RESUMEN
Antibody-producing plasma cells fuel humoral immune responses. They also contribute to autoimmune diseases such as systemic lupus erythematosus or IgA nephropathy. Interleukin-6 and the tumor necrosis factor (TNF) family ligands BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) participate in plasma cell survival. BAFF binds to three receptors, BAFFR (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B cell maturation antigen), while APRIL binds to TACI, BCMA, and proteoglycans. However, which ligand-receptor pair(s) are required to maintain plasma cells in different body locations remains unknown. Here, by combining mouse genetic and pharmacological approaches, we found that plasma cells required BCMA and/or TACI but not BAFFR. BCMA responded exclusively to APRIL, while TACI responded to both BAFF and APRIL, identifying three self-sufficient ligand-receptor pairs for plasma cell maintenance: BAFF-TACI, APRIL-TACI, and APRIL-BCMA. Together, these actors accounted for 90% of circulating antibodies. In BAFF-ko mice, the reduction of plasma cells upon APRIL inhibition indicated that APRIL could function in the absence of BAFF-APRIL heteromers. No evidence was found that in the absence of BCMA and TACI, binding of APRIL to proteoglycans would help maintain plasma cells. IL-6, alone or together with BAFF and APRIL, supported mainly splenic plasmablasts and plasma cells and contributed to circulating IgG but not IgA levels. In conclusion, survival factors for plasma cells can vary with body location and with the antibody isotype that plasma cells produce. To efficiently target plasma cells, in particular IgA-producing ones, dual inhibition of BAFF and APRIL is required.
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Factor Activador de Células B , Receptor del Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Interleucina-6 , Proteína Activadora Transmembrana y Interactiva del CAML , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Animales , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Factor Activador de Células B/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Antígeno de Maduración de Linfocitos B/inmunología , Antígeno de Maduración de Linfocitos B/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Interleucina-6/metabolismo , Interleucina-6/inmunología , Ratones , Receptor del Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/genética , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Ratones Noqueados , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Ratones Endogámicos C57BLRESUMEN
Background: Chronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear. Methods: We generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo. Results: Our findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo. Conclusions: Our study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output.
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Leucemia Linfocítica Crónica de Células B , Animales , Humanos , Ratones , Linfocitos B/metabolismo , Supervivencia Celular/genética , Leucemia Linfocítica Crónica de Células B/patologíaRESUMEN
Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.
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Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.
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Hipergammaglobulinemia , Trastornos Linfoproliferativos , Humanos , Apoptosis/genética , Centro Germinal , Trastornos Linfoproliferativos/genética , Serina-Treonina Quinasas TORRESUMEN
Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.
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Escherichia coli , Metiltransferasas , Escherichia coli/metabolismo , Metiltransferasas/metabolismo , Metilación , S-Adenosilmetionina/química , MetioninaRESUMEN
The BAFF/APRIL-system with the two cytokines BAFF and APRIL and their three receptors, transmembrane activator and CAML interactor (TACI), BAFF receptor, and B-cell maturation Ag, is important for B cell maintenance. The BAFF/APRIL system is a therapeutic target in B cell-derived malignancies and autoimmune diseases. However, unexpected outcomes of clinical trials with atacicept (TACI-Fc) underline our incomplete understanding of this system. Shedding of the three receptors is one important regulatory element. In humans, TACI exists in two isoforms generated through alternative splicing in their extracellular portion: TACI-long (l) has two cysteine-rich domains, whereas TACI-short (s) lacks the first low-affinity one. In this study, we discriminated soluble (s) forms of TACI-l and TACI-s with newly generated mAbs and found that both were spontaneously released from activated human B cells, with a predominance of sTACI-l. Furthermore, sTACI-l was also the dominant isoform in human serum. Vaccination with the mRNA vaccine from BioNTech does not significantly affect the serum levels of sTACI-l. Both TACI-s and TACI-l were shed by a disintegrin and metalloproteinase domain-containing protein 10. TACI-l and TACI-s formed homo- and hetero-oligomers in soluble and membrane-bound forms. Both sTACI-l and sTACI-s acted as decoy receptors for BAFF, but only sTACI-l also efficiently inhibited APRIL. Dimerization of sTACI-l enhanced its decoy functions only slightly. Together, we extend our knowledge of the complexity of the BAFF/APRIL system by identifying and characterizing the two soluble isoforms of TACI.
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Linfocitos B , Proteína Activadora Transmembrana y Interactiva del CAML , Humanos , Empalme Alternativo , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/genética , Citocinas/genética , Isoformas de Proteínas/genética , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12-60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose-response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible.
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Displasia Ectodermal Anhidrótica Tipo 1 , Displasia Ectodérmica , Niño , Embarazo , Femenino , Lactante , Humanos , Masculino , Preescolar , Displasia Ectodermal Anhidrótica Tipo 1/genética , Displasia Ectodermal Anhidrótica Tipo 1/terapia , Ectodisplasinas/genética , Displasia Ectodérmica/genética , Sudoración , Cabello , Proteínas RecombinantesRESUMEN
BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.
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Lesiones Encefálicas , Conexina 43 , Animales , Ratones , Ratas , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Conexina 43/metabolismo , Gliosis/metabolismo , Inflamación/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Integrina beta3/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba , Antígenos Thy-1/metabolismo , Integrina alfa5/metabolismoRESUMEN
BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.
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Síndrome Linfoproliferativo Autoinmune , Humanos , Síndrome Linfoproliferativo Autoinmune/genética , Proteína Ligando Fas/genética , Mutación , Biomarcadores , Vitaminas , Receptor fas/genética , Apoptosis/genéticaRESUMEN
PURPOSE: Binding of the B cell activating factor (BAFF) to its receptor (BAFFR) activates in mature B cells many essential pro-survival functions. Null mutations in the BAFFR gene result in complete BAFFR deficiency and cause a block in B cell development at the transition from immature to mature B cells leading therefore to B lymphopenia and hypogammaglobulinemia. In addition to complete BAFFR deficiency, single nucleotide variants encoding BAFFR missense mutations were found in patients suffering from common variable immunodeficiency (CVID), autoimmunity, or B cell lymphomas. As it remained unclear to which extent such variants disturb the activity of BAFFR, we performed genetic association studies and developed a cellular system that allows the unbiased analysis of BAFFR variants regarding oligomerization, signaling, and ectodomain shedding. METHODS: In addition to genetic association studies, the BAFFR variants P21R, A52T, G64V, DUP92-95, P146S, and H159Y were expressed by lentiviral gene transfer in DG-75 Burkitt's lymphoma cells and analyzed for their impacts on BAFFR function. RESULTS: Binding of BAFF to BAFFR was affected by P21R and A52T. Spontaneous oligomerization of BAFFR was disturbed by P21R, A52T, G64V, and P146S. BAFF-dependent activation of NF-κB2 was reduced by P21R and P146S, while interactions between BAFFR and the B cell antigen receptor component CD79B and AKT phosphorylation were impaired by P21R, A52T, G64V, and DUP92-95. P21R, G64V, and DUP92-95 interfered with phosphorylation of ERK1/2, while BAFF-induced shedding of the BAFFR ectodomain was only impaired by P21R. CONCLUSION: Although all variants change BAFFR function and have the potential to contribute as modifiers to the development of primary antibody deficiencies, autoimmunity, and lymphoma, P21R is the only variant that was found to correlate positively with CVID.
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Receptor del Factor Activador de Células B , Inmunodeficiencia Variable Común , Humanos , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Linfocitos B , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/metabolismo , Ligandos , Transducción de SeñalRESUMEN
The scutes of the turtle shell are epidermal shields that begin their formation during the early stages of shell development. Like other skin appendages, turtle scutes are hypothesized to be patterned by reaction-diffusion systems. We have previously established ex vivo and in silico systems to study these mechanisms experimentally and have further shown that mathematical models can explain the dynamics of the induction of turtle scute primordia and the generation of final scute architecture. Using these foundations, we expand our current knowledge and test the roles of ectodysplasin and activin signaling in the development of turtle scutes. We find that these molecules play important roles in the prepatterning of scute primordia along the carapacial ridge and show that blocking Edar signaling may lead to a complete loss of marginal scute primordia. We show that it is possible to reproduce these observations using simple mathematical modeling, thereby suggesting a stabilizing role for ectodysplasin within the reaction-diffusion mechanisms. Finally, we argue that our findings further entrench turtle scutes within a class of developmental systems composed of hierarchically nested reaction-diffusion mechanisms, which is conserved across ectodermal organs.
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Tortugas , Animales , Ectodisplasinas , Epidermis , Transducción de Señal , Desarrollo EmbrionarioRESUMEN
Mutations within the ectodysplasin A (EDA) gene have been associated with congenital hypotrichosis and anodontia (HAD/XHED) in humans, mice, dogs and cattle. We identified a three-generation family of Fleckvieh cattle with male calves exhibiting clinical and histopathological signs consistent with an X-linked recessive HAD (XHED). Whole genome and Sanger sequencing of cDNA showed a perfect association of the missense mutation g.85716041G>A (ss2019497443, rs1114816375) within the EDA gene with all three cases following an X-linked recessive inheritance, but normal EDAR and EDARADD. This mutation causes an exchange of glycine (G) with arginine (R) at amino acid position 227 (p.227G>R) in the second collagen triple helix repeat domain of EDA. The EDA variant was associated with a significant reduction and underdevelopment of hair follicles along with a reduced outgrowth of hairs, a complete loss of seromucous nasolabial and mucous tracheal and bronchial glands and a malformation of and reduction in number of teeth. Thermostability of EDA G227R was reduced, consistent with a relatively mild hair and tooth phenotype. However, incisors and canines were more severely affected in one of the calves, which correlated with the presence of a homozygous missense mutation of RNF111 (g.51306765T>G), a putative candidate gene possibly associated with tooth number in EDA-deficient Fleckvieh calves.
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Displasia Ectodermal Anhidrótica Tipo 1 , Displasia Ectodérmica , Hipotricosis , Deformidades Congénitas de las Extremidades , Animales , Bovinos , Masculino , Ratones , Displasia Ectodérmica/genética , Displasia Ectodermal Anhidrótica Tipo 1/genética , Mutación , Mutación MissenseRESUMEN
BAFF (B cell activation factor of the TNF family/B lymphocyte stimulator, BLyS) and APRIL (a proliferation-inducing ligand) are targeted by atacicept, a decoy receptor consisting of the extracellular domain of TACI (transmembrane activator and calcium-modulator and cyclophilin (CAML) interactor) fused to the Fc portion of human IgG1. The purpose of the study was to characterize free and ligand-bound atacicept in humans. Total and active atacicept in serum of healthy volunteers receiving a single dose of subcutaneous atacicept or in patients treated weekly for one year were measured by ELISA, Western blot, or cell-based assays. Pharmacokinetics of free and bound atacicept were predicted based on total atacicept ELISA results. Persistence of complexes of purified atacicept bound to recombinant ligands was also monitored in mice. Results show that unbound or active atacicept in human serum exceeded 0.1 µg/ml for one week post administration, or throughout a 1-year treatment with weekly administrations. After a single administration of atacicept, endogenous BAFF bound to atacicept was detected after 8 h then increased about 100-fold within 2 to 4 weeks. Endogenous heteromers of BAFF and APRIL bound to atacicept also accumulated, but atacicept-APRIL complexes were not detected. In mice receiving intravenous injections of purified complexes pre-formed in vitro, atacicept-BAFF persisted longer (more than a week) than atacicept-APRIL (less than a day). Thus, only biologically inactive BAFF and BAFF-APRIL heteromers accumulate on atacicept in vivo. The measure of active atacicept provides further support for the once-weekly dosing regimen implemented in the clinical development of atacicept.
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Inmunoglobulina G , Activación de Linfocitos , Humanos , Ratones , Animales , Ligandos , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Deficiency of ectodysplasin A1 (EDA1) due to variants of the gene EDA causes X-linked hypohidrotic ectodermal dysplasia (XLHED), a rare genetic condition characterized by abnormal development of ectodermal structures. XLHED is defined by the triad of hypotrichosis, hypo- or anhidrosis, and hypo- or anodontia. Anhidrosis may lead to life-threatening hyperthermia. A definite genetic diagnosis is, thus, important for the patients' management and amenability to a novel prenatal treatment option. Here, we describe five familial EDA variants segregating with the disease in three families, for which different prediction tools yielded discordant results with respect to their significance. Functional properties in vitro and levels of circulating serum EDA were compared with phenotypic data on skin, hair, eyes, teeth, and sweat glands. EDA1-Gly176Val, although associated with relevant hypohidrosis, still bound to the EDA receptor (EDAR). Subjects with EDA1-Pro389LeufsX27, -Ter392GlnfsX30, -Ser125Cys, and an EDA1 splice variant (c.924+7A > G) showed complete absence of pilocarpine-induced sweating. EDA1-Pro389LeufsX27 was incapable of binding to EDAR and undetectable in serum. EDA1-Ter392GlnfsX30, produced in much lower amounts than wild-type EDA1, could still bind to EDAR, and so did EDA1-Ser125Cys that was, however, undetectable in serum. The EDA splice variant c.924+7A > G resulted experimentally in a mix of wild-type EDA1 and EDA molecules truncated in the middle of the receptor-binding domain, with reduced EDA serum concentration. Thus, in vitro assays reflected the clinical phenotype in two of these difficult cases, but underestimated it in three others. Absence of circulating EDA seems to predict the full-blown phenotype of XLHED, while residual EDA levels may also be found in anhidrotic patients. This indicates that unborn subjects carrying variants of uncertain significance could benefit from an upcoming prenatal medical treatment even if circulating EDA levels or tests in vitro suggest residual EDA1 activity.
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Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.
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Apoptosis , Linfocitos B , Polisacáridos , Ácidos Siálicos , Animales , Receptor del Factor Activador de Células B/metabolismo , Linfocitos B/fisiología , Supervivencia Celular , Eliminación de Gen , Ratones , N-Acilneuraminato Citidililtransferasa/genética , Polisacáridos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismoRESUMEN
Binding of BAFF to BAFFR activates in mature B cells PI3K/AKT signaling regulating protein synthesis, metabolic fitness, and survival. In humans, naive and memory B cells express the same levels of BAFFR, but only memory B cells seem to survive without BAFF. Here, we show that BAFF activates PI3K/AKT only in naive B cells and changes the expression of genes regulating migration, proliferation, growth, and survival. BAFF-induced PI3K/AKT activation requires direct interactions between BAFFR and the B cell antigen receptor (BCR) components CD79A and CD79B and is enhanced by the AKT coactivator TCL1A. Compared to memory B cells, naive B cells express more surface BCRs, which interact better with BAFFR than IgG or IgA, thus allowing stronger responses to BAFF. As ablation of BAFFR in naive and memory B cells causes cell death independent of BAFF-induced signaling, BAFFR seems to act also as an intrinsic factor for B cell survival.
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Receptor del Factor Activador de Células B , Células B de Memoria , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Receptores de Antígenos de Linfocitos B , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/metabolismo , Humanos , Células B de Memoria/inmunología , Células B de Memoria/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
We present TIMo (Time-of-flight Indoor Monitoring), a dataset for video-based monitoring of indoor spaces captured using a time-of-flight (ToF) camera. The resulting depth videos feature people performing a set of different predefined actions, for which we provide detailed annotations. Person detection for people counting and anomaly detection are the two targeted applications. Most existing surveillance video datasets provide either grayscale or RGB videos. Depth information, on the other hand, is still a rarity in this class of datasets in spite of being popular and much more common in other research fields within computer vision. Our dataset addresses this gap in the landscape of surveillance video datasets. The recordings took place at two different locations with the ToF camera set up either in a top-down or a tilted perspective on the scene. Moreover, we provide experimental evaluation results from baseline algorithms.
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Algoritmos , HumanosRESUMEN
Desorption/ionization induced by neutral clusters (DINeC) in combination with mass spectrometry (MS) was used for the investigation of the molecular composition of the surface of ionic liquids (IL). Based on the surface sensitivity of DINeC-MS, accumulation of either cations or anions was discriminated on the surface of bulk IL depending on the molecular structure of the IL components. In particular, cations with long alkyl chains aggregate on the surface, but this tendency is more reduced the larger the respective anion is; in the case of larger anions and smaller cations, it can be even reversed. For thin layers of IL, the ratio between cations and anions as detected in the mass spectra was found to be further influenced by the substrate surface.
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Soft cluster-induced desorption/ionization of polystyrene oligomers was investigated with respect to application in mass spectrometry. Clear peak progressions corresponding to intact polystyrene molecules were observed in the mass spectra, and no fragmentation was detected; efficient desorption was deduced from quartz crystal microbalance measurements. Molecular dynamics (MD) simulations of the process revealed that even in the case of the nonpolar polystyrene molecules cluster-induced desorption proceeds via dissolvation in the polar clusters. Experimentally, a significantly lower desorption efficiency was observed for polystyrene molecules with larger chain length. Taking into account MD simulations and further experiments with mixed samples consisting of long- and short-chain polystyrene oligomers, the reduced desorption efficiency for longer chain polystyrene molecules was attributed to a stronger entanglement of the larger polystyrene molecules.
RESUMEN
In mature B cells, TACI controls class-switch recombination and differentiation into plasma cells during T cell-independent antibody responses. TACI binds the ligands BAFF and APRIL. Approximately 10% of patients with common variable immunodeficiency (CVID) carry TACI mutations, of which A181E and C172Y are in the transmembrane domain. Residues A181 and C172 are located on distinct sides of the transmembrane helix, which is predicted by molecular modeling to spontaneously assemble into trimers and dimers. In human B cells, these mutations impair ligand-dependent (C172Y) and -independent (A181E) TACI multimerization and signaling, as well as TACI-enhanced proliferation and/or IgA production. Genetic inactivation of TACI in primary human B cells impaired survival of CpG-activated cells in the absence of ligand. These results identify the transmembrane region of TACI as an active interface for TACI multimerization in signal transduction, in particular for ligand-independent signals. These functions are perturbed by CVID-associated mutations.