Delayed hypersensitivity against staphylococcal antigens in guinea pigs (author's transl)]

Delayed type hypersensitivity against staphylococcal antigens could be induced in guinea pigs by injecting the animals with a staphylococcal homogenate in Freund's adjuvant in all four foot pads and the nuchal skin. Maximal skin reactivity, tested intracutaneously, was observed 21 days after sensitization. Highest stimulation of lymph node cells and peripheral blood lymphocytes was also obtained 21 days after sensitization. When comparing cell walls, cell materials (nonsoluble material obtained after the separation of the cell wall fraction) and soluble fraction (obtained after sedimentation of all nonsoluble material, the strongest skin test reactions occurred after testing intracutaneously with the cell wall fraction.     

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcus aureus antigens

Summary The development of delayed hypersensitivity against staphylococcal antigens in guinea pigs was observed from day 3 to day 50 after sensitization with staphylococcal homogenate in Freund's incomplete (FIA) and Freund's complete adjuvant (FCA). Skin test reactivity, stimulation of lymph node lymphocytes, and peripheral blood lymphocytes and the titre of precipitating antibodies were followed during this period. Maximal skin test reactivity as well as maximal lymphocyte responsiveness occurred at day 21 after sensitization in FCA-sensitized guinea pigs. In FIA-sensitized animals highest skin reactivity was observed at day 14 and maximal lymphocyte stimulation 35 days after sensitization. Precipitating antibodies reached a plateau at day 20 in plasma of animals sensitized with FCA and at day 35 in FIA-sensitized animals.These investigations were performed under the supervision of the retired director Prof. Dr. H. Storck     

Mixed skin cell lymphocyte culture reaction (MSLR) in psoriasis

The Mixed Skin Cell Lymphocyte Culture Reaction (MSLR) was studied as an in vitro approach of lympho-epidermal interactions in psoriasis. The ability of peripheral blood lymphocytes from patients with psoriasis to proliferate in response to stimulation by epidermal cells and the capacity of psoriatic epidermal cells to stimulate T cells were investigated and compared to results with cells from controls. While stimulation of control lymphocytes by autologous epidermal cells was observed, although weaker than the proliferation in response to allogeneic control epidermal cells, no stimulation was observed in autologous MSLR using psoriatic cells. The ability of epidermal cells from psoriatic skin, either uninvolved or involved skin, to induce proliferation of control lymphocytes in allogeneic MSLR did not differ from that of control epidermal cells. In contrast with results obtained with control lymphocytes and epidermal cells in allogeneic MSLR, peripheral blood cells from psoriatic subjects failed to react to stimulation by control allogeneic epidermal cells. These results indicate a normal capacity of psoriatic epidermal cells to stimulate in MSLR and a functional inability of peripheral blood lymphocytes from patients with psoriasis to react in MSLR which is in agreement with previous reports of abnormal T cell functions in the disease.     

CCR4 Expression by atypical T cells in systemic pilotropic lymphoma: its behavior under treatment with interferon gamma, topical PUVA and systemic retinoid

We describe an 88-year-old Japanese patient with pilotropic T cell lymphoma involving the peripheral blood as well as lymph nodes. This patient presented with multiple red follicular papules, confluent, infiltrated erythematous plaques and nodules. Moreover, he was conspicuous for the presence of total alopecia of the scalp and eyebrows. Histopathologically, the lesional skin showed dense follicular and perifollicular infiltrates of atypical lymphocytes. The flow cytometry disclosed the presence of weakly CD4+ CCR4+ cell populations that would not be detected in the peripheral blood from healthy controls. The patient responded well to topical PUVA and systemic etretinate (retinoid-PUVA) and intravenous IFN-gamma. Parallel with the decrease in atypical cells in the peripheral blood, the percentage of weakly CD4+ CCR4+ T cells declined. However, about 1 week after we discontinued this treatment because of the side effects, the lymph node swelling became prominent, and, 4 weeks later, the patient died before restarting any specific chemotherapy.     

Sézary syndrome with early immunoblastic transformation

Summary Two patients with clinical manifestations of Sézary syndrome are reported. In both cases from an early stage of the disease in addition to characteristic Sézary cells large numbers of immunoblasts were present in skin lesions and peripheral lymph nodes and in one case also in the blood. Their relationship to the characteristic Sézary cells was shown by morphological, cytochemical and immunological methods. The infiltrates in the skin were epidermotropic in one case and nonepidermotropic in the other. Lymph node structure was effaced by diffuse infiltration of abnormal lymphoid cells. These were found to proliferate in the skin as well as in lymph nodes. Cytogenetical studies of blood lymphocytes indicated an abnormal hypodiploid clone in both cases. Immunologically the tumour cells had properties of peripheral T-lymphocytes but whereas all abnormal cells exhibited inducer/helper cell characteristics in one case, only a minority of the lymphocytes revealed these characteristics in the other case. In this case the tumour cell population changed into a more pleomorphic type. The classification of the cases is discused.     

Adult T-cell leukemia/lymphoma (ATL) –clinical, histopathological, immunological and immunohistochemical characteristics

Twenty-one patients with ATL were assessed. The predominant physical findings were lymph node and bone marrow involvement, skin involvement, hepatosplenomegaly and leukemic manifestations. The predominant histopathological findings in both skin and lymph node specimens were the diffuse medium-sized cell type and the diffuse mixed cell type. Some phenotypic discrepancy was found between the neoplastic cells in the peripheral blood, lymph nodes and skin of patients with ATL with respect to CD45RA and CD45RO, and CD7, CD29, CD25 and HLA-DR. That is, the predominant neoplastic cell phenotype was the helper T-cell, which was CD3+, CD4+, CD7+, CD25+, CD45RA+ and HLA-DR+, and CD29- and CD45RO- in peripheral blood and lymph nodes, and CD3+, CD4+, CD7+, CD29+, CD45RO+ and HLA-DR+, and CD45RA- in the skin. In other words, we have described the phenotypic heterogeneity of ATL cells and demonstrated the heterogeneity of CD45R isoform expression on ATL cells in different organs--the skin, peripheral blood and lymph nodes--of the same patient.     

Studies on the dinitrophenylated lymphocytes in guinea pig painted with DNCB

The number of DNP group-bearing lymphocytes in the regional lymph node, thoracic duct and peripheral blood was determined at various time intervals after painting normal guinea pigs with DNCB by the immunofluorescent method using anti-DNP antibody. The incidence of such cells in the regional node was maximal at 12 hours whereas in the thoracic duct and peripheral blood the maximum incidence was found at 0.1-2 hours after painting. Unreacted DNCB was demonstrated in both the thoracic lymph duct and the blood at least for 12 or 24 hours respectively following exposure to DNCB. The authors therefore suggest that DNCB reacts directly in vivo with the lymphocyte cell membrane of guinea pig following epicutaneous application of the chemical.     

Establishment of T and B cell lines from patients with mycosis fungoides

Eighteen patients with mycosis fungoides were studied in order to establish cell lines that might be associated with the human T cell leukaemia-lymphoma virus (HTLV). Three T cell lines were established, two from affected skin and one from a lymph node showing dermatopathic lymphadenopathy. The T cells expressed OKT3 and OKT4 antigens. They temporarily expressed an HTLV p19-like antigen in up to 5% of the cells during culture. None of our patients had lymphocytosis or abnormal lymphocytes, except one patient with Sézary's syndrome. We could not establish T cell lines from peripheral blood, but five B lymphoblastoid cell lines were obtained, all positive for the Epstein-Barr virus nuclear antigen. Our finding that T cell lines can be established from skin biopsies and lymph nodes of patients with mycosis fungoides, but not from the blood, supports the concept of a malignant T lymphocyte primarily localized in the skin. The temporary expression of HTLV p19 antigen may indicate the presence of retrovirus, but further studies are needed.     

Interleukin 2 and granulocyte-macrophage colony-stimulating factor induce a perivascular lymphocytic infiltrate in a skin explant model.

Cutaneous eruptions displaying perivascular inflammatory cell infiltrates histologically may develop with the intravenous administration of cytokines. Similar findings are seen spontaneously in some patients on recovery of peripheral blood lymphocytes after profound marrow aplasia. To investigate the production of a cutaneous perivascular infiltrate further, the ability of several cytokines to induce a perivascular lymphocytic infiltrate was studied in vitro using a skin explant model. A skin biopsy specimen obtained at the time of peripheral blood lymphocyte recovery after chemotherapy-induced marrow aplasia (n = 10) was divided and incubated for 3 days with and without a series of cytokines plus various peripheral blood mononuclear cell populations. Skin incubated with interleukin 2 and granulocyte-macrophage colony-stimulating factor induced a perivascular lymphocytic infiltrate, while control samples did not. Immunophenotypic analysis revealed that the lymphocytes were predominantly CD3+/CD4+. An infiltrate was not observed when skin was incubated with cytokines alone, without the addition of simultaneously isolated peripheral lymphocytes. A perivascular pattern was not observed with the addition of interferon gamma. Only interferon gamma induced keratinocyte intercellular adhesion molecule 1 expression in experimental tissue. Certain cytokines that affect a range of cell types are capable of inducing a common cutaneous histologic pattern, the perivascular lymphocytic infiltrate.     

UVB irradiation decreases the magnitude of the Th1 response to hapten but does not increase the Th2 response

Exposure of murine skin to low doses of ultraviolet-B (UVB) radiation before sensitization with hapten reduces the ability of antigen presenting cells (APC) in the draining lymph nodes to initiate contact hypersensitivity responses in vivo and results in the induction of hapten-specific suppressor T cells. In the present study, we tested the hypothesis that exposure of skin to UVB radiation suppresses T cell responses to hapten in vivo by altering the functions of APC, resulting in decreased stimulation of Th1 lymphocytes, which mediate contact hypersensitivity responses, and preferential activation of Th2 cells. C3H/HeN mice were exposed to either a single 2 kJ/m² dose of UVB or to 400 J/m² of UVB daily from FS40 sunlamps for four consecutive days and sensitized with fluorescein isothiocyanate on UV-irradiated skin. Draining lymph node cells were collected 18 h after sensitization and co-cultured with nylon wool-purified T cells from naive or fluorescein-immunized mice. Unseparated lymph node cells or sorter-purified fluorescein-bearing APC from UV-irradiated mice induced less T cell proliferation than APC from non-UV-exposed mice. Lymph node cells produced less Th1 and Th2-associated cytokines, interferon-gamma and interleukin-4, respectively, in response to APC from UV-irradiated animals compared with APC from unirradiated, fluorescein-sensitized mice. Thus, low doses of UV radiation do not result in preferential stimulation of Th2 response in lymph nodes, and results from cloned cell lines may incompletely reflect T cell responses in vivo.     

Immune sensitization against epidermal antigens in polymorphous light eruption

To get further insight into the pathogenesis of polymorphous light eruption, we studied nine patients with polymorphous light eruption and six healthy persons. Two skin biopsy specimens were obtained from each person, one from previously ultraviolet light-irradiated skin and another one from unirradiated skin. An epidermal cell suspension, skin homogenate, or both were prepared from each specimen. Autologous cultures were made with peripheral blood mononuclear cells combined with irradiated or unirradiated skin homogenate and peripheral blood mononuclear cells combined with irradiated or unirradiated epidermal cell suspension. Cell proliferation was assessed by 3H-thymidine incorporation assay. The response of peripheral blood mononuclear cells to unirradiated epidermal cells or unirradiated skin homogenate was similar in both patients and controls. However, peripheral blood mononuclear cells from patients with polymorphous light eruption showed a significantly increased proliferative response to both irradiated epidermal cells and irradiated skin homogenate. Our results indicate that ultraviolet light increases the stimulatory capability of polymorphous light eruption epidermal cells in a unidirectional mixed culture with autologous peripheral blood mononuclear cells. This suggests that an immune sensitization against autologous ultraviolet light-modified skin antigens occurs in polymorphous light eruption.     

Detection of a peripheral blood T cell clone is an independent prognostic marker in mycosis fungoides

T cell receptor gene analysis is a sensitive method for assessment of peripheral blood involvement in mycosis fungoides. This study uses polymerase chain reaction/single-strand conformational polymorphism (PCR/SSCP) analysis of the T cell receptor gamma gene and relates the results to skin stage and outcome in mycosis fungoides. Seventy-five peripheral blood samples from 66 patients were obtained from 1990 onwards and subjected to PCR/SSCP. Both Southern blot analysis and PCR/SSCP analysis were performed on 63 samples from 56 patients. Fourteen patients had T1 disease (12 IA, two IIA), 20 T2 (14 IB, five IIA, one IVA), 29 T3 (24 IIB, two IVA, three IVB, two patients tested at both T2 and T3), and five T4 (all III). The percentage of positive samples was higher with PCR/SSCP than with Southern blot analysis (29 of 63 vs eight of 63 samples, p < 0.001), and the percentage of positive samples increased with each stage (21% at T1, 35% at T2, 58% at T3, and 71% at T4). Proportional hazards analysis corrected for age, skin, and lymph node stage showed that the presence of a peripheral blood clone is associated with a worse outcome (p = 0.03, CI 1.1-6.03). These results indicate that the presence of a peripheral blood clone is an independent prognostic variable in patients with mycosis fungoides after correcting for age, skin, and lymph node stage, and that peripheral blood involvement is present in a large proportion of patients with early stage mycosis fungoides. Keywords: polymerase chain reaction/single-strand conformational polymorphism/T cell receptor gene rearrangement. J Invest Dermatol 114:117-121, 2000     

Hydroa vacciniforme-like eruptions in a patient with chronic active EB virus infection

We report a case of chronic active Epstein-Barr (EB) virus infection (CAEBV) associated with skin eruptions mimicking hydroa vacciniforme (HV) in a 4-year-old boy. The patient had repeated episodes of vesiculo-necrotic eruptions on the face, scalp, and bilateral forearms one year before the first visit to our department. General symptoms including fever, hepatosplenomegaly, abnormal liver function, and cervical lymph node swelling were noted three months before the first visit. At the first visit, small, bean-sized, erythemic papules with central necrosis were observed on the face and anterior chest wall. Thumb-sized ulcers with crust were present on the bilateral forearms. Histopathological examination of an erythematous lesion in the submandibular area revealed parakeratosis with a thick crust, mild spongiosis in the epidermis, and a dense infiltration of lymphoid cells into the dermis and perivascular space. Laboratory examination showed EBNA x 40, EBV VCA IgG x 1,280, and EBV DNA (PCR) 8 x 10(4). EBV-encoded small nuclear RNA (EBER) positive cells were detected in the dermis by an in situ hybridization (ISH) method. Large granular lymphocytes (65%) with the NK cell phenotype were found in the peripheral blood. A real time PCR method showed 171,741 copies/ micro g DNA in CD 16 positive cells. Although latent EBV infection-associated eruptions have been documented, detailed skin manifestations in CAEBV are less well known.     

Cutaneous involvement by angioimmunoblastic T-cell lymphoma with remarkable heterogeneous Epstein-Barr virus expression

INTRODUCTION: Initially described as an abnormal immune reaction, most cases of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-like T-cell infiltrates are now regarded as a peripheral T-cell lymphoma (AILD T-NHL). AILD T-NHL is characterized clinically with constitutional symptoms, generalized lymphadenopathy, hepatosplenomegaly, skin rash, and polyclonal hypergammaglobulinemia. Epstein-Barr virus (EBV) is frequently detected in involved lymph nodes, but the presence of EBV in cutaneous infiltrates of AILD T-NHL has rarely been examined. We present a patient with AILD T-NHL with cutaneous involvement that shows marked heterogeneity of EBV expression in the lymph node and skin biopsies, and review the histological findings of AILD T-NHL in the skin. METHODS: Two skin biopsies of a diffuse maculopapular rash and a lymph node were examined and immunophenotyped. In situ hybridization for detection of EBV in the lymph node and skin biopsies was utilized. In order to attempt to delineate which lymphocytes were EBV positive, skin biopsies were dual labeled with CD3, CD45RO, CD20 and EBV. The skin biopsies and lymph node were submitted for gene rearrangement studies by polymerase chain reaction (PCR). Capillary electrophoresis of fluorescently labeled PCR products was utilized for PCR product quantitation. RESULTS: The histological features of the lymph node were diagnostic of AILD T-NHL and a T-cell clone was identified by PCR. The skin biopsies showed an atypical superficial and deep perivascular polymorphous infiltrate consistent with cutaneous involvement by AILD T-NHL. Both skin biopsies showed the same clonal T-cell receptor gene rearrangement as the lymph node. In situ hybridization of the lymph node and one skin biopsy showed a few scattered EBV-positive lymphocytes (<1% of the infiltrate). A second skin biopsy revealed 40-50% of the lymphocytes as EBV positive. Dual staining for CD20 and EBV identified a minority of EBV-infected lymphocytes as B-cells, but most of the EBV-positive cells lacked staining for CD3 and CD45RO. CONCLUSIONS: In our patient, the same T-cell receptor gene rearrangement was found by PCR in all three biopsy sites. Most cases of AILD T-NHL contain only a few EBV-positive cells, but in our patient the extent of EBV expression ranged from <1% to 40-50% of the AILD T-NHL cutaneous infiltrate. To our knowledge, this case is the most extensive and heterogeneous expression of EBV in cutaneous AILD T-NHL to date.     

Clonal disease in extracutaneous compartments in cutaneous T-cell lymphomas. A comparative study between cutaneous T-cell lymphomas and pseudo lymphomas

Extracutaneous involvement is a sign of poor prognosis in cutaneous T-cell lymphomas (CTCL). Unfortunately it becomes clinically and histologically manifest only late in the course of the disease. It was the purpose of this study to detect clonality in peripheral blood, lymph nodes and bone marrow samples at times when extracutaneous involvement cannot other-wise be demonstrated. In addition to skin biopsies, peripheral blood, lymph node and bone marrow samples from a total of 25 patients were analysed by Southern blotting for clonal gene rearrangement of the T-cell receptor β-chain. Six of the patients were suffering from mycosis fungoides (MF), four from non-MF CTCL (pleomorphic T-cell lymphomas), seven from Sézary syndrome (SS), eight from pseudolymphoma (insect bites) (PSL), and one from lymphomatoid papulosis (LP). Clonal TcR b gene rearrangements were found in patients with MF in four of five skin probes as well as in two of two lymph node samples and in one of two peripheral blood samples. In SS patients, all skin probes (seven of seven), lymph node samples (six of six), peripheral blood samples (six of six) and one bone marrow specimen had a clonal TcR β gene rearrangement. In patients with non-MF CTCL, two of four skin, zero of two peripheral blood and one of one bone marrow samples with clonal T cells were detected. All investigated patients showed exactly the same rearrangement pattern at extranodal sites and in the skin, which is proof for the same clone in all compartments. In contrast, no rearrangements were detected in LP and PSL (zero of eight skin probes, zero of two peripheral blood samples). Our results provide strong evidence for an early systemic spread of neoplastic cells in CTCL. However, an initial tumour burden has to be reached in order to lead to a clinically and prognostically relevant manifestation.     

Basal Cell Carcinoma of the Vulva with Lymph Node and Skin Metastasis

A 79-year-old Japanese woman who had basal cell carcinoma presenting as a large ulcer on her vulva with lymph node and skin metastasis is described. Histological examination revealed that tumor nests with peripheral palisading invaded deeply into the subcutaneous tissue and were accompanied by marked mucinous changes and fibrous reaction. Vascular invasion was also observed. There were inguinal lymph node metastases and two papular skin metastases on her right thigh. The primary tumor and the metastases were excised. The defect was repaired by bilateral gracilis musculo cutaneous flaps and a skin graft. We surveyed the literature and found 20 cases of metastasizing basal cell carcinoma in Japan.     

Epidermotropic precursor T-cell lymphoma with highly aggressive clinical behavior simulating localized pagetoid reticulosis

We describe a 43-year-old patient with a solitary, eczematous plaque on his right nipple that had developed during the previous 6 weeks. Histopathology revealed a superficial band-like infiltrate of medium to large-sized pleomorphic lymphocytes with striking epidermotropism. The tumor cells expressed a T-phenotype (CD3+, CD20-) and were CD30-, CD4-, and CD8-negative. A diagnosis of localized pagetoid reticulosis (Woringer-Kolopp disease) with possible large cell transformation was proposed. A cervical lymph node excised 2 months later showed features of a highly aggressive blastoid precursor T-cell lymphoma. Reevaluation of the skin lesion and of a tonsil specimen previously interpreted as benign hyperplasia showed features consistent with those observed in the lymph node. The disease was rapidly progressive, and the patient died 15 months after the first skin biopsy. This case represents a unique cutaneous presentation of aggressive precursor lymphoma, showing the protean nature of lymphoproliferative disorders and the overlapping clinical and histopathologic features of different entities.     

Antigen presenting cells and T lymphocytes in the induction phase of contact hypersensitivity

When a sensitizing substance that induces contact hypersensitivity, fluorescein isothiocyanate (FITC), was painted on abdominal skin of mice, FITC+ cells appeared in the inguinal lymph node after 24 hours. The FITC+ cell in the lymph node was relatively large in size, and it did not appear to be a T lymphocyte. When FITC was painted on either murine tail skin or skin pre-treated by tape stripping, the number of FITC+ cells in the inguinal lymph node was significantly less than that in the positive control. In the mesenteric lymph nodes, which have a different lymph flow from that of the skin regional lymph node, FITC+ cells did not increase in number, and the few FITC+ cells were not significantly different in number among above-mentioned experimental systems. In the inguinal lymph nodes on the 4th day after painting of picryl chloride (PCl) on the abdominal skin of mice, L3T4+ cells, which expressed an interleukin 2 receptor (IL-2R), increased in number. On the other hand, when PCl was painted on either tail skin or skin treated by tape stripping, L3T4+ IL-2R+ cells did not increase in the skin regional lymph nodes. The number of L3T4+ IL-2R+ cells in the mesenteric lymph nodes did not increase in any of the experimental systems mentioned above. These results suggest some relationship between antigen presenting cells and T lymphocytes, as well as one between the skin and the regional lymph nodes, in an induction phase of sensitization in contact hypersensitivity.     

Frequency and prognostic significance of clonal T-cell receptor beta-gene rearrangements in the peripheral blood of patients with mycosis fungoides.

BACKGROUND--Previous studies have shown that peripheral blood involvement is a poor prognostic sign in patients with cutaneous T-cell lymphoma. However, evaluation of the results of these studies is difficult. In this study peripheral blood mononuclear cells from 45 patients with various stages of mycosis fungoides (MF) were investigated for the presence of clonal T-cell populations by T-cell receptor beta (TCR beta)-gene rearrangement analysis. RESULTS--Clonal TCR beta-gene rearrangements were found in five (11%) of 45 patients with MF, including one (3%) of 31 patients without MF and four (27%) of 15 patients with histologically confirmed lymph node involvement. With respect to skin stage, clonal T-cell populations were detected in one (4%) of 23 patients with plaque stage disease, two (10%) of 19 patients with tumor stage disease, and two (50%) of four patients with erythrodermic MF. In the group of patients with lymph node involvement the median survival of patients with detectable clonal T-cell rearrangements in the peripheral blood was much shorter (3 months) than that of patients without clonal rearrangements (16 months). CONCLUSIONS--The results of this study indicate that clonal TCR beta-gene rearrangements, as detected by Southern blot analysis, are uncommon in the peripheral blood of patients with MF, in particular in patients without histologically documented lymph node involvement. The presence of clonal T-cell populations in the peripheral blood of MF patients with lymph node involvement is usually associated with rapidly fatal disease.