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The rising debt-to-GDP ratios of the eurozone member states result not least from the coronavirus crisis. Without external support, especially with regard to Italy, but also for other Mediterranean states, access to the capital market could be seriously threatened in the medium run. The recovery fund ‘Next Generation EU’ likely directs the fundamental structures of the European Union (EU) towards a fiscal union with considerable transfer elements, while the Pandemic Emergency Purchase Programme (PEPP), which is declared as a monetary policy instrument, is even discussed as a violation of the prohibition of monetary financing. As an alternative, this contribution analyses a debt relief by the European System of Central Banks (ESCB), implemented via an EU debt agency. This construction would avoid a negative equity position of the central banks and also enable a legal integration into the EU system. The question remains: What would be the consequences of such a non-recurring step?

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)—in a strip format—that generate force under auxotonic stretch conditions. 10–15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

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Engineered heart tissue (EHT) from primary heart cells contains endothelial cells (ECs), but the extent to which ECs organize into vessel-like structures or even functional vessels remains unknown and is difficult to study by conventional methods. In this study, we generated fibrin-based mini-EHTs from a transgenic mouse line (Cdh5-CreERT2 × Rosa26-LacZ), in which ECs were specifically and inducibly labeled by applying tamoxifen (ECiLacZ). EHTs were generated from an unpurified cell mix of newborn mouse hearts and were cultured under standard serum-containing conditions. Cre expression in 15-day-old EHTs was induced by addition of o-hydroxytamoxifen to the culture medium for 48 h, and ECs were visualized by X-gal staining. ECiLacZEHTs showed a dense X-gal-positive vessel-like network with distinct tubular structures. Immunofluorescence revealed that ECs were mainly associated with cardiomyocytes within the EHT. ECiLacZEHT developed spontaneous and regular contractility with forces up to 0.1 mN. Coherent contractility and the presence of an extensive vessel-like network were both dependent on the presence of animal sera in the culture medium. Contractile ECiLacZEHTs successfully served as grafts in implantation studies onto the hearts of immunodeficient mice. Four weeks after implantation, EHTs showed X-gal-positive lumen-forming vessel structures connected to the host myocardium circulation as they contained erythrocytes on a regular basis. Taken together, genetic labeling of ECs revealed the extensive formation of vessel-like structures in EHTs in vitro. The ECiLacZEHT model could help simultaneously study biological effects of compounds on cardiomyocyte function and tissue vascularization.

In engineered heart tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitroperfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm3). Thin dry alginate fibers (17×0.04×0.04 mm) were embedded into the cell–fibrin–thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the tissue. Perfusion rapidly increased it to 10%–12% in the immediate vicinity of the microchannel. Continuous perfusion (20 μL/h, for 3 weeks) of the tubular lumina (100–500 μm) via hollow posts of the silicone rack increased mean dystrophin-positive cardiomyocyte density (36%±6% vs. 10%±3% of total cell number) and cross sectional area (73±2 vs. 48±1 μm2) in the central part of the tissue compared to nonperfused EHTs. The channels were populated by endothelial cells present in the reconstitution cell mix. In conclusion, we developed a novel approach to generate small tubular structures suitable for perfusion of spontaneously contracting and force-generating EHTs and showed that prolonged perfusion improved cardiac tissue structure.

Differentiation arrest is a hallmark of acute leukemia. Genomic alterations in B cell differentiation factors such as PAX5, IKZF1, and EBF-1 have been identified in more than half of all cases of childhood B precursor acute lymphoblastic leukemia (ALL). Here, we describe a perturbed epigenetic and transcriptional regulation of ZNF423 in ALL as a novel mechanism interfering with B cell differentiation. Hypomethylation of ZNF423 regulatory sequences and BMP2 signaling result in transactivation of ZNF423α and a novel ZNF423β-isoform encoding a nucleosome remodeling and histone deacetylase complex–interacting domain. Aberrant ZNF423 inhibits the transactivation of EBF-1 target genes and leads to B cell maturation arrest in vivo. Importantly, ZNF423 expression is associated with poor outcome of ETV6-RUNX1–negative B precursor ALL patients. Our work demonstrates that ALL is more than a genetic disease and that epigenetics may uncover novel mechanisms of disease with prognostic implications.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Description

Patients with chronic inflammatory rheumatic diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and especially primary Sjögren's syndrome (SS), are at higher risk than the general population of developing B-cell non-Hodgkin lymphoma (NHL). Analyses of the association between various lymphoma subtypes and specific disease entities suggest that this association might be mediated by disease-specific mechanisms, as well as by mechanisms unique to lymphoma subtype. These specific associations can provide important information about abnormal B-cell stimulation in these conditions. Patients with primary SS, SLE and RA are at high risk of developing diffuse large B-cell lymphomas, a group of high-grade NHLs with remarkable heterogeneity. Patients with primary SS are at particularly high risk of developing marginal-zone B-cell lymphomas. The risk factors of lymphoma development in primary SS seem to be closely related to the underlying mechanisms of abnormal stimulation and/or impaired censoring mechanisms of B cells. In patients with RA and SLE, more intense disease activity and/or long-lasting disease might be indications of a higher risk of lymphoma development. This Review will focus on the risk of lymphoma, common and disease-specific mechanisms of B-cell lymphoma development, and on the clinical consequences of lymphoma in patients with inflammatory rheumatic diseases.

Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathological features. A hallmark of pSS is B-cell hyperactivity as manifested by the production of autoantibodies, hypergammaglobulinemia, formation of ectopic lymphoid structures within the inflamed tissues, and enhanced risk of B-cell lymphoma. Changes in the distribution of peripheral B-cell subsets and differences in post-recombination processes of immunoglobulin variable region (IgV) gene usage are also characteristic features of pSS. Comparison of B cells from the peripheral blood and salivary glands of patients with pSS with regard to their expression of the chemokine receptors CXCR4 and CXCR5, and their migratory capacity towards the corresponding ligands, CXCL12 and CXCL13, provide a mechanism for the prominent accumulation of CXCR4+CXCR5+memory B cells in the inflamed glands. Glandular B cells expressing distinct features of IgV light and heavy chain rearrangements, (re)circulating B cells with increased mutations of cμ transcripts in both CD27-and CD27+memory B-cell subsets, and enhanced frequencies of individual peripheral B cells containing IgV heavy chain transcripts of multiple isotypes indicate disordered selection and incomplete differentiation processes of B cells in the inflamed tissues in pSS. This may possibly be related to a lack of appropriate censoring mechanisms or different B-cell activation pathways within the ectopic lymphoid structures of the inflamed tissues. These findings add to our understanding of the pathogenesis of this autoimmune inflammatory disorder and may result in new therapeutic approaches.

Published reports in 2006 on systemic lupus erythematosus are reviewed with regard to preclinical and clinical studies on disturbances of the immune system including co-stimulation, cytokines and recent insights into new therapeutic approaches. Increasing knowledge of components of the innate immune system, such as certain receptors (Toll-like receptors, Fc receptors and complement receptors) and cytokines as well as immune cells (dendritic cells, plasmacytoid cells and neutrophils) supports their immunopathogenic relevance and enhance our understanding of the pathogenic complexity of lupus. Although it remains to be shown which of those could be targets for therapy or biomarkers, lymphocyte-directed therapy is currently under promising clinical investigation.

Recent studies have broadened our understanding of the etiopathogenesis and immunopathology of primary Sjögren's syndrome. This review highlights recent advances in understanding the underlying mechanisms of the disease as well as their implications for clinical handling and therapeutic options.

Maintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19+/CD27high/intracellular immunoglobulin Ghigh(IgGhigh)/HLA-DRhigh/CD38high/CD20–/CD95+tetanus toxin–specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19+/CD27high/intracellular IgGhigh/HLA-DRlow/CD38+/CD20–/CD95+cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.

Maintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19+/CD27high/intracellular immunoglobulin Ghigh (IgGhigh)/HLA-DRhigh/CD38high/CD20–/CD95+ tetanus toxin–specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19+/CD27high/intracellular IgGhigh/HLA-DRlow/CD38+/CD20–/CD95+ cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.

Although a modified European–American consensus classification of Sjögren syndrome has been introduced during the last year, the etiopathogenesis of this disease characterized by chronic lymphocytic inflammation, impaired function, and, finally, destruction of the salivary and lacrimal glands as well as systemic manifestations remains to be elucidated. Recent insights into the pathogenesis of Sjögren syndrome resulting from immunogenetic, hormonal, and epidemiologic evaluations as well as animal and in vitrostudies are highlighted by this review. Evidence confirms that lymphocytic disturbances, including ectopic germinal center formation and aberrations of cellular signaling play a significant role in Sjögren syndrome. Although some of these features are unique to Sjögren syndrome, others are also found in a number of systemic autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. The underlying cause of Sjögren syndrome remains largely enigmatic. However, distinct characteristics may provide the basis for the classification of the disease entities. Finally, an enhanced risk of lymphomagenesis is a well-known hallmark of primary Sjögren syndrome, indicating the central role of derangement of lymphocyte regulation. As demonstrated by the introduction of the new targeted therapeutic approaches in rheumatoid arthritis, solid insights into the pathogenesis of Sjögren syndrome may pave the way toward new therapeutic approaches.

Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription–polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, theIgVκgenes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgVHgenes were invariant. Thus, the light-chain–restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population.

Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription–polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, theIgVκ genes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgVH genes were invariant. Thus, the light-chain–restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population.

To delineate the mechanism of the abnormalities in B cell biology found in patients with primary Sjögren's syndrome (SS). The distribution of peripheral B cell subpopulations in 21 patients with primary SS was analyzed by immunofluorescence labeling and flow cytometry. Immunoglobulin rearrangements were analyzed in single B cells isolated from the peripheral blood and parotid glands by fluorescence-activated cell sorting. A significant reduction in the number of peripheral CD27+ memory B cells was found in SS patients, including a significantly reduced number of CD27+/IgD+/IgM+/CD5+ memory B cells. Remarkably, SS patients with secondary lymphoma uniquely exhibited an increase in CD27-expressing peripheral B cells, including CD27^high plasmablasts. Molecular analysis for mutated Ig gene rearrangements confirmed that CD27 expression distinguished naive and memory cells in SS. In contrast to the peripheral blood, the majority of parotid B cells from 1 patient examined exhibited both the mutational status and phenotype of memory B cells. Accordingly, the mutational frequencies of VH rearrangements were significantly greater in parotid B cells than in peripheral blood B cells, whereas the VH gene repertoire appeared to be very similar between the compartments. These data indicate that there is an accumulation/retention of memory B cells in the inflamed salivary glands of SS patients. It is possible that preferential accumulation of CD27+ memory B cells in the inflamed parotid gland explains their reduction in the peripheral blood.

To determine whether patients with Sjögren's syndrome (SS) have abnormalities in Ig Vλand Jλgene usage, differences in somatic hypermutation, defects in selection, or indications for perturbations of B cell maturation. Individual peripheral B cells from SS patients were analyzed for their Vλgene usage by single‐cell polymerase chain reaction amplification of genomic DNA and compared with those from normal controls. Molecular differences from controls in Vλ–Jλrecombination were identified that were reflected by findings in the nonproductive Vλrepertoire of the patients, including enhanced rearrangement of Vλ10A and Jλ2/3 gene segments. In addition, a number of abnormalities in the productive repertoire were identified, indicating disordered selection. A greater usage of 4 Vλgenes (2A2, 2B2, 2C, and 7A), representing 56% of all productive Vλrearrangements, was observed, suggesting positive selection of these genes. Overutilization of Jλ2/3 and underutilization of Jλ7 in both nonproductive and productive Vλrearrangements of SS patients compared with controls suggested decreased receptor editing in SS. The mutational frequency did not differ from that in controls, and positive selection of mutations into the productive V gene repertoire was found, similar to that in controls, although mutational targeting toward RGYW/WRCY motifs, typically found in controls, was not found in SS patients. Disturbed regulation of B cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting may contribute to the emergence of autoimmunity in SS.

To determine whether patients with Sjögren's syndrome (SS) have abnormalities in Ig Vλ and Jλ gene usage, differences in somatic hypermutation, defects in selection, or indications for perturbations of B cell maturation. Individual peripheral B cells from SS patients were analyzed for their Vλ gene usage by single-cell polymerase chain reaction amplification of genomic DNA and compared with those from normal controls. Molecular differences from controls in Vλ–Jλ recombination were identified that were reflected by findings in the nonproductive Vλ repertoire of the patients, including enhanced rearrangement of Vλ10A and Jλ2/3 gene segments. In addition, a number of abnormalities in the productive repertoire were identified, indicating disordered selection. A greater usage of 4 Vλ genes (2A2, 2B2, 2C, and 7A), representing 56% of all productive Vλ rearrangements, was observed, suggesting positive selection of these genes. Overutilization of Jλ2/3 and underutilization of Jλ7 in both nonproductive and productive Vλ rearrangements of SS patients compared with controls suggested decreased receptor editing in SS. The mutational frequency did not differ from that in controls, and positive selection of mutations into the productive V gene repertoire was found, similar to that in controls, although mutational targeting toward RGYW/WRCY motifs, typically found in controls, was not found in SS patients. Disturbed regulation of B cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting may contribute to the emergence of autoimmunity in SS.

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltration of the salivary glands with a previously reported predominance of Vκ‐bearing B cells and produce a variety of autoantibodies, indicating that there is a humoral autoimmune component in this syndrome. This study was undertaken to determine whether there are primary deviations of immunoglobulin V gene usage, differences in somatic hypermutation, defects of selection, or indications for perturbances of B cell maturation in SS.Individual peripheral B cells from patients with SS were analyzed for their Ig V gene usage, and the findings were compared with results in normal controls.Molecular differences, as reflected by findings in the nonproductive Vκ repertoire of the patients, were identified by an enhanced usage of Jκ2 gene segments and a lack of mutational targeting toward RGYW/WRCY sequences compared with controls. A greater usage of Vκ1 family members and a reduced frequency of Vκ3 gene segments in the productive repertoire suggested differences in selection, possibly driven by antigen. Overall positive selection for mutations, especially for replacements in the complementarity‐determining region and for mutations in RGYW/WRCY, similar to that found in controls, was detected.Disturbances of strictly regulated B cell maturation, during early B cell development as indicated by prominent Jκ2 gene usage and during germinal center reactions as indicated by a lack of targeting of the hypermutation mechanism, might contribute to the emergence of autoimmunity in SS.

To determine the impact of somatic hypermutation and selective influences on the VH gene repertoire in SLE, the mutational frequency and pattern of mutations in nonproductively and productively rearranged VH genes obtained from genomic DNA of individual CD 19+ B cells were analyzed in a patient with SLE.The mutational frequencies of nonproductive (6.54×10-2) as well as of productive (4.38×10-2) VH rearrangements were significantly higher in the SLE patient than in normal controls (3.8×10-2 p<0.001 and 3.3×10-2; p<0.001, respectively). Analysis of nonproductive rearrangements documented only minor abnormalities of the targeting of the mutator in the SLE patient. The majority of “mutational hot spots”, although different than in normals, appeared in the CDRs and an increased frequency of mutations in RGYW/WRCY sequences was observed. Moreover, no biases in base pair changes were found in the nonproductive repertoire.In contrast, there was a selection against A and T mutations and towards G mutations within the productive repertoire. Importantly, there were no significant differences in the R/S ratios of mutations within the FRs between the nonproductive and productive repertoire consistent with abnormalities in elimination of B cells expressing VH genes with these mutations. The result of this abnormality was a significantly higher R/S ratio of the VHgenes in the productive repertoire of the SLE patient compared to normals (p<0.05).These data indicate that the mutational machinery was markedly enhanced in this SLE patient but exhibited nearly normal targeting, whereas selective influences were abnormal. These findings suggest that both enhanced mutational activity and disturbances in selection may have played a role in the emergence of autoreactivity in this SLE patient

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8×10−10mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 μg/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Ohne Zusammenfassung:

The reproducibility of stem cell research relies on the constant availability of quality‐controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch‐to‐batch variation. Here, we provide a cost‐efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT‐PCR Support Protocol 4: Identity: Short tandem repeat analysis

Despite a considerable growth in space related industry and scientific research over the past few decades, space related education has largely been neglected in our country. NAROM - the National Centre for Space Related Education - was formed last year to organize space related educational activities, to promote recruitment, to promote appreciation for the benefits of space activities, and to stimulate interest for science in general. This year, nine students from Narvik Engineering College have participated in the Hotel Payload Project (HPP) at Andøya Rocket Range. They have thus played an active and essential role in an ongoing engineering project.

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Vasopressin stimulates erythropoiesis.

Human engineered heart tissue derived from induced pluripotent stem cells improves cardiac function in guinea pigs.

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27negative B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related VLchain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P< 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of VLchain genes caused by selection and clonal expansion of B cells expressing particular VLgenes. In addition, the data document an accumulation of B cells bearing mutated VLgene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.

To assess the impact of somatic hypermutation and selective influences on the Vλ light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of mutations were analyzed in individual CD19+B cells from a patient with previously undiagnosed SLE. The mutational frequency of nonproductive and productive rearrangements in the SLE patient was greater (3.1 × 10-2vs 3.4 × 10-2, respectively) than that in normal B cells (1.2 × 10-2vs 2.0 × 10-2, both P< 0.001). The frequencies of mutated rearrangements in both the nonproductive and productive repertoires were significantly higher in the patient with SLE than in normal subjects. Notably, there were no differences in the ratio of replacement to silent (R/S) mutations in the productive and nonproductive repertoires of the SLE patient, whereas the R/S ratio in the framework regions of productive rearrangements of normal subjects was reduced, consistent with active elimination of replacement mutations in this region. The pattern of mutations was abnormal in the SLE patient, with a significant increase in the frequency of G mutations in both the productive and nonproductive repertoires. As in normal subjects, however, mutations were found frequently in specific nucleotide motifs, the RGYW/WRCY sequences, accounting for 34% (nonproductive) and 46% (productive) of all mutations. These data are most consistent with the conclusion that in this SLE patient, the mutational activity was markedly greater than in normal subjects and exhibited some abnormal features. In addition, there was decreased subsequent positive or negative selection of mutations. The enhanced and abnormal mutational activity along with disturbances in selection may play a role in the emergence of autoreactivity in this patient with SLE.