IntroductionSomatic hypermutation (SHM) is a specialized process that takes place in germinal center (GC) B cells in response to T celldependent antigen stimulation. 1,2 This process introduces single nucleotide substitutions, with occasional deletions and duplications, primarily into the variable region of the immunoglobulin (IgV) heavy and light chain genes, resulting in the production of high-affinity antibodies and allowing affinity maturation of the humoral immune response. 3 SHM requires active transcription of the target locus but is not IgV sequence specific and does not depend on the V region promoter. [4][5][6][7] In fact, at least 3 non-Ig genes, including BCL6, the FAS/CD95 gene, and the genes encoding the 2 components of the BCR (B29 and mb1) have been shown to acquire somatic mutations during the normal GC reaction, indicating that this mechanism may target more genes than originally suspected. [8][9][10][11][12] The molecular basis of SHM remains largely unknown. However, studies from the past 4 years have identified the AID gene, encoding for activation-induced cytidine deaminase, as an absolute requirement for both SHM and class switch recombination (CSR) in humans and mice. 13,14 Activation-induced cytidine deaminase (AID) expression is also sufficient to initiate both events in fibroblasts expressing transcribed artificial constructs. 15,16 Because of the high homology with the RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1), it has been proposed that AID may function as a cytidine deaminase to modify a preexisting mRNA into a new one, possibly encoding an endonuclease. 14,17 However, experimental evidence in Escherichia coli indicated that AID may act directly on DNA and convert deoxycytidines to uracils, which are then processed by uracil-DNA glycosylase (UNG) and endonucleases. 18 AID would thereby lead to the creation of abasic sites, which may be repaired by base excision repair and putative error-prone mechanisms. Indeed, in vitro AID exhibits cytidine deamination activity on single-stranded DNA, with a base specificity similar to that reported for SHM. [19][20][21][22] Recently, the SHM process has been shown to malfunction in about 50% of diffuse large B-cell lymphomas (DLBCLs) as well as in about 20% of AIDS-related non-Hodgkin lymphomas (NHLs) and in a significant fraction of primary central nervous system lymphomas derived from non-HIV patients. [23][24][25] In these tumors, multiple somatic mutations are introduced into the 5Ј region, including coding sequences, of several genes that do not represent physiologic SHM targets. These comprise the well-known protooncogenes PIM1, PAX5, RhoH/TTF, and cMYC, all of which have Two of the authors (R.P. and J.M.) are employed by a company (Cell Signaling Technology, Inc, Beverly, MA) whose potential product was studied in the present work.The online version of this article contains a data supplement.An Inside Blood analysis of this article appears in the front of this issue.Reprints: Laura Pasqualucci, Institute for...

Non-Hodgkin's lymphomas (NHL) form a heterogeneous group of diseases, with diffuse large B-cell lymphoma (DLBCL) comprising the largest subgroup. The commonest chromosomal translocations found in DLBCL are those affecting band 3q27. In 35% of DLBCL cases, as well as in a small fraction of follicular lymphomas, the normal transcriptional regulation of Bcl-6 is disrupted by these chromosomal translocations. In addition, about three-quarters of cases of DLBCL display multiple somatic mutations in the 5' non-coding region of Bcl-6, which occur independently of chromosomal translocations and appear to be due to the IgV-associated somatic hypermutation process. Bcl-6 is a 95-kD nuclear phosphoprotein belonging to the BTB/POZ (bric-a-brac, tramtrack, broad complex/Pox virus zinc finger) zinc finger family of transcription factors. It has been suggested that Bcl-6 is important in the repression of genes involved in the control of lymphocyte activation, differentiation, and apoptosis within the germinal center, and that its down-regulation is necessary for normal B-cells to exit the germinal center. Bcl-6 remains constitutively expressed in a substantial proportion of B-cell lymphomas. Recently, acetylation has been identified as a mode for down-regulating Bcl-6 activity by inhibition of the ability of Bcl-6 to recruit complexes containing histone deacetylases (HDAC). The pharmacologic inhibition of two recently identified deacetylation pathways, HDAC- and silent information regulator (SIR)-2-dependent deacetylation, results in the accumulation of inactive acetylated Bcl-6 and thus in cell cycle arrest and apoptosis in B-cell lymphoma cells. These results reveal a new method of regulating Bcl-6, with the potential for therapeutic exploitation. These studies also indicate a novel mechanism by which acetylation promotes transcription, not only by modifying histones and activating transcriptional activators, but also by inhibiting transcriptional repressors.

IL-12 activates murine and human B cells, but little information is available as to the expression and function of IL-12R on human B lymphocytes. Here we show that the latter cells, freshly isolated from human tonsils, expressed the transcripts of both β1 and β2 chains of IL-12R and that β2 chain mRNA was selectively increased (4- to 5-fold) by incubation with Staphylococcus aureus Cowan I bacteria or IL-12. B cell stimulation with IL-12 induced de novo expression of the transcripts of the two chains of IL-18R, i.e., IL-1 receptor-related protein and accessory protein-like. Functional studies showed that both IL-12 and IL-18 signaled to B cells through the NF-κB pathway. In the case of IL-12, no involvement of STAT transcription factors, and in particular of STAT-4, was detected. c-rel and p50 were identified as the members of NF-κB family involved in IL-12-mediated signal transduction to B cells. IL-12 and IL-18 synergized in the induction of IFN-γ production by tonsillar B cells, but not in the stimulation of B cell differentiation, although either cytokine promoted IgM secretion in culture supernatants. Finally, naive but not germinal center or memory, tonsillar B cells were identified as the exclusive IL-12 targets in terms of induction of NF-κB activation and of IFN-γ production.

This study shows that SDF-1 substantially enhances the migration of follicular center lymphoma B cells but not the migration of freshly purified germinal center B cells. This difference may be related to the extended survival of follicular center lymphoma versus germinal center B cells. SDF-1 produced in follicular center lymphoma lymph nodes may play a role in the local dissemination of tumor cells.

A 10-month-old male maremma shepherd dog was presented with chronic diarrhoea, moderate polyuria/polydipsia, lethargy, dysorexia and stiffness. Pain was elicited in the distal parts of all four limbs. Radiographs of the limbs showed increased endomedullary radiopacity and lysis, with thick periosteal proliferations at the metadiaphyseal areas of each radius-ulna and tibia and of the distal metacarpus on one side. Polyclonal hypergammaglobulinaemia was documented and a similar electrophoretic protein pattern was also found in the synovial fluid. Leishmania amastigotes were found in the macrophages in a bone marrow aspirate performed at the level of a distal radius and in a synovial fluid sample obtained from a carpal joint. An indirect immunofluorescence test confirmed the infection. Treatment with N-methyl-glucamine antimoniate was successful and the osteoarticular changes progressively disappeared.

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Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191-18_c.2213del) that affects the splicing of intron 22 and generates multiple out-of-frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C-ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PV's cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes.

ABSTRACT