Immunohistological analysis of delayed-type hypersensitivity in man.
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Animals↗
Biomedical subjects
G Janossy
Publications and source records attributed to G Janossy.
Nuclear terminal deoxynucleotidyl transferase in leukaemic infiltrates of testicular tissue.
Early detection of testicular leukaemia and the identification of residual leukaemic cells in treated patients are important aims in the management of males with acute lymphoblastic leukaemia (ALL). In most cases of ALL ( greater than 95%) the blast cells express terminal deoxynucleotidyl transferase (TdT), a nuclear enzyme. We have therefore standardized the immuno-fluorescence and -peroxidase techniques (using anti-Tdt antibodies) for identifying TdT cells in the normal thymus, as well as in samples of testis with heavy leukaemic infiltrates (positive controls). TdT cells can be identified in formalin (but not in Bouin's or Carnoy's) fixed paraffin-embedded tissues, and the preservation of morphological details is excellent. The method is nevertheless difficult to standardize and also requires the use of deoxyribonuclease (DNase) for the digestion of sections. However, in frozen tissue sections, stronger staining of TdT cells was found, even without DNase treatment. Good morphology was preserved when cut sections were fixed immediately in the cryostat. In the second part of the study 15 samples from treated boys were analysed to see whether the technique is suitable to identify residual minimal leukaemic infiltrates. In 5 patients scanty disseminated TdT cells were detected, and in 2 patients small clumps of TdT cells were seen. The results indicate that the immunohistological identification of TdT ALL blasts may be the method of choice.
Cell Nucleus↗
The relationship between T lymphocyte subsets and Ia-like antigen positive nonlymphoid cells in early stages of cutaneous T cell lymphoma.
Immunofluorescence studies were carried out in cutaneous T cell lymphoma (mycosis fungoides) in order to analyse the microanatomical relationship of the different T lymphocyte subsets (inducer and suppressor/cytotoxic cell populations) to large nonlymphoid Langerhans-type and so-called "indeterminate" or interdigitating cells. The conventional and mouse (monoclonal) antibodies were used in various combinations using fluorescein and rhodamine labeled second layers. In 5 of the 7 cases studied the dermal infiltrate consisted of numerous T (HuTLA+) lymphocytes, 80-90% of which expressed the inducer phenotype (HuTLA+,OKT4+). Most of these cells formed close contact with large cells exhibiting large amounts of Ia-like antigens. These cells corresponded to the interdigitating and indeterminate cells in the sections. By contrast, only small numbers (10-20%) of T cells of suppressor/cytotoxic type (HuTLA+,OKT8+) were seen. These did not show a close affinity to the Ia-like antigen positive nonlymphoid component but appeared to have a predilection for the epidermis. Epidermal Langerhans cells, also strongly Ia-like antigen positive, were further defined by 2 monoclonal antibodies reacting with a cortical thymocyte antigen HTA-1. Although Langerhans cells are probably related to the Ia-like antigen positive dermal cells only a few of the abundant latter population were HTA-1+. In the remaining 2 cases, larger populations of OKT8+ (suppressor/cytotoxic) cells were seen and could be heralding a particularly benign course. These observations indicate a close functional relationship between the lymphoid and Ia-like antigen positive dermal cells during the pre-malignant phase of cutaneous T cell lymphoma.
Aged↗
Immuno-histological analysis of bone marrow involvement in lymphoproliferative disorders.
Cryostat sections from bone marrow biopsies submerged in gum-sucrose solution and snap-frozen can be stained with various antisera to detect both the membrane and cytoplasmic antigens on normal and malignant populations in their tissue environments. This technique seems to be the method of choice to analyse the degree of BM involvement in cases of non-Hodgkin's lymphoma where conventional histology provides equivocal results. The uniform expression of only one type of light chain in B lymphoma and the analysis of the T cell infiltrate in T-CLL using monoclonal antibodies is demonstrated.
Aged↗
Elimination of T-lymphocytes from human bone marrow with monoclonal T-antibodies and cytolytic complement.
Complement-mediated cytolysis has been used to remove T-lymphocytes from suspensions of human peripheral blood and bone marrow. Selective T-cell removal was investigated by three monoclonal antibodies. OKT3, MBG6 and OKT11A. All three removed greater than 90% of T-cells but combinations were necessary to kill greater than 99% of T-cells in vitro. The macrophage-granulocyte and erythroid colony forming cells of the bone marrow were spared. The method can be applied on bulk BM samples during clinical BM transplantation and will be useful to establish whether the virtually complete removal of T-lymphocytes totally prevents transplant associated graft-versus-host disease in man.
Antibodies, Monoclonal↗
A technique for rapid isolation of bone marrow mononuclear cells using Ficoll-Metrizoate and the IBM 2991 blood cell processor.
Marrow from seven normal donors and patients has been layered onto Ficoll-Metrizoate (FM) under pressure in the IBM 2991 blood cell processor to isolate the mononuclear cell (MNC) population prior to allogeneic transplantation or cryopreservation. This separation method, which takes less than 90 min, is a further development since our previous report detailing the use of the IBM 2991 to produce a concentrated marrow 'buffy coat' for infusion (Gilmore & Prentice, 1981). By adding FM to the system, marrow stem cells are further concentrated in a small volume with removal of unwanted granulocytes and red blood cells. This facilitates in in vitro treatment of marrow with monoclonal antibodies (Granger et al. 1982) or drugs, for either the selective elimination of malignant cells prior to autologous bone marrow transplantation (BMT), or T lymphocytes in an attempt to prevent graft versus host disease (GvHD) in allogeneic BMT. Five of the seven marrows processed by this procedure have thus far been infused into lethally irradiated recipients with engraftment (allogeneic); the other two marrows have been cryopreserved.
Bone Marrow Cells↗
Enzyme histochemical analysis on cryostat sections of human bone marrow.
A recently developed procedure, that has been shown to be suitable for detailed immunohistological analysis, has been used to prepare cryostat sections of bone marrow to investigate whether enzyme-histochemical techniques are also feasible on such material. A selected group of enzymes, some of which are inhibited or destroyed in paraffin- or plastic-embedded samples, have been demonstrated. The morphological details obtained were satisfactory in the preparations. The enzymes were dipeptidyl(amino)peptidase IV (for T lymphocytes); tartrate-resistant acid phosphatase (for hairy cell leukaemia); acid phosphatase and non-specific esterase (for macrophages and monocytes); ATPase and 5'nucleotidase (for B lymphocytes); and peroxidase or chloroacetate esterase (for granulocytic cells). In these preparations strong enzyme activities were shown. In adjacent sections the immunological analysis of membrane markers could also be performed contributing to a comprehensive study of the normal and malignant bone marrow cells.
Bone Marrow↗
Combined immunological and histochemical analysis of skin and lymph node lesions in histiocytosis X.
The immunological phenotype of the cells involved in skin and lymph node lesions from two cases of histiocytosis X (H-X) were analysed by immunofluorescence techniques using combinations of heterologous and monoclonal antisera to Ia-like antigen and human cortical thymocyte (HTA-1) determinant. These cells were also characterised by a new technique using simultaneous immunofluorescence and enzyme histochemistry for acid phosphatase (ACPase). The major cell type in the lesions was found to express the same Ia+, HTA-1+ phenotype as normal epidermal Langerhans' cells (LC) and was unreactive for ACPase. Additional cell types included Ia-, HTA-1- multinucleate giant cells and residual lymphoid populations. These findings endorse previous concepts that H-X is a proliferation of abnormal LC and emphasise the heterogeneous nature of the cells involved in the disease.
Acid Phosphatase↗
Histochemical discrimination of HLA-DR positive cell populations in the normal and arthritic synovial lining.
A combination of immunochemical staining for HLA-DR antigens and the histochemical demonstration of enzyme activity has been used to identify specific cell populations in the normal and arthritic synovial lining layers. Such combined staining has revealed that the normal synovial lining contains a proportion of HLA-DR + ve cells, all of which show strong lysosomal enzyme activity. This population is greatly expanded in biopsies from patients with osteoarthritis and these positive cells also express strong ATPase activity. In the rheumatoid synovium five distinct cell types can be identified; all of which are HLA-DR + ve but differ in their morphology and pattern of enzyme activity. Of special interest was the discovery that a small but significant proportion of these cells have the characteristics of the interdigitating cells of the lymph node paracortex. The relationship between the emergence of these heterogeneous populations and the immunological basis of this inflammatory response is discussed.
Acid Phosphatase↗
Distribution of terminal deoxynucleotidyl transferase and purine degradative and synthetic enzymes in subpopulations of human thymocytes.
Terminal deoxynucleotidyl transferase (TdT) and purine metabolic enzymes were examined in subsets of human infant thymocytes (defined by surface cell antigens) and normal peripheral T lymphocytes. Putative prothymocytes (RFB-1+, HTA-1+/- large blast-like cells), medium and high density cortical thymocytes (RFB-1+, HTA-1+), and medullary thymocytes (RFB-1-, HTA-1-, OKT3+) were isolated by density gradient centrifugation, monoclonal antibody and complement-mediated cytolysis, and cell-antibody affinity chromatography. Peripheral T lymphocytes were isolated from normal adult mononuclear cells using nylon fiber technique. Adenosine deaminase (ADA) and TdT were highest in prothymocytes 48.8 +/- 14.7 mumol/hr/10(8) cells (mean +/- SE) and 22.9 +/- 1.4 U/10(8) cells, respectively. Both enzymes decreased progressively down the maturation pathway. In peripheral T lymphocytes, ADA was 3.9 +/- 1.5 mumol/hr/10(8) cells, and TdT was undetectable. Purine nucleoside phosphorylase (PNP) and ecto-5'nucleotidase (5'NT) were lowest in cortical thymocytes (27.5 +/- 11.0 nmol/hr/10(6) cells and 2.8 +/- 1.3 nmol/hr/10(6) cells, respectively) and increased with T cell maturation. The PNP level was 124.9 +/- 17.2 nmol/hr/10(6) cells and 5'NT was 30.1 +/- 3.9 nmol/hr/10(6) cells in peripheral T lymphocytes. The deoxynucleoside kinases (deoxyguanosine, deoxyadenosine, and deoxycytidine kinases) paralleled the changes in ADA and TdT activity among the different T subsets. The proliferative activity (labeling index) was highest in the prothymocyte fraction and lowest in peripheral T cells. Variation in the distribution of these enzymes in T cell subsets may explain their different sensitivities to deoxyadenosine and deoxyguanosine toxicity and the different effects on T cell development of ADA or PNP deficiency.
Adenosine Deaminase↗
T cell subset abnormalities in tissue lesions developing during autoimmune disorders, viral infection, and graft-vs.-host disease.
The authors review a large body of contemporary immunohistologic findings on the tissue distribution of T lymphocytes in normal and pathological conditions. The suggestions for technological advances in this field are: signal amplification using mixtures of monoclonal antibodies directed against different epitopes on the same antigen (e.g. OKT4A+B+D), triple layer amplification systems using hapten-labelled antibodies, and informative double staining methods with combinations of antibodies labelled with different fluorochromes or enzymes. Review of histological observations in a series of human diseases suggests that imbalances of OKT4+ and OKT8+ subsets of T lymphocytes may represent different types of immunoregulatory disorders. Rheumatoid arthritis and sarcoidosis appear to involve a high level of OKT4+ subpopulation response coupled with an associated appearance of a special type of HLA-DR+ macrophages. It remains to be seen whether normal or self-limited immunological responses (early stages of bacterial infection or delayed-type hypersensitivity reactions) produce OKT4+ and macrophage responses that are characteristically different. Meanwhile, excessive levels of OKT8+ cells have been found in a wide range of recognized or presumed immunoregulatory disorders including: graft-vs.-host reaction and viral infections. These disorders, as well as primary biliary cirrhosis and lichen planus, appear to possess both overlapping and disparate clinical characteristics, and the immunohistological observations may reflect the functional heterogeneity of OKT8+ populations in these diseases. These studies show that histologically meaningful heterogeneity can already be demonstrated for the OKT8+ lymphocyte group.
Animals↗
Enzymes of purine metabolism in human peripheral lymphocyte subpopulations.
Ecto-5'nucleotidase (5'NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and deoxycytidine (CdR), deoxyguanosine (GdR), deoxyadenosine (AdR) and adenosine (AR) kinases have been measured in subpopulations of peripheral blood lymphocytes of eight healthy volunteers. The separation of B, T, T helper/inducer and T suppressor/cytotoxic cells was performed by means of density gradient centrifugation, E rosetting, passage through a nylon-wool column and antibody affinity chromatography utilising OKT8 and OKT4 monoclonal antibodies. ADA was significantly higher in T lymphocytes and 5'NT in B lymphocytes. Among T cell subpopulations, 5'NT activity was significantly higher (P less than 0 . 01) in T suppressor/cytotoxic (OKT8+) cells (32 . 9 units/10(6) cells than in T helper/inducer (OKT4+) cells (9 . 7 units/10(6) cells). Indeed, the 5'NT activity in T suppressor cells was similar to that in B cells. T helper cells tended, however, to have higher PNP and ADA activities than T suppressor cells but the differences were not statistically significant. No major differences were noted in kinase activities between any of the lymphocyte subpopulations.
5'-Nucleotidase↗
An immunohistological analysis of lymphocyte subpopulations and their microenvironment in the synovial membranes of patients with rheumatoid arthritis using monoclonal antibodies.
We have used monoclonal antibodies of the orthoclone (OKT) series to identify T cell subsets in an immunohistological analysis of the synovial membranes obtained from normal individuals and patients with osteoarthritis or rheumatoid arthritis. T cells of the inducer and the suppressor/cytotoxic subsets were identified by the OKT4 and OKT8 antibodies respectively while HLA-DR (Ia-like) antigens were recognized by a conventional antiserum. In the normal and osteoarthritic synovial membranes, virtually no lymphocytes were identified whereas the mononuclear cell infiltrates of the rheumatoid synovial membranes were composed predominantly of T cells expressing the OKT4 inducer phenotype with few OKT8+ suppressor/cytotoxic cells. The OKT4+ cells were found to be intimately related to B cells and strongly HLA-DR+ cells which morphologically resembled the interdigitating cells of lymph nodes. The micro-anatomical arrangement of these different cell types in the mononuclear infiltrates of the rheumatoid synovial membranes closely resembled that of the paracortical or T cell dependent area of normal lymph nodes except few OKT8+ lymphocytes were present. These findings are explained in terms of rheumatoid arthritis as a disease of altered T lymphocyte/macrophage immunoregulation.
Antibodies, Monoclonal↗
Immunological analysis of the skin in graft versus host disease.
Immunohistochemical analysis was performed on skin biopsies from patients suffering from graft versus host disease (GVHD) following bone marrow transplantation for aplastic anaemia and leukaemia. Lymphoid cells infiltrating the skin were exclusively T cells with the OKT8+ phenotype and these are probably cytotoxic T cells. Langerhans cells were reduced in number in all specimens and a variable number of HLA-DR positive macrophages were seen in the dermis. In several cases HLA-DR antigens were expressed on keratinocytes. These results show consistent features which may help discriminate rashes in the skin in the post-transplant period.
Adolescent↗
T cell subpopulations, monoclonal antibodies and their therapeutic applications.
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Antibodies, Monoclonal↗
Distinct T lymphocyte subsets affect granulo-monocytic differentiation and proliferation.
Bone marrow culture techniques using semi-solid support-media provide an opportunity to observe granulocyte-macrophage progenitor cells proliferating and differentiating in vitro. Cells from the bone marrow and peripheral blood produce specific growth factors which stimulate both the proliferation and differentiation processes (collectively termed colony-stimulating factors or activity, CSF/CSA). Even highly purified CSA induces proliferation and concomitant differentiation suggesting that the two processes are inseparable. We here present evidence to suggest that the two processes are, at least in part, individually regulated. We observed delayed differentiation by morphological, cytochemical and functional criteria in granulocyte-macrophage clones formed in cultures in which the growth stimulus was supplied by marrow or blood cells which have been depleted of T lymphocytes bearing the OKT3/MBG6 antigen(s) by complement lysis. The proliferation stimulus was unaffected. T cell depletion using another monoclonal antibody, OKT11a (which removed an overlapping but not identical population of T cells), did not produce the same effect but did increase the level of CSA produced by the remaining marrow cells. Selective replacement experiments using OKT4+ or OKT8+ cell preparations suggested that removal of an MBG6+, OKT3+, OKT8+, OKT4-lymphocyte subset is responsible for the effect on differentiation.
Antibodies, Monoclonal↗
Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody.
A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.
Animals↗
Lymphoid infiltrates in skin in graft-versus-host disease.
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Adolescent↗