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1.
Front Immunol ; 10: 2434, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708916

RESUMO

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

2.
Am J Med Genet A ; 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729179

RESUMO

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

3.
Cytometry A ; 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31577065

RESUMO

Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.

4.
J Immunol Methods ; : 112680, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31655051

RESUMO

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.

5.
Front Immunol ; 10: 2194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620126

RESUMO

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in SAMD9 (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel SAMD9 mutation was confirmed experimentally. Expression of mutant SAMD9 caused a significant decrease in proliferation and increase in cell death of the transfected cells. Conclusion: We describe a novel SAMD9 mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with SAMD9 mutations and multisystem involvement.

6.
Cytometry A ; 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31593368

RESUMO

There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry.

7.
Front Immunol ; 10: 1271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263462

RESUMO

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

8.
J Immunol Methods ; : 112618, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181212

RESUMO

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.

9.
J Neurooncol ; 143(1): 15-25, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30827009

RESUMO

PURPOSE: The aim of this study was to test the possibility of using specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) in flow and mass cytometry investigations of pediatric brain tumors. METHODS: CUSA specimens obtained from 19 pediatric patients with brain tumors were investigated. Flow and mass cytometry methods were applied to analyze the composition of material collected using the CUSA. Cell suspensions were prepared from CUSA aspirates. Then sample viability was assessed by conventional flow cytometry and subsequently stained with a panel of 31 metal-labeled antibodies. RESULTS: Viability assessment was performed using conventional flow cytometry. Viability of cells in the acquired samples was below 50% in 16 of 19 cases. A mass cytometry investigation and subsequent analysis enabled us to discriminate brain tumor cells from contaminating leukocytes, whose proportions varied across the specimens. The addition of the viability marker cisplatin directly into the mass cytometry panel gave the means to selecting viable cells only for subsequent analyses. The proportion of non-viable cells was higher among tumor cells compared leukocytes. CONCLUSIONS: When the analysis of the tumor cell immunophenotype is performed with markers for determining viability, the expression of the investigated markers can be evaluated. Suitable markers can be selected by high-throughput methods, such as mass cytometry, and those that are diagnostically relevant can be investigated using flow cytometry, which is more flexible in terms of time.


Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Sobrevivência Celular , Cisplatino/metabolismo , Citometria de Fluxo , Humanos , Leucócitos/metabolismo , Leucócitos/patologia , Procedimentos Neurocirúrgicos/instrumentação , Análise de Célula Única , Terapia por Ultrassom/instrumentação
10.
J Allergy Clin Immunol ; 144(3): 809-824, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30826363

RESUMO

BACKGROUND: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. OBJECTIVE: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. METHODS: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. RESULTS: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA+ PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA+ PCs with mild versus severe smIgA+ MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA+ and smIgG+ MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27+ MBCs with almost normal IgG3+ MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG2+ MBCs; and (6) with IgA1+ MBCs. CONCLUSION: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles.

11.
Front Immunol ; 10: 246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30886612

RESUMO

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.

12.
J Immunol Res ; 2019: 6705949, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30886872

RESUMO

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.


Assuntos
Imunofenotipagem/métodos , Linfócitos/fisiologia , Células Mieloides/fisiologia , Neoplasias/imunologia , Anticorpos Monoclonais/metabolismo , Separação Celular , Colagenases/metabolismo , Código de Barras de DNA Taxonômico , Desoxirribonuclease I/metabolismo , Citometria de Fluxo , Humanos , Antígenos Comuns de Leucócito/imunologia , Neoplasias/diagnóstico , Paládio/metabolismo , Análise de Célula Única
13.
Nat Methods ; 15(11): 909-912, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30377371

RESUMO

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

15.
PLoS One ; 13(10): e0205812, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335807

RESUMO

While stress is a widely utilized concept, no direct methods facilitating its measurement are currently available. In our previous work we proposed stress entropic load (SEL) as a potential new marker of stress response in the human body. However, at that time no method for SEL measurement existed. In this pilot study we devised and then tested methodology for SEL measurement. Healthy male participants were monitored by indirect calorimetry and thermography while resting and subsequently while under prolonged mental effort. The acquired data was then used to calculate the temporal development of SEL for each participant. Our results show that SEL production increased significantly in participants subjected to prolonged mental effort. Furthermore, we observed that the calculated development of SEL over time may be used to accurately determine the time point at which participants started performing stressful tasks.

16.
J Immunol Methods ; 2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30237053

RESUMO

EuroFlow Quality Assessment was designed to provide a feedback on the quality of the standardization effort in executing the EuroFlow protocols for sample preparation and instrument setup. It was first beta-tested by the members of the EuroFlow consortium internally (2010-2013) and opened to the external participants from 2015 onwards. The goal of participation in the EuroFlow QA is to evaluate whether the technical quality of the data generated by the laboratory is comparable to the data of the EuroFlow members and thus if a non-EuroFlow member participant can use the EuroFlow reference sample database for his own patient evaluation. Also it assesses whether data are sufficiently standardized for automated population gating and alarm notification. By spring 2018, a total 87 laboratories from 32 countries on five continents have registered for the EuroFlow QA program. We evaluated 163 results of 2015-2016 QA rounds, where we noted clear improvement in the score of first-time participants (median score of 91% correct) when they participated second time or later (median score of 94% correct, p = 0,017), which was comparable to EuroFlow member scores (median score of 97% correct). Among frequent mistakes, we found non-adherence to the EuroFlow protocols (improper reagent used), improper gating and some compensation issues. In summary, we show that EuroFlow QA has a positive impact on improvement of standardized data quality of non-member laboratories adhering to the EuroFlow standard operating procedures and reagent panels.

17.
Am J Med Genet A ; 2018 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-30194816

RESUMO

Danon disease (DD) is an X-linked disorder caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene (Xq24). DD is characterized by cognitive deficit, myopathy, and cardiomyopathy in male patients. The phenotype is variable and mitigated in females. The timely identification of de-novo LAMP2 mutated family members, many of whom are heterozygous females, remains critical for their treatment and family counseling. DD laboratory testing builds on minimally invasive quantification of the LAMP2 protein in white blood cells and characterization of the specific mutation. This integrative approach is particularly helpful when assessing suspect female heterozygotes. LAMP2 exon-copy number variations (eCNVs) were so far reported only in X-hemizygous male DD probands. In heterozygous female DD probands, the wild-type allele may hamper the identification of an eCNV even if it results in the complete abolition of LAMP2 transcription and/or translation. To document the likely underappreciated rate of occurrence and point out numerous potential pitfalls of detection of the LAMP2 eCNVs, we present the first two DD heterozygote female probands who harbor novel multi-exon LAMP2 deletions. Critical for counseling and recurrence prediction, we also highlight the need to search for somatic-germinal mosaicism in DD families.

19.
J Clin Immunol ; 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29934865

RESUMO

PURPOSE: Signal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We aim to report the effect of oral ruxolitinib, the Janus kinase (JAK) family tyrosine kinase inhibitor, on clinical and immune status of a 12-year-old boy with severe CMC due to a novel STAT1 GOF mutation. METHODS: Clinical features and laboratory data were analyzed, particularly lymphocyte subsets, ex vivo IFNγ- and IFNα-induced STAT1, 3, 5 phosphorylation dynamics during the course of JAK1/2 inhibition therapy, and Th17-related, STAT1- and STAT3-inducible gene expression before and during the treatment. Sanger sequencing was used to detect the STAT1 mutation. Literature review of ruxolitinib in treatment of CMC is appended. RESULTS: A novel STAT1 GOF mutation (c.617T > C; p.L206P), detected in a child with recalcitrant CMC, was shown to be reversible in vitro with ruxolitinib. Major clinical improvement was achieved after 8 weeks of ruxolitinib treatment, while sustained suppression of IFNγ- and IFNα-induced phosphorylation of STAT1, STAT3, and STAT5, as well as increased STAT3-inducible and Th17-related gene expression, was demonstrated ex vivo. Clinical relapse and spike of all monitored phosphorylated STAT activity was registered shortly after unplanned withdrawal, decreasing again after ruxolitinib reintroduction. No increase of peripheral CD4+ IL17+ T cells was detected after 4 months of therapy. No adverse effects were noted. CONCLUSION: JAK1/2 inhibition with ruxolitinib represents a viable option for treatment of refractory CMC, if HSCT is not considered. However, long-term administration is necessary, as the effect is not sustained after treatment discontinuation.

20.
Front Oncol ; 8: 151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29951370

RESUMO

The aim of this work was to offer an ecological alternative to conventional observational studies and identify factors potentially associated with cancer incidence in Europe. The incidence of 24 types of cancer in 39 European countries (2012) was compared with a long-term mean supply of 68 food items from the FAOSTAT database (1993-2011) and some other variables such as smoking, body mass index, raised cholesterol, and socioeconomic indicators. In addition to simple Pearson linear correlations, the data were analyzed via factor analyses and penalized regression methods. This comparison identified two main groups of cancers that are characteristically associated with the same variables. The first group consists of cancers of the prostate, breast, white blood cells, and melanoma. Their incidence increases with rising gross domestic product (GDP) per capita, a prevalence of raised cholesterol and a high intake of animal products. The second group includes primarily cancers of the digestive tract and is most consistently correlated with alcoholic beverages, lard, and eggs. In addition, we found specific correlations between certain variables and some other types of cancer (smoking-lung and larynx cancer; low GDP per capita and high carbohydrate consumption-stomach and cervical cancer; tea drinking-esophageal cancer; maize consumption and wine drinking-liver cancer). The documented findings often remarkably agree with the current scientific consensus, and when combined with evidence based on different methodologies, they can further extend our knowledge of the etiology of cancer. In addition, our study also identifies several foods with possible preventive effects and indicates that various dairy products may markedly differ in their relationship to cancer incidence. All these data can potentially be of fundamental importance for clinical practice and the survival of cancer patients.

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