A ribosome-related signature in peripheral blood CLL B cells is linked to reduced survival following treatment.

We have used polysome profiling coupled to microarray analysis to examine the translatome of a panel of peripheral blood (PB) B cells isolated from 34 chronic lymphocytic leukaemia (CLL) patients. We have identified a 'ribosome-related' signature in CLL patients with mRNAs encoding for ribosomal proteins and factors that modify ribosomal RNA, e.g. DKC1 (which encodes dyskerin, a pseudouridine synthase), showing reduced polysomal association and decreased expression of the corresponding proteins. Our data suggest a general impact of dyskerin dysregulation on the translational apparatus in CLL and importantly patients with low dyskerin levels have a significantly shorter period of overall survival following treatment. Thus, translational dysregulation of dyskerin could constitute a mechanism by which the CLL PB B cells acquire an aggressive phenotype and thus have a major role in oncogenesis.

Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus.

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin.

Poly-(ADP-ribose) polymerase-1 (Parp-1) binds in a sequence-specific manner at the Bcl-6 locus and contributes to the regulation of Bcl-6 transcription.

Bcl-6 is a transcription factor that is normally expressed in germinal centre B cells. It is essential for the formation of germinal centres and the production of high-affinity antibodies. Transcriptional downregulation of Bcl-6 occurs on terminal differentiation to plasma cells. Bcl-6 is highly expressed in B-cell non-Hodgkin's lymphoma and, in a subset of cases of diffuse large cell lymphoma, the mechanism of Bcl-6 overexpression involves interruption of normal transcriptional controls. Transcriptional control of Bcl-6 is, therefore, important for normal antibody responses and lymphomagenesis, but little is known of the cis-acting control elements. This report focuses on a region of mouse/human sequence homology in the first intron of Bcl-6, which is a candidate site for such a control element. We demonstrate that poly-(ADP-ribose) polymerase-1 (Parp-1) binds in vitro and in vivo to specific sequences in this region. We further show that PARP inhibitors, and Parp-1 knockdown by siRNA induce Bcl-6 mRNA expression in Bcl-6 expressing cell lines. We speculate that Parp-1 activation plays a role in switching off Bcl-6 transcription and subsequent B-cell exit from the germinal centre.

Characterization of a novel calcium phosphate composite bone cement: flow, setting, and aging properties.

The flow, setting, and aging characteristics of a newly developed calcium phosphate/calcium aluminate composite orthopaedic cement were studied. The effect of vibration on the flow of the cement paste was studied and found to greatly enhance placement. The setting times of this cement were dependent on temperature and decreased with increasing temperatures. At 37 degrees C, the working and setting times were 6.3 +/- 0.3 and 12.8 +/- 0.4 minutes, respectively. Hydration and conversion of the cement phases continued while specimens were stored under simulated, physiological conditions. A cumulative increase in mass of 8.23 +/- 0.65% was observed over a 14 month test period. During this time, the cement was found to expand slightly, 0.71 +/- 0.39%. X-ray diffraction was used to characterize the crystalline phases present during hydration and conversion. The calcium aluminate in the cement hydrated and formed calcium aluminate chloride hydrates, while no changes were observed in the beta-tricalcium phosphate during the testing period.

A peptide aptamer to antagonize BCL-6 function.

BCL-6 is a transcription factor essential for germinal centre B-cell development. The BCL-6 gene is involved in diffuse large-cell lymphoma and overexpressed in other types of non-Hodgkin's lymphoma and in high-grade breast cancer. BCL-6 is a transcriptional repressor whose N-terminal POZ domain mediates protein-protein interactions to exert its effects. Reasoning that disruption of POZ domain-mediated interactions may be an effective route to antagonizing the effects of BCL-6 in lymphoma, we screened a library for peptide aptamers that specifically bind to BCL-6 POZ and not the POZ domains of related proteins and describe here the first of these reagents, Apt48. Apt48 binds BCL-6 POZ in a manner distinct from the transcriptional corepressor SMRT, yet was found to prevent BCL-6-mediated repression of a luciferase reporter gene. Apt48 also reproduced several previously validated effects of BCL-6 inhibition. Notably, expression of the differentiation markers CD69, Blimp-1 and cyclin D2 was increased in B-cell lines when Apt48 was expressed. We also show that expression of Apt48 restores cytokine-mediated growth arrest to BCL-6 overexpressing cells. Thus, we have identified a peptide aptamer that affects a function of BCL-6 that is required to prevent differentiation of proliferating B cells.

Novel calcium phosphate composite bone cement: strength and bonding properties.

A new high-strength cement prepared from calcium phosphate and calcium aluminate has been developed and was evaluated for potential use in bone and joint repair applications. Cement specimens were aged under simulated physiological conditions. The compressive strength of the cement was determined at time intervals 1 h after setting up to 52 weeks. A compressive strength of 111.6+/-12.9 MPa was measured at 4 weeks, with the cement attaining 64% of this maximum strength within 4 h of preparation. Compressive strength greater than 90 MPa was maintained up to 52 weeks. The strength of the cement-prosthesis interface was studied using a pull-out test. Polished, 316L stainless steel rods were implanted in canine cadaver femurs to simulate a cemented hip prosthesis. At 4, 24 h, and 60 days post implantation, the force required to displace the rod was measured. Mean interfacial shear strengths of 1.17+/-0.25, 1.11+/-0.21, and 1.11+/-0.32 MPa were observed at respective time-periods.

Arrested differentiation, the self-renewing memory lymphocyte, and vaccination.

Vaccination for persistent viral or bacterial infections must program the immune system for a lifelong need to generate antigen-specific effector lymphocytes. How the immune system does this is not known, but recent studies have shown that a subset of B lymphocytes, the germinal center B cell, is capable of self-renewal because it expresses a transcriptional repressor, BCL6, that blocks terminal differentiation. If a similar mechanism for arresting differentiation exists for long-lived, antigen-selected lymphocytes, a stem cell-like capacity for self-renewal could be the basis for the continual generation of effector lymphocytes from the memory pool. Understanding how to regulate the terminal differentiation of lymphocytes will improve immunotherapeutic approaches for chronic infectious diseases and cancer.

Suppression of signal transducer and activator of transcription 3-dependent B lymphocyte terminal differentiation by BCL-6.

Lymphocytes usually differentiate into effector cells within days after antigen exposure, except in germinal centers where terminal differentiation is delayed while somatic hypermutation creates high-affinity antibody mutants. Here we investigate whether arrest of terminal differentiation can be mediated by BCL-6, a transcriptional repressor that is expressed by germinal center B cells and is required for this phase of B cell development. We find that BCL-6 suppresses the differentiation of transformed and primary B cells to plasma cells by inhibiting the signal transducer and activator of transcription 3-dependent expression of the major regulator of plasma cell development, the B lymphocyte-induced maturation protein (Blimp-1). This function of BCL-6 as a repressor of B lymphocyte differentiation may also underlie the association between chromosomal translocations of its gene and B cell lymphomas.

Preferential selection of receptor-binding variants of influenza virus hemagglutinin by the neutralizing antibody repertoire of transgenic mice expressing a human immunoglobulin mu minigene.

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 G-->R, 225 G-->D) abrogated neutralization by each of the MAbs; nevertheless V-21.2 was recognized by its own selecting MAb in enzyme-linked immunosorbent assay and therefore qualified as an adsorptive mutant rather than an antigenic variant. We consider that a low-affinity neutralizing antibody response may preferentially select for receptor-binding variants of influenza virus HA.

Antibody expression from the core region of the human IgH locus reconstructed in transgenic mice using bacteriophage P1 clones.

Mice carrying transgenic human immunoglobulin gene miniloci can be used for the production of human monoclonal antibodies. The human variable region (V) gene segments in these miniloci undergo productive rearrangement in mouse lymphoid tissue to yield a population of B lymphocytes expressing a repertoire of antibodies. Many of the miniloci studied to date have included only a small number of germline gene segments in an artificially compact configuration. Here we describe the use of the bacteriophage P1 cloning system to create mice carrying the core region of the human immunoglobulin heavy chain (IgH) locus. Three P1 clones carrying overlapping regions of the human IgH locus (spanning the five JH-proximal VH segments, the entire DH and JH clusters, and the C mu and C delta constant regions) were injected into mouse eggs and appear to have reconstituted the core region of the locus (> 180 kb) following homologous recombination with each other. While this translocus yielded a titer of serum immunoglobulin similar to that obtained with a smaller plasmid-based minilocus, the P1-based locus gave rise to substantially greater diversification by somatic hypermutation. Such diversification is important for obtaining high-affinity antibodies. The results show the usefulness of the P1 system in facilitating the manipulation and recreation of large transgenes.

The targeting of somatic hypermutation.

Somatic hypermutation does not occur randomly within immunoglobulin V genes but, rather, is preferentially targeted to certain nucleotide positions (hot spots) and away from others (cold spots). Cold spots often coincide with residues essential for V gene folding. Hotspots, which appear to be strategically located to favour affinity maturation, are most frequently located in the CDRs (particularly CDR1) though conserved hotspots are also found at the base of FR3. Hotspots are in part created by local DNA sequence and the strong biases of codon usage in V genes indicate that the genes have evolved such that somatic hypermutation is targeted to those parts of the V where it is likely to prove most useful. These features of mutational hotspots and biased codon usage are also evident in V genes of lower animals suggesting that diversification by strategic targeting of non-templated mutation may have evolved early in antigen receptor evolution.

Somatic hypermutation of Ig genes in patients with xeroderma pigmentosum (XP-D).

Antibody diversification by somatic hypermutation occurs by the introduction of nucleotide substitutions in and around the rearranged Ig V gene segments. Several characteristics of the process suggest that the introduction of mutations is linked to Ig gene transcription. Since there is a connection between mutation and repair with indications that both processes might show linkage to transcription, we asked whether defects in a component of the transcription factor TFIIH which lead to an inability to carry out nucleotide excision repair also affect somatic hypermutation. A PCR strategy was devised that required small samples of peripheral blood and enabled us to monitor hypermutation of a single, abundantly used VH gene. However, the results showed that in xeroderma pigmentosum patients (complementation group D), somatic hypermutaton appears to take place unaffected as regard both extent and distribution.

Somatic hypermutation of immunoglobulin genes.

The relationship between somatic hypermutation and affinity maturation in the mouse is delineated. Recent work on the anatomical and cellular site of this process is surveyed. The molecular characteristics of somatic hypermutation are described in terms of the region mutated and the distinctive patterns of nucleotide changes that are observed. The results of experiments utilizing transgenic mice to find out the minimum cis-acting sequences required to recruit hypermutation are summarized. The hypothesis that V gene sequences have evolved in order to target mutation to certain sites but not others is discussed. The use that different species make of somatic hypermutation to generate either the primary or secondary B cell repertoire is considered. Possible molecular mechanisms for the hypermutation process and future goals of research are outlined.

The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci.

An approach to the preparation of antigen-specific human monoclonal antibodies focuses on mice transgenic for human immunoglobulin gene miniloci; the V gene segments in these miniloci undergo productive rearrangement to yield mouse B cells expressing human immunoglobulin (Ig) chains. The general usefulness of this strategy hinges on whether it is feasible to obtain specific, high-affinity antibodies following immunization of such animals with a variety of antigens. To test this, we have investigated the antigen-specific responses in mice which carry human IgH miniloci (constaining just one or two VH segments) instead of a functional mouse IgH locus. Although serum responses were relatively weak, monoclonal antibodies were readily obtained to all immunogens tested (a hapten, foreign proteins and human lymphoma cells). The affinities of two of the hapten-specific (anti-2-phenyl-oxazol-5-one) antibodies were 60 and 160 nM, values intermediate between what is typically obtained in the primary and secondary response of normal mice. Sequence analysis of the rearranged V genes revealed that junctional events made a major contribution to diversity with a considerable amount of apparently non-templated sequence at the V-D and D-J borders. Somatic hypermutation was also evident within the expressed V gene segments of many of the antigen-specific hybridomas. These findings augur well for the general usefulness of the transgenic approach for the isolation of high-affinity human antibodies to a wide range of antigens and suggests that the miniloci need not be particularly large.

Similar patterns of V kappa gene usage but different degrees of somatic mutation in hairy cell leukemia, prolymphocytic leukemia, Waldenstrom's macroglobulinemia, and myeloma.

To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from patients with lymphoproliferative disorders, we have polymerase chain reaction-amplified and sequenced a total of 26 V kappa genes from a total of 55 cases. Six sequences were obtained both from six cases of prolymphocytic leukemia (PLL) and from nine cases of hairy cell leukemia (HCL). Seven sequences were obtained both from 11 cases of Waldenström's macroglobulinemia (WM) and 29 cases of multiple myeloma (MM). Eleven different germline genes have been used in this series, indicating a wide but nonrandom usage of germline Ig gene rearrangements in these disorders. Comparison of the nucleotide sequences of V kappa genes obtained from B-cell malignancies with germline V kappa genes shows that somatic mutation is rare in PLL and HCL and common in WM and MM. Analysis of the pattern of mutations suggests that WM and MM are derived from B cells that have been selected by antigen at a relatively late stage of differentiation.

Antibodies generated from human immunoglobulin miniloci in transgenic mice.

One approach to the production of human monoclonal antibodies focusses on the creation of transgenic mice bearing human immunoglobulin gene miniloci. Whilst such loci undergo lymphoid-specific gene rearrangement, only a small proportion of mouse B cells express the human immunoglobulin chains; the miniloci thus contribute poorly to serum immunoglobulin. Attributing this poor performance to competition between the transgenic and endogenous immunoglobulin loci, we crossed mice bearing a human immunoglobulin heavy-chain (HulgH) minilocus with animals that had been rendered B cell-deficient by disruption of their endogenous heavy-chain locus. The results were dramatic: the human minilocus rescued B cell differentiation such that effectively all B cells now expressed human mu chains. The concentration of antibody in the mouse serum recognised by anti-human mu increased to a concentration about one sixth that in human serum. The HulgH antibodies are heterogenous with diversity being generated by both combinatorial and junctional processes. Following antigen challenge, specific antibody is elicited but at low titre.

V kappa gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the V kappa locus and do not show somatic mutation.

The structure of the human V kappa locus has been thoroughly investigated, but how the germ-line V kappa gene segment repertoire is actually sampled in kappa chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged V kappa genes from 26 kappa-expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four V kappa gene families were represented amongst rearranged genes. In 25 out of 32 cases, the sequence of the rearranged gene matches perfectly that of 1 of 11 different known germ-line V kappa genes, indicating that no somatic mutation has occurred. Of the remaining 7 rearranged V kappa genes, 4 differ from known germ-line genes by only one or two amino acid residues; and 3 differ from each other and from all known sequences by 5 or more residues, suggesting that somatic mutation has occurred in these 3 cases. We conclude that: (a) in at least three-quarters of cases the rearranged genes are unmutated; (b) there is preferential usage of individual V kappa genes but not of V kappa gene families; and (c) the V kappa genes used are widely dispersed in the V kappa locus.