Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components

Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms

Compósito para a construção civil a partir de resíduos industriais

RESUMO Os estudos que visam à utilização dos resíduos industriais vêm se intensificando diante da pressão das organizações ambientais, escassez de recursos naturais, busca de certificações para ganho de mercado e minimização de impacto ambiental. O presente trabalho desenvolveu um novo compósito a base de resíduos industriais como cinzas de madeira, lodo de estação de tratamento de água (ETA) e resíduos de produção de cal com propriedades mecânicas que atendem às exigências da ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - NBR 15270-1/2005, NBR 15270-2/2005 e NBR 5739/2007, objetivando a sua utilização na construção civil sem o acréscimo de cimento Portland. As resistências à compressão variaram de 2,35 a 16,48 MPa. O índice de absorção de água das amostras testadas também atendeu às normas aplicadas, demonstrando que ao longo do tempo de cura houve diminuição da porosidade com possível hidratação da cal. Os resultados do ensaio de resistência à compressão apresentaram variações durante o tempo de cura que podem ser justificadas pela presença de material orgânico no lodo de ETA e pelo tamanho das partículas de cinza de madeira que durante a homogeneização não foram destruídas completamente. Apesar das variações observadas nos resultados, as resistências dos compósitos se enquadram na classificação para blocos cilíndricos de concreto e blocos cerâmicos para alvenaria

Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host

Background: Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from reinfection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157: H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls. Results: In tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed. Conclusions: Although the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157: H7 (Stx(-)) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function

Functional analysis of Ig-α signaling in the context of B cell development and response

The B cell antigen receptor (BCR) signaling cascade is initiated by phosphorylation of tyrosine residues in the Immunoreceptor Tyrosine based Activation Motif (ITAM) of Ig-α and Ig-β. Signals initiated by the pre-BCR in pre-B cells and the BCR in immature and mature cells can lead to cell maturation, selection, terminal differentiation or cell death. It is still unclear how a single receptor can mediate such a variety of cellular responses. Our aim is to develop a system for the structural and functional analysis of Ig-α in vivo and to understand its role in B cell development, selection and response. We use retroviral gene transfer to introduce different mb-1 (Ig-α encoding gene) mutants into hematopoietic stem cells of Ig-α-deficient mice. Transduced cells are reinjected into irradiated recipient mice where they can differentiate into B cells. Here we have focused on the serine and threonine residues of the cytoplasmic region of Ig-α. These residues are phosphorylated upon BCR engagement and studies in cell lines indicated their potential negative regulatory role in BCR signaling. We found that Ig-α deficient hematopoietic stem cells transduced with retroviral vectors containing either the wild-type or a serine/threonine mutated mb-1 sequence were able to differentiate into IgM expressing cells in vivo. Thus, our data indicate that the serine and threonine residues do not play a dominant role in pre-BCR signaling as pre-B cells and immature B cells expressing serine and treonine mutant Ig-α develop. We are in the process of testing the effect of these mutations in B cell negative selection and B cell response

B Cell Progenitors Are Arrested in Maturation but Have Intact VDJ Recombination in the Absence of Ig-α and Ig-β¹

Ig-α and Ig-β mediate surface expression and signaling of diverse B cell receptor complexes on precursor, immature, and mature B cells. Their expression begins before that of the Ig chains in early progenitor B cells. In this study, we describe the generation of Ig-α-deficient mice and their comparative analysis to mice deficient for Ig-β, the membrane-IgM, and recombination-activating gene 2 to determine the requirement of Ig-α and Ig-β in survival and differentiation of pro-B cells. We find that in the absence of Ig-α, B cell development does not progress beyond the progenitor stage, similar to what is observed in humans lacking this molecule. However, neither in Ig-α- nor in Ig-β-deficient mice are pro-B cells impaired in V(D)J recombination, in the expression of intracellular Ig μ-chains, or in surviving in the bone marrow microenvironment. Finally, Ig-α and Ig-β are not redundant in their putative function, as pro-B cells from Ig-α and Ig-β double-deficient mice are similar to those from single-deficient animals in every aspect analyzed

Efficient generation of B lymphocytes by recognition of self-antigens

Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock-in model system of the 3-83 autoreactive B-cell antigen receptor (BCR) in combination with precursor-BCR (pre-BCR) deficiency, we show here that the 3-83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto-antigen. Experiments with mixed bone marrow chimeras showed that combining the 3-83 BCR with the corresponding auto-antigen resulted in efficient reconstitution of B-cell development in immune-deficient mice. These results suggest that B cells are positively selected by recognition of self-antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre-BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains

Canonical NF-κB Activity, Dispensable for B Cell Development, Replaces BAFF-Receptor Signals and Promotes B Cell Proliferation upon Activation

The maintenance of mature B cells hinges on signals emitted from the BAFF-R cell-surface receptor, but the nature of these signals is incompletely understood. Inhibition of canonical NF-κB transcription factor activity through ablation of the essential scaffold protein NEMO arrests B cell development at the same stage as BAFF-R deficiency. Correspondingly, activation of this pathway by constitutively active IκB Kinase2 renders B cell survival independent of BAFF-R:BAFF interactions and prevents proapoptotic PKCδ nuclear translocation. In addition, canonical NF-κB activity mediates differentiation and proper localization of follicular and marginal zone B cells in the absence of BAFF-R, but not CD19. By replacing BAFF-R signals, constitutive canonical NF-κB signaling, a hallmark of various B cell lymphomas, causes accumulation of resting B cells and promotes their proliferation and survival upon activation, but does not per se induce lymphomagenesis. Therefore, canonical NF-κB activity can substitute for BAFF-R signals in B cell development and pathogenesis