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O Pritsch

Publications and source records attributed to O Pritsch.

At least 19 recordsLinked to original sources
PubMed2002

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells.

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete V(H) nucleotide sequence and the presence of RNA transcripts from different C(H) domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Adult↗
PubMed2000

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy.

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

Animals↗
PubMed2000

Negative regulation of autoreactive B cells in transgenic mice expressing a human pathogenic cold agglutinin.

Cold agglutinins (CA) are autoantibodies that bind to erythrocyte carbohydrates at low temperatures and induce complement-mediated cell lysis, thus causing hemolytic anemia. Tolerance mechanisms towards CA-expressing B cells and the factors inducing pathogenic CA production are unknown. In order to develop an animal model for CA disease, we have produced transgenic mice expressing the heavy or the light chain of a human CA, previously shown to be pathogenic to the mouse. Expression of the human H chain alone resulted in a B cell maturation block at the pro-B stage, and did not induce allelic exclusion. In double-transgenic mice, co-expression of the human H and L chains restored B cell development but the majority of bone marrow cells expressing the human IgM were eliminated by deletion. In the periphery, B cells were depleted, and a large proportion of the remaining cells co-expressed a human and a murine H chain, secreting "mixed" IgM. A few autoreactive cells, predominating in the peritoneal cavity, escaped tolerance mechanisms and secreted transgenic IgM. The autoreactive B cells are amenable to polyclonal stimulation, making these transgenic mice a suitable model for a human autoimmune disease.

Agglutinins↗
PubMed2000

Can isotype switch modulate antigen-binding affinity and influence clonal selection?

Four different monoclonal Ig (MIg) (IgA1kappa, IgG1kappa, IgG2kappa and IgG4kappa) displaying anti-tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical V(H) and V(L) domains and the presence of mutations compatible with an antigen-driven process. Surprisingly, despite complete homology in their variable domains, IgA1kappa, IgG1kappa, or their Fab fragments bound to a common motif recognized in beta tubulin, with significant differences in affinity (IgA1kappa 1.52x10(-8) M, and IgG1kappa 2.09x10(-7) M). To substantiate these results, the V(H) and V(L) domains from IgA1kappa were cloned and introduced into expression vectors containing the constant kappa exon and either the mu or the gamma1 constant exon, and complete recombinant IgMkappa and IgG1kappa were obtained. Like the IgA1kappa, the IgMkappa construction bound to the tubulin epitope with consistent affinity (7.7x10(-9) M), whereas the IgG1kappa construction displayed a significantly lower affinity (3.28x10(-7) M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

Amino Acid Sequence↗
PubMed2000

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Animals↗
PubMed1999

VH gene usage by family members affected with chronic lymphocytic leukaemia.

The excess risk of chronic lymphocytic leukaemia (CLL) in the first-degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non-random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B-cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members. Among familial cases, VH gene segment utilization proved non-random and diverged from the frequencies previously reported among unrelated patients with CLL. Specifically, although the 4-34 and 5-51 gene segments were found repeatedly, the 1-69 and 4-39 gene segments were used sparingly and the 3-23 gene segment presented with increased frequency. Following the pattern detected in studies of unrelated patients, the single 1-69 expressing CLL contained an unmutated H chain sequence and included a long HCDR3 interval. In contrast, 3-23 containing H chains all used JH4, retained at most 93% homology with germline sequence, and included only short HCDR3 intervals. The vast majority of the CLL variable domains contained a high degree of somatic mutation and exhibited an excess of replacement mutations in the CDR intervals. These findings suggest that familial CLL cases may preferentially derive from B-cell progenitors that have responded to antigen.

Adult↗
PubMed1998

Basic biology of autoimmune phenomena in chronic lymphocytic leukemia.

There is evidence indicating that autoreactive B cells constitute a substantial part of the B cell repertoire. This autoreactive repertoire secretes the so-called natural autoantibodies characterized by their broad reactivity mainly directed against well-conserved public epitopes. Their germinal origin is suggested by their early appearance during ontogeny, their expression of cross-reactive idiotopes, and structural studies of their sequence. As for the physiological role of the repertoire, it may play a major role as a first barrier of defense. It is presently unknown whether these polyreactive B cells could constitute a pre-immune template which, through an antigen-driven process, may be involved in the production of immune high-affinity antibodies. This autoreactive B cell repertoire frequently undergoes malignant transformation, although there is controversy concerning the reasons accounting for this. It has been postulated that the continuous challenge of this autoreactive repertoire by self-antigens could create propitious conditions for malignant transformation to occur. However, this hypothesis still needs to be substantiated. Chronic lymphocytic leukemia (CLL), the most frequent form of leukemia in western countries, is characterized by constant expression of the CD5 marker and low expression of surface membrane immunoglobulin (Ig) in B lymphocytes. CLL B lymphocyte is frequently committed for natural autoantibody secretion. Despite expressing the Epstein-Barr virus (EBV) receptor CLL B cells cannot be infected by the EBV virus, they overexpress the bcl-2 protein and they are unable to adequately respond when stimulated through the B cell receptor pathway. Autoimmune-associated phenomena are frequently observed in B-cell CLL. These autotoxic manifestations are mainly directed against hematopoietic cells. In most cases, autoantibodies against red blood cells are warm reactive polyclonal IgG. Immune thrombocytopenia is observed in about 2% of cases, but higher frequencies of increased platelet associated Igs have been reported. Pure red cell aplasia and autoantibodies against neutrophils are only rarely observed. This pattern is similar to that observed in primary immunodeficiency syndromes, in which immune thrombocytopenia, autoimmune hemolytic anemia, and pure red cell aplasia are frequently observed. The potential role of T cell defects in inducing autoimmune complications in B-cell CLL has been stressed by recent publications showing increased frequency of autoimmune hemolytic anemia in patients treated with purine nucleoside analogues. However, evidence is presently scarce concerning a functional impairment of T cells after administration of these drugs.

Agammaglobulinemia↗
PubMed1997

Structural and affinity studies of IgM polyreactive natural autoantibodies.

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.

Amino Acid Sequence↗
PubMed1997

Minimal residual disease detection in B-cell malignancies by assessing IgH rearrangement.

In B-cell malignancies, the uniqueness of the immunoglobulin heavy chain locus (IgH) clonal rearrangement provides a useful marker for the detection of minimal residual disease (MRD) after treatment. During the last decade, several techniques have been proposed and used for detecting MRD. In this review, we report the current PCR based techniques dealing with amplification of the VDJ segment since the CDR3 region is unique to each IgH rearrangement. The sensitivity of these techniques varies considerably with a detection level of one tumoral cell in 10(-2) to 10(-6) normal cells. Accurate and sensitive assessment of MRD may have profound impact in the clinical management of patients with hematologic malignancies. Although, a majority of studies have shown a good correlation between the rapidity or extent of the reduction in the number of tumoral cells and the subsequent relapse, other studies demonstrated substained positivity of PCR in patients in long term remission. Thus, current clinical studies of MRD should establish whether MRD predicts relapse uniformly and, therefore, justifies intensification of therapy in positive cases, or whether it simply detects leukemic cell populations whose proliferative potential has been altered by chemotherapy.

B-Lymphocytes↗
PubMed1997

A murine model of human cold agglutinin disease.

Progress has been limited in the treatment of cold agglutinin (CA) disease by the absence of an animal model. We have recently studied at the molecular level one CA displaying the rare anti-Sia-1b specificity (CAGAS), CAGAS displays strong CA activity and is able to haemolyse mouse RBC in the presence of complement, thus constituting a suitable Ab for creating a murine model of CA disease. In the present work we introduced CAGAS VH and VL domains into eukaryotic expression vectors and transfected them into the non-secreting mouse myeloma X63 cell line. Clones expressing complete engineered pentameric IgM kappa CAGAS (eCAGAS) recapitulating the characteristics of serum CA (sCAGAS) could be obtained. The i.p. injection of eCAGAS to normal BALB/c mice induced a typical haemolytic anaemia, as demonstrated by the presence of spontaneous cold agglutination of RBC, induction of anaemia and significant reticulocytosis. Of interest, conspicuous bilateral ear loss was observed in one of these animals. In addition, i.p. injection of X63 transfected line into BALB/c nude mice induced ascites, typical haemolytic anaemia, and shortening of the mean RBC survival. These findings validate the practical interest of constructing a transgenic mouse model expressing eCAGAS.

Anemia, Hemolytic, Autoimmune↗
PubMed1997

Molecular cloning of a monoclonal anti-tumor antibody specific for the Tn antigen and expression of an active single-chain Fv fragment.

We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.

Amino Acid Sequence↗
PubMed1997

VH gene expression in CD5 positive and CD5 negative B cell chronic lymphoid malignancies.

In this review, we report analyses of VH genes in mature B cell malignancies generally or occasionally bearing CD5 antigen such as B CLL, MCL, SLVL and PLL. In the majority of cases, B CLL and MCL use VH genes in germline configuration. However in some cases a higher rate of random mutations is observed. These differences are not related to CD5 expression but are accounted by Ig phenotype, since less mutations are observed in CLL cases expressing membrane mu delta, when compared to forms exclusively expressing membrane mu. PLL and SLVL cases display mutated V genes independently of CD5 expression. Although there is some evidence that CD5+ B cells constitute a separate lineage, the possibility that CD5 constitutes an activation marker cannot be ruled out. Indeed, CD5- B cells can be induced to differentiate into CD5+ B cells and VH gene analyses showed no significative differences between CD5+ and CD5- B cell lymphoproliferative disorders. In this review we have tried to examine B cell chronic malignancies on the basis of phenotype and VH gene usage. Thus we propose a tentative classification where these disorders are allocated according to these characteristics.

Animals↗
PubMed1996

Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.

Amino Acid Sequence↗
PubMed1996

High frequency of somatic mutations in the VH genes expressed in prolymphocytic leukemia.

Prolymphocytic leukemia (PLL) is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed lg heavy chain variable region (VH) genes in eleven cases of de novo PLL Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%), with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed VH genes diverged from their putative germline counterpart, and in eight cases the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and VH4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of lg VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.

Adult↗
PubMed1996

Complete nucleotide sequence of Ig V genes in three cases of Burkitt lymphoma associated with AIDS.

To investigate the role of polyclonal stimulation and antigen driven selection in the pathogenesis of acquired immunodeficiency syndrome (AIDS) related lymphomas, we studied the variable region nucleotide sequence of heavy (VH) and light (VL) chains expressed by 3 Burkitt lymphomas (BL) associated with HIV infection. Two cases expressed the VH3-30P1 gene with 88.6% and 86.7% homology when compared to their germinal counterpart, whereas the VH4-18 was rearranged in the third one (89% identity). All these genes displayed high numbers of mutations (27, 22, 28 respectively), predominating in CDR regions. The encoded light chain genes determined for cases 1 and 2 expressed the same V kappa I-018 gene. These results indicate that: 1) Although, it is difficult to address the issue of VH usage based on the limited number of cases studied, Burkitt's lymphoma associated with AIDS may use a restricted repertoire of Ig genes. 2) Mutations and/or replacements predominated in CDR regions, which might suggest the occurrence of an antigen driven selection process, at least in some AIDS associated lymphomas. However, the high ratio of mutations observed in framework (FW) regions also favors the possibility that the antigen selection process is associated with polyclonal B cell stimulation.

Acquired Immunodeficiency Syndrome↗