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Results for “Complementarity Determining Regions”

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PubMed2002

Salivary gland B cell lymphoproliferative disorders in Sjögren's syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins's lymphomas.

Sjögren's syndrome (SS) represents a pathological model of the evolution from polyclonal B lymphocyte activation to oligoclonal/monoclonal B cell expansion, which may culminate in the development of a malignant lymphoproliferative disease. The different phases of this process are usually marked by the appearance of antigen-driven activated B cell clones, which are commonly IgM-positive and with rheumatoid factor (RF) activity. However, the agent(s) able to trigger B cell proliferation is still unknown. A similar pathogenetic mechanism exist in mixed cryoglobulinemia, another autoimmune disease that often evolves to non-Hodgkin's lymphoma (NHL) and in which hepatitis C virus (HCV) infection has been demonstrated to play an etiopathogenetic role. In the present study, we cloned and sequenced the antigen receptor (IgR) variable region genes of SS-associated monoclonal non-neoplastic lymphoproliferations and compared them with those of our previous reported HCV-associated NHL, to derive clues on the antigen(s) that sustains SS. The results obtained showed remarkable homologies between the antigen combinatory regions of the IgR expressed by both diseases. These homologies concern: a) the specific combinations of heavy and light variable region genes; b) the limited length of complementarity-determining regions (CDR3); c) the homology with antibodies with RF activity; d)the amino acid sequences of CDR3 in which common somatic mutations are present that possibly determine the antigen-binding specificity. In conclusion, although there are significant differences between SS and HCV-associated lymphoproliferative diseases, they share many molecular characteristics, which suggest an immunological cross-reactivity or molecular mimicry among the agents that underlie these disorders.

Adult↗

PubMed1993

Ex vivo clonotype primer-directed gene amplification to identify malignant T cell repertoires.

A novel strategy that utilizes input genomic DNA and overcomes limitations encountered with traditional RNA reverse transcription-polymerase chain reaction (PCR) amplification methodology is described to screen for T cell receptor (TCR) repertoires. The methodology has been developed to identify individual T cell clonotypes with regard to their unique receptor beta chain variable/diversity/joining (VDJ) region gene rearrangement. The technique avoids preselection for a given antigen specificity and is therefore independent of artificial bias introduced by in vitro cell population expansion. This technique was used to detect and identify genetically of malignant clones from heterogeneous mononuclear cell populations from an array of hemato-oncological disorders, including mycosis fungoides/Sézary Syndrome, adult T cell leukemia, and large granular lymphoproliferative disease. An initial primary PCR, directed by a TCR-J beta generic primer and a complement of family-specific TCR-V beta primers, defines predominant T cell receptor variable gene usage. Use of a TCR-J beta generic primer supplants the use of a constant region primer anchor and thus eliminates the need to target mRNA. The process of variable gene screening also expedites gene sequencing. By sequencing through the VDJ juxtaposed region, i.e., the third complementarity determinant region, clonotype-specific primers are developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, unique T cell clonal repertoires. Analysis of the products of the CPD-PCR permits the detection of a single malignant cell among one million polyclonal cells and supercedes the constraints of prior studies that provide a limited evaluation of family variable gene repertoire usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones.

Base Sequence↗

PubMed1996

Diversity of human T cell receptor sequences of T cell clones with specificity for Bet v 1 peptide/MHC II complexes.

T cell clones (TCC) were raised from the peripheral blood of patients suffering from tree pollen allergy. All TCC were restricted by HLA-DR molecules. In order to investigate possible intervention targets in Type I allergic diseases, we have examined T cell receptor (TCR) alpha- and beta-chain nucleotide sequences of several allergen-reactive human CD4+ TCC specific for four frequently found epitopes of Bet v 1, the major birch pollen allergen. In general, TCC specific for the 4 epitopes investigated, used diverse TCRAV and TCRBV gene segments. Moreover, the junctional regions encoding the third complementarity determining regions (CDR3) of the TCR showed striking heterogeneities in length and amino acid composition. A more restricted use of two J gene segments (TCRBJ1S4 and 2S7) was only observed in the beta-chain of TCR used by TCC specific for epitope 1. In addition, all TCC specific for epitope 4 showed an arginine residue in the N-terminal region of their TCRBV CDR3 loops despite their sequence diversities. In view of the striking heterogeneities found, therapeutical strategies aimed at the clonal deletion of allergen-specific T cell clones, providing help for IgE synthesis, may not be feasible. Moreover, our results cast a doubt on the theory, that the CDR3 exclusively provides the primary contact with the peptide bound in the major histocompatibility (MHC) groove, and suggest additional interaction with MHC class II.

Allergens↗

PubMed1991

Evolution of a VH gene family in low vertebrates.

Antibody genes represent one of the most complex receptor gene systems which have evolved in the vertebrate lineage. One of the central questions is how antibody variable region genes diversify the complementarity determining region (CDR) in evolution while they are subject to forces for sequence homogenization operating in multigene families. Information on germ line antibody genes in lower vertebrates is still fragmentary and it would be fruitful to gain insight into this question. We have studied the evolutionary stability of a rainbow trout VH family (RTVH 431) by Southern blot hybridization in various fish species. Comparison of our results with paleontological/zoological evidence suggests that this VH family has been preserved in the genomes of many fish species over a time span of 150 million years. We also show that there exist species-specific residues common to rainbow trout VH genes, which strongly supports the presence of genetic processes for homogenization. These results are compatible with the view that antibody V genes evolve at a fairly constant rate and that the homogenization process (e.g. gene duplication) may be slow enough to allow diversification of CDR by mutation, selection and drift.

Amino Acid Sequence↗

PubMed2001

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

Amino Acid Sequence↗

PubMed2004

The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates gammadelta T cell clones that express unique T cell receptors.

Major surface protein 2 (MSP2) of the bovine rickettsial pathogen Anaplasma marginale is an abundant, serologically immunodominant outer membrane protein. Immunodominance partially results from numerous CD4+ T cell epitopes in highly conserved amino and carboxy regions and the central hypervariable region of MSP2. However, in long-term cultures of lymphocytes stimulated with A. marginale, workshop cluster 1 (WC1)+ gammadelta T cells and CD4+ alphabeta T cells proliferated, leading to a predominance of gammadelta T cells. As gammadelta T cells proliferate in A. marginale-stimulated lymphocyte cultures, this study hypothesized that gammadelta T cells respond to the abundant, immunodominant MSP2. To test this hypothesis, gammadelta T cell clones were isolated from MSP2 vaccinates and assessed for antigen-specific proliferation and interferon-gamma secretion. Seven WC1+ gammadelta T cell clones responded to A. marginale and MSP2, and three of these proliferated to overlapping peptides from the conserved carboxy region. The gammadelta T cell response was not major histocompatibility complex-restricted, although it required antigen-presenting cells and was blocked by addition of antibody specific for the T cell receptor (TCR). Sequence analysis of TCR-gamma and -delta chains of peripheral blood lymphocytes identified two novel TCR-gamma chain constant (Cgamma) regions. It is important that all seven MSP2-specific gammadelta T cell clones used the same one of these novel Cgamma regions. The TCR complementarity-determining region 3 was less conserved than those of MSP2-specific CD4+ alphabeta T cell clones. Together, these data indicate that WC1+ gammadelta T cells recognize A. marginale MSP2 through the TCR and contribute to the immunodominant response to this protein.

Anaplasma marginale↗

PubMedDate not supplied

HIV-1 gp120 and immune network.

It has been demonstrated that the immunodominant V3 loop of HIV-1 gp120 and its flanking regions bear sequence and structural homology to the framework and complementarity-determining regions of human immunoglobulins. It has been proposed that the Ig-like domain of gp120 might encode idiotypes and in this way permit HIV-1 entry into the immune regulatory network. This notion is strongly supported by results demonstrating that the anti-V3 loop and anti-Ig antibodies of healthy individuals share complementary structure and that V3 reactive antibodies are present in HIV-negative sera. This might be the mechanism by which HIV induces immunological abnormalities, and it should be taken into consideration in AIDS vaccine development.

Complementarity Determining Regions↗

PubMed1987

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits.

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.

Amino Acid Sequence↗

PubMed1990

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.

Animals↗

PubMed2000

Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia.

Analysis of the immunoglobulin receptor (IGR) variable heavy- and light-chain sequences on 17 hepatitis C virus (HCV)-associated non-Hodgkin lymphomas (NHLs) (9 patients also had type II mixed cryoglobulinemia [MC] syndrome and 8 had NHL unrelated to MC) and analysis of intraclonal diversity on 8 of them suggest that such malignant lymphoproliferations derive from an antigen-driven pathologic process, with a selective pressure for the maintenance of a functional IgR and a negative pressure for additional amino acid mutations in the framework regions (FRs). For almost all NHLs, both heavy- and light-chain complementarity-determining regions (CDR3) showed the highest similarity to antibodies with rheumatoid factor (RF) activity that have been found in the MC syndrome, thus suggesting that a common antigenic stimulus is involved in MC syndrome and in HCV-associated lymphomagenesis. Moreover, because HCV is the recognized pathologic agent of MC and the CDR3 amino acid sequences of some HCV-associated NHLs also present a high homology for antibody specific for the E2 protein of HCV, it may be reasonable to speculate that HCV E2 protein is one of the chronic antigenic stimuli involved in the lymphomagenetic process. Finally, the use of specific segments, in particular the D segment, in assembling the IgH chain of IgR seems to confer B-cell disorders with the property to produce antibody with RF activity, which may contribute to the manifestation of an overt MC syndrome.

Aged↗

PubMed1993

Microheterogeneity in the recognition of a HLA-DR2-restricted T cell epitope from a meningococcal outer membrane protein.

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3+CD4+CD8-TcR alpha/beta+, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, Ile 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since Ile 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V alpha and V beta genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.

Adult↗

PubMed2003

Skew in T-cell receptor usage and clonal T-cell expansion in patients with chronic rejection of transplanted kidneys.

BACKGROUND: It is not known whether alloreactive T cells are involved in chronic rejection of transplanted kidneys. The aim of the present study was to determine the involvement of T cells in the chronic graft rejection. METHODS: T-cell receptor (TCR) variable region alpha-chain and TCR variable region beta-chain repertoires were analyzed in peripheral blood mononuclear cells. T-cell clonalities were analyzed by complementarity-determining region 3 size spectratyping. RESULTS: A significant increase in the frequencies of one or more TCR variable region alpha-chain and TCR variable region beta-chain segments was detected in 13 and 15 of the 24 kidney transplant recipients, respectively. The extent of the skew in the TCR usage was correlated with the levels of clonal T-cell expansion, indicating that the clonally expanding T cells were responsible for the skew in the TCR usage. The levels of the skew in the TCR usage and clonal T-cell expansion were significantly greater in the recipients with a graft failure than in those with a stable graft function ( P=0.0081 and P=0.012, respectively). These results indicate that the clonally expanding T cells in the periphery may be related to graft rejection. The percent increase in the serum creatinine levels, which reflected the deterioration of the kidney functions, was significantly higher in the recipients who showed high levels of clonal T-cell expansion than in those who did not ( P=0.021). CONCLUSIONS: The results demonstrate that clonal T-cell expansion in the periphery has a negative impact on the long-term graft functions, and that analysis of the clonal T-cell expansion in peripheral blood mononuclear cells provide significant information on the fate of the transplanted kidney.

Adolescent↗

PubMed2004

Genomic organization of the chicken T-cell receptor beta chain D-J-C region.

We examined overlapping genomic clones containing the chicken T cell receptor (TCR) Dbeta-Jbeta-Cbeta complex, which contains a single diversity segment, four joining segments and four exons that encode the constant region. This sequence comprised 18.3 kb. All four Jbeta sequences possessed typical recombination signal sequences (RSS) with intervening 12-bp spacers at their 5'-ends and splice sites at their 3'-ends. No Jbeta-pseudogenes were identified. TGTG sequences in the RSS heptamer sequences were well conserved, as is the case in mammals. A chicken repeat 1-like sequence was found in the intron region between Jbeta-1336 and Cbeta, and several small repeat sequences were identified in intron regions throughout this cloned genome. As germline sequences revealed complete Jbeta sequences, the CDR3 (complementarity-determining region) sequences of TCRbeta from non-immunized splenocytes were analyzed. Non-coding (N) and palindromic (P) nucleotides were frequently observed at the Dbeta-Jbeta recombination sites. There were differences in length of deletion at the 5'-end of each Jbeta. Deletion of the 5'-end of Jbeta-1280 was particularly short when compared with that of Jbeta-1336, but there were no changes in the length of the CDR3 using any of the four Jbeta sequences.

Amino Acid Sequence↗

PubMed1993

The human immunoglobulin VH7 gene family consists of a small, polymorphic group of six to eight gene segments dispersed throughout the VH locus.

In this report we describe the analysis and mapping of members of the human immunoglobulin VH7 gene family. VH7 and VH1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from VH1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The VH7 gene family was found to be a small but discrete VH gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different VH7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four VH7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel VH families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these VH7 elements. Individual VH7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other VH gene families. The polymorphism of the VH7 gene segments and their scattered location throughout the VH locus makes them potentially useful markers for mapping and linkage studies.

Amino Acid Sequence↗

PubMed1993

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis.

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or rho and L2 or sigma, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80% identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J-C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.

Amino Acid Sequence↗

PubMed1996

Transition-state theory and secondary forces in antigen--antibody complexes.

Secondary forces, defined as those interactions between the antigen (epitope including the surrounding environment) and areas immediately adjacent to the antibody active site, were investigated using monofluorescein-derivatized synthetic peptides of varying electrostatic properties. Secondary forces were quantitated by measuring the unimolecular rate constants at two different temperatures using the high-affinity anti-fluorescein monoclonal antibody 4-4-20 complexed with fluorescein-derivatized synthetic peptides. Unimolecular rate constants were correlated with transition-state theory to explain secondary effects. An acidic peptide produced a large temperature-dependent effect upon binding including a significant enthalpic factor (+33.28 kcal/mol) relative to the binding of fluorescein ligand (+23.96 kcal/mol). Binding of a basic peptide produced both a relatively smaller temperature effect and enthalpy factor than fluorescein ligand. The antibody-ligand binding results were interpreted invoking the concepts of thermally averaged metatypic (liganded) states of the antibody as well as potential biochemical interactions between the antigen and accessible surface regions of the antibody's complementarity determining regions.

Antibodies, Monoclonal↗

PubMed2000

Immunoglobulin heavy chain variable region gene replacement As a mechanism for receptor revision in rheumatoid arthritis synovial tissue B lymphocytes.

Mature B cells can alter their antibody repertoires by several mechanisms, including immunoglobulin heavy chain variable region (V(H)) replacement. This process changes the antigen combining site by replacing a portion of the original V(H)/diversity/heavy chain joining region (V(H)DJ(H)) rearrangement with a corresponding portion of a new V(H) segment. This exchange can involve cryptic heptamer-like sequences embedded in the coding regions of V(H) genes. While studying the B lymphocytes that expand in the synovial tissues of patients with rheumatoid arthritis (RA), clones with V(H)DJ(H) variants that were apparently generated by V(H) replacement were identified with surprising frequency (approximately 8%). Examples of multiple independent V(H) replacement events occurring in distinct progeny clones were also identified. These secondary V(H) rearrangements were documented at both the cDNA and genomic DNA levels and involved several heptamer-like sequences at four distinct locations within V(H) (three sites in framework region 3 and one in complementarity determining region 2). The identification of blunt-ended double-stranded DNA breaks at the embedded heptamers and the demonstration of recombinase activating gene (RAG) expression suggested that these rearrangements could occur in the synovial tissues, presumably in pseudo-germinal centers, and that they could be mediated by RAG in a recognition signal sequence-specific manner. The presence of V(H) mutations in the clones that had undergone replacement indicated that these B cells were immunocompetent and could receive and respond to diversification signals. A relationship between these secondary V(H) gene rearrangements and the autoimmunity characteristic of RA should be considered.

Adult↗

PubMed2002

Analysis of immunoglobulin H gene rearrangement by polymerase chain reaction in primary central nervous system lymphoma.

OBJECT: Diagnosing primary central nervous system lymphoma (PCNSL) may be difficult either because of a paucity of tumor cells in the brain biopsy specimens or a failure to demonstrate monoclonality on immunomorphological studies. Monoclonality can also be demonstrated by amplification of the rearranged immunoglobulin H genes by polymerase chain reaction (PCR) to the framework region (FR)3 and FR2 complementarity determining region (CDR)-III and CDR-II of these genes. The PCR method is feasible with formalin-fixed, paraffin-embedded biopsy material and has proven to be helpful in the diagnosis of non-Hodgkin lymphoma on biopsy samples obtained from various locations in the body. Nevertheless, few studies have addressed the value of this method in the context of PCNSL. In the present study, the contribution of both FR3 single and FR2 seminested PCR procedures for confirming the diagnosis of PCNSL was estimated retrospectively in 30 cases of PCNSL and in three cases of epidural lymphoma. METHODS: Twenty-eight cases of immunophenotypically confirmed PCNSL and two of suspected lymphoma were studied. Tissue specimens obtained in 22 cases of other cerebral diseases, among which were various inflammatory conditions. were used as negative controls. In 18 (60%) of 30 cases the results of FR3 PCR demonstrated monoclonality, whereas FR2 PCR showed monoclonality in 12 cases (40%). In 11 cases FR3 PCR yielded monoclonal patterns and FR2 PCR did not, whereas reversibly in five cases FR2 PCR proved monoclonality and FR3 PCR failed to do so. Adding the results of FR3 to those of FR2 PCR, monoclonal patterns were obtained in 23 (77%) of 30 cases. In both cases in which lymphoma was suspected but not proven immunomorphologically, FR3 PCR revealed monoclonality, as did FR2 PCR in one case. In all 22 control lesions either polyclonal patterns were seen or no consistent patterns were obtained. In the PCNSL group, older age of patients and multifocal presentation of lesions on neuroimaging were significantly associated with worse survival. No correlation between histological subtype and clinical outcome was elucidated. CONCLUSIONS: The application of FR3 and FR2 PCR is a useful additional tool in making the diagnosis of PCNSL. Moreover, in some cases the PCR method may be essential in distinguishing neoplasia from reactive conditions.

Adolescent↗