◽  
P. Holly ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Solid Tumors ◽  
Kinase Activity ◽  
Mononuclear Cells ◽  
Maximal Inhibition ◽  
Peripheral Blood Mononuclear ◽  
First Order ◽  
Patients With Cancer ◽  
Clinical Responses ◽  
Dose Dependent

3046 Background: Dasatinib - an inhibitor of several oncologenic kinases including BCR-ABL and SRC - has demonstrated efficacy in CML and Ph+ ALL, and is being evaluated in patients with solid tumors. Studies CA180–002, and CA180–003 were multiple ascending dose studies of dasatinib in patients with refractory Ph+ leukemia, and solid tumors, respectively. We have analyzed the preliminary results of pharmacokinetics (PK) and pharmacodynamic (PD) biomarker phospho-SRC (pSRC) combined across the CA180002 and CA180003 studies. Methods: Dasatinib was administered orally at doses of 15 - 180 mg QD or 25 - 160 mg BID. Dasatinib PK was determined by LC MS/MS. The level of phospho-SRC in peripheral blood mononuclear cells (PBMCs) was determined by an ELISA and was used as a surrogate biomarker of the level of kinase activity of the SRC family members. PK-PD modeling was performed using a two-compartment PK model with first-order absorption kinetics and an indirect inhibitory Emax model as the PD model. Results: On the BID regimen, pSRC inhibition was approximately dose-dependent across the dosing range. Inhibition also appeared to be directly correlated with dasatinib plasma concentration, with maximal inhibition achieved at ∼2.5 hr. Based on PK-PD modeling, the estimated EC50 (the plasma concentration required to achieve 50% inhibition of pSRC) is 14.4 ng/mL. Conclusions: SRC family kinase activity is substantially inhibited at the exposures of dasatinib achieved to date. The optimal regimens of dasatinib to achieve both target inhibition and solid tumors efficacy will be determined based on biomarkers, clinical responses, and toxicity profiles. [Table: see text]

scholarly journals Tonsils of the Soft Palate Do Not Mediate the Response of Pigs to Oral Vaccination with Heat-Inactivated Mycobacterium bovis

2014 ◽  
Vol 21 (8) ◽  
pp. 1128-1136 ◽  
Author(s):  
Beatriz Beltrán-Beck ◽  
Beatriz Romero ◽  
Mariana Boadella ◽  
Carmen Casal ◽  
Javier Bezos ◽  
...  
Keyword(s):  
Wild Boar ◽  
Mycobacterium Bovis ◽  
Mononuclear Cells ◽  
Mammalian Species ◽  
Serum Antibody ◽  
Control Group ◽  
Mrna Levels ◽  
Oral Vaccination ◽  
Peripheral Blood Mononuclear ◽  
Content Type

ABSTRACTMycobacterium boviscauses animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivatedM. bovisand challenge with a virulentM. bovisfield strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests forin vivoTB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivatedM. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. MassiveM. bovisgrowth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased afterM. bovischallenge. This information is relevant for pig production in regions that are endemic forM. bovisand for TB vaccine research.

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