ABSTRACT
Body cavity-based lymphoma, also known as primary effusion lymphoma, is a newly recognized acquired immunodeficiency syndrome (AIDS)-related lymphoma that has been linked to the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). To date, direct visualization of the virus in a clinical sample has not been demonstrated. We have performed an extensive clinical, histologic, immunophenotypic, ultrastructural, and molecular genetic correlative study on multiple tissue samples obtained premortem and at autopsy from an patient with AIDS with Kaposi's sarcoma and body cavity-based lymphomas. We demonstrate the presence of human herpesvirus-8 in a primary clinical sample at the ultrastructural and molecular level, as well as document multiple lymphomatous tumor masses at autopsy.
Subject(s)
Herpesvirus 4, Human/isolation & purification , Herpesvirus 8, Human/isolation & purification , Lymphoma, AIDS-Related/pathology , Lymphoma, AIDS-Related/virology , Sarcoma, Kaposi/virology , Skin Neoplasms/virology , Adult , Cytomegalovirus/isolation & purification , DNA Nucleotidyltransferases/analysis , DNA, Viral/analysis , Fatal Outcome , HIV Infections/complications , Humans , Immunohistochemistry , In Situ Hybridization , Lymphoma, AIDS-Related/chemistry , Lymphoma, AIDS-Related/complications , Lymphoma, AIDS-Related/ultrastructure , Male , Microscopy, Electron , Polymerase Chain Reaction , Proto-Oncogene Proteins c-bcl-2/analysis , RNA, Messenger/analysis , RNA, Viral/analysis , Sarcoma, Kaposi/complications , Skin Neoplasms/complications , VDJ RecombinasesABSTRACT
Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Tnp overproduction causes cell filamentation, abnormal chromosome segregation, and an increase in anucleated cell formation. There are two simple explanations for the observed phenotype: induction of the SOS response or of the heat shock response. The data presented here show that overproduction of Tnp neither induces an SOS response nor a strong heat shock response. However, our experiments do indicate that induction of some sigma32-programmed function(s) (either due to an rpoH mutation, a deletion of dnaK, or overproduction of sigma32) suppresses Tnp overproduction killing. This effect is not due to overproduction of DnaK, DnaJ, or GroELS. In addition, Tnp but not deltall Tnp (whose overproduction does not kill the host cells) associates with the inner cell membrane, suggesting a possible correlation between cell killing and Tnp membrane association. These observations will be discussed in the context of a model proposing that Tnp overproduction titrates an essential host factor(s) involved in an early cell division step and/or chromosome segregation.
Subject(s)
DNA Nucleotidyltransferases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/genetics , Sigma Factor/genetics , Suppression, Genetic , Transcription Factors , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Cell Membrane/chemistry , Cell Nucleus/metabolism , Chaperonin 10/biosynthesis , Chaperonin 60/biosynthesis , Chromosome Mapping , Chromosomes, Bacterial/physiology , DNA Nucleotidyltransferases/analysis , DNA Nucleotidyltransferases/biosynthesis , DNA Nucleotidyltransferases/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/physiology , Genes, Bacterial , Genes, Suppressor , HSP70 Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/physiology , Phenotype , SOS Response, Genetics , Sigma Factor/physiology , TransposasesABSTRACT
Multiple members of the NF-kappa B/Rel protein family are induced during B cell differentiation and have been implicated in transcriptional activation of the immunoglobulin kappa (Ig kappa) locus. Despite these findings, normal numbers of Ig kappa + B lymphocytes are produced by mice bearing targeted mutations in individual NF-kappa B/Rel genes. In the present study, precursor B lymphocytes were engineered to express a trans-dominant form of I kappa B alpha that simultaneously impairs the c-Rel and RelA transactivating subunits of NF-kappa B. This dual block in NF-kappa B/Rel signaling led to potent inhibition of germline Ig kappa transcription and rearrangement, whereas recombinase activity was unaffected. These findings suggest that c-Rel and RelA serve compensatory functional roles in the developmental mechanisms that govern Ig kappa gene assembly.
Subject(s)
B-Lymphocytes , Gene Expression Regulation, Developmental , Genes, Immunoglobulin , Hematopoietic Stem Cells , Immunoglobulin kappa-Chains/genetics , Integrases , Animals , Cells, Cultured , DNA Nucleotidyltransferases/analysis , Enhancer Elements, Genetic , Gene Rearrangement, B-Lymphocyte , Genes, Reporter , Germ Cells , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Binding , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-rel , Recombinases , Transcription Factor RelA , Transcription, Genetic , Transformation, GeneticABSTRACT
The immunohistochemical evaluation of acute leukemia specimens has been limited in the past due of the inability to detect many lineage-related antigens in paraffin sections. With the improvement in immunohistochemical methods as well as the introduction of new antibodies, these limitations are now reduced. To evaluate the diagnostic utility of paraffin section immunohistochemistry in the lineage determination of acute leukemias, 77 previously immunophenotyped acute leukemias were studied with a panel of antibodies that included antibodies directed against CD3, CD20, CD34, CD43, CD68, CD79a, HLA-DR, myeloperoxidase (MPX), and terminal deoxynucleotidyl transferase (TdT). The cases included 48 acute myeloid leukemias, 18 precursor B-cell acute lymphoblastic leukemias, 6 T-cell acute lymphoblastic leukemias, and 5 mixed precursor B/myeloid leukemias. This immunohistochemical panel correctly identified the lineage of 96% of both acute myeloid leukemias and acute lymphoblastic leukemias and identified evidence of mixed lineage in 60% of mixed lineage leukemias. Antibodies directed against CD3, CD79a, MPX, and TdT were found to be the most useful, although the latter three alone were not entirely lineage specific. These findings suggest a role for paraffin section immunohistochemistry in the lineage determination of some cases of acute leukemia.
Subject(s)
Bone Marrow/immunology , Bone Marrow/pathology , Immunophenotyping/methods , Leukemia/immunology , Acute Disease , Adolescent , Adult , Aged , Antigens, CD/analysis , Antigens, CD20/analysis , Antigens, CD34/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Biopsy, Needle , CD3 Complex/analysis , CD79 Antigens , Child , Child, Preschool , DNA Nucleotidyltransferases/analysis , Female , Flow Cytometry , HLA-DR Antigens/analysis , Humans , Immunohistochemistry , Leukemia/diagnosis , Leukemia/pathology , Leukosialin , Male , Middle Aged , Paraffin Embedding , Peroxidase/analysis , Receptors, Antigen, B-Cell/analysis , Sialoglycoproteins/analysisABSTRACT
This analysis of B cell development as a function of age reveals a relatively widespread distribution of progenitor B (pro-B), pre-B, and B cells in fetal tissues, and thus supports the idea of a multifocal origin of B lineage cells during embryonic development. From mid-gestation onward, the bone marrow is the major site of B cell generation in humans. A relatively constant ratio of bone marrow precursors to B cells of immature phenotype (CD24highCD10+CD20lowIgD-) is maintained from mid-gestation through the eighth decade of life. The persistence of recombinase gene activity in pro-B cells further attests the sustained production of B cells in bone marrow. Interestingly, a subpopulation of B cells with mature phenotype (CD24lowCD10-CD20highIgD+) accumulates in the bone marrow during childhood, and this becomes the predominant B cell subpopulation in adult bone marrow. This mature population of bone marrow B cells may represent a subpopulation of recirculating B cells that have undergone selection in the periphery.
Subject(s)
Aging/immunology , B-Lymphocytes/cytology , Hematopoiesis/physiology , Hematopoietic System/growth & development , Adult , B-Lymphocytes/enzymology , Base Sequence , Biomarkers , Bone Marrow/embryology , Bone Marrow/growth & development , Bone Marrow Cells , CD5 Antigens/analysis , Cell Lineage , Child , Clonal Deletion , DNA Nucleotidylexotransferase/analysis , DNA Nucleotidyltransferases/analysis , Gestational Age , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/enzymology , Humans , Immunophenotyping , Molecular Sequence Data , Receptors, Antigen, B-Cell/analysis , VDJ Recombinases , Viscera/cytology , Viscera/embryologyABSTRACT
A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.
Subject(s)
DNA Restriction Enzymes/analysis , Bacteriophage lambda/genetics , DNA Nucleotidyltransferases/analysis , DNA Nucleotidyltransferases/metabolism , DNA Restriction Enzymes/metabolism , DNA, Circular/chemistry , Deoxyribonuclease EcoRI/physiology , Electrophoresis, Agar Gel , Nucleic Acid Denaturation , Plasmids/genetics , Recombination, Genetic , TransposasesABSTRACT
A mammalian expression vector designed for production of HIV-1 integrase was found to enhance the stability of a linear reporter plasmid in COS-7 cells. The effect is strictly dependent on coexpression of the HIV-1 rev gene and on the inclusion of U3 and U5 portions of the HIV-1 LTR in the reporter plasmid. Integrase point mutations P109S and D116N drastically reduced stabilization whereas T115A and D64A had little or no effect. Immunoblot analysis revealed the presence of a 32-34kDa integrase protein in extracts of transfected COS-7 cells and of wild type and mutant integrase proteins at comparable levels. We conclude that integrase acts in trans in COS-7 cells, possibly by binding to the HIV-1 LTR in the plasmid. This transfection system may be useful for studying factors that stabilize the HIV-1 DNA genome prior to its integration into the host cell chromosome.
Subject(s)
DNA Nucleotidyltransferases/metabolism , Genes, rev , HIV Long Terminal Repeat , HIV-1/enzymology , HIV-1/genetics , Plasmids/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , DNA Nucleotidyltransferases/analysis , DNA Nucleotidyltransferases/biosynthesis , Humans , Integrases , Molecular Sequence Data , Molecular Weight , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Point Mutation , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Restriction Mapping , Transfection , Virus IntegrationABSTRACT
Cyclophosphamide, an alkylating agent belonging to the family of nitrogen mustards, is commonly used to treat progressive autoimmune diseases in humans. At the molecular level, its cytotoxicity results from DNA double strand crosslinks and, at higher concentrations, from DNA strand breaks. At the cellular level, cyclophosphamide may selectively affect mature lymphocytes with relative sparing of the respective precursor cells. In this study, we show that 4-hydroxycyclophosphamide (4-OH-CP), the active metabolite of cyclophosphamide, induces apoptosis in mature human lymphocytes at concentrations that are achieved in vivo. Since cyclophosphamide requires enzymatic conversion in the liver to yield its active metabolite, 4-OH-CP was generated in vitro by non-enzymatic hydrolysis of mafosfamide. Apoptotic cell death of lymphocytes was characterized by typical morphological changes, nucleosomal DNA fragmentation, and quantified by 3'-OH end labeling of fragmented DNA. The percentage of apoptotic cells both depended on drug concentration and time of exposure. Cycloheximide or ZnSO4 did not suppress 4-OH-CP induced apoptosis. Etoposide, a topoisomerase II inhibitor known to induce apoptosis in human tumor cell lines like 4-OH-CP, did induce detectable DNA fragmentation in only a minor proportion of T-lymphocytes but suppressed T-cell proliferation.
Subject(s)
Apoptosis/immunology , Cyclophosphamide/analogs & derivatives , DNA Damage/immunology , Growth Inhibitors/pharmacology , Immunosuppressive Agents/toxicity , T-Lymphocytes/drug effects , Adult , Alkylation , Cycloheximide/pharmacology , Cyclophosphamide/toxicity , DNA Nucleotidyltransferases/analysis , Electrophoresis, Polyacrylamide Gel , Humans , Lymphocyte Activation/drug effects , Male , Sulfates/pharmacology , Zinc Compounds/pharmacology , Zinc SulfateSubject(s)
DNA Nucleotidyltransferases/analysis , Gene Rearrangement, B-Lymphocyte , Genetic Vectors , Immunoglobulin Class Switching , Retroviridae/genetics , Virus Integration , Animals , Base Sequence , Cell Line , DNA Nucleotidyltransferases/metabolism , Genes, Reporter , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Proviruses/genetics , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Retroviridae/physiology , VDJ RecombinasesABSTRACT
Episomal vectors have been developed which are useful for studying V(D)J recombination both after transient transfections and in stably transfected cells. In contrast to recombination substrates previously described for transient assays, rearrangement of these vectors results in expression of beta-galactosidase which can be visualized directly in the transfected cell, shortening the time required for the assay to 1-2 days instead of 3-4 days. When these substrates are stably integrated into a preB cell line, subclones are found which show no beta-galactosidase staining, although the substrate is properly integrated, transcriptionally active and the transfectants still possess recombinase activity. This finding suggests that, at least in some chromosomal locations, transcription through a locus bearing recombination signal sequences is not sufficient for V(D)J recombination. Using these same vectors, we estimate that the frequency with which V(D)J recombination-negative preB variants arise is less than 10(-4) per generation.
Subject(s)
DNA Nucleotidyltransferases/analysis , Recombination, Genetic/immunology , Animals , Blotting, Southern , Cells, Cultured , Genetic Vectors , Lac Operon/genetics , Mice , Mice, Inbred BALB C , Mice, Transgenic , Transfection , VDJ RecombinasesABSTRACT
Eleven murine hybridoma clones were selected for their ability to produce anti-HIV-1 integrase (IN) antibodies. Competition and epitope mapping studies allowed segregation of the monoclonal antibodies (MAbs) into four distinct classes. The five MAbs that comprise the first class showed high affinity for epitopes within an N-terminal domain of 58 amino acids that includes a conserved zinc finger motif. The second class, with two MAbs, showed high affinity for epitopes within 29 amino acids at the C terminus. Another two MAbs, which constitute the third class, displayed moderate affinities for epitopes that mapped to regions within the highly conserved catalytic core referred to as the D,D(35)E domain. One of these MAbs showed significant cross-reactivity with HIV-2 IN and weak, but detectable, cross-reactivity with RSV IN. The remaining two MAbs, which comprise the fourth class, exhibited fairly low binding affinities and appeared to recognize epitopes in the zinc finger motif domain as well as the C-terminal half of the IN protein. The MAbs can be used for immunoprecipitation and immunoblotting procedures as well as for purification of HIV-1 IN protein by affinity chromatography. We show that several can also be used to immunostain viral IN sequences in HIV-1-infected T cells, presumably as a component of Gag-Pol precursors. Finally, analysis of our mapping and competition data suggests a structure for mature IN in which the C terminus approaches the central core domain, and the N and C termini touch or are proximal to each other. These MAbs should prove useful for further analyses of the structure and function of IN both in vitro and in vivo.
Subject(s)
Antibodies, Monoclonal , DNA Nucleotidyltransferases/analysis , DNA Nucleotidyltransferases/immunology , HIV-1/enzymology , T-Lymphocytes/virology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/isolation & purification , Conserved Sequence , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Epitopes/analysis , Female , HIV-1/genetics , HIV-2/enzymology , Hybridomas , Immunoblotting , Immunoglobulin G/classification , Immunoglobulin G/isolation & purification , Integrases , Mice , Mice, Inbred BALB C/immunology , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/immunology , Recombinant Proteins/analysis , Recombinant Proteins/immunology , Sequence Deletion , T-Lymphocytes/immunology , Virus IntegrationABSTRACT
We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.
Subject(s)
B-Lymphocytes/chemistry , DNA Nucleotidyltransferases/analysis , DNA-Binding Proteins , Gene Rearrangement, T-Lymphocyte/genetics , Integrases , Proteins/genetics , T-Lymphocytes/chemistry , Animals , Antibodies, Monoclonal , Base Sequence , Mice , Mice, Mutant Strains , Mice, Transgenic , Molecular Sequence Data , Phenotype , Proteins/analysis , RecombinasesABSTRACT
We developed a highly specific and sensitive polymerase chain reaction (PCR) assay to measure V(D)J recombinase activity using extrachromosomal plasmids and PCR. Extrachromosomal plasmids were prepared by eukaryotic replication origin, and a combination of the DQ52 and JH2 regions of the murine IgH gene, or of the D beta 2-1 and J beta 2.6 regions of the murine TCR beta gene, both with recombination signal sequences. Plasmids, transfected into cells to be examined and recovered after 48 h, were processed to detect recombined molecules by PCR with primers for the expected sequences produced by the precise signal joint. The PCR assay, when compared with a Camr assay that we prepared with the DQ52 and JH2 regions of the murine IgH gene, seems to have the following advantages. It detects only the recombined products produced by V(D)J recombinase activity and is therefore highly specific. It detects V(D)J recombinase activity in cells, including those with low replication frequency, which our Camr assay failed to do. This also enables detection of the recombinase activity not only in murine cell lines, but also in cells of murine lymphoid organs. The assay detects V(D)J recombinase activity in cell lines of human origin by replacing the eukaryotic replication origin of plasmids. High V(D)J recombinase activity was detected in bone marrow cells followed by thymic cells, and apparently lower activity was detected in cells of the lymph node and spleen of normal mice.
Subject(s)
DNA Nucleotidyltransferases/analysis , DNA-Binding Proteins , Homeodomain Proteins , Immunoglobulins/genetics , Integrases , Lymphoid Tissue/enzymology , Polymerase Chain Reaction/methods , Animals , Cells, Cultured , Cloning, Molecular , DNA, Recombinant/genetics , Drug Resistance/genetics , Genetic Vectors , Humans , Mice , Nuclear Proteins , Plasmids/genetics , Proteins/genetics , Recombinases , Recombination, Genetic , Regulatory Sequences, Nucleic Acid/genetics , Sensitivity and Specificity , Transfection/geneticsABSTRACT
A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.
Subject(s)
Avian Myeloblastosis Virus/enzymology , DNA Nucleotidyltransferases/analysis , Deoxyribonucleases/analysis , Base Sequence , DNA Nucleotidyltransferases/chemistry , DNA Nucleotidyltransferases/genetics , DNA, Viral , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Hydrogen-Ion Concentration , Integrases , Kinetics , Methods , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Solubility , Substrate SpecificityABSTRACT
In E. coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-1, three vitally encoded proteins were produced: an 11-kDa protein and a 32-kDa protein identified by immunoassays as the mature PR and IN protein, respectively, and an additional protein 15-kDa in size that reacted strongly with an antiserum recognizing a region in the carboxyl half of the IN protein. The kinetics of its synthesis indicated that it was not a degradation product of p32-IN, rather it probably arose from internal initiation at an AUG codon in the middle of the IN gene. Amino terminal sequence analysis of the first 70 residues demonstrated a perfect match with those predicted from the nucleotide sequence, beginning with the methionine codon at position 154 of the integrase gene.
Subject(s)
DNA Nucleotidyltransferases/biosynthesis , Escherichia coli/genetics , HIV-1/enzymology , Transformation, Genetic , Amino Acid Sequence , Base Sequence , DNA Nucleotidyltransferases/analysis , DNA Nucleotidyltransferases/genetics , DNA, Recombinant/analysis , Endopeptidases/biosynthesis , Endopeptidases/genetics , Escherichia coli/metabolism , HIV-1/genetics , Integrases , Molecular Sequence Data , PlasmidsABSTRACT
We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.
Subject(s)
Antibody Diversity , DNA Nucleotidyltransferases/analysis , Genes, Immunoglobulin/genetics , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Lymphocytes/immunology , Animals , B-Lymphocytes/immunology , Cell Line , Clone Cells , Flow Cytometry/methods , Gene Rearrangement , VDJ RecombinasesABSTRACT
The morphology, membrane markers and ultrastructural cytochemistry of 39 cases of acute myeloid leukaemia (AML) with variable proportion (10-99%) of terminal deoxynucleotidyl transferase (TdT) positive blasts was compared with that of 134 cases of TdT negative AML. The incidence of TdT positive AML was 22.5% and this was significantly higher in poorly differentiated myeloblastic (M0 and M1) types (54%) than in all other FAB subtypes (10%; P less than 0.001). Our findings suggest heterogeneity among TdT positive cases. Whilst the majority correspond to genuine TdT positive AML in which evidence for exclusive myeloid nature was demonstrated by phenotypic, cytochemical and ultrastructural markers, a distinct minority (22%) of cases had mixtures of lymphoid and myeloid blasts. A change in phenotype occurred in three out of six cases studied in relapse. There was no difference in the incidence of immunoglobulin (Ig) gene rearrangement between TdT positive (two out of 12) and TdT negative (one out of 11) cases, although published data suggests that Ig gene rearrangement is significantly more common in TdT positive cases. The determination of TdT in AML allows the identification of cases of mixed acute leukaemia which probably represent proliferations of multipotent progenitor cells. The majority of TdT positive cases, nevertheless, correspond to immature types of myeloblastic leukaemia which may constitute a clinically distinct subgroup.
Subject(s)
DNA Nucleotidylexotransferase/analysis , DNA Nucleotidyltransferases/analysis , Leukemia, Myeloid, Acute/enzymology , Adult , Aged , Antigens, Surface/analysis , Bone Marrow/pathology , Child , Humans , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Middle Aged , PhenotypeABSTRACT
This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.
Subject(s)
Clinical Enzyme Tests , DNA Nucleotidylexotransferase/analysis , DNA Nucleotidyltransferases/analysis , Lymphoma, Non-Hodgkin/enzymology , B-Lymphocytes , Deoxyribonucleases , Formaldehyde , Humans , Immunohistochemistry , Lymphoma, Non-Hodgkin/pathology , Paraffin , T-LymphocytesABSTRACT
Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The probes were then detected with either streptavidin-alkaline phosphatase complex or anti-fluorescein antibodies and alkaline phosphatase-conjugated secondary antibodies. Tailing conditions were optimized and the probes were tested for detection of Escherichia coli ST1a enterotoxin DNA and rotavirus RNA.