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Biomedical subjects

W M Comans-Bitter

Publications and source records attributed to W M Comans-Bitter.

At least 19 recordsLinked to original sources
PubMed2002

Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL.

Sensitive and quantitative detection of minimal residual disease (MRD) in bone marrow (BM) samples of children with acute lymphoblastic leukemia (ALL) is essential for evaluation of early treatment response. In this study, we evaluated whether the traumatic BM samplings can be replaced by peripheral blood (PB) samplings. MRD levels were analyzed in follow-up samples of 62 children with precursor-B-ALL (532 paired BM-PB samples) and 22 children with T-ALL (149 paired BM-PB samples) using real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T cell receptor gene rearrangements with sensitivities of 10(-3) to 10(-5) (one ALL cell in 10(3) to 10(5) normal cells). In 14 of the 22 T-ALL patients, detectable MRD levels were found in 67 paired BM-PB samples: in 47 pairs MRD was detected both in BM and PB, whereas in the remaining pairs very low MRD levels were detected in BM (n = 11) or PB (n = 9) only. The MRD levels in the paired BM-PB samples were very comparable and strongly correlated (r(s) = 0.849). Comparable results were obtained earlier by immunophenotyping in 26 T-ALL patients (321 paired BM-PB samples), which also showed a strong correlation between MRD levels in paired BM and PB samples (r(s) = 0.822). In 39 of the 62 precursor-B-ALL patients, MRD was detected in 107 BM-PB pairs: in 48 pairs MRD was detected in both BM and PB, in 47 pairs MRD was solely detected in BM (at variable levels), and in 12 pairs only the PB sample was MRD-positive at very low levels (</=10(-4)). Furthermore, in the 48 double-positive pairs, MRD levels in BM and PB varied enormously with MRD levels in BM being up to 1000 times higher than in the corresponding PB samples. Consequently, BM samples cannot easily be replaced by PB sampling for MRD analysis in childhood precursor-B-ALL, in line with their BM origin. In T-ALL, which are of thymic origin, BM sampling might be replaced by PB sampling, because the dissemination of T-ALL cells to BM and PB appears to be comparable.

Basic Helix-Loop-Helix Proteins↗
PubMed2000

Neonatal blood lymphocyte subpopulations: a different perspective when using absolute counts.

We compared the absolute counts of lymphocyte subpopulations in 15 neonates, and 9 adults using the whole lysed blood technique with 15 different triple immunostainings. To obtain accurate absolute lymphocyte counts in neonatal cord blood samples, the flow cytometric 'lympho-gate' was corrected for the erythroid cell contamination by normoblasts and unlysed erythrocytes. In contrast to earlier studies where relative frequencies were reported, we found that the major difference between neonatal and adult lymphocyte subpopulations concerned the much larger pool of naive 'untriggered' cells in neonates, standby for participation in primary immune responses.

Adult↗
PubMed2000

Autoimmune lymphoproliferative syndrome (ALPS) in a child from consanguineous parents: a dominant or recessive disease?

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by autoimmune features and lymphoproliferations and is generally caused by defective Fas-mediated apoptosis. This report describes a child with clinical features of ALPS without detectable Fas expression on freshly isolated blood leukocytes. Detection of FAS transcripts via real-time quantitative PCR made a severe transcriptional defect unlikely. Sequencing of the FAS gene revealed a 20-nucleotide duplication in the last exon affecting the cytoplasmic signaling domain. The patient was homozygous for this mutation, whereas the consanguineous parents and the siblings were heterozygous. The patient reported here is a human homologue of the Fas-null mouse, inasmuch as she carries an autosomal homozygous mutation in the FAS gene and she shows the severe and accelerated ALPS phenotype. The heterozygous family members did not have the ALPS phenotype, indicating that the disease-causing FAS mutation in this family is autosomal recessive.

Autoimmune Diseases↗
PubMed2000

Longitudinal survey of lymphocyte subpopulations in the first year of life.

Age-matched reference values for lymphocyte subpopulations are generally obtained via cross-sectional studies, whereas patients are followed longitudinally. We performed a detailed longitudinal analysis of the changes in lymphocyte subpopulations in a group of 11 healthy infants followed from birth up to 1 y of age, with special attention for early developmental markers, markers of maturation, and markers of activation. We found that T and B lymphocytes increased at 1 and 6 wk of age, respectively. In contrast, NK cells showed a sharp decline directly after birth, suggesting that they are more important during pregnancy than thereafter. CD45RA+--mainly CD4+--naive T lymphocytes were high at birth, and increased further during the first year of life; they form a large expanding pool of cells, ready for participation in primary immune responses. The absolute counts of CD45RO+ memory T lymphocytes were similar in infants and adults, albeit with a lower level of expression of CD45RO on infant T lymphocytes. Almost all infant T lymphocytes expressed CD38 throughout the first year of life. The abundant expression of CD38 on an infant's T lymphocytes might be related to a greater metabolic need of the large population of naive untriggered cells that are continually involved in primary immune responses during the first year of life. The high B lymphocyte counts in infants mainly concerned CD38+ B lymphocytes throughout the first year of life. Also, the relative frequencies of CD1c+ and CD5+ B lymphocytes were higher throughout the first year of life than in adults. Therefore, CD1c, CD5, and CD38 could be markers of untriggered B lymphocytes. In conclusion, our longitudinal survey of T and B lymphocytes, NK cells, and their subpopulations during the first year of life helps to complete the picture of lymphocyte development in infants. This information contributes to the correct interpretation of data from infants with possible immune disorders.

Antigens, CD↗
PubMed1999

Analysing the developing lymphocyte system of neonates and infants.

UNLABELLED: Technical developments in immunophenotyping and function testing have greatly facilitated studies on the developing lymphocyte system in the past decade and contributed to a better interpretation of the data obtained in these studies. This is important for the correct interpretation of data obtained in paediatric patients with possible immunological diseases. The age-related differences in lymphocyte subpopulations and function imply that the available adult reference values cannot be used in children. CONCLUSION: In this review we give an outline of the technical developments, their influence upon the interpretation of data, and the available literature about age-related changes in the developing lymphocyte system.

Humans↗
PubMed1999

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.

Antigens, CD↗
PubMed1997

Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations.

OBJECTIVE: Immunophenotyping of blood lymphocytes is an important tool in the diagnosis of hematologic and immunologic disorders. Because of maturation and expansion of the immune system in the first years of life, the relative and the absolute size of lymphocyte subpopulations vary during childhood. Therefore we wished to obtain reference values for the relative and the absolute size of all relevant blood lymphocyte subpopulations in childhood. STUDY DESIGN: We used the lysed whole blood method for analysis of lymphocyte subpopulations in 429 blood samples from neonates (n = 20), healthy children (n = 358), and adults (n = 51). The following age groups were used: 1 week to 2 months (n = 13), 2 to 5 months (n = 46), 5 to 9 months (n = 105), 9 to 15 months (n = 70), 15 to 24 months (n = 33), 2 to 5 years (n = 33), 5 to 10 years (n = 35), and 10 to 16 years (n = 23). RESULTS: Our results show that the absolute number of CD19+ B lymphocytes increases twofold immediately after birth, remains stable until 2 years of age, and subsequently gradually decreases 6.5-fold from 2 years to adult age. The CD3+ T lymphocytes increase 1.5-fold immediately after birth and decrease threefold from 2 years to adult age. The absolute size of the CD3+/CD4+ T-lymphocyte subpopulation follows the same pattern as the total CD3+ population, but the CD3+/CD8+ T lymphocytes remain stable from birth up to 2 years of age, followed by a gradual threefold decrease toward adult levels. In contrast to B and T lymphocytes, the absolute number of natural killer cells decreases almost threefold in the first 2 months of life and remains stable thereafter. Our study also showed that changes in the absolute size of lymphocyte subpopulations are not always consistent with changes in their relative size. This demonstrates that the relative counts of lymphocyte subsets do not reflect their actual size and are therefore of limited value. CONCLUSION: On the basis of this study we strongly recommend that immunophenotyping of blood lymphocytes for the diagnosis of hematologic and immunologic disorders be based on the absolute rather than on the relative size of lymphocyte subpopulations. Our data can be used as age-matched reference values for blood lymphocyte immunophenotyping.

Adolescent↗
PubMed1995

Southern blot detection of immunoglobulin lambda light chain gene rearrangements for clonality studies.

Southern blot analysis of immunoglobulin (Ig) genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig lambda (Ig lambda) gene rearrangements, we carefully determined the precise restriction map of the J-C lambda gene region, developed a suitable C lambda probe (IGLC3), and evaluated relevant restriction enzymes in combination with the IGLC3 probe. For the latter purpose, we selected 75 B cell malignancies with proven expression of Ig lambda protein chains in order to be sure that each malignancy contained at least one clonally rearranged IG lambda allele. Our extensive Southern blot analyses with the IGLC3 probe in EcoRI and/or HindIII digests revealed that combined EcoRI/HindIII digestion detected Ig lambda gene rearrangements in 95% of the 75 patients and 94% of the 98 rearranged alleles. In contrast, HindIII and EcoRI single digests allowed detection of rearrangements in only 78% and 83% of the patients, and 67% and 79% of rearranged alleles, respectively. We conclude that the use of the IGLC3 probe in combined EcoRI/HindIII digests is superior to EcoRI and HindIII single digests. This probe/enzyme combination is informative for clonality studies in approximately 95% of patients.

Alleles↗
PubMed1993

Transcription and protein expression of mb-1 and B29 genes in human hematopoietic malignancies and cell lines.

The transmembrane forms of all immunoglobulin (Ig) classes are associated with two glycoproteins, mb-1 and B29, that are crucial for signal transduction following antigen binding to the Ig molecule. We have investigated the transcription and protein expression of mb-1 and B29 genes during B-cell development. Sixty human continuous cell lines (35 B-lineage, 11 T-lineage, 11 myeloid-lineage and three non-hematopoietic) and 75 hematopoietic malignancies (55 B-lineage, 12 T-lineage and eight myeloid-lineage), were tested for RNA expression by Northern blotting experiments with the mb-1 pRA3 cDNA probe, and a newly isolated B29 cDNA probe. Protein expression was analyzed by immunofluorescence microscopy of cytocentrifuge preparations, which were labeled with the anti-mb-1 HM57 monoclonal antibody (mAb) and an anti-B29 polyclonal antiserum, directed against intracellular epitopes of these polypeptides. Except for two early precursor B-cell lines, mb-1 and B29 transcripts and proteins were detected in all B-cell lines and B-cell malignancies, i.e. from immature to more mature B cells, irrespective of their Ig class expression. Transcription of mb-1 genes seems to be down-regulated at the plasma cell stage, because no mb-1 transcripts and mb-1 proteins could be detected in the four plasma cell lines and two plasma cell leukemias tested. B29 transcripts were detectable in these cell samples, but low levels of B29 proteins were only detected in one plasma cell line. The HM57 mAb gave strong labeling on fresh cytocentrifuge preparations of all B-cell samples, and this mb-1 protein expression appeared to be B-cell specific. We therefore conclude that the HM57 mAb is well suited for the detection of the mb-1 molecule as a pan-B-cell marker for the diagnosis of immature and mature B-cell malignancies. The expression pattern of the mb-1 protein is comparable to that of the CD19 and CD22 antigens, but has the advantage of being B-lineage specific. Although B29 protein expression was restricted to B-lineage cells, the anti-B29 antiserum is less suitable for diagnosis of B-cell malignancies, because of the variable and generally weak signals on cytocentrifuge preparations.

Antigens, CD↗
PubMed1992

The B29 and mb-1 polypeptides are differentially expressed during human B cell differentiation.

Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb-1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37-kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti-B29 antibodies was compared with those of antibodies to the mb-1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb-1 whereas B29 was expressed in only 13 cases. Most of these B29-positive ALL expressed immunoglobulin mu heavy chain in their cytoplasm (pre-B ALL). In lymphoid tissue sections, anti-B29-labeled B cell follicles in a similar fashion to anti-mb-1, with the striking exception that plasma cells were unreactive for B29, but positive for mb-1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb-1, and ceases before the terminal plasmacyte phase.

Animals↗
PubMed1991

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).

Animals↗
PubMed1990

BMA031, a monoclonal antibody suited to identify the T-cell receptor alpha beta/CD3 complex on viable human T lymphocytes in normal and disease states.

Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross-react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

Animals↗
PubMed1990

Antibody L26 recognizes an intracellular epitope on the B-cell-associated CD20 antigen.

Monoclonal antibody L26 is a highly selective marker of B cells and B-cell neoplasms in paraffin-embedded tissues, but it suffers from the drawback that the target molecule has not been identified. In this paper we provide evidence by two independent techniques that antibody L26 recognizes an intracellular epitope on the CD20 antigen (a pan B-cell marker). When this antigen was redistributed on the surface of unfixed viable B cells by incubation with monoclonal anti-CD20 followed by anti-mouse Ig, the diffuse cytoplasmic staining of L26 was abolished and replaced by coincident dotlike labeling for antibody L26 and the CD20 antigen. None of the other antibodies tested (covering 10 different B-cell-associated antigens) had this effect on the L26 staining pattern. Furthermore, COS-1 cells transfected with cDNA encoding the CD20 molecule gave positive staining with antibody L26 and with two other CD20 reagents, but not with antibodies to other pan B-cell markers (eg, CD19 and CD22).

Antibodies, Monoclonal↗
PubMed1989

Non-random expression of T cell receptor gamma and delta variable gene segments in functional T lymphocyte clones from human peripheral blood.

Human T cell receptor (TcR) gamma delta displays a variety of protein forms. Disulfide-linked (type 1) or non disulfide-linked (type 2) receptors occur, with gamma chains encoded by the C gamma 1 or the C gamma 2 gene segment, respectively. Exon 2 of C gamma 2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR gamma chains differ in molecular mass and charge between type 1 and type 2 receptors. The delta chains as well as the gamma chains have different structural properties between receptor types. This cannot be due to the use of different C delta gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in gamma/delta combinations, rearrangement and expression of V gamma, J gamma, C gamma and V delta gene segments were determined in TcR gamma/delta+ clones derived randomly from peripheral blood of normal donors. Most clones used C gamma 1, a minority C gamma 2. The different protein properties of receptor types could be explained by the non-random expression of V gamma (J gamma) and V delta gene segments. Type 1 receptors preferentially used gamma chains encoded by the V gamma 9 and J gamma 1.2 gene segments together with delta chains encoded by V delta 2. In type 2a receptors, V gamma 9 was not predominant; often other V gamma gene segments were employed, but then in high frequency in coordination with V delta 1. Reactivity of the clones with monoclonal antibodies anti-Ti gamma A, BB3 and delta-TCS-1 correlated with the expression of the V gamma 9, V delta 2 and V delta 1 gene segments, respectively. Therefore, V gamma and V delta use in TcR gamma/delta+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the V gamma 9-V delta 2 combination was predominant, while the V gamma 9-V delta 1 and particularly the V gamma 9-"V delta other" combination was rare. These data indicate that the TcR gamma delta repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.

Antibodies, Monoclonal↗
PubMed1988

Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies.

The expression of cytoplasmic CD3 (CyCD3) was analyzed in 45 leukemias, five thymus cell samples, five peripheral blood (PB) samples, and ten cell lines. All T cell acute lymphoblastic leukemias (T-ALL) that did not express surface membrane CD3 (SmCD3) appeared to express CyCD3. Furthermore, the majority of SmCD3+ T-ALL also expressed CyCD3. Analogous results were obtained with thymus cell samples in that about 95% of the thymocytes expressed CyCD3 whereas 60% to 75% of the thymocytes also expressed SmCD3. In normal peripheral blood only prominent SmCD3 expression was found. These data indicate that immature T cells express CyCD3 only, that the combined expression of CyCD3 and SmCD3 is characteristic for intermediate differentiation stages, and that mature T cells express prominent SmCD3. All (precursor) B cell leukemias, acute myeloid leukemias, and non-T cell lines tested did not express CyCD3. On the basis of these data, we conclude that CyCD3 expression is restricted to the T cell lineage and can be used as a diagnostic marker for immature SmCD3- T cell malignancies. Therefore, we evaluated which fixative is optimal for CyCD3 staining, and we determined by immunofluorescence staining and Western blotting which anti-CD3 monoclonal antibody (MoAb) can be used for the detection of CyCD3. In our opinion, acid ethanol was the best fixative for the cytocentrifuge preparations. Furthermore, we demonstrated that CyCD3 can be easily detected by use of MoAbs raised against denaturated CD3 chains such as those of the SP series (SP-6, SP-10, SP-64, and SP-78). In addition we tested 22 anti-CD3 MoAbs of the Oxford CD3 panel that were raised against native SmCD3, and it appeared that only four (UCHT1, VIT-3b, G19-41 and SK7/Leu-4) of them were able to detect CyCD3. In Western blot analysis all four MoAbs recognized the CD3-epsilon chain only.

Acute Disease↗
PubMed1987

T cell receptor-CD3 complex during early T cell differentiation. Analysis of immature T cell acute lymphoblastic leukemias (T-ALL) at DNA, RNA, and cell membrane level.

T cell acute lymphoblastic leukemias (T-ALL) can be regarded as the malignant counterparts of cells in various T cell differentiation stages. To study the expression of the human T cell receptor (TcR)-CD3 complex during the early stages of T cell differentiation, we have analyzed 22 T-ALL at the cell membrane level and the DNA level and 12 of them at the RNA level. According to their immunologic phenotype, the T-ALL could be divided into three main groups: 10 immature T-ALL (CD1-/CD3-), seven common thymocytic T-ALL (CD1+/CD3-or+), and five mature T-ALL (CD1-/CD3+). Among the 10 immature T-ALL three appeared to express the immunologic phenotype of the putative prothymocyte (TdT+/HLA-DR+/CD7+/CD2+/CD5-/CD1-/CD3-), whereas the other seven T-ALL appeared to be immature thymocytic (TdT+/HLA-DR-/CD7+/CD2+/CD5+/CD1-/CD3-). Transcripts of the CD3-delta and CD3-epsilon genes were present in all CD3- and CD3+ T-ALL tested, including prothymocytic T-ALL. However, prothymocytic T-ALL had germline TcR-beta genes and were not rearranged to the characterized TcR-gamma joining regions. The presence of CD3 transcripts and absence of TcR gene rearrangements in prothymocytic T-ALL supports their immature T cell character. Two immature thymocytic T-ALL also had germline TcR-gamma genes and one of them had germline TcR-beta genes. In all other T-ALL the TcR-gamma and TcR-beta genes were rearranged. The presumptive functional 1.3-kilobase TcR-beta transcripts were detected in the majority of T-ALL with rearranged TcR-beta genes. Distinct levels of TcR-gamma transcripts appeared to be present only in some thymocytic T-ALL, i.e., some immature thymocytic T-ALL and common thymocytic T-ALL. TcR-alpha mRNA could only be detected in CD3+ mature T-ALL, but was absent in all CD3+ common thymocytic T-ALL tested. Our data indicate that CD3 gene transcription is one of the earliest events during T cell differentiation and already occurs in prothymocytes. The TcR-gamma and TcR-beta genes rearrange early during thymocytic differentiation and can subsequently be transcribed. High levels of TcR-gamma gene transcription may only occur in a part of the T cells during thymic differentiation, while TcR-beta gene transcription continues during further differentiation. TcR-alpha gene transcription may be the final step in the production of the complete set of TcR and CD3 proteins, resulting in the expression of the TcR alpha beta-CD3 complex at the cell surface of mature T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗