Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Lineage Reconstruction of In Vitro Identified Antigen-Specific Autoreactive B Cells from Adaptive Immune Receptor Repertoires.

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis.

Care of patients with inborn errors of immunity in thirty J Project countries between 2004 and 2021.

Introduction: The J Project (JP) physician education and clinical research collaboration program was started in 2004 and includes by now 32 countries mostly in Eastern and Central Europe (ECE). Until the end of 2021, 344 inborn errors of immunity (IEI)-focused meetings were organized by the JP to raise awareness and facilitate the diagnosis and treatment of patients with IEI. Results: In this study, meeting profiles and major diagnostic and treatment parameters were studied. JP center leaders reported patients' data from 30 countries representing a total population of 506 567 565. Two countries reported patients from JP centers (Konya, Turkey and Cairo University, Egypt). Diagnostic criteria were based on the 2020 update of classification by the IUIS Expert Committee on IEI. The number of JP meetings increased from 6 per year in 2004 and 2005 to 44 and 63 in 2020 and 2021, respectively. The cumulative number of meetings per country varied from 1 to 59 in various countries reflecting partly but not entirely the population of the respective countries. Altogether, 24,879 patients were reported giving an average prevalence of 4.9. Most of the patients had predominantly antibody deficiency (46,32%) followed by patients with combined immunodeficiencies (14.3%). The percentages of patients with bone marrow failure and phenocopies of IEI were less than 1 each. The number of patients was remarkably higher that those reported to the ESID Registry in 13 countries. Immunoglobulin (IgG) substitution was provided to 7,572 patients (5,693 intravenously) and 1,480 patients received hematopoietic stem cell therapy (HSCT). Searching for basic diagnostic parameters revealed the availability of immunochemistry and flow cytometry in 27 and 28 countries, respectively, and targeted gene sequencing and new generation sequencing was available in 21 and 18 countries. The number of IEI centers and experts in the field were 260 and 690, respectively. We found high correlation between the number of IEI centers and patients treated with intravenous IgG (IVIG) (correlation coefficient, cc, 0,916) and with those who were treated with HSCT (cc, 0,905). Similar correlation was found when the number of experts was compared with those treated with HSCT. However, the number of patients treated with subcutaneous Ig (SCIG) only slightly correlated with the number of experts (cc, 0,489) and no correlation was found between the number of centers and patients on SCIG (cc, 0,174). Conclusions: 1) this is the first study describing major diagnostic and treatment parameters of IEI care in countries of the JP; 2) the data suggest that the JP had tremendous impact on the development of IEI care in ECE; 3) our data help to define major future targets of JP activity in various countries; 4) we suggest that the number of IEI centers and IEI experts closely correlate to the most important treatment parameters; 5) we propose that specialist education among medical professionals plays pivotal role in increasing levels of diagnostics and adequate care of this vulnerable and still highly neglected patient population; 6) this study also provides the basis for further analysis of more specific aspects of IEI care including genetic diagnostics, disease specific prevalence, newborn screening and professional collaboration in JP countries.

Increased insulin-like growth factor 1 production by polyploid adipose stem cells promotes growth of breast cancer cells.

BACKGROUND: Adipose-tissue stem cells (ASCs) are subject of intensive research since their successful use in regenerative therapy. The drawback of ASCs is that they may serve as stroma for cancer cells and assist tumor progression. It is disquieting that ASCs frequently undergo genetic and epigenetic changes during their in vitro propagation. In this study, we describe the polyploidization of murine ASCs and the accompanying phenotypical, gene expressional and functional changes under long term culturing. METHODS: ASCs were isolated from visceral fat of C57BL/6 J mice, and cultured in vitro for prolonged time. The phenotypical changes were followed by microscopy and flow cytometry. Gene expressional changes were determined by differential transcriptome analysis and changes in protein expression were shown by Western blotting. The tumor growth promoting effect of ASCs was examined by co-culturing them with 4 T1 murine breast cancer cells. RESULTS: After five passages, the proliferation of ASCs decreases and cells enter a senescence-like state, from which a proportion of cells escape by polyploidization. The resulting ASC line is susceptible to adipogenic, osteogenic and chondrogenic differentiation, and expresses the stem cell markers CD29 and Sca-1 on an upregulated level. Differential transcriptome analysis of ASCs with normal and polyploid karyotype shows altered expression of genes that are involved in regulation of cancer, cellular growth and proliferation. We verified the increased expression of Klf4 and loss of Nestin on protein level. We found that elevated production of insulin-like growth factor 1 by polyploid ASCs rendered them more potent in tumor growth promotion in vitro. CONCLUSIONS: Our model indicates how ASCs with altered genetic background may support tumor progression.

[Cardiomyopathy and ion channel diseases registry: the Szeged CardioGen Registry]. / Cardiomyopathiás és ioncsatorna-betegek regisztere: a Szegedi CardioGen Regiszter.

The Szeged cardiomyopathy and ion channel diseases registry aims to establish a representative disease-specific registry based on the recruitment of patients with different cardiomyopathies and ion channel diseases followed at the Cardiology Center, University of Szeged. The registry collects patient data on the main forms of primary cardiomyopathies (hypertrophic, dilated, restrictive, arrhythmogenic right ventricular, left ventricular non-compact, tako-tsubo cardiomyopathy) and ion channel diseases (long QT syndrome, short QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia). Patients with hypertrophic cardiomyopathy (388 patients) make up the largest group of patients in the registry. Patients with dilated cardiomyopathy (310 patients) and patients with the long QT syndrome (111 patients) form two other sizable groups. Analyzed data of the group of patients with hypertrophic cardiomyopathy indicate similar figures with regard to disease related mortality and morbidity and clinical parameters. Orv. Hetil., 2017, 158(3), 101-105.

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion.

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.

Engineered chromosomes in transgenics.

Horizontal gene transfer or simply transgenic technology has evolved much since 1980. Gene delivery strategies, systems, and equipments have become more and more precise and efficient. It has also been shown that even chromosomes can be used besides traditional plasmid and viral vectors for zygote or embryonic stem cell transformation. Artificial chromosomes and their loadable variants have brought their advantages over traditional genetic information carriers into the field of transgenesis. Engineered chromosomes are appealing vectors for gene transfer since they have large transgene carrying capacity, they are non-integrating, and stably expressing in eukaryotic cells. Embryonic stem cell lines can be established that carry engineered chromosomes and ultimately used in transgenic mouse chimera creation. The demonstrated protocol describes all the steps necessary for the successful production of transgenic mouse chimeras with engineered chromosome bearer embryonic stem cells.