A new monoclonal antibody (3A5) that recognises a fixative resistant epitope on tissue macrophages and monocytes.

AIMS--To develop a monoclonal antibody specific for human macrophages in routinely processed material. METHODS--The monoclonal antibody was derived from a mouse popliteal lymph node after subcutaneous immunisation in the footpad with fragments of human spleen depleted of lymphocytes and erythrocytes. RESULTS--3A5 is a monoclonal antibody reactive with macrophages, monocytes, and histiocytes in routinely processed (formalin fixed, paraffin wax embedded) human tissue specimens. Unlike the well known panmacrophage marker KP1 (CD68), neither dendritic cells (interdigitating cells, Langerhans' cells, and microglia) nor myeloid, lymphoid, or epithelial cells stained with 3A5. CONCLUSION--As the staining pattern of 3A5 is restricted, compared with other macrophage markers and the recognised epitope survives common fixation and embedding procedures, 3A5 is a valuable marker for histiocytes and macrophages in routine diagnostic applications.     

Angiomyolipoma of the kidney. Immunoreactivity with HMB-45. Light- and electron-microscopic findings

Immunoreactivity with HMB-45 has recently been described in renal angiomyolipoma, a tumour of smooth muscle cells. HMB-45 is a monoclonal antibody that reacts specifically with melanosomes. In order to determine whether the tumour cells contain melanosomes and synthesize melanin, seven tumours were studied by light microscopy and immunohistochemically with the antibodies HMB-45, KP1 (CD68), PG-M1 (CD68), Ki-M1P, anti-lysozyme, anti-smooth-muscle actin, anti-vimentin, anti-S100 protein and KL1 (anti-keratin). Two tumours were also studied by electronmicroscopy and one by immuno-electronmicroscopy. Histochemical investigation for dopa oxidase was performed on cryostat sections. The tumours contained varying numbers of HMB-45-positive muscle cells. Reactivity was noted in lysosomal granules and rough endoplasmic reticulum. Typical premelanosomes were found in the tumour cells by electronmicroscopy. Groups of tumour cells stained for dopa oxidase. The tumour cells were not reactive for lysozyme, but reacted with KP1, PG-M1 and Ki-M1P. Immuno-electronmicrosopy showed that reactivity for KP1 was located within lysosomal granules. The findings show that the tumour cells of renal angiomyolipoma contain premelanosomes and that they are able to synthesize melanin, because they contain dopa oxidase. Immunoreactivity with KP1, PG-M1 and Ki-M1P can be attributed, in the absence of staining for lysozyme, to the large number of lysosomal granules. The tumour cells were not found to be related to macrophages or myeloid cells.     

Use of monoclonal antibody KP1 for identifying normal and neoplastic human mast cells.

The monoclonal antibody KP1 (CD68) was used to stain normal and neoplastic monocytes and macrophages in routinely processed, paraffin wax embedded tissue: mast cells also exhibited strong, consistent cytoplasmic immunoreactivity. Light microscopic findings were corroborated by electron microscopical and immunocytochemical findings. The predominant sites of immunoreactivity were the specific intracytoplasmic granules of the mast cells. All mast cell subtypes--that is, normal and reactive mast cells, such as those in lymph nodes exhibiting chronic non-specific lymphadenitis, and malignant or neoplastic mast cells in various types of mastocytosis--reacted with this antibody. This finding is of diagnostic importance, because mast cell proliferation could be mistaken for histiocyte proliferation. It also supports the hypothesis that mast cells derive from the bone marrow.     

An immunohistological study of the cellular constituents of Hodgkin''s disease using a monoclonal antibody panel

Cryostat sections of lymphoid tissue from 44 cases of Hodgkin's disease were analysed by immunoperoxidase staining using a panel of monoclonal antibodies which included reagents reactive with T cells and their subsets, B cells, HLA-DR, Ig, dendritic reticulum cells and C3b receptor. A wide spectrum of immunohistological patterns was observed ranging from cases in which T cells were numerous (B cells being absent or present in only small numbers) to cases in which very prominent B cell follicles were present. These follicles contained a meshwork of dendritic reticulum cells and were composed of polyclonal B cells (as assessed by light chain expression). T cells were present in small numbers within these B cell follicles, often clustered in a thin rim around individual Reed-Sternberg and Hodgkin's cells. All B cell-rich cases were examples of lymphocyte predominant Hodgkin's disease. Assessment of the T cell helper/suppressor ratios was hindered by the fact that both anti-helper antibodies (OKT4 and anti-Leu 3a) reacted with macrophages. However the majority of cases appeared to contain a normal excess of T helper cells. HLA-DR was strongly expressed in T cell rich areas, on Reed-Sternberg and Hodgkin's cells, on vascular endothelium and on numerous infiltrating cells in the fibrous tissue areas in cases of nodular sclerosing disease. Reed-Sternberg and Hodgkin's cells were not labelled by either anti-fibronectin or by antibodies reactive with dendritic reticulum cells (anti-C3b receptor and antibody R4/23).     

Expression and cell distribution of the intercellular adhesion molecule, vascular cell adhesion molecule, endothelial leukocyte adhesion molecule, and endothelial cell adhesion molecule (CD31) in reactive human lymph nodes and in Hodgkin''s disease.

The immunocytochemical expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), endothelial leukocyte adhesion molecule (ELAM-1), endothelial cell adhesion molecule (EndoCAM CD31), and HLA-DR antigens was investigated in sections of 24 reactive lymph nodes and in 15 cases of Hodgkin's disease. ICAM-1 was detected in sinus macrophages, follicular dendritic reticulum cells (FDRCs), interdigitating reticulum cells (IDRCs), epithelioid macrophages, Hodgkin's cells (HCs), and vascular endothelium. ICAM-1 expression was often associated with that of HLA-DR antigens. VCAM-1 was detected in FDRCs, in fibroblast reticulum cells (FRCs), in macrophages, and in rare blood vessels. EndoCAM (CD31) was constitutively expressed in all types of endothelial cells, sinus macrophages, and in epithelioid granulomas. ELAM-1 was selectively expressed by activated endothelial cells of high endothelium venules (HEVs). When expression of the inducible adhesion molecules ICAM-1, VCAM-1 and ELAM-1 was comparatively evaluated in HEVs, it was found that ICAM-1 + HEVs were present in all reactive and HD nodes, whereas ELAM-1 and/or VCAM-1 were expressed only in those pathologic conditions characterized by high levels of interleukin-1/tumor necrosis factor (IL-1/TNF) production, such as granulomatosis and Hodgkin's disease. In Hodgkin's disease, the expression of ELAM-1/VCAM-1 was more pronounced in cases of nodular sclerosis and was associated with a significantly higher content of perivascular neutrophils.     

Monoclonal antibody JC1: new reagent for studying cell proliferation.

AIM: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. METHODS: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. RESULTS: Biochemical investigation indicated that the antigen recognised by JC1 gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. CONCLUSION: Antibody JC1, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined.     

Cytokine Production (IL-1α, IL-1β, and TNFα) and Endothelial Cell Activation (ELAM-1 and HLA-DR) in Reactive Lymphadenitis, Hodgkin's Disease, and in Non-Hodgkin's Lymphomas: An Immunocytochemical Study

Cryostat sections of 58 lymph nodes were immunostained with a polyclonal rabbit serum against IL-1 alpha, and with monoclonal antibodies directed to IL-1 alpha (Vmp18), IL-1 beta (Vhp20 and BRhC3), and tumor necrosis factor alpha (TNF alpha) (B154.7). Furthermore the presence of cytokine-containing cells was correlated with the expression of endothelial leukocyte adhesion molecule (ELAM-1; 29F2) and of human leukocyte antigen (HLA-DR) (OKIa-1) by endothelial cells. Cells containing IL-1 and/or TNF alpha were detected mainly in pathologic conditions characterized by reactive or neoplastic expansion of the lymph node paracortex. Cells positive for IL-1 were detected in 16 of 21 cases of Hodgkin's disease, in 4 of 4 cases of T-NHL, and in 5 cases of diffuse or mixed lymphadenitis. Interleukin-1 alpha was detected in macrophages, interdigitating reticulum cells (IDRCs), endothelial cells, and neoplastic Hodgkin's and Reed-Sternberg (H-RS) cells. Cells positive for IL-1 beta were much fewer and consisted mainly of macrophages. Hodgkin's Reed-Sternberg cells were negative for IL-1 beta even after in vitro stimulation with bacterial endotoxin. Tumor necrosis factor alpha (TNF alpha) was present in macrophages and H-RS cells. Endothelial leukocyte adhesion molecule-1 expression by endothelial venules was detected in 17 of 20 cases of Hodgkin's disease, in 2 of 4 cases of T-NHL, and in 5 of 5 cases of diffuse lymphadenitis. In these pathologic conditions, HLA-DR antigens also were expressed frequently by endothelial cells. Cytokine-containing cells and ELAM-1-positive high endothelial venules (HEV) were extremely rare in lymph nodes involved by follicular lymphadenitis (12 cases) or B-NHL (16 cases). In cases of reactive or neoplastic B-cell proliferations, HLA-DR-positive HEVs still were present often. Our results indicate that IL-1/TNF alpha production at tissue level is often associated with ELAM-1 expression by HEVs, but is less well correlated with expression of HLA-DR antigens by endothelial cells.     

Monocyte/macrophage infiltrate in 41 oligodendrogliomas. Paraffin-wax study

We have studied wax-embedded tissue from 41 (33 low and 8 high grade) oligodendrogliomas with KP 1, a new monoclonal anti-monocytes/macrophages (M/Ms) antibody. Away from areas of degeneration, M/Ms cells were detected in 63% of low grade and 87% of high grade tumours. The latter had, on average, at least more than twice as many infiltrating M/Ms. It is concluded that our findings may indicate the presence of a host M/Ms immune response and that KP 1 antibody is suitable for the study of such infiltrate, thus lessening (but not obviating) the need for frozen tissue for immunohistological studies.     

CD163: a specific marker of macrophages in paraffin-embedded tissue samples

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody's usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163- with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163-. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.     

Plasmacytoid T cells: a cell population normally present in the reactive lymph node. An immunohistochemical and electronmicroscopic study

Plasmacytoid T cells (PTCs) are medium-sized cells characterized by abundant rough endoplasmic reticulum. They occur in the thymic-dependent area in human lymph nodes. PTCs are hardly identified in routinely stained sections. We studied their occurrence in 100 reactive lymph nodes with the use of monoclonal antibodies MB2, MT1, LN1, LN2, reactive on paraffin-embedded tissue, and with electron microscopy in nine selected cases. PTCs strongly reactive with MT1 and LN2 were found in 87 of 100 lymph nodes. They were observed in clusters, loose aggregates, and as singular cells. An association between PTCs, postcapillary venules, small T lymphocytes, and interdigitating reticulum cells (IDRCs) was found. Our results indicate that PTCs are normally present in the human lymph node. Their immunophenotype suggests a relationship with a monocyte/macrophage lineage, but does not rule out a T cell origin. If the various distribution patterns represent the morphologic substrate of functional stages of PTCs it can be assumed that PTCs play a role in T cell-mediated immune response.     

Macrophage infiltration in non-Hodgkin's lymphomas: a quantitative in situ study

The frequency and distribution pattern of macrophages within 93 non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry using stereological methods. For the identification of macrophages (M phi), several antibodies (Mono 1, Mono 2, OKM 1) reactive with surface antigens on cells of the monocyte-macrophage series and cytochemical staining for acid phosphatase were applied. The average number of macrophages within lymph node tissue of NHL was 6,299 +/- 760 cells/microliter (similar to reactive lymphatic tissue: 6,559 +/- 1,027). The highest number of infiltrating macrophages was detected in immunoblastic NHL (17,306 +/- 2,773), differing significantly from other histological subtypes and reactive lymphatic tissue (p less than 0.005). The possible impact of tumor-infiltrating macrophages on lymphoma cell proliferation and differentiation is discussed.     

IFN-beta1b induces kynurenine pathway metabolism in human macrophages: potential implications for multiple sclerosis treatment.

Interferon-beta(1b) (IFN-beta(1b)) has limited efficacy in the treatment of relapsing-remitting multiple sclerosis (RRMS). The kynurenine pathway (KP) is chiefly activated by IFN-gamma and IFN-alpha, leading to the production of a variety of neurotoxins. We sought to determine whether IFN-beta(1b) induces the KP in human monocyte-derived macrophages, as one explanation for its limited efficacy. Serial dilutions of IFN-beta(1b) (at concentrations comparable to those found in the sera of IFN-beta(1b)-treated patients) were added to human macrophage cultures. Supernatants were collected at various time points and assayed for the KP end product, quinolinic acid (QUIN). The effect of IFN-beta(1b) on the KP enzymes indoleamine 2,3-dioxygenase (IDO), 3-hydroxyanthranilate dioxygenase (3HAO), and quinolinate phosphoribosyltransferase (QPRTase) mRNA expression was assessed by semiquantitative RT-PCR. IFN-beta(1b) (> or =10 IU/ml) led to increased mRNA expression of both IDO and QUIN production (7901 +/- 715 nM) after 72 h at 50 IU/ml IFN-beta(1b) (p < 0.0001). This study demonstrates that IFN-beta(1b), in pharmacologically relevant concentrations, induces KP metabolism in human macrophages and may be a limiting factor in its efficacy in the treatment of MS. Inhibitors of the KP may be able to augment the efficacy of IFN-beta in MS.     

Macrophages and interdigitating reticulum cells in normal human thymus and thymomas: immunoreactivity for interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha

Pairs of monoclonal/polyclonal antibodies directed against interleukin-1 (IL-1) alpha, IL-1 beta and tumour necrosis factor (TNF) alpha were used for immunocytochemical identification of cytokine-containing cells in cryostat sections of human fetal thymuses and thymomas. In the fetal thymus immunoreactivity for IL-1 alpha was mainly confined to the medulla and was detected in S-100 positive interdigitating reticulum cells. The pattern of immunoreactivity for IL-1 beta was similar to that for IL-1 alpha, but the number of positive cells was much lower. Cells positive for TNF alpha were extremely rare in the fetal thymus. In 11 thymomas macrophages were constantly present and were regularly distributed throughout the tumour, whereas S-100 positive interdigitating reticulum cells were fewer and were characterized by a zonal distribution. Thymoma-associated macrophages were negative for IL-1 beta and were poorly reactive for IL-1 alpha, only a few positive cells being detected in five of the cases. Some macrophages with immunoreactivity for TNF alpha were detected in seven cases; they formed rosettes with surrounding lymphocytes or were located in a perivascular position. A marked immunoreactivity for TNF alpha was constantly detected in mast cell granules, which were observed in nine thymomas but not in fetal thymus. Positive immunoreactivity of interdigitating reticulum cells for IL-1 alpha was confirmed in five reactive lymph nodes and was also observed in Langerhans' cells in dermatopathic lymphadenitis. Our findings suggest that IL-1 alpha is a crucial molecule for interdigitating reticulum cell and Langerhans' cell function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cells of the human mononuclear phagocyte system with rat monoclonal antibodies

Five rat monoclonal antibodies (McAbs) to human macrophages are described: YTH 8.18, YTH 25.7, YTH 51.1, YTH 85.12.1, and YHB 65.5. These McAbs are divided into three groups, since YTH 8.18, YTH 51.1, and YHB 65.5 are thought to identify the same antigen. These McAbs react with some bone marrow blast cells, granulocytes, and different percentages of peripheral blood monocytes. When studied on different body tissues, they were found to identify all members of the mononuclear phagocyte system (MPS), except Langerhans cells of skin and epithelium and in the case of one group (YTH 8.18/YTH 51.1/YHB 65.5) osteoclasts. In nine reactive lymph nodes the anti macrophage McAbs identified germinal centre macrophages, sinus macrophages, and interdigitating cells, but not dendritic reticulum cells. They also identified epithelioid macrophages and Langhans-type multi-nucleated giant cells in lymph nodes involved in granulomatous lesions (sarcoidosis and toxoplasmosis). In 24 cases of non-Hodgkin's lymphoma, the antimacrophage McAbs identified reactive macrophages in cases of B- or T-lymphocyte origin, whereas in three selected cases of true histiocytic lymphoma all the McAbs were found to be reactive with the vast majority of neoplastic macrophages as they were with the cells of a neoplastic macrophage line (U937). The possible use of these McAbs in the identification of benign and malignant macrophages in different systems is discussed.     

Ki-M2R, a new specific monoclonal antibody, discriminates tissue macrophages from reticulum cells and monocytes in vivo and in vitro

Utilizing rat peritoneal macrophages as the immunogen, a new monoclonal antibody enabling differential monitoring of the mononuclear phagocyte system (MPS) by immunohistochemistry has been raised. Designated Ki-M2R, this antigen could be detected with the immune alkaline phosphatase reaction on all macrophages including those of bone marrow, lymphatic sinuses, lymphoid follicles, splenic red pulp, and von Kupffer cells of the liver, as well as on macrophages of connective tissue, renal interstitial tissue, serous cavities, and gastrointestinal tract. Langerhans cells--the MPS-derived reticulum cells of the epidermis--interdigitating reticulum cells, and dendritic reticulum cells of lymphoid follicles were invariably negative. Blood monocytes were rendered positive only after evolving into macrophages upon appropriate stimulation. Thus, Ki-M2R selectively labels monocytes after transformation into macrophages.     

VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections.

AIMS--To characterise a new mouse monoclonal antibody, VS38, which recognises an intracytoplasmic antigen of 64 kilodaltons present in normal and neoplastic plasma cells; and to establish its value as a diagnostic reagent for routine pathological practice. METHODS--A range of normal and neoplastic tissue sections, both frozen and routinely fixed, were immunostained, using the microwave method of antigen retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range of human cell lines. The molecular weight of the antigen recognised by the antibody was obtained by western blot analysis. FACS analysis was used to demonstrate the cellular location of the antigen and its presence on tonsil cell suspensions and myeloma cases. RESULTS--VS38 recognised normal and neoplastic plasma cells in all of the tissues, including all routinely fixed plasma cell neoplasms tested. The antibody also weakly stained epithelial elements within the tissue but was absent from haemopoietic cells of other lineages. CONCLUSION--Antibody VS38 is of potential value in identifying myeloma or plasmacytoma in bone marrow or other tissues. It differentiates lymphoplasmacytoid lymphoma from lymphocytic and follicular lymphoma. It also subdivides large cell lymphomas into two groups which may be a more reliable method of separating these tumours than morphology alone.     

The p150,95 molecule is a marker of human mononuclear phagocytes: comparison with expression of class II molecules

A new monoclonal antibody (mAb), named 3.9, is described that is specific for the p150,95 molecule, a member of the LFA-1, CR3, p150,95 family of human leukocyte differentiation antigens. The LFA-1 molecule participates in a variety of T cell interactions and the CR3 molecule is the receptor for the complement component iC3b, but little is known about the p150,95 molecule. Here we show that the expression of p150,95 is confined to myeloid cells. mAb 3.9 reacts variably with neutrophils, more strongly with monocytes and is most strongly expressed on tissue macrophages. Using this mAb and others, we have examined the heterogeneity of tissue macrophages. Cells such as Langerhans' cells, dendritic reticulum cells and osteoclasts failed to react with these mAb and thus, probably do not belong to the mononuclear phagocyte lineage. Using a new double-labeling technique, we investigated lymphoid tissue for dendritic cells bearing class II molecules which might function in interactions with T cells. In T cell areas macrophages expressing class II markers were seen but there was no evidence for other types of dendritic or interdigitating cells which expressed class II molecules but not macrophage epitopes. The conclusion from this survey was that the most prominent cell with dendritic morphology found in the T cell areas of lymphoid tissue was a macrophage.     

Phenotype for activated tissue macrophages in histiocytic necrotizing lymphadenitis

Macrophage polarization is divided into M1 and M2 type based on membrane receptors, cytokines, and chemokines. M1 expresses CD80, interleukin (IL)-6, IL-12, and chemokine receptor (CCR)7, while M2 expresses CD163, IL10, and chemokine ligand (CCL)22. The aim of the present study was to identify the properties of infiltrating tissue macrophages in histiocytic necrotizing lymphadenitis (HNL). Twenty patients with HNL were studied, and immunohistochemistry for CD68 (KP1), CD163, CCL22, CCR7, and CD123 was done, along with myeloperoxidase (MPO). To evaluate the phenotypes of tissue macrophages in HNL, the number of cells stained positively for CD163, CCL22, CCR7, CD123 and MPO concurrently with CD68 was counted, and the ratio was calculated for each antibody to CD68+ cells. There was a high rate of co-expression for CD163 (median, 78%) or CCL22 (80%) and a low rate for CCR7 (5%) in CD68+ cells. It is therefore conceivable that infiltration by M2 macrophages is dominant in HNL. Furthermore, some CD68+ tissue macrophages in HNL co-express MPO or CD123 (range, 5–80%; median, 23% and 40%, respectively). It is suggested that these characteristic tissue macrophages may be associated with the pathogenesis of HNL and that M2 macrophages may infiltrate to repair the lymphoid tissue injured by cytotoxic T cells in HNL.     

Distribution of a formalin-resistant myelomonocytic antigen (L1) in human tissues. I. Comparison with other leukocyte markers by paired immunofluorescence and immunoenzyme staining

L1 is an approximately 36-kd protein recently identified as a cytoplasmic and surface marker antigen of virtually all resting peripheral blood neutrophils and monocytes. This study of its tissue distribution showed that L1 is particularly well preserved in formalin-fixed and paraffin-embedded routine material. It had a restricted distribution within the monocyte-derived cell lineage, being mainly confined to reactive histiocytes (infiltrating macrophages). L1 was a much more reliable marker for such cells than lysozyme, except that the latter was better expressed by epithelioid and giant cells. L1 was lacking in HLA-DR-positive interdigitating, Langerhans', and most intestinal histiocytic cells. The same was true for Kupffer cells in normal livers; but in livers of three patients with malignant histiocytosis or histiocytic medullary reticulosis infiltrating histiocytes and putative Kupffer cells stained positively. Follicular dendritic cells and tangible body macrophages were always questionably L1 positive.     

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.