Monoclonal antibodies: powerful new tools for the clinician.
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Publications and source records attributed to C L Berger.
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A population of cells showing the surface phenotype of Langerhans cells (LCs) was identified in the dermal infiltrates of cutaneous T cell lymphoma (CTCL). Peroxidase-conjugated OKT6, a monoclonal antibody reactive with epidermal LCs, was used to directly label frozen tissue sections of diseased skin from twenty-three patients with CTCL, two patients with secondary cutaneous involvement by a B cell lymphoma, and three patients with lymphocytoma cutis. OKT6-reactive cells represented a significant although minor population in the dermal infiltrate of twenty-two of the twenty-three CTCL biopsies, accounting for up to 5% of the cells. Double-labeling studies revealed that the OKT6-positive cells also exhibited Ia but not T cell antigens. Since OKT6-reactive cells were not found in either the B cell lymphomas or lymphocytoma cutis, their presence in a malignant infiltrate is suggestive of a T cell neoplasm.
Two murine monoclonal antibodies (BE1 and BE2), produced by using leukemic helper T cells from a patient with cutaneous T-cell lymphoma (CTCL) as immunogens, reacted selectively with CTCL lymphocytes and some transformed cultured lymphocytes, as determined by radioimmunoassay (RIA) and indirect immunofluorescence (IIF). BE1 reacted significantly (P less than or equal to 0.001) with leukemic CTCL lymphocytes and with CTCL cells from infiltrated lymph nodes (RIA, mean +/- SD = 776 +/- 275 cpm), as compared with background counts (263 +/- 68). BE1 binding to normal blood mononuclear cells (RIA, mean +/- SD = 283 +/- 58 cpm) was indistinguishable from background. BE1 also reacted with Epstein-Barr virus (EBV)-transformed B-cell lines (RIA, mean +/- SD = 794 +/- 230) and some long-term T-cell lines. BE1 did not react with the majority of lymphoid cell lines or tumor cell lines tested. BE1 also did not react with any normal tissues screened by IIF. BE1 precipitated a molecule from CTCL cells that, under reducing conditions, has two components with molecular mass of 27,200 and 25,800 D. BE2 also reacted significantly (P less than or equal to 0.001) with CTCL cells from two of four patients (RIA, mean +/- SD = 519 +/- 113 cpm). The binding of BE2 to normal mononuclear cells was indistinguishable from background (309 +/- 38 cpm). BE2 also reacted with an antigen present on EBV-B-cell lines (RIA, mean +/- SD = 654 +/- 194) and MOLT 3 and HUT 78 T-cell lines. BE2 reacted with an antigen expressed on a subpopulation of lymphocytes from five of eight patients with B-cell CLL studied by IIF (mean +/- SD = 18 +/- 6). Other long-term T-cell lines and tumor cell lines studied by IIF were unreactive with BE2. BE2 did not react with any of the normal tissues studied. BE2 precipitated a molecule (78,000 D) from CTCL cells and EBV-B cells with a single component under reducing conditions. Immunoperoxidase-labeled BE1 and BE2 reacted with CTCL cells in frozen sections of infiltrated lymph nodes and skin. In addition, BE1 and BE2 reacted with blood lymphocytes from 16 of 21 patients whose CTCL had otherwise been considered localized to skin. These two monoclonal antibodies react with tumor antigens associated with CTCL and appear to be useful in the diagnosis of this disorder.
Previous immunofluorescent studies have shown that differentiation antigens recognized by the monoclonal antibody (OKT6) are present on the external membranes of human epidermal Langerhans cells, cortical thymocytes and some cultured T cell lines. In the present investigation, the biochemical characteristics of the OKT6 recognized antigens derived from these three sources were compared. Following immunoprecipitation with OKT6, a single band with an approximate molecular weight of 52,000 daltons was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (under both reducing and nonreducing conditions) in the detergent lysate of radioiodinated normal epidermal cells. A molecule with the same apparent molecular weight was immunoprecipitated from thymocytes and cultured MOLT-3 (T cell-acute lymphoblastic leukemia) cells. However, a low molecular weight protein of approximately 10,000 daltons was coprecipitated from these MOLT-3 cells. No electrophoretically identifiable antigens were precipitated from peripheral lymphocytes or monocytes with OKT6. These observations further distinguish Langerhans cells from classical monocytes, indicate that these cells express a membrane antigen otherwise characteristic of cortical thymocytes, and suggest the potential usefulness of the monoclonal antibody, OKT6, in further investigations of the functions and ontogeny of Langerhans cells.
Monoclonal antibodies recognizing human T cell differentiation antigens were used to study lymphocyte populations in three cutaneous diseases. Neoplastic lymphocytes from patients with varying phases of cutaneous T cell lymphoma (mycosis fungoides, Sezary syndrome and related presentations) were reactive with OKT1 and OKT3 (pan T cell reagents) and OKT4 (an antibody defining the functional "helper" T cell subset). The malignant cells lacked membrane antigens reactive with OKT5 and OKT8 (markers of "suppressor" T cells). The presence of an OKT1+, OKT3+, OKT4+, OKT5-, OKT8- phenotype on the neoplastic T lymphocytes of cutaneous T cell lymphoma (CTCL) supports the clinical impression that all phases of CTCL represent a single disease entity. A patient with pemphigus vulgaris, a disease of autoreactive, antiepidermal antibodies was shown to consistently have a marked expansion of the peripheral blood OKT4 reactive T lymphocyte population. These findings suggest that autoantibodies in pemphigus vulgaris may occur in the context of a profound OKT4/OKT5 immunoregulatory imbalance. Peripheral blood lymphocytes from patients ith extensive psoriasis vulgaris had a normal profile of reactivity with the OKT antibodies. In addition, OKT6 (marker of intrathymic T cells) has been shown to react with Ia+ dendritic cells in the epidermis suggesting that this antibody may recognize Langerhans' cells.
Previous studies of E rosette-forming cells in the blood of patients with psoriasis vulgaris have demonstrated conflicting results. Availability of a battery of monoclonal antibodies permitted quantitation of individual T cell populations in patients with psoriasis. Peripheral blood mononuclear leukocytes from twelve patients with extensive, active psoriasis and from fifteen normal controls were studied by indirect immunofluorescence. No statistically significant differences from control values were identified (p values were all greater than or equal to 0.4). These monoclonal antibodies provide highly specific reagents, previously not available, for identifying different lymphocyte phenotypes. Analysis of the data indicates that shifts in major T lymphocyte subpopulations are not present in patients with active psoriasis.
The association of hormonal syndrome and APUD (amine precursor uptake, decarboxylase) features with small cell carcinoma of the lung (SCC) has suggested that SCC has a separate cell origin from other major forms of lung cancer. Recently, however, both SCC and non-SCC lung cancers have been found to contain small polypeptide hormones and APUD enzymes. The present study quantitates, in 50 samples of human lung cancer tissue, relationships among the 4 major types of lung cancer and endocrine-related properties. Among 4 parameters measured (dopa decarboxylase, histaminase, beta-endorphin, and calcitonin), no single marker clearly separated SCC from non-SCC lung cancer. The high activity of dopa decarboxylase (the "D" in "APUD") best separated SCC from non-SCC, but significant overlap existed even for this critical APUD property. In fact, 2 adenocarcinomas had among the highest concentrations of dopa decarboxylase, histaminase, and calcitonin of any tumor tissue studied. The simultaneous appearance of high levels of 2 or more markers favored SCC. This was quantitated by deriving an index unit based upon the product of the values for the 4 markers in each lesion. This index separated all SCC from all non-SCC lung carcinomas, with the exception of the above 2 adenocarcinomas. Endocrine-related properties thus occur throughout the spectrum of human lung cancer. Biochemical differences between the major histopathological types are quantitative rather than qualitative and probably reflect the fact that the major forms of lung cancer represent a continuum of differentiation within a common cell lineage which includes both SCC and non-SCC lung tumors.
Monoclonal antibodies to human T cells permit the characterization of the surface phenotype of cutaneous T cell lymphoma (CTCL). The majority of CTCL cells are reactive with OKT1 and OKT3 monoclonals, which identify peripheral T cells and mature thymocytes. The neoplastic cells also react with OKT4, which recognizes the inducer T cell subset; they are, however, unreactive with OKT5 monoclonal, which identifies cytotoxic/suppressor T cell subsets. These data are in agreement with previous functional studies demonstrating that CTCL is a neoplasm of inducer (helper) T cells.
Monoclonally-derived neoplastic T-cells from a patient with cutaneous T-cell lymphoma respond to multiple human HLA-D antigens in mixed lymphocyte culture. The implications of this phenomenon relevant to normal T-cell function and to malignancy are discussed.
Mononuclear leukocytes were separated from whole blood by ficoll-hypaque flotation and by elutriation (counterflow centrifugation). Lymphocytes isolated from 6 control subjects by elutriation showed a 30% greater response to stimulation with phytohemagglutinin and 130% greater response to streptokinase-streptodornase stimulation than did autologous lymphocytes obtained by ficoll-hypaque separation. Cell yields of major mononuclear cell subpopulations and cell viability were comparable after separation of leukocytes by both techniques. These results indicate that ficoll-hypaque flotation may diminish lymphocyte responses, and that elutriation offers a useful alternative to ficoll-hypaque separation. In addition, elutriation may be the preferable method for evaluation of lymphocytes from patients with suspected immunologic dysfunction and may be valuable in the isolation of mononuclear cells from infiltrated skin lesions.
Neoplastic lymphocytes from 3 patients with widespread cutaneous T cell lymphoma were karyotyped, using a chromosome banding technique. None of the patients had previously received chemotherapy. Morphologically homogeneous populations of abnormal T cells were available from 2 distinct body regions of 2 of the individuals and from the bone marrow of the third. Karyotypes from each of the 3 patients indicated monoclonality of their lymphoma. These observations suggest that extracutaneous dissemination of cutaneous T cell lymphoma involves spread of neoplastic cells derived from a single clone. If future studies can demonstrate that those neoplastic T cells actually localized in the skin are also progeny of a single malignant cell, a widely accepted concept that cutaneous T cell lymphoma is of multifocal origin will have to be reexamined.
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Four patients with cutaneous T cell lymphoma were treated with iv administered horse antithymocyte globulin. Evidence of a beneficial response was obtained in three of the four patients. Limited sensitivity of neoplastic T cells to complement-mediated lysis in the presence of the antithymocyte globulin was identified, suggesting that other mechanisms may be responsible for the observed clinical responses.
The effect of 310 courses of corticotropin, methylprednisolone, prednisone, and dexamethasone were studied in 62 patients with generalized myasthenia gravis who were poorly responsive to anticholinesterase medication and most of whom required assisted respiration. Improvement in strength and response to anticholinesterase medication occurred in 91% of the courses, and was moderate or marked in 63%. The incidence, degree, and duration of improvement appeared to be dose related. High doses of dexamethasone (20 mg orally each day for 10 days, repeated if necessary), which had 75% more glucocorticoid effect than any other regimen studied, produced the highest incidence of both improvement (100%) and moderate-to-marked improvement (75%), and the greatest duration of improvement (more than 3 months after 40% of the courses). The duration of improvement following intensive courses of any of the corticosteroids was approximately doubled by the subsequent administration of smaller doses of dexamethasone or prednisone on alternate days. Most patients with severe disease relapsed after 3 to 6 months of corticosteroid treatment, but increase in the dose of corticosteroid, and daily administration, which was more effective than alternate-day administratin, almost always again resulted in improvement. Corticotropin and corticosteroids were equally effective before and after thymectomy. High doses of corticotropin and corticosteroid produced an initial exacerbation of the disease in 80% of the courses, which was moderate or marked in 57%. Reduction in dose reduced the incidence of severe exacerbation, but did not prevent it, and also resulted in slower and less marked improvement. Withholding anticholinesterase medication did not prevent exacerbation or increase improvement, and afforded no advantage, though it was usually helpful to reduce the dose of this medication. Because of the hazard of initial exacerbation and the occurrence of other serious side effects in 15% of the patients. (bleeding ulcer, vertebral compression, aseptic necrosis of the femoral head or tibia, and subcapsular cataracts), it is recommended that corticosteroid treatment be limited to myasthenic patients who are not responding satifactorily to anticholinesterase medication, that smaller doses be employed in patients whose disease is not life threatening, and that higher doses be reserved for patients who are critically ill and are being managed, at least initially, in an intensive care unit.
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Cycloheximide (up to 0.4 g/liter) was significantly more inhibitory to the growth of three isolates of Aspergillus fumigatus at 23 C than at 37 or 45 C. Preincubation of the media for 7 days at 45 C did not alter the inhibitory effect of the cycloheximide at 23 C. Neither mutation nor adaptation in the fungus seems to be the reason for its growth on the antibiotic at the higher temperature. The mechanism for the differences in sensitivity as related to temperature of incubation cannot be explained at this time.
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Heart transplant recipients in whom high levels of lymphocytotoxic antibodies directed towards a spectrum of histocompatibility antigens develop frequently represent difficult management problems. Recipients of multiple transplants and multiparous females generally form higher levels of panel reactive antibodies, which have been associated with fatal rejection episodes and accelerated graft atherosclerosis. In this study, two multiple transplant patients with preexistent high levels of panel reactive antibodies and two multiparous women who were considered at risk of sensitization were treated with a new form of immunotherapy termed photochemotherapy in addition to conventional immunosuppression. High levels of panel reactive antibodies have been reduced, and patients have suffered few rejection episodes and no infectious complications. This preliminary experience shows that the addition of photochemotherapy to conventional regimens may improve the clinical course of hypersensitized transplant patients without additional immunosuppressive risk.