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Ki-M4, a new IgG3 monoclonal antibody, selectively recognizes dendritic reticulum cells (DRC; follicular dendritic cells) in all human lymphatic organs, as tested by the immunoperoxidase method on the light and electron microscopic level. This antibody was raised against separated lysosomes of the 12–0-tetradecanoyl phorbol-13-acetate (TPA) stimulated permanent cell line, U-937, derived from a human histiocytic lymphoma. No cross-reactivity was encountered in epithelial and mesenchymal cells, including macrophages and other cells detectable in bronchial and peritoneal lavages. In the nonadherent fraction of the mononuclear blood cells collected from the interphase of a Ficoll- Urografin gradient (density = 1.077 g/ml), 0.1 per million of the cells hitherto not classified as monocytes or lymphocytes showed a strong reaction. All other separated blood cell types were devoid of any reactivity. The observation that DRC share a highly restricted, and thus specific, antigen with a small but distinct subpopulation of mononuclear leukocytes implies their blood derivation.

In this study, 32 cases of T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for their association with Epstein-Barr virus (EBV). For this purpose, three different approaches were applied: polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER), and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced almost identical results, showing that all but one case of AILD-TCL contained EBV genomes. Three distinctive patterns of EBV infection were observed after immunophenotypical characterization of EBER-positive cells: (1) in 26% of the cases, B and T cells were infected, the majority of which were B cells of immunoblastic morphology located in the remnants of lymphoid follicles; (2) in 42% of the cases, the vast majority of infected cells were neoplastic T cells diffusely distributed in the lymph nodes, but infected B cells were also present; and (3) in 32% of the cases, there were only a few infected small lymphoid cells. Detectable LMP was frequent in cases exhibiting patterns 1 and 2. These findings suggest that in AILD-TCL patients, B cells and especially T cells are highly susceptible to a persistent EBV infection, which often leads to a growth advantage of the infected cells. Thus EBV, in conjunction with genetic abnormalities and selective defects of the immune system, might be involved in the pathogenesis of AILD-TCL.

A monoclonal antibody (MoAb), Ki-M8, that reacts specifically with cells of the monocyte/macrophage system is described. On light and electron microscopic immunohistochemistry, Ki-M8 recognizes intracytoplasmatically localized antigens of mol wt 30,000 and 32,000, increasingly expressed during differentiation of monocytes into macrophages. Ki-M8 antigen is detectable on almost all known tissue macrophages and monocyte/macrophage-related cell lines after appropriate stimulation. In functional terms Ki-M8 significantly impairs the generation of oxygen radicals during an induced respiratory burst. Applied to acute nonlymphoblastic leukemias, a clear-cut differentiation of the monocytic phenotype and differentiation is possible on the basis of Ki-M8 immunoreactivity. Ki-M8 represents a reagent specific for the monocyte/macrophage system with regard to antigen distribution in normal and neoplastic cells as well as with regard to its influence on a typical monocyte/macrophage-related function.

Myelofibrosis with myeloid metaplasia (MMM) belongs to the group of myeloproliferative syndromes. It is characterized by a sustained proliferation of megakaryocytes and increased medullary reticulin fibers. Until now the cellular phase at onset of the disease has not been analyzed for clonality of the hematopoietic cells. In this study we used X-linked restriction length polymorphism (RFLP) analysis to investigate the clonality of granulocytes and bone marrow cells from the cellular phase and advanced stages of the disease. In each of 12 heterozygous females, monoclonality of granulocytes or total bone marrow cells could be demonstrated. These results show that the cellular phase represents a monoclonal, and hence a probably neoplastic, proliferation of a pluripotent stem cell. The monoclonality of granulocytes present at the onset of disease should allow analysis of DNA of these easily accessible peripheral cells for the detection of specific clonal aberrations.

Human monocyte/macrophage serine esterase (HMSE), commonly known as acid esterase or alpha-naphthylacetate esterase, comprises a group of five enzyme variants that can be distinguished by their isoelectric points from esterase variants of the other normal human blood cell populations. A cDNA for one of the monocytic enzyme variants (HMSE1) was cloned from a U-937 lambda gt11 cDNA library by screening with an oligonucleotide mixture designed according to amino acid sequence data of the purified enzyme. The cDNA contains 1,727 bp with an open reading frame of 1,512 bp coding for a protein of 503 amino acid residues. HMSE1 cDNA represents the first cloned monocyte/macrophage-specific serine esterase and its sequence shows up to 77% homology to other known serine esterases of different species. The amino acid composition of the putative active site of HMSE1 as deduced from the nucleotide sequence corresponds with the active sites of other serine esterases but not with the active sites of serine proteases. Hybridization of the cDNA with RNA of separated normal blood cell populations and hematopoietic cell lines shows restricted expression within the monocyte/macrophage lineage.

In the search for immunoreagents appropriate for the histopathologic diagnosis of malignant B-cell lymphomas in routinely processed paraffin sections, a new monoclonal antibody, Ki-B5, was generated using a high- grade B-cell lymphoma as the immunogene. Ki-B5 is a mouse IgG1/kappa that recognizes five protein fractions of about 84, 82, 55, 48, and 27 Kd after biosynthetic radiolabeling and immunoprecipitation. Protein fractions with the molecular weights of approximately 84 and 82 Kd were expressed on the cell surface and show that Ki-B5 is probably unrelated to CD45. It was possible through electron microscopy to visualize the membrane-bound portion of Ki-B5. Extensive immunohistologic studies on normal human tissue and various neoplasias demonstrated the high specificity of Ki-B5 to normal human B cells and a minor subgroup of plasma cells. Except for ML-2, which is a myelomonocytic human cell line, Ki-B5 exclusively recognized the B-cell lineage, including EB-3, BALL-1, and NALM-1. All carcinomas, sarcomas, and malignant melanomas tested with Ki-B5 were negative. Although normal granulocytes and monocytes were constantly negative, three of eight myelomonocytic leukemias coreacted with this antibody. Eight of the 57 T-cell lymphomas studied were positive to Ki-B5. Five were classified as lymphoblastic, two represented T8-CLL, and one was classified as immunoblastic T-cell lymphoma. Only 3 of 126 cases of B-cell lymphoma, including rare types not considered in the current classifications, were negative to Ki-B5. Plasmacytomas were also negative, except for one case. Irrespective of the cases of lymphoblastic lymphoma and plasmacytoma, Ki-B5 represents a new monoclonal antibody appropriate for the diagnosis and immunophenotyping of malignant lymphomas in routinely processed paraffin sections.

Total cellular RNA of highly purified normal human blood cell populations was analyzed for the expression of the protooncogene c-fos, the cellular counterpart of the transforming FBJ virus. In marked contrast to previous findings based on in vitro studies with permanent leukemic cell lines, c-fos transcription was restricted to granulocytes. Neither blood monocytes nor blood lymphocytes or alveolar macrophages revealed detectable levels of c-fos transcription. Whereas this cellular oncogene is constitutively expressed in granulocytes, the monocytic cell line U-937 showed a transient c-fos transcription only after induction of differentiation. The contradiction between the results found in vivo and in vitro is discussed.

A new monoclonal antibody Ki-B3 produced by a fusion with leukemic cells of a centroblastic/centrocytic lymphoma (m.l. follicular) is introduced. This antibody predominantly recognizes B cells of follicular mantle and germinal center cells, as well as plasma cells in normal lymphoid tissue. Furthermore, 80% of all low- and high-grade B cell lymphomas are stained, whereas among T cell lymphomas, only four of 15 T lymphoblastic lymphomas were positive to Ki-B3. All peripheral T cell lymphomas showed a negative reaction. Additionally, Ki-B3 detects a small percentage of monocytes and some myelomonocytic leukemias. All epithelial tissues as well as all sarcomas tested were invariably negative. Ki-B3 precipitates a 220 kiloDalton (kD) molecular weight antigen similar to the leukocyte common antigen. Presumably Ki- B3 detects a subtype of the leukocyte common antigen that is predominantly expressed on mature and immature B cells. As the antigen is formalin resistant, Ki-B3 can be used in routine hematology on paraffin sections for the detection and differential diagnosis of B cell lymphomas.

The striking difference in the cytochemical pattern of acid esterase between blood monocytes and T lymphocytes initiated the present study to determine whether or not the cytochemical difference is related to a cell-specific polymorphism of the isoenzymes. Enzyme assays and isoelectric focusing were performed using detergent-treated lysosomes from viable monocytes, granulocytes, T lymphocytes, platelets, and erythrocytes isolated from peripheral blood. B lymphocytes were separated from tonsils. Thymocytes were obtained from thymus glands excised during cardiac surgery. Except for monocytes, all cell suspensions showed a purity of more than 98%. The mean enzyme activity in monocytes amounted to 39 mU/107cells. This value was 7 times higher than the activity level of lymphocytes, which showed values of 5.5 mU for 107B lymphocytes, 5.3 mU for 107T lymphocytes, and 8.1 mU for 107thymocytes. Granulocytes exhibited the lowest enzyme activity. The isoelectric focusing pattern of monocytes disclosed 4 isoenzymes, with the anodic one accounting for more than 85% of the total activity. T lymphocytes had 13-16 bands distributed in 3 complexes between pH 7.9 and 4.5. Thymocytes displayed a similar pattern, with only 11 bands. B lymphocytes showed 7 isoenzymes between pH 6.4 and 5.5. Platelets revealed 10 bands (pH 7.5-5.8), and erythrocytes, 5 ill-defined bands (pH 5.6-4.9). These data illustrate the diversity of the lysosomal acid esterase isoenzymes of the different types of blood cells. The characteristic isoenzyme pattern of acid esterase in T lymphocytes and monocytes is well in line with the cytochemical staining pattern and indicates the existence of cell-specific enzyme variants.

We investigated 81 cases of peripheral pleomorphic T-cell lymphoma (PMTCL) occurring in human immunodeficiency virus-negative Europeans for the presence of Epstein-Barr virus (EBV)-DNA through polymerase chain reaction (PCR) for the presence of EBV-encoded small nuclear RNAs (EBER) and immediate early mRNAs (Bam H-fragment, lower strand frame [BHLF]) by in situ hybridization (ISH) and for EBV-encoded latent membrane protein (LMP) and nuclear antigen 2 (EBNA2) by immunohistology (IH). EBER-ISH, which could be applied on all cases, showed an overall incidence of EBV-infected cells in 38 of 81 cases (47%) of PMTCL. These data could be confirmed by PCR, which produced congruent results in the cases with amplifiable DNA. By EBER-ISH, the virus was located in the tumor cells in 30 of the 38 EBV-positive cases, with the proportion of the infected cells ranging from 1% to 100%. In 18 of these cases and in the 8 cases without EBV-infected tumor cells, the virus was, respectively, either additionally or exclusively detectable in occasional nonmalignant lymphoid bystander cells. An LMP expression was observed in several of the EBER-expressing tumor cells in 18 cases, whereas EBNA2 was detectable only in one case, which also displayed signs of viral replication. Some nonmalignant EBV-infected B immunoblasts also expressed LMP in several cases. Primary cutaneous and enteropathy-associated PMTCL displayed less frequent EBV infection when compared with other extranodal or nodal manifestations.

There is considerable interest in immunohistochemical markers of proliferation which are suitable for use on routinely fixed clinical material. The novel proliferation-associated antibody Ki-S1 shows promise in this respect. In this study we have: (i) defined the pattern of Ki-S1 labelling relative to the cell cycle phase; (ii) investigated the labelling pattern with Ki-S1 on a human breast cell line (ZR75) under varying proliferative conditions induced by serum deprivation and refeeding; (iii) examined in a flow cytometric study Ki-S1 staining in archival, clinical breast carcinoma samples. In exponentially growing cells Ki-S1 showed a marked cell cycle phase-specific variation in staining intensity which increased linearly through the S-phase, was high in G2 and reached its peak in mitosis. Ki-S1 staining intensity mirrored the changes in proliferative activity of ZR75 cells during serum deprivation and refeeding. In a small series of human breast carcinoma, Ki-S1 staining intensity correlated with S-phase fraction (SPF) derived from DNA profiles. The antigen labelled by Ki-S1 is extremely robust, resisting degradation by fixation and by an aggressive enzymic tissue disaggregation method. Ki-S1 warrants further investigation as a proliferation-related marker, particularly for routine clinical application.