Febe van Maldegem

Differential expression of interleukin-17 family cytokines in intact and complicated human atherosclerotic plaques†

In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL‐17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL‐17A, ‐E and ‐F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL‐17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL‐17A, ‐E and ‐F were expressed in the majority of plaques under investigation. IL‐17A/F was expressed by mast cells in all stages of plaque development. IL‐17A/F+ neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL‐17A/F+ Tcells (‘TH17’) were never observed. IL‐17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL‐17E and its functional receptor (IL‐17RB). The constitutive expression of IL‐17E by resident plaque cells, and the additional presence of IL‐17E+ B cells and IL‐17A/F+ neutrophils in advanced and complicated plaques indicates a complex contribution of IL‐17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease. Copyright

Germinal centers in human lymph nodes contain reactivated memory B cells

To reveal migration trails of antigen-responsive B cells in lymphoid tissue, we analyzed immunoglobulin (Ig)M-VH and IgG-VH transcripts of germinal center (GC) samples microdissected from three reactive human lymph nodes. Single B cell clones were found in multiple GCs, one clone even in as many as 19 GCs. In several GCs, IgM and IgG variants of the same clonal origin were identified. The offspring of individual hypermutated IgG memory clones were traced in multiple GCs, indicating repeated engagement of memory B cells in GC reactions. These findings imply that recurring somatic hypermutation progressively drives the Ig repertoire of memory B cells to higher affinities and infer that transforming genetic hits in non-Ig genes during lymphomagenesis do not have to arise during a single GC passage, but can be collected during successive recall responses.

Effects of processing delay, formalin fixation, and immunohistochemistry on RNA recovery from formalin-fixed paraffin-embedded tissue sections

Contemporary pathology involves an emerging role for molecular diagnostics. Current tissue handling procedures [ie, formalin fixation and paraffin embedment (FFPE)] have their origin in the aim to obtain good tissue morphology and optimal results within immunohistochemistry. Unfortunately, FFPE is notorious for its poor RNA conservation capacities. In this study, we have examined the impact of the individual steps in tissue handling processes on the RNA extractability, quality, and usability for reverse-transcription polymerase chain reaction. It was found that a prolonged prefixation time (ie, the time between tissue dissection and fixation) has a measurable impact on RNA integrity when analyzed with the Agilent Bioanalyzer. Surprisingly, however, the deteriorated RNA quality hardly had any consequences for reverse-transcription polymerase chain reaction yields. Furthermore, we assessed the optimal fixation time for RNA preservation, and we found that an RNA heating step, preceding copy DNA synthesis, significantly increases the RNA template length. Finally, we provide a protocol for RNA isolation from immunohistochemically stained FFPE tissue sections. Thus, by applying alterations to tissue handling procedures, archival FFPE tissues become well suitable for RNA-based molecular diagnostics.

The majority of cutaneous marginal zone B-cell lymphomas expresses class-switched immunoglobulins and develops in a T-helper type 2 inflammatory environment

Extranodal marginal zone B-cell lymphomas (MZBCLs) arise on a background of chronic inflammation resulting from organ-specific autoimmunity, infection, or by unknown causes. Well-known examples are salivary gland MZBCL in Sjögrens sialadenitis and gastric MZBCL in Helicobacter pylori gastritis. MZBCLs express CXCR3, a receptor for interferon-gamma-induced chemokines highly expressed in the chronic inflammatory environment. The immunoglobulin (Ig) variable heavy/light chain (IgV(H)/IgV(L)) gene repertoire of salivary gland and gastric MZBCL appears restricted and frequently encodes B-cell receptors with rheumatoid factor reactivity. Primary cutaneous marginal zone B-cell lymphomas (PCMZLs) are regarded as the skin-involving counterparts of extranodal MZBCLs. Although PCMZLs have been associated with Borrelia burgdorferi dermatitis, PCMZLs generally arise because of unknown causes. We studied an extensive panel of PCMZLs and show that PCMZLs do not conform to the general profile of extranodal MZBCL. Whereas most noncutaneous MZBCLs express IgM, PCMZLs in majority express IgG, IgA, and IgE and do not show an obvious immunoglobulin repertoire bias. Furthermore, the isotype-switched PCMZLs lack CXCR3 and seem to arise in a different inflammatory environment, compared with other extranodal MZBCLs.

AID splice variants lack deaminase activity

To the editor: With particular attention we read the article by Wu et al[1][1] dealing with regulation of activation-induced cytidine deaminase (AID) by alternative splicing. We have also studied 3 alternative AID splice forms identical to the variants AID-ΔE4, AID-ΔE4a, and AID-ivs3 described by

Deficiency in spliceosome-associated factor CTNNBL1 does not affect ongoing cell cycling but delays exit from quiescence and results in embryonic lethality in mice.

CTNNBL1 is an armadillo-repeat protein that associates with the CDC5L/Prp19 complex of the spliceosome. Unlike the majority of spliceosomal proteins (and despite having no obvious homologs), CTNNBL1 is inessential for cell viability as revealed by studies in both vertebrate B cell lines and in fission yeast. Here, however, we show that ablation of CTNNBL1 in the mouse germline results in mid-gestation embryonic lethality but that lineage-specific CTNNBL1 ablation in early B cell precursors does not affect the production and abundance of mature B lymphocytes. However, CTNNBL1-deficient resting B lymphocytes show sluggish exit from quiescence on cell activation, although once entry into cycle has initiated, proliferation and differentiation in response to mitogenic stimuli continue largely unaffected. A similar sluggish exit from quiescence is also observed on reprovision of nutrients to nitrogen-starved CTNNBL1-deficient yeast. The results indicate that, whereas other RNA splicing-associated factors have been connected to cell cycle progression, CTNNBL1 plays no essential role in cycling cells but does fulfill an evolutionarily conserved function in helping cells to undergo efficient exit from quiescence following activation.

Activation-Induced Cytidine Deaminase Splice Variants Are Defective Because of the Lack of Structural Support for the Catalytic Site

Recently, conflicting results were reported on the hypermutation activity of activation-induced cytidine deaminase (AID) splice variants. With the generation of single point mutations, we studied the structure-function relationship of the amino acids that are commonly absent from all described splice variants. The results from this analysis pointed to several amino acids that are required for class switch recombination (CSR), without perturbing cellular localization or nucleocytoplasmic shuttling. A defect in deaminase activity was found to underlie this CSR deficiency. Interestingly, the most debilitating mutations concentrated on hydrophobic amino acids, suggesting a structural role for this part of the protein. Indeed, by generating homologous amino acid replacements, CSR activity could be restored. These results are in agreement with recent reports on the protein structure of the AID homolog APOBEC3G, suggesting a similar protein composition. In addition, the findings underscore that AID splice variants are unlikely to have preservation of catalytic activity.

Structural and mutational analysis reveals that CTNNBL1 binds NLSs in a manner distinct from that of its closest armadillo-relative, karyopherin α.

CTNNBL1 is a spliceosome‐associated protein that binds nuclear localization signals (NLSs) in splice factors CDC5L and Prp31 as well as the antibody diversifying enzyme AID. Here, crystal structures of human CTNNBL1 reveal a distinct structure from its closest homologue karyopherin‐α. CTNNBL1 comprises a HEAT‐like domain (including a nuclear export signal), a central armadillo domain, and a coiled‐coil C‐terminal domain. Structure‐guided mutations of the region homologous to the karyopherin‐α NLS‐binding site fail to disrupt CTNNBL1–NLS interactions. Our results identify CTNNBL1 as a unique selective NLS‐binding protein with striking differences from karyopherin‐αs.

CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex

In order to catalyse the splicing of messenger RNA, multiple proteins and RNA components associate and dissociate in a dynamic highly choreographed process. The Prp19 complex is a conserved essential part of the splicing machinery thought to facilitate the conformational changes the spliceosome undergoes during catalysis. Dynamic protein interactions often involve highly disordered regions that are difficult to study by structural methods. Using amine crosslinking and hydrogen–deuterium exchange coupled to mass spectrometry, we describe the architecture of the Prp19 sub-complex that contains CTNNBL1. Deficiency in CTNNBL1 leads to delayed initiation of cell division and embryonic lethality. Here we show that in vitro CTNNBL1 enhances the association of CWC15 and CDC5L, both core Prp19 complex proteins and identify an overlap in the region of CDC5L that binds either CTNNBL1 or CWC15 suggesting the two proteins might exchange places in the complex. Furthermore, in vivo, CTNNBL1 is required to maintain normal levels of the Prp19 complex and to facilitate the interaction of CWC15 with CDC5L. Our results identify a chaperone function for CTNNBL1 within the essential Prp19 complex, a function required to maintain the integrity of the complex and to support efficient splicing.