ORAI1 mutations abolishing store-operated Ca2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency.

BACKGROUND: Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. OBJECTIVE: We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. METHODS: We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. RESULTS: We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. CONCLUSION: ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).

Accelerating antibody discovery using transgenic animals overexpressing the neonatal Fc receptor as a result of augmented humoral immunity.

In recent years, there has been an increasing demand for the development of faster and more efficient technologies for the generation of monoclonal antibodies against challenging targets that are weakly immunogenic or available only in limited amounts. Typical classes of such targets are cell surface antigens such as G-protein related receptors (GPCRs) or ion channels. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn), resulting in an augmented humoral immune response even if challenging antigens are used for immunization. The impressively enhanced FcRn-mediated immune reactions are characterized by improved IgG protection and enhanced antigen presentation leading to greater number of antigen-specific T-helper and B-cell activation in lymphoid organs. Notably, these animals do not show any sign of autoimmunity and can be efficiently bred. FcRn overexpression thus leads to a number of practical benefits for improved generation of monoclonal and polyclonal antibodies against multiple antigens, including weakly immunogenic epitopes or tiny amounts of proteins. This review summarizes our current understanding about the mechanisms by which FcRn overexpression leads to such a significantly enhanced humoral immune response.

Neonatal FcR overexpression boosts humoral immune response in transgenic mice.

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

A novel monoclonal antibody reveals the enrichment of NADPH oxidase 5 in human splenic endothelial cells.

Members of the NOX/DUOX family of NADPH oxidases are responsible for regulated ROS production in diverse cells and tissues. Detection of NOX/DUOX proteins at the protein level remains an important challenge in the field. Here we report the development and characterization of a novel anti-NOX5 monoclonal antibody, which recognizes the human NOX5 protein in both Western blot, immunocytochemistry, and histochemistry applications. With the help of the antibody we could successfully detect both heterologously and endogenously expressed NOX5 in mammalian cells. Furthermore, we could also detect NOX5 protein in the human spleen, testis, and ovary. Immunohistochemical studies on human testis revealed that NOX5 localized to spermatogenic cells. This expression pattern was also supported by the result of in silico analysis of single-cell RNA sequencing data that indicated that NOX5 protein is present in developing spermatids and spermatocytes. Mature spermatozoa, however, did not contain detectable NOX5. In the human ovary, both immunostaining and single-cell RNA sequencing suggest that NOX5 is expressed in interstitial fibroblasts and theca cells. We also analyzed vascular cells for the presence of NOX5 and we found that NOX5 expression is a fairly specific feature of splenic endothelial cells.

Context-Dependent Role of Glucocorticoid Receptor Alpha and Beta in Breast Cancer Cell Behaviour.

Background. The dual role of GCs has been observed in breast cancer; however, due to many concomitant factors, GR action in cancer biology is still ambiguous. In this study, we aimed to unravel the context-dependent action of GR in breast cancer. Methods. GR expression was characterized in multiple cohorts: (1) 24,256 breast cancer specimens on the RNA level, 220 samples on the protein level and correlated with clinicopathological data; (2) oestrogen receptor (ER)-positive and -negative cell lines were used to test for the presence of ER and ligand, and the effect of the GRß isoform following GRα and GRß overexpression on GR action, by in vitro functional assays. Results. We found that GR expression was higher in ER- breast cancer cells compared to ER+ ones, and GR-transactivated genes were implicated mainly in cell migration. Immunohistochemistry showed mostly cytoplasmic but heterogenous staining irrespective of ER status. GRα increased cell proliferation, viability, and the migration of ER- cells. GRß had a similar effect on breast cancer cell viability, proliferation, and migration. However, the GRß isoform had the opposite effect depending on the presence of ER: an increased dead cell ratio was found in ER+ breast cancer cells compared to ER- ones. Interestingly, GRα and GRß action did not depend on the presence of the ligand, suggesting the role of the "intrinsic", ligand-independent action of GR in breast cancer. Conclusions. Staining differences using different GR antibodies may be the reason behind controversial findings in the literature regarding the expression of GR protein and clinicopathological data. Therefore, caution in the interpretation of immunohistochemistry should be applied. By dissecting the effects of GRα and GRß, we found that the presence of the GR in the context of ER had a different effect on cancer cell behaviour, but independently of ligand availability. Additionally, GR-transactivated genes are mostly involved in cell migration, which raises GR's importance in disease progression.

Liquid biopsy-based monitoring of residual disease in multiple myeloma by analysis of the rearranged immunoglobulin genes-A feasibility study.

The need for sensitive monitoring of minimal/measurable residual disease (MRD) in multiple myeloma emerged as novel therapies led to deeper responses. Moreover, the potential benefits of blood-based analyses, the so-called liquid biopsy is prompting more and more studies to assess its feasibility. Considering these recent demands, we aimed to optimize a highly sensitive molecular system based on the rearranged immunoglobulin (Ig) genes to monitor MRD from peripheral blood. We analyzed a small group of myeloma patients with the high-risk t(4;14) translocation, using next-generation sequencing of Ig genes and droplet digital PCR of patient-specific Ig heavy chain (IgH) sequences. Moreover, well established monitoring methods such as multiparametric flow cytometry and RT-qPCR of the fusion transcript IgH::MMSET (IgH and multiple myeloma SET domain-containing protein) were utilized to evaluate the feasibility of these novel molecular tools. Serum measurements of M-protein and free light chains together with the clinical assessment by the treating physician served as routine clinical data. We found significant correlation between our molecular data and clinical parameters, using Spearman correlations. While the comparisons of the Ig-based methods and the other monitoring methods (flow cytometry, qPCR) were not statistically evaluable, we found common trends in their target detection. Regarding longitudinal disease monitoring, the applied methods yielded complementary information thus increasing the reliability of MRD evaluation. We also detected indications of early relapse before clinical signs, although this implication needs further verification in a larger patient cohort.

Measuring peroxidasin activity in live cells using bromide addition for signal amplification.

Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

Creation of the first monoclonal antibody recognizing an extracellular epitope of hABCC6.

Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.

FcRn Overexpression Expands Diversity of the Humoral Immune Response in bFcRn Transgenic Mice.

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In this study we analyzed the diversity of the humoral immune response of bFcRn Tg mice, using a length distribution analysis (spectratyping) and next generation sequencing (NGS) of the immunoglobulin heavy chain variable regions. Our analysis showed that in response to immunization with ovalbumin or transfected cells that expressed a unique membrane protein, our Tg animals developed a more diverse plasma cell repertoire than controls, which manifested in greater numbers of different clones, and clusters with fewer highly expanded large clones, as identified by the variable region (CDR3) of the immunoglobulin heavy chain. The increased antibody diversity in Tg mice after immunization was observed at both IgM and IgG levels, indicating that the increased humoral immune diversity in Tg mice is due to a higher number of both activated, antigen-specific naïve and isotype switched B cells. We thus demonstrated that the BCR repertoire of the immunized bFcRn Tg animals is more diverse compared to wild type mice, which likely makes these Tg mice a better choice for monoclonal antibody production against challenging antigens, including the extracellular regions of cell membrane proteins.

ABHD4-dependent developmental anoikis safeguards the embryonic brain.

A specialized neurogenic niche along the ventricles accumulates millions of progenitor cells in the developing brain. After mitosis, fate-committed daughter cells delaminate from this germinative zone. Considering the high number of cell divisions and delaminations taking place during embryonic development, brain malformations caused by ectopic proliferation of misplaced progenitor cells are relatively rare. Here, we report that a process we term developmental anoikis distinguishes the pathological detachment of progenitor cells from the normal delamination of daughter neuroblasts in the developing mouse neocortex. We identify the endocannabinoid-metabolizing enzyme abhydrolase domain containing 4 (ABHD4) as an essential mediator for the elimination of pathologically detached cells. Consequently, rapid ABHD4 downregulation is necessary for delaminated daughter neuroblasts to escape from anoikis. Moreover, ABHD4 is required for fetal alcohol-induced apoptosis, but not for the well-established form of developmentally controlled programmed cell death. These results suggest that ABHD4-mediated developmental anoikis specifically protects the embryonic brain from the consequences of sporadic delamination errors and teratogenic insults.

Porcine IgG: structure, genetics, and evolution.

Eleven genomic porcine Cgamma gene sequences are described that represent six putative subclasses that appear to have originated by gene duplication and exon shuffle. The genes previously described as encoding porcine IgG1 and IgG3 were shown to be the IgG1(a) and IgG1(b) allelic variants of the IGHG1 gene, IgG2a and IgG2b are allelic variants of the IGHG2 gene, while "new" IgG3 is monomorphic, has an extended hinge, is structurally unique, and appears to encode the most evolutionarily conserved porcine IgG. IgG5(b) differs most from its putative allele, and its C(H)1 domain shares sequence homology with the C(H)1 of IgG3. Four animals were identified that lacked either IgG4 or IgG6. Alternative splice variants were also recovered, some lacking the C(H)1 domain and potentially encoding heavy chain only antibodies. Potentially, swine can transcribe >20 different Cgamma chains. A comparison of mammalian Cgamma gene sequences revealed that IgG diversified into subclasses after speciation. Thus, the effector functions for the IgG subclasses of each species should not be extrapolated from "same name subclasses" in other species. Sequence analysis identified motifs likely to interact with Fcgamma receptors, FcRn, protein A, protein G, and C1q. These revealed IgG3 to be most likely to activate complement and bind FcgammaRs. All except IgG5(a) and IgG6(a) should bind to FcgammaRs, while all except IgG6(a) and the putative IgG5 subclass proteins should bind well to porcine FcRn, protein A, and protein G.

The neonatal Fc receptor plays a crucial role in the metabolism of IgG in livestock animals.

The role of the FcRn is fundamental in IgG metabolism. It is involved in transporting maternal immunity and protects IgG from fast degradation throughout life. While the acquisition of the humoral immunity through the transfer of IgG from mother to offspring shows species-specific differences, the mechanism how FcRn protects IgG from degradation is highly similar in all species analyzed so far. This review summarizes the current understanding of the FcRn-mediated IgG metabolism in livestock animals (cattle, sheep and pig) and point out those aspects that remain to be exposed for better understanding the function of this system in these species and also to take advantages of it for economical purposes.

Association of FcRn Heavy Chain Encoding Gene (FCGRT) Polymorphisms with IgG Content in Bovine Colostrum.

In this study, we evaluated haplotypes of the bovine FCGRT (encoding the FcRn heavy chain) and their relationship to the IgG concentration in bovine colostrum. Four single nucleotide polymorphism (SNP), classified into five haplotypes, were identified in a total of 49 Holstein-Frisians cows. Haplotype 5 was found to be significantly associated with a high IgG level ([OR] = 9.90, 95%CI = 1.11-88.34, p = 0.016) and haplotype 2 exhibited a similar trend ([OR] = 2.89, 95%CI = 1.17-7.11, p = 0.019).

Cloning, expression and characterization of the bovine p65 subunit of NFkappaB.

The full length coding sequence of the cattle transcription factor p65 was isolated and cloned. The cloned bovine p65 was expressed in mammalian cells, and it induced the NF-kappaB-specific luciferase reporter gene expression. Using gel retardation techniques, we demonstrated that the cloned bovine p65 bound to the consensus kappaB sequence. The comparison of the bovine p65 with its human and mouse orthologues at amino acid level showed high homology in both the DNA-binding domain, known as Rel homology domain (RHD) and the transactivation domain (TAD). The phylogenetic analysis at DNA level provided a new insight in the evolution of the NF-kappaB family, and it could resolve the topology of the mammalian p65molecules. Although, the RHD was conserved in vertebrates, the TAD sequences deviated from each other, and showed faster molecular evolution than RHD sequences, which may indirectly result in the modification of NF-kappaB immune functions.

Characterization of the interactions of rabbit neonatal Fc receptor (FcRn) with rabbit and human IgG isotypes.

Despite the increasing importance of rabbit as an animal model in pharmacological studies like investigating placental transfer of therapeutic IgGs, little is known about the molecular interaction of the rabbit neonatal Fc receptor (FcRn) with rabbit and human IgG molecules. We analyzed the interactions of the rabbit and human FcRn with rabbit and human IgG isotypes using surface plasmon resonance assay. Similar to FcRn of other species, rabbit FcRn functions in pH-dependent manner, as it binds IgGs at pH 6.0, but no binding occurs at pH 7.4. We also showed that rabbit FcRn binds rabbit IgG and human IgG1 with nearly identical affinity, whereas it has stronger interactions with the other human IgG isotypes. The similar affinity of rabbit IgG and human IgG1 for rabbit FcRn was confirmed by in vitro FcRn-mediated recycling assay. These data verify that rabbit is an appropriate animal model for analyzing the pharmacokinetics of human therapeutic monoclonal antibodies.

ORAI2 modulates store-operated calcium entry and T cell-mediated immunity.

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is critical for lymphocyte function and immune responses. CRAC channels are hexamers of ORAI proteins that form the channel pore, but the contributions of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2 double-deficient mice have impaired T cell-dependent antiviral immune responses, and are protected from T cell-mediated autoimmunity and alloimmunity in models of colitis and graft-versus-host disease. Our study demonstrates that ORAI1 and ORAI2 form heteromeric CRAC channels, in which ORAI2 fine-tunes the magnitude of SOCE to modulate immune responses.

Characterization of the nuclear factor-kappa B responsiveness of the human dio2 gene.

Type 2 iodothyronine deiodinase (D2) activates T4 by deiodination to T3, a process being the source of most T3 present in the brain. In the mediobasal hypothalamus, expression of the dio2 gene is potently activated by administration of bacterial lipopolysaccharide (LPS), which in turn mediates the modifications in thyroid homeostasis typically observed in patients with nonthyroidal illness syndrome. Here we show that LPS-induced D2 expression is also observed in human MSTO-211H cells that endogenously express D2. Exposure to LPS rapidly doubled D2 activity by a mechanism that was partially blocked by the nuclear factor-B (NF-B) inhibitor sulfasalazine. Next, the human dio2 5'-flanking region promoter assay was used in HC11 cells and the p65/NF-kappa B responsiveness mapped to the 3' approximately 600-bp region of hdio2 5'-flanking region, with an approximately 15-fold induction. Semiquantitative EMSA identified the strongest NF-B binding sites at the positions -683 bp (called no. 2) and -198 bp (no. 5) 5' to the transcriptional starting site. Despite the very similar NF-kappa B binding affinity of these two sites, site-directed mutagenesis and promoter assay indicated that only site no. 5 possessed transactivation potency in the presence of the p65 subunit of NF-kappa B. Other cytokine mediators such as signal transducer and activator of transcription-3 (STAT3) or signal transducer and activator of transcription-5 (STAT5) did not induce transcription of the dio2 gene. Our results indicate that inflammatory signals regulate D2 expression predominantly via the NF-kappa B pathway in a direct transcriptional manner and could contribute to the changes in thyroid economy observed in nonthyroidal illness syndrome during infection.

Cloning and characterization of the dromedary (Camelus dromedarius) neonatal Fc receptor (drFcRn).

The full length cDNA of the dromedary neonatal Fc receptor (drFcRn) alpha chain was isolated and found that it is similar to the neonatal Fc receptor (FcRn) of other species with a high overall similarity to ruminant FcRn alpha chains. The drFcRn/Fc contact residues are highly conserved and predicted to bind both conventional (IgG1) and heavy chain (IgG2a, IgG3) antibodies. Using immunohistochemistry, we detected its expression in the hepatocytes and in epithelial cells of portal bile ductuli and also in the mammary gland acini and ducti. Remarkably, Ser313, that was identified to be crucial for apical to basolateral transcytosis, is substituted in the drFcRn alpha chain. The full length of the dog and orangutan FcRn alpha chains was also identified from databases. Analyzing the phylogenetic relatedness of this gene we found that dromedary clustered together with artiodactyls, dog is located between artiodactyls and primates, where the orangutan was branched, reflecting the accepted evolutionary relationships.

Isolation of the gene encoding the bovine neonatal Fc receptor.

The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. Here we report our preliminary characterization of a clone encoding the alpha chain of the bovine FcRn from a BAC library. The recombinant BAC DNA was digested, analyzed by Southern blot hybridization and a bovine FcRn positive 9 kb long fragment was subcloned and partially sequenced. The exon/intron organization of the bovine FcRn alpha chain gene was deduced by comparison with its cDNA sequence. The sequence revealed a similar organization to the human and mouse FcRn alpha chain genes. The bovine FcRn alpha chain gene has acquired several repetitive sequences in its 5'-flanking region, including multiple SINE and LINE elements. Potential binding sites for transcription factors within the 5'-flanking sequence were identified using TESS and TFSEARCH programs. The 5'-flanking region of the bFcRn alpha chain gene was analyzed for its ability to directly express the luciferase reporter gene in bovine mammary gland epithelial cells. Transient transfection of a luciferase construct revealed that there was promoter activity in the region -1787 to +92 of the 5'-flanking sequence.