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Cryostat sections of skin biopsies from 83 patients with benign or malignant cutaneous lymphoid infiltrates were examined immunohistologically for reactivity with Ki-67 (a monoclonal antibody recognizing a nuclear antigen expressed by cycling cells) using the APAAP immunoalkaline phosphatase labeling method and a double immunoperoxidase/alkaline phosphatase staining technique. Ki-67-positive neoplastic lymphocytes were plentiful in all large-cell lymphoma cases and in one-third of the small-cell lymphomas. Some benign disorders (patch test biopsies, cutaneous lymphocytoma, lichen planus) also contained many Ki-67-positive lymphocytes. These data indicate the value of using Ki-67 to assess the proliferative capacities of the lymphoid cells in cutaneous infiltrates. Use of this technique in the future may have important prognostic and therapeutic implications in the management of cutaneous lymphoid malignancies.

The antigenic properties of epidermal Langerhans cells (LC) were determined and compared with those of non-lymphoid dendritic dermal cells (DDC), interdigitating reticulum cells (IRC), dendritic reticulum cells (DRC), and histiocytic reticulum cells (HRC) by examination of serial and double immunoenzymatic and -fluorescence stained frozen sections of skin and lymph node biopsies. All of these cell types expressed leucocyte common antigen. LC, DDC, and IRC demonstrated similar antigenic phenotypes (HLA-DR+, Leu3+, OKT6+/-, anti-C3 receptor-, R4/23-, Ig-complex-, MO2-), whereas the antigenic properties on DRC (HLA-DR-, Leu3-, OKT6-, anti-C3 receptor+, R4/23+, Ig-complex+, MO2-) and HRC (HLA-DR+/-, Leu3-, OKT6-, anti-C3 receptor+ R4/23-, Ig-complex+, MO2+) were markedly different. These data suggest that LC, DDC, and IRC are closely interrelated cell types, and support the concept that DRC and HRC are unique cell types which do not appear to be related to LC, DDC, or IRC. The lack of labelling of LC with monoclonal anti-C3b receptor antibody, and polyclonal antiserum recognizing C3b, C3bi, and C3d receptors strongly indicate that the EAC-rosetting of LC previously described is not due to the presence of C3 receptors on these cells. Alternatively, LC may express C3 receptor molecules different from those previously identified (C3b, C3bi, and C3d).

Based on the literature and 2 patients studied, we suggest that at least 2 different clinical entities are included in the concept of T CLL: (i) a clinical variant characterized by a relatively benign course, splenomegaly without lymphadenopathy, low lymphocyte count and granulocytopenia; the proliferating lymphocyte is morphologically mature, of medium size and a cytoplasm with azurophilic granules staining positively for acid phosphatase and corresponding to parallel tubular arrays as demonstrated by electron microscopy. The cells form E-rosettes, have no surface-membrane-bound Ig, but Fc-receptors for IgG. With monoclonal antibodies, the phenotype is OKT3+, OKT4- and OKT8+, theoretically corresponding to the suppressor/cytotoxic T lymphocyte subset, but functionally the cells demonstrate killer cell (responsible for ADCC), but not natural or suppressor cell activity. (ii) another clinical variant with an aggressive course, massive hepato-splenomegaly, lymph node enlargement and very high lymphocyte counts; the lymphocytes are small without cytoplasmic granules; their immunological and functional characteristics have not been determined, but morphologically the cells correspond to the T helper/inducer lymphocyte subset. Thus, involvement of different T lymphocyte subsets may be the reason for the clinical variation in T CLL.

Skin biopsy specimens from normal skin and from 115 patients with benign dermatoses, pre- or pseudo-malignant disorders or malignant cutaneous lymphomas have been examined immunohistologically for expression of the Reed-Sternberg cell associated antigen CD30 detected by monoclonal antibodies Ki-1 and Ber-H2. The antibodies stained the atypical cells in lymphomatoid papulosis, a proportion of the neoplastic cells in some cases of mycosis fungoides and most of the neoplastic cells in six large cell anaplastic/pleomorphic non-Hodgkin's lymphomas. The lymphoid cells in all other specimens were Ki-1- and Ber-H2-negative. In all cases, expression of the Ki-1/Ber-H2 antigen was accompanied by expression of activation and proliferation associated markers (i.e., HLA-DR, IL-2 receptor, transferrin receptor and the Ki-67 nuclear antigen). These data indicate the value of antibodies Ki-1 and Ber-H2 in distinguishing between lymphomatoid papulosis and other types of pre- or pseudo-malignant disorders and support the view that lymphomatoid papulosis, Hodgkin's disease and some types of non-Hodgkin's lymphoma constitute a spectrum of related disorders, originated from activated lymphoid cells.

Peripheral blood lymphocytes from 48 consecutive, newly diagnosed CLL patients were examined by light- and electron-microscopy for the presence of nuclear cleaving or folding. On this basis, the patients could be subdivided into 2 easily separable morphological groups. In 42 patients the lymphocytes had regular round nuclei. In 6 patients (13%) the lymphocytes of peripheral blood had a highly irregular nuclear outline characterized by pronounced cleaving or folding of the nuclear membrane. In 4 of these 6 patients immunological investigations demonstrated membrane markers consistent with B-cell monoclonality. Age, sex, distribution between stages as well as mode of clinical presentation were all alike in the 2 groups of patients. In spite of this a statistically significant, highly increased mortality was observed in patients with lymphocyte nuclear irregularities. Thus, 50% of all deaths occurred in this small group of patients. These findings indicate that lymphocyte nuclear cleaving or folding represents a stage-independent prognostic parameter, which permits selection of a small but easily identified high-risk group of patients.

It has been reported that the monoclonal antibody BE2, raised against leukaemic T cells, reacts specifically with malignant lymphoid cells and represents a valuable reagent for the early identification of cutaneous T-cell lymphomas. To test this hypothesis, a comprehensive range of nodal and cutaneous biopsies have been examined immunohistologically using single and double immunoenzymatic and immunofluorescent staining methods. BE2 showed a broad range of reactivity, consistently labelling normal endothelial cells, B-lymphocytes in mantle and marginal zones, T-lymphocytes in the paracortex of lymph nodes and medulla of thymus, as well as a variety of different macrophage types, including Langerhans cells and dermal macrophages. Furthermore, although BE2-positive T-lymphocytes were most frequent in malignant lymphomas, they were also found in four of 19 benign dermatoses. We conclude that BE2 is neither T-cell nor tumour-cell specific, and that use of this reagent on tissue sections is unlikely to improve the diagnosis of cutaneous lymphomas.

The gene encoding the beta-chain of the T-cell antigen receptor (TCR) has been analyzed for evidence of rearrangement in skin, blood, and lymph node specimens from 23 cases of known or suspected cutaneous T-cell lymphoma (CTCL). Two cutaneous large cell lymphomas, 4 cases of Sézary syndrome, and 5 cases of advanced (tumor) stages of mycosis fungoides showed clonal rearrangement of the TCR beta-chain gene in all samples, including lymph nodes in which histologic examination revealed only dermatopathic lymphadenitis. These results indicate that DNA analysis provides a valuable means for improving the diagnosis of extracutaneous disease in advanced stages of CTCL. In contrast, the gene was in a germline configuration in all samples from 12 patients with plaque stages of mycosis fungoides or suspected early CTCL, suggesting that in these 2 conditions the T-cell proliferation is either polyclonal or contains very few monoclonal (i.e., neoplastic) cells.

DNA histograms of skin and blood specimens from 64 patients with known or suspected cutaneous T-cell lymphoma (CTCL) have been examined and compared with normal blood mononuclear cells and skin biopsy samples from 50 patients with various benign cutaneous conditions (i.e. patch test infiltrates, eczema, psoriasis, lichen planus, atopic dermatitis) in an attempt to establish whether DNA measurements by flow cytometry may improve the early recognition of CTCL. The results indicate that right-skewed G0/G1 peaks are seen frequently in both benign disorders and known and suspected CTCL. Such peaks may reflect increased stainability of DNA due to chromatin dispersion during cell activation and/or cell proliferation and do not constitute reliable evidence of malignancy. In contrast, discrete aneuploid DNA peaks are confined to malignant lesions, but are seen almost exclusively in the advanced stages in which the diagnosis can be established easily based on routine histological criteria. These data indicate that DNA measurements by flow cytometry is of only limited help in the early recognition of CTCL and support the view that the lymphoid infiltrate in early CTCL may be reactive (rather than neoplastic) or alternatively may contain only minor reactive (rather than neoplastic) or alternatively may contain only minor populations of abnormal (malignant) cells which cannot be detected by currently available DNA measurement techniques.

Cryostat sections of skin biopsies from five patients with chronic photosensitivity dermatitis with actinic reticuloid syndrome (PDAR) have been examined immunohistologically by the alkaline phosphatase:anti-alkaline phosphatase staining technique using a panel of 24 monoclonal antibodies against lymphoid cells and their subsets. The lymphoid infiltrates in all cases had an essentially identical cellular composition, containing a mixture of T-lymphocytes, T-cell accessory cells (Langerhans cells) and other types of HLA-DR positive dermal macrophages. In two patients there was an excess of T-helper/inducer cells relative to T-suppressor cells, while in the other three patients the numbers of T-cells in these two subsets were approximately equal. Many of the infiltrating T-cells expressed activation (HLA-DR, interleukin-2 receptor) or proliferation (the Ki67 nuclear antigen, transferrin receptor) associated markers. These data indicate that a T-cell immune response is operative in cutaneous PDAR lesions.

Biopsies from 21 lymph nodes with benign hyperplasia were examined by immuno-enzymatic labelling of frozen sections with a panel of monoclonal antibodies. B-cells (B1+, HLA-DR+, C3b receptor+/-) localized in primary follicles, secondary follicles, and areas adjacent to the subcapsular sinus. The B-cells in primary follicles and mantle zones of secondary follicles were indistinguishable ( SmIgD +, SmIgM +, CyIg -, T10-, CALLA-). B-cells adjacent to the subcapsular sinus demonstrated a higher density of SmIgM , and a weaker expression of SmIgD . The germinal centre cells showed a more differentiated phenotype ( SmIgD -, SmIgM +, CyIgM +/-), and also expressed T10 and CALLA. T-cells ( Lyt3 +, Lyt2+, Leu4 +, OKT6-, OKT10 -) localized in paracortial and interfollicular areas, and demonstrated a relative predominance of T-helper/inducer cells ( Leu3 +). T-helper/inducer cells were also identified in secondary follicles. The B-cell areas contained dendritic reticulum cells (R4/23+, C3b-receptor+). Interdigitating reticulum cells (HLA-DR+, OKT6+/-) localized in T-cell regions. The cells in sinuses demonstrated monocyte/macrophage properties (MO2+, Ig+, C3b-receptor+, HLA-DR+/-).

Skin biopsies from positive allergic patch tests were analysed by immunoenzymatic labelling of frozen sections with monoclonal antibodies. In seventeen patients the cellular infiltrate consisted of T cells admixed with Langerhans cells/indeterminate cells, but in two patients there were also many B lymphocytes. The B cells were accompanied by dendritic reticulum cells forming B-cell follicles, indistinguishable from those of normal and hyperplastic lymph nodes. There was no correlation between these two immunohistological staining patterns and the sensitizing antigen, the extent of local reaction or the time from epicutaneous application of allergen to examination (2 to 16 days). The ratio between T-helper and T-suppressor cells varied considerably, and showed no correlation with these variables. In all patients the infiltrating T cells expressed HLA-DR antigen. Transferrin receptors were identified on the infiltrating T cells in biopsies from nine patients. These data indicate activation of T cells in the infiltrate from positive patch tests, and support the functional significance of Langerhans cells in the initiation and maintenance of cutaneous contact allergy. An involvement of B cells and B-cell accessory cells in the pathogenesis of contact allergic reactions is also suggested. The presence of dendritic reticulum cells in skin infiltrates from positive patch tests may reflect a functional implication of the skin in the development of B-cell memory.

Urokinase and tissue-type plasminogen activators (u-PA and t-PA) were identified immunohistochemically in normal and inflamed human appendices by means of polyclonal and monoclonal antibodies. In addition, extracts of the tissues were analyzed for u-PA and t-PA by ELISA. Twelve appendices (five normal and seven with acute inflammation) were analyzed. In the normal appendices, there was a strong staining of the endothelial cells for t-PA, whereas there was negative staining for u-PA. In contrast, the endothelial cells in the inflamed appendices showed u-PA immunoreactivity, and negative or very weak reactions for t-PA. In the inflamed appendix, there was also a labeling of u-PA in fibroblast-like cells and in interstitial areas. The specificity of the staining was supported by a variety of staining controls and also by analysis of tissue extracts with ELISA, showing that on the average the inflamed appendices contained more than twice as much mu-PA per mg of protein as the normal appendices and less than one third of the amount of t-PA.

Phenotypic properties of the neoplastic cells in skin, blood and lymph node specimens from 5 patients with the Sézary syndrome were examined by immuno-enzymatic and -fluorescence labelling of cells and tissue sections with a monoclonal antibody panel. In 3 cases, the in vitro functional properties of the neoplastic cells (isolated from blood specimens) were also analysed using a reverse plaque-forming cell assay. 3 different immunological categories were identified as follows: T-helper/inducer neoplasms (3 patients); T-suppressor/cytotoxic neoplasms (1 patient); and neoplastic T-cells demonstrating characteristics consistent with a concept of their derivation from inducible suppressor T-cells (1 patient). These data provide conclusive evidence that Sézary syndrome is heterogeneous with respect to the immunological characteristics of the neoplastic cells.

Altogether, 209 skin biopsies from 103 patients with mycosis fungoides (MF), large plaque parapsoriasis (LPP), and benign chronic dermatoses (BCD) have been examined immunohistologically with the use of a panel of 21 monoclonal antibodies against lymphoid cells and their subsets. All the infiltrates contained a mixture of T-lymphocytes, Langerhans cells, and other types of HLA-DR-positive dermal macrophages. The neoplastic T-cells in MF lesions expressed proliferation-(transferrin receptor) and activation-(the OKT10 antigen) associated markers more frequently than the T-cells in LPP and BCD. In other respects, the neoplastic T-cells in plaque lesions of MF resembled those seen in LPP and BCD; and most of these cases demonstrated a clear predominance of T-cells of helper/inducer type. The neoplastic T-cells in tumor lesions of MF were much more heterogeneous in phenotype. Only eight of these cases could be classified as T-helper neoplasms. In the remaining ten tumor cases, the neoplastic cells expressed either suppressor/cytotoxic or aberrant T-cell phenotypes. There were no phenotypic differences between the "classical" tumor stages and MF d'emblee cases. The data indicate that the early lesions of MF show an immunohistologic reaction pattern common to many immune responses of the skin and that the neoplastic cells in the advanced stages are more heterogeneous in phenotype than previously recognized.

The antigenic properties of normal epidermal Langerhans cells and their neoplastic counterparts (histiocytosis X cells) were compared with those of other types of macrophages, by immunohistochemical staining of skin and lymph node biopsies with a panel of mono-clonal antibodies. The phenotype of Langerhans cells, histiocytosis X cells, dermal macrophages and interdigitating reticulum cells were similar (HLA-DR+, OKT 6+/-, Leu3+/-, C3-receptor-, Ig-complex-, R4/23-, MO2-), and differed markedly from those of follicular dendritic cells (HLA-DR-, OKT6-, Leu3-, C3 receptor+, Ig+, R4/23+, MO2-, and histiocytic reticulum cells (HLA-DR+/-, OKT6-, Leu3+/-, C3-receptor+, Ig-complex+, R4/23-, MO2+/-). This indicates that Langerhans cells are related to the interdigitating reticulum cells found in the T cell areas of lymphoid organs, and demonstrates that Langerhans cells do not express C3b, C3bi or C3d receptor molecules. Our data also emphasise that the Leu3 antigen is not restricted to cells of the T cell lineage.

Skin biopsies from nine patients with a histological and/or clinical diagnosis of cutaneous lymphocytoma, lymphoplasia or Jessner's lymphocytic infiltrate were examined by immunoenzymatic labelling with a panel of monoclonal antibodies against lymphocytes and accessory cells. Similar cellular constituents were demonstrated in the biopsies from three patients with lymphocytoma, two with lymphoplasia and two with atypical lymphocytes, but a protracted benign clinical course. The infiltrates from these patients consisted of T cells, Langerhans cells, related HLA-DR positive dendritic dermal cells and clusters of polyclonal B cells. In four patients, the B cell clusters contained B cell accessory follicular dendritic cells, and thereby closely resembled the B cell follicles seen in lymphoid organs. The T cells were predominantly T helper/inducer cells and in all patients the T cells expressed HLA-DR. One patient diagnosed as lymphocytoma cutis differed from the other patients by having no detectable B cells. One patient with Jessner's lymphocytic infiltrate differed from the other patients by having a marked relative predominance of T suppressor/cytotoxic cells. These data suggest that cutaneous lymphocytoma and lymphoplasia are basically similar disorders which may be considered to be exaggerated immune responses, whereas Jessner's lymphocytic infiltrate may be a separate entity. Immunological analysis may assist in establishing a definite diagnosis in cases of lymphocytoma or lymphoplasia with atypical cytological features.

Cryostat sections from fully developed papular lesions of lymphomatoid papulosis (histologic subtype A or B) have been examined by immunoenzymatic staining with 24 monoclonal antibodies against lymphoid cells and their subsets. The lesions demonstrated essentially identical cellular compositions and consisted of T-lymphocytes with a peripheral phenotype (Lyt3+, anti-Leu-4+, OKT6-), macrophages (HLA-DR+, EB11+, OKM1+), and Langerhans cells (HLA-DR+, OKT6+). T-helper/inducer cells (anti-Leu-3+) usually dominated over T-suppressor/cytotoxic cells (anti-Leu-2+). In all cases, proportions of the infiltrating T-cells expressed markers associated with activation (HLA-DR, the OKT1O antigen, interleukin-2 receptor) or proliferation (transferrin receptor, the Ki-67 antigen) of lymphoid cells. Furthermore, the infiltrates contained clusters and/or sheets of large cells reactive with antibodies (Ki-1, Ki-24, Ki-27), which recognize Hodgkin's and Reed-Sternberg cells. These data indicate an origin of the cellular infiltrate from transformed or activated lymphoid cells and suggest an interrelationship of lymphomatoid papulosis to Hodgkin's disease.

A new monoclonal antibody, KP1, against the CD68 antigen, which labels macrophages and other members of the mononuclear phagocyte lineage in routinely processed tissue sections, has been used to stain a range of lymphoid, histiocytic, and myelomonocytic proliferations. All 20 neoplasms of myeloid, myelomonocytic, and presumed macrophage derivation reacted with antibody KP1. None of the 22 cases of T cell neoplasia had positive reactions. Although 14 of 41 B lineage lymphomas and leukaemias were stained by antibody KP1, staining was usually confined to small dots of reactivity, in contrast to the strong and extensive cytoplasmic staining seen in the neoplasms of myeloid and macrophage/monocyte origin. Furthermore, positive B cell neoplasms were almost all small cell proliferations, which are unlikely to be confused with myelomonocytic malignancies. It was concluded that antibody KP1 is a valuable addition to a panel of monoclonal antibodies for phenotyping lymphomas, particularly in routinely fixed tissues. It should assist the pathologist in the recognition of extramedullary presentation of leukaemia, aid in the diagnosis of suspected cases of true histiocytic neoplasia, and allow for quantitation of macrophages infiltrating lymphomas and other solid tumors.

Using a reverse plaque forming cell (PFC) assay the production of immunoglobulin (Ig) by peripheral blood mononuclear cells (MNCs) in vitro was studied in 12 patients with Wegener's granulomatosis (WG). Spontaneous IgG production was increased in two of six untreated patients. The IgG response of MNCs from eight untreated patients to pokeweed mitogen (PWM) and Epstein-Barr virus (EBV) stimulation was significantly depressed. The IgM and IgA production followed the individual pattern of IgG. Blood B-cell and T-cell subset concentrations were normal before therapy, whereas the monocyte concentration was increased in four of six patients. Titers of anti-neutrophil cytoplasm autoantibodies (ANCAs) did not correlate with spontaneous or induced Ig production nor with blood leukocyte subset concentrations. Biopsy specimens from upper respiratory tract lesions in seven untreated patients showed numerous macrophages, activated T lymphocytes, and plasma cells, suggesting a pathogenetic role of these cells in the development of lesions and local production of ANCAs.

Cushing syndrome due to primary adrenocortical nodular dysplasia was diagnosed in two patients, aged 3 years 9 months and 9.5 years. Subsequently, adrenalectomy was performed and followed by steroid replacement. In both cases, the adrenals were normal or only slightly enlarged and showed adrenocortical nodular dysplasia histologically. Small lymphocytic infiltrates consisting of T-cells and class II MHC positive macrophages were present in adrenal specimens of both the patients. Samples of protein A sepharose purified serum immunoglobulins from both children stimulated adrenocortical DNA synthesis and cortisol production in cultured guinea-pig adrenal segments in vitro in a dose dependent fashion. Adrenal stimulating immunoglobulins were also demonstrated in serum specimens of both patients' mothers. However, none of them had overt signs of adrenal disease. Our data support the view that autoimmune mechanisms may be involved in primary adrenocortical nodular dysplasia.

A case of the disseminated variant of pagetoid reticulosis is described which progressed after many years of disease to a large cell anaplastic (Ki-1) lymphoma of T-cell type. The lymphoma cells showed abnormal cellular DNA and a high proliferative rate, as revealed by immunophenotypic examination and single-cell DNA measurements. The cells were positive for activation associated antigens and expressed a T-helper/inducer phenotype. A similar phenotype was expressed by the neoplastic cells in the co-existing lesions of pagetoid reticulosis. These findings support the view that pagetoid reticulosis is a variant of the cutaneous T-cell lymphomas which originates from activated lymphoid cells and which may on occasion progress to a potentionally more aggressive lymphoid malignancy.

Skin biopsies from 150 patients with cutaneous lymphoma, premalignant or "pseudolymphomatous" conditions and benign dermatoses were examined by immunoenzymatic staining of frozen sections with a panel of monoclonal antibodies against lymphocytes, macrophages and accessory cells. Two patterns of infiltration were identified. In one of these T cells associated with Langerhans cells and HLA-DR-positive macrophages of dendritic morphology, were seen. In the other B cell follicles were present in addition to the above type of infiltrate. The latter pattern was restricted to patch tests biopsies, cutaneous lymphocytomas, and malignant lymphomas of B-cell origin. It is concluded that lymphocytic skin infiltrates are divisible into different patterns of immunological reactions similar to those seen in lymphoid organs.

Single-cell DNA measurements obtained using flow cytometry have previously been used as a diagnostic tool for various malignant diseases. We used this technique to characterize the dermal infiltrates of 14 patients with psoriatic skin lesions and 22 patients with various benign skin disorders. The DNA histograms of all of the patients exhibited an increased number of cells showing propidium-iodide fluorescence in the hyperdiploid region as compared to those seen in control DNA histograms of normal blood mononuclear cells. The calculated DNA indices revealed one hyperdiploid (G0/G1) peak in 17 cases and two hyperdiploid (G0/G1) peaks in 13 cases. The results suggest that the dermal lymphocytic infiltrate consists of (1) lymphocytes in the cell-division cycle, (2) non-cycling lymphocytes (G0) and (3) activated lymphocytes that may either remain in a non-cycling state or enter the cell cycle.

The kinetics of expression of the intercellular adhesion molecule-1 (ICAM-1) were studied on keratinocytes in skin biopsy specimens of sensitive persons in whom the haptens were applied in a standardized format for allergic contact dermatitis testing. There was no ICAM-1 expressed on keratinocytes of normal skin; ICAM-1 was induced as early as 4 hours after the application of the patch in some subjects. By 48 hours after the application of the patch, all specimens contained ICAM-1-positive keratinocytes. This was concurrent with a heavy mononuclear cell dermal infiltrate and maximum clinical manifestations. Expression of human lymphocyte antigen (HLA)-DR or other inducible surface proteins on keratinocytes under these conditions was much less frequent. When specimens from primary irritant dermatitis were used, only 1 of 14 cases had keratinocytes expressing ICAM-1 at 48 hours, the time of maximum clinical manifestation. Among benign inflammatory lesions, most cases resembled the allergic patch test specimens in that ICAM-1 was expressed to a large degree on keratinocytes. Again, the expression of HLA-DR was variable. Malignant skin lesions, on the other hand, were much less consistent and generally lower in terms of ICAM-1 expression on keratinocytes. Furthermore, in contrast to the benign cutaneous conditions, some malignant skin lesions contained keratinocytes that expressed class II antigens or other inducible surface proteins in the absence of ICAM-1. These data suggest that ICAM-1 plays a role in the specific immune response by facilitating either antigen presentation or lymphocytic infiltration.

Biopsies of involved and uninvolved skin from psoriatic patients and of normal skin were stained immunocytochemically with monoclonal antibodies against urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activator using a multilayer peroxidase technique. Epidermis from psoriatic lesions showed focal staining for u-PA in and between the basal keratinocytes in the suprapapillary epidermal areas, while t-PA was found in the superficial keratinizing cells, including both stratum spinosum and the parakeratotic layer. No staining of keratinocytes was observed in uninvolved and normal skin. The specificity of the staining was supported by the finding that 3 different monoclonal antibodies and polyclonal antibodies against each of the plasminogen activators gave identical staining, while monoclonal antibodies of irrelevant specificity gave no staining. The present findings suggest abnormalities in the regulation of both types of plasminogen activators in psoriatic epidermis.

The immunological phenotypes of the lymphoid cells in 39 cutaneous malignant melanomas have been investigated by staining cryostat sections with a panel of 20 monoclonal antibodies against lymphoid cells and their subsets. Staining was performed by the alkaline phosphatase: anti-alkaline phosphatase (APAAP) method in which the substrate label (red) is easily distinguishable from melanin. The lymphoid infiltrates had an essentially identical composition in all cases, consisting of T-lymphocytes associated with both Langerhans cells and HLA-DR-positive tissue macrophages. B-lymphocytes and natural killer cells were either absent or only present in low numbers. The ratio between T8 (suppressor/cytotoxic) and T4 (helper/inducer) lymphocytes varied and showed no correlation with melanoma subtype, level of invasion or magnitude of lymphocytic response. Examination for markers associated with T-cell activation and/or with cell proliferation revealed that all lesions contained HLA-DR-positive T-lymphocytes, whereas expression of the transferrin receptor and the interleukin-2 receptor (Tac-antigen) occurred mainly in melanomas with a significant inflammatory infiltrate. These data support the concept that malignant melanomas are capable of evoking autologous T-cell immune reactions.

Peripheral blood from 90 CLL patients was examined by light-and electron-microscopy for the occurrence of crystalline inclusions in lymphocytes. Inclusions were demonstrated in 10 patients (11%). In these patients the inclusions were present in 5-45% of peripheral blood lymphocytes. In the light microscope the inclusions appeared as rectangular, unstained structures in May-Grünewald Giemsa and PAS stains. In the electron microscope the inclusions appeared as intracytoplasmic, completely partially membrane-bound bodies, which were often associated with dilated profiles of rough endoplasmic reticulum. The ultrastructure of the inclusions was granular. In immunofluorescence staining the inclusions were found to contain immunoglobulin of the same type and class as the surface membrane-bound immunoglobulin of the neoplastic lymphocytes, most frequently IgM-lambda. The lymphocytes of one case with kappa light chains at the cell surface membrane contained inclusions of the same ultrastructural morphology as those of the other cases with lambda light chains. The presence of inclusions was not associated with any specific clinical or prognostic features. the inclusions persisted during antileukaemic therapy. Their formation may be related to a dysfunction in the synthesis of surface membrane-bound immunoglobulins.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.

Cellular DNA in skin biopsies from eighteen patients with positive patch tests and eleven patients with benign lymphocytic skin infiltates (cutaneous lymphocytoma, lymphoplasia or Jessner's lymphotic infiltrate) was estimated by flow cytometry of propidium iodide-stained dermal cells. Specimens from three patients with patch tests and one with cutaneous lymphocytoma had DNA histograms similar to those of normal blood mononuclear cells. The remaining patients demonstrated DNA histogram abnormalities in the form of hyperdiploid peaks in forty-two of forty-five specimens. The estimated proportion of cells in the S-phase was similar in patch tests (median: 9.2%) and in benign lymphocytic skin infiltrates (median: 9.6%). The median number of cells in the G2/M-phase was 3.1% in both groups of disorders. These data indicate DNA content heterogeneity of the dermal cells in benign lymphocytic skin infiltrates. This may be attributed to DNA replication and/or chromatin dispersion during cellular activation and proliferation. The data also emphasize that hyperdiploid peaks are not necessarily indicative of the presence of separate cell clones.

Urokinase- and tissue-type plasminogen activators (u-PA and t-PA) were identified immunohistochemically during reepithelialization of mouse and human skin wounds, by means of polyclonal and monoclonal antibodies. In incised mouse skin wounds u-PA immunoreactivity was found in keratinocytes at the edge of the wound after 12 h, and at days 2 to 10 after wounding it was found in virtually all keratinocytes of the epithelial outgrowth that gradually covered the wound. At day 14, the epidermis appeared normal and no u-PA immunoreactivity was detected. t-PA immunoreactivity was found from day 5 to day 10 in some keratinocytes located superficially in the epidermal outgrowths near the edge of the mouse wounds. In 3- and 5-day old human skin wounds, u-PA immunoreactivity was found in keratinocytes in the epithelial outgrowths, whereas no t-PA immunoreactivity was detected. No u-PA and no t-PA immunoreactivity was found in normal mouse and human epidermis. The specificity of the staining was supported by a variety of controls, including absorption of the polyclonal antibodies with highly purified u-PA and t-PA preparations and zymographic analysis of extracts of wound tissue. The function of the plasminogen activators during reepithelialization is discussed and it is suggested that the keratinocytes use plasmin activated by u-PA for dissecting their way through the provisional matrix in the upper part of the granulation tissue.