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

In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin

Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin zymography that the tumors contained MMP-2 and MMP-9, but only MMP-2 was present in the active form. Immunolocalization of MMP-2 showed that the protein was localized at basement membranes of colon cancer cells and in intratumor stroma, associated with extracellular matrix (ECM) components. However, zymography and immunohistochemistry (IHC) do not provide information on the localization of MMP activity. Therefore, we developed an in situ zymography technique using the quenched fluorogenic substrate DQ-gelatin in unfixed cryostat sections. The application of DQ-gelatin in combination with a gelled medium allows precise localization of gelatinolytic activity. Fluorescence due to gelatinolytic activity was found in the ECM of tumors and was localized similarly to both MMP-2 protein and collagen type IV, its natural substrate. The localization of MMP-2 activity and collagen type IV at similar sites suggests a role of MMP-2 in remodeling of ECM of stroma in colon cancer metastases in rat live

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. The Journal of Immunology, 2010, 184: 2487-249

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 diagnostic

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 Sjorgren's 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 counter-parts 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

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

AbstractCTNNBL1 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