Pax5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. To better understand the functional importance of Pax5 at the later stages of B cell differentiation, we established a Pax5-deficient DT40 B cell line. The Pax5(-/-) cells exhibited slower growth, decreased surface IgM expression, and total loss of B cell receptor signaling. Moreover, the expression of the plasma cell-characteristic transcription factors Blimp-1 and XBP-1 were significantly upregulated and the expression of Bcl-6 diminished in the Pax5(-/-) cells, and this alteration was normalized by restored Pax5 expression. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results indicate that downregulation of Pax5 function promotes the plasma cell differentiation of B cells.

The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cc2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.

The Ikaros family transcription factor Aiolos is important for B cell function, since B cells of Aiolos‐null mutant mice exhibit an activated phenotype, enhanced B‐cell receptor (BCR) signalling response and develop a systemic lupus erythematosus (SLE) type autoimmune disease. Aiolos has also been reported to interact with anti‐apoptotic Bcl‐2 and Bcl‐xL in T cells, but whether Aiolos regulates cell death has not been studied in B cells. Here we show that the disruption of Aiolos in the DT40 B cell line induces a cell death sensitive phenotype, as the Aiolos−/− cells are more prone to apoptosis by nutritional stress, BCR cross‐linking, UV‐ or γ‐irradiation. Furthermore, the Aiolos−/− cells have defective Ig gene conversion providing evidence that Aiolos is needed for the somatic diversification of the BCR repertoire. The re‐expression of DNA‐binding isoform Aio‐1 was able to restore the gene conversion defect of the Aiolos‐deficient cells, whereas the introduction of dominant negative isofom Aio‐2 had no effect on gene conversion, thus demonstrating the functional importance of alternative splicing within Ikaros family. Although the Aiolos−/− cells exhibit reduced expression of activation‐induced cytidine deaminase (AID), ectopic AID overexpression did not restore the gene conversion defect in the Aiolos−/− cells. Our findings indicate that Aiolos may regulate gene conversion in an AID independent manner.

The purpose of the study was to examine the response of the endoneurium of the rat sciatic nerve after removal of the epi- and perineurium. For this purpose, segments (4-5 mm long) of the whole epi- and perineurium around the rat sciatic nerve were microsurgically removed (the peel-off area) and the endoneurium was left intact. The post-operative changes were followed up to 5 weeks post-operatively (PO) by histo- and immunohistochemical studies. Additionally, neuromorphometric analyses considering the number of Schwann cells, axons, macrophages and endothelial cells were examined in the peel-off area. The results showed that at the operative area the central part of the endoneurium (65% of the total area of the endoneurium) remained morphologically intact, but the outer part of the endoneurium (35% of the total area) reacted strongly and showed Wallerian type of degeneration. The number of axons and Schwann cells decreased 3 days PO. However, after 2 weeks the number of Schwann cells increased markedly and the highest number was noted 5 weeks PO. A great number of capillaries were observed in the outer part 1 week PO. A rapid invasion of macrophages was noted at the outer part of the endoneurium immediately after the operation. During the regeneration the endoneurial fibroblasts in the peripheral zone started to form minifascicle-like formations, which resulted in a distinct dense outer part of the endoneurium. This dense outer zone was preserved up to 5 weeks PO and participated in the formation of a new perineurium-like structure, but no distinct new perineurium was formed. At the border zone, areas beside the normal epi- and perineurium proliferation of preserved perineurial cells were noted, which fused to the outer part of the dense endoneurium. On focal areas, an attachment of the operated area to the adjoining muscle was observed. This study shows for the first time that despite the microsurgical removal of epi- and perineurium, the inner part of the endoneurium stays intact, but in the outer part of the endoneurium marked reactive changes ensue, probably to protect the injured peripheral nerve.

Flow cytometry (FCM) has provided an efficient method to determine DNA content in cells, thus providing a feasible alternative to microscopy for the estimation of ploidy for population level studies requiring large sample sizes. In cytological studies, searching for example chromosomal abnormalities, samples are commonly treated in separate tubes to ensure the capture of sufficient number of separate cells to meet the required resolution (nuclei number > 5000 and coefficient variation (CV) < 5%). In this study, we tested whether FCM can be expedited by using 96-well plate-based FCM sample preparation and automated data analysis using Flowing Software without compromising the resolution required for ploidy level detection in population biology. We estimated ploidy levels in red fescue (Festuca rubra L.) and viviparous sheep's-fescue (F. vivipara Sm.) plants. A tube-based procedure gave more tightly defined peaks compared to a plate-based procedure. However, our results suggest that the plate-based higher throughput FCM procedure gives equally reliable estimation of the number of sets of chromosomes (ploidy) in plant with three times less reagent consumed and over 20 times more time-efficiently compared to the tube-based FCM procedure. We propose that the integration of plate-based FCM with Flowing Software provides a time-and cost-efficient and less error-prone procedure to determine ploidy levels in taxonomy, evolutionary ecology and plant breeding.