Mechanisms of apoptosis: UVA1-induced immediate and UVB-induced delayed apoptosis in human T cells in vitro.

OBJECTIVE: The decreased number of lymphocytes combined with the induction of apoptosis and necrosis seems to be the key mechanism of many phototherapeutic agents. The purpose of our study was to determine the regulating pathway, time course and dose dependence of UVA1- vs. UVB-induced cell death in human T lymphocytes. METHODS: In our study we applied an in vitro method using single-laser flow cytometry differentiating between intact (Annexin V-FITC-/PI-), apoptotic (Annexin V-FITC+/PI-) and necrotic T cells (Annexin V-FITC+/PI+) following UVA1 (340-400 nm) or UVB (280-320 nm) irradiation. Additionally, fluorescence microscopy of apoptotic cells was performed using acridine orange and ethidium bromide. RESULTS: Compared to DNA-binding fluorescent microscopy, the flow cytometric method revealed similar, but more precise, results concerning apoptosis and necrosis. Our data indicate that UVB irradiation exerts its effects by the induction of delayed apoptosis within 24-48 h. In contrast, UVA1 irradiation acts via the dose-dependent induction of immediate apoptosis and necrosis within 6 h. CONCLUSIONS: Our findings demonstrate that UVA1 irradiation may effect structural and functional modifications leading to immediate initiation of apoptosis followed by early membrane rupture, whereas UVB irradiation leads to DNA damage followed by delayed apoptosis, obviously without initial membrane alteration.

Efficacy of ultraviolet A1 phototherapy on the expression of bcl-2 in atopic dermatitis and cutaneous T-cell lymphoma in vivo: a comparison study.

BACKGROUND/PURPOSE: Atopic dermatitis (AD) is characterized immunohistochemically by a high number of skin infiltrating T-helper cells (CD4 +). In most cases cutaneous T-cell lymphoma (CTCL) is characterized by a malignant proliferation of CD4+ T-helper lymphocytes. The purpose of our study was to evaluate the extent of anti-apoptotic effects in patients suffering from AD or CTCL, respectively, which may contribute to the prolonged inflammation. Furthermore, we investigated whether medium-dose ultraviolet A1 (UVA1) phototherapy is able to modulate the expression of bcl-2 within the dermal inflammatory infiltrate. METHODS: In order to enumerate bcl-2+ cells pre- and post-therapeutic punch skin biopsies of ten patients with AD and five patients with CTCL were stained immunohistochemically for features of apoptosis using a monoclonal antibody detecting bcl-2. RESULTS: Both AD and CTCL sections revealed a high percentage of bcl-2+ cells within the dermal perivascular infiltrate before therapy. After the successful treatment using medium-dose UVA1 phototherapy this percentage could be decreased significantly. CONCLUSION: Both T-cell-derived skin diseases exhibit an increased pre-therapeutic number of bcl-2+ cells. After medium-dose UVA1 phototherapy the substantial improvement of the skin condition was linked to a significant decrease of the dermal bcl-2+ cell count. Moreover, we could demonstrate a remarkable correlation referring to the decrease and staining pattern of bcl-2 between these two groups as well as within each group. Because the bcl-2 protein is known to act as an apoptosis inhibitor, its pre-therapeutic increase may provide the persistent cutaneous inflammatory reaction in T-cell-derived skin diseases. Additionally, the post-therapeutic reduction of bcl-2+ cells might represent a key mechanism of medium-dose UVA1 phototherapy.

Mononuclear cells in atopic dermatitis in vivo: immunomodulation of the cutaneous infiltrate by medium-dose UVA1 phototherapy.

INTRODUCTION: Recently, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great therapeutic success within the treatment of severe atopic dermatitis (AD). Histologically, AD is recognised by a pathological perivascular dermal infiltrate including T lymphocytes, eosinophils and Langerhans cells. The purpose of our study was to investigate the extent to which UVA1 irradiation is able to modulate the mononuclear dermal inflammatory infiltrate using different monoclonal antibodies. PATIENTS AND METHODS: Biopsy specimens before and after treatment with medium-dose UVA1 irradiation (cumulative dose: 750 J/cm2) from 15 patients suffering from severe AD were analysed immunohistochemically concerning the presence of CD4+ and CD8+ T lymphocytes, CD1a+ Langerhans cells and EG2+ activated eosinophils. RESULTS: Compared to lesional skin of patients with AD before UVA1 irradiation, the relative number of CD4+ cells, CD1a+ dendritic cells and activated EG2+ eosinophils within the dermal infiltrate could be decreased significantly after treatment. In contrast, medium-dose UVA1 phototherapy led to a significant increase of the percentage of dermal CD8+ cells. These alterations were closely linked to a decrease of the absolute skin-infiltrating cells and a substantial clinical improvement of the skin. CONCLUSIONS: In summary, our findings demonstrate that medium-dose UVA1 irradiation leads to a remarkable modulation of the dermal mononuclear infiltrate in patients with severe atopic dermatitis referring to a decrease of dermal Langerhans cells, activated eosinophils and CD4 cell count as well as to a relative increase of CD8+ lymphocytes. The immunomodulation of the cutaneous infiltrate is associated with a depletion of cytotoxic agents, the defective IgE overproduction and the aberrant presence of T lymphocytes combined with the pathological cytokine pattern.

[Acute generalized cat scratch disease in myelodysplastic syndrome]. / Akute generalisierte Katzenkratzkrankheit bei myelodysplastischem Syndrom.

In patients with normal immunity, cat scratch disease typically develop a papule at the portal of entry and no other cutaneous features. A 73 year old male patient with a myelodysplastic syndrome developed generalized petechial, papular and, vasculitic skin lesions in association with cat scratch disease. After the diagnosis was established by identifying the causative organism in a lymph node biopsy, the patient was treated with erythromycin for three weeks resulting in progressive clearance of the skin lesions. Apart from the soluble IL-2 receptor, no other serologic inflammatory parameters were elevated. IgG antibodies against Bartonella henselae and Bartonella quintana increased only slightly during acute exacerbation of the disease, but significantly increased some months later. The diagnosis was established by the positive staining of the lymph node biopsy using the Warthin-Starry stain.

[Generalized pustulous psoriasis: A novel extraintestinal manifestation of Crohn's disease?]. / Psoriasis pustulosa generalisata: neue extraintestinale Manifestation des Morbus Crohn?

A 66-year-old female patient suffering for 10 years from Crohn's disease firstly presented with a parallel outbreak of generalized pustulous psoriasis and Crohn's disease. A second synchronous exacerbation of both disorders occurred after discontinuation of treatment with prednisolone, methotrexate, and mesalazine. As to their pathogenetic concepts, both disease entities reveal similar immunologic alterations, i. e. comparable patterns of cytokines, chemokines, and inflammatory cells (T cells and neutrophils). Generalized pustulous psoriasis, therefore, might develop as hitherto undescribed, more rare extraintestinal manifestation of Crohn's disease.

Effects of low dose ultraviolet A-1 phototherapy on morphea.

AIM: The effects of low dose ultraviolet A-1 (UVA-1) phototherapy on different clinical stages of morphea (localized scleroderma) were analyzed in this case study. Based on these data, the different types of phototherapy described in the literature and currently used for treatment of morphea are compared. METHODS: Three patients with severe plaque type morphea in different stages were studied: one patient with late-stage lesions having stable sclerotic plaques; another patient with active inflammatory lesions; and a third patient with late-stage lesions associated with overlying lichen sclerosus et atrophicus (LSA). The treatment given was low dose UVA-1 phototherapy with single doses of 20 J/cm2 administered four times a week for 6 weeks, and once a week for another 6 weeks. RESULTS: Following UVA-1 phototherapy, the sclerotic plaques resolved, leaving smooth and soft tanned skin with normal structure, consistency and folding capability. In morphea with overlying LSA the elastic fibers did not completely return to the superficial papillary dermis despite the clinical clearance of both morphea and LSA. These data suggest that low dose UVA-1 phototherapy may improve, but not completely reverse, the histopathological changes of LSA. No side effects were observed during or after treatment. CONCLUSION: Our observations show in three patients that low-dose UVA-1 phototherapy is highly effective for treatment of all stages of morphea, including early inflammatory and late sclerotic lesions, and morphea with overlying lichen sclerosus et atrophicus. Because of its safety and efficacy, low dose UVA-1 phototherapy appears to be the treatment modality of choice.

Combined treatment with calcipotriol ointment and low-dose ultraviolet A1 phototherapy in childhood morphea.

Various therapies for morphea have been used with limited success, including ones with potentially hazardous side effects. When morphea occurs in childhood it may lead to progressive and long-lasting induration of the skin and subcutaneous tissue, growth retardation, and muscle atrophy. We report an open prospective study in which the efficacy of a combined treatment with calcipotriol ointment and low-dose ultraviolet A1 (UVA1) phototherapy in childhood morphea was investigated. Nineteen children (mean age 8.5 years, range 3-13 years) with morphea were exposed to UVA1 (340-400 nm) phototherapy at a dose of 20 J/cm(2) four times a week for 10 weeks. Forty phototherapy sessions resulted in a cumulative dose of 800 J/cm(2) UVA1. In addition, calcipotriol ointment (0.005%) was applied twice a day. After 10 weeks, palpation and inspection showed a remarkable softening and repigmentation of formerly affected skin resulting in a highly significant (p < 0.001) decrease of the mean clinical score from 7.3 +/- 0.9 at the beginning to 2.4 +/- 0.9 (relative reduction 67.1%) at the end of combined therapy. Our results indicate that a combined therapy with calcipotriol ointment and low-dose UVA1 phototherapy is highly effective in childhood morphea. Further controlled studies are necessary to investigate whether this combined therapy is superior to UVA1 phototherapy alone.

Opposing effects of UVA1 phototherapy on the expression of bcl-2 and p53 in atopic dermatitis.

Recently, medium-dose UVA1 phototherapy (50 J/cm2) has been introduced as an effective treatment for severe atopic dermatitis (AD). In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement in skin status in patients with AD, biopsy specimens from ten patients before and after treatment were analysed immunohistochemically for features of apoptosis. We sought to determine the extent to which UVA1 irradiation was able to modulate the balance between p53 and bcl-2 expression in vivo using monoclonal antibodies labelling these proteins. As compared with lesional skin of patients with AD before UVA1 irradiation, the number of dermal cells, apparently lymphocytes, that were positive for p53 had significantly increased after treatment and, in addition, some basal keratinocytes showed slight positive staining for p53. An increased expression of the bcl-2 gene before treatment in predominately dermal lymphocytes was significantly downregulated by UVA1 therapy. The increase in p53+ cells and the decrease in bcl-2+ cells were closely linked to a significant reduction in dermal T cells (CD3+) and a substantial clinical improvement in skin condition. In summary, medium-dose UVA1 irradiation led to a marked modulation of the expression of p53 and bcl-2, and this plays a key role in regulating UVA1-induced apoptosis.

Low-dose ultraviolet-A1 phototherapy for lichen sclerosus et atrophicus.

Lichen sclerosus et atrophicus (LSA) is a chronic inflammatory skin disease characterized by white porcelain-like sclerotic skin lesions. It is most commonly seen in adult females and usually affects the genitoanal area. Extragenital LSA appears in 15-20% of cases. We report a 9-year-old Caucasian girl suffering from extragenital LSA that was resistant to conventional treatment. After 40 treatment sessions with low-dose UVA1 phototherapy, all skin lesions were resolved completely. Moreover, the improvement of skin status has been sustained during 6-months of follow-up. Long-wave UVA irradiation has been shown to induce intensively collagenase activity in human dermal fibroblasts. We suggest that UVA1 irradiation could be an effective treatment in patients suffering from extragenital LSA.

[Ultraviolet-A1 (UVA1) phototherapy in lichen sclerosus et atrophicus]. / UVA1-Phototherapie bei Lichen sclerosus et atrophicus.

Two patients, a nine year old girl and a 59 year old woman, presented with extensive and recalcitrant lichen sclerosus et atrophicus (LSA). Both patients were treated with low-dose ultraviolet-A1 (UVA1) phototherapy (340-400 nm) for ten weeks. The cumulative UVA1 dose was 800 J/cm(2), the single UVA1 dose was 20 J/cm(2). After 40 treatment sessions, the previously sclerotic skin lesions had almost completely cleared in both patients. In addition 20-MHz ultrasound examination and histological specimen revealed no further signs of sclerosis. UVA1 phototherapy seems to be a new and effective treatment for LSA with optimal patients' acceptance due to the absence of systemic side effects. UVA1 should be therefore considered as therapeutic option for LSA.

UVA1 irradiation induces deoxyribonuclease dependent apoptosis in cutaneous T-cell lymphoma in vivo.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T-cells, predominantly of the helper phenotype, that primarily invade the skin. Different photo- and chemotherapeutic treatments are known to be beneficial in early-stage CTCL. This observation has initiated prospective investigations into the efficacy of phototherapeutic regimens. The purpose of our study was to investigate the ability of medium-dose UVA1 phototherapy (60 J/cm2) to induce apoptosis (programmed cell death) in skin infiltrating T-cells of CTCL in vivo. We describe the results of three different staining methods for formalin-fixed, paraffin-embedded tissue sections. The in situ end-labeling (ISEL) procedure, nuclear staining using the DNA-binding fluorochrome Hoechst 33342, and immunohistochemistry using polyclonal antibodies against recombinant mouse deoxyribonuclease I (DNase I) demonstrated that UVA1 irradiation was able to induce marked apoptosis in CTCL. Thereby, ISEL and Hoechst staining clearly revealed DNA-condensation and nuclear fragmentation, accompanied by the formation of typical "apoptotic bodies". The accumulation of DNase I immunoreactivity in the cytoplasm of lymphocytes in UVA1 irradiated skin indicated that DNase I or DNase I-related endonucleases may have acted as apoptotic endonuclease(s) which were synthesized after UVA1 irradiation prior to their apoptotic elimination.

Medium-dose UVA1 cold-light phototherapy in the treatment of severe atopic dermatitis.

BACKGROUND: Recently, conventional high-dose UVA1 phototherapy (340-400 nm) has been shown to be more effective than combined UVA-UVB therapy in the treatment of severe atopic dermatitis (AD). However, there are limitations of this treatment, such as intense sweating caused by the immense heat load during therapy and the high cumulative UVA1 doses that are required. For this reason, lavish UVA1 equipment was developed containing an advanced filtering and cooling system resulting in almost complete absence of heat load and sweating during therapy. OBJECTIVE: In this study we compared the monotherapeutic efficacy of conventional medium-dose UVA1, medium-dose UVA1 cold-light, and combined UVA-UVB phototherapy in the treatment of severe AD. METHOD: The study involved 120 patients with severe AD. Fifty patients each received conventional UVA1 or UVA1 cold-light phototherapy (15 days, 50 J/cm(2)/day), and 20 patients were treated with combined UVA-UVB (15 days, minimal erythema dose dependent). Severity of AD was scored by means of the SCORAD score, and clinical improvement was additionally monitored by serologic cytokine markers. RESULTS: Six (12%) of 50 patients treated with UVA1, 2 (4%) of 50 patients treated with UVA1 cold-light therapy, and 4 (20%) of 20 patients treated with combined UVA-UVB therapy discontinued treatment prematurely because of an unsatisfactory clinical outcome or adverse reactions. Skin status improved or even cleared completely in 77.3% of the patients treated for 3 weeks with conventional UVA1 therapy and in 85.4% of the patients treated for 3 weeks with UVA1 cold-light therapy, resulting in a significant decrease in the SCORAD score in both UVA1 groups (P <.05 each). In the group treated with combined UVA-UVB, the SCORAD score also decreased but significantly less than in both groups treated with UVA1 photo-therapy (P <.05 each). At follow-up after 4 weeks, the patients treated with UVA1 displayed a more prolonged therapeutic benefit than the patients treated with UVA-UVB therapy. Plasma levels of soluble interleukin 2 receptors and soluble interleukin 4 receptors significantly decreased under both UVA1 and UVA1 cold-light phototherapy but not under combined UVA-UVB phototherapy. CONCLUSION: Our study demonstrates that medium-dose UVA1 cold-light phototherapy displays advantages compared with conventional UVA1 phototherapy caused by the almost complete absence of heat load and intense sweating and is more effective than UVA-UVB phototherapy in the treatment of severe AD.

[Balneologic photochemotherapy of prurigo simplex subacuta]. / Balneophotochemotherapie der Prurigo simplex subacuta.

Seventeen patients with extensive subacute prurigo, often resistant to different regimens of prior external and internal therapy, were treated with PUVA bath photochemotherapy (psoralen bath followed by UVA irradiation) using bath water containing 0.5 mg/l of 8-methoxypsoralen (8-MOP). In 15 of 17 patients treated, the skin lesions showed a significant improvement or even complete clearance within 8 weeks of therapy. In two patients, only limited clinical improvement was achieved. Follow-up after 6 weeks revealed sustained improvement in the successfully treated patients. The mean cumulative UVA dose given until clearance was 30.3 (SD +/- 12.6) J/cm2. Therefore, a mean of 24.1 (SD +/- 5.3) PUVA bath treatments was necessary. No side effects were seen except phototoxic reactions in two patients manifested as slight erythema. The results of this trial show that PUVA bath photochemotherapy with 8-MOP is an effective therapeutic alternative to other known therapies in subacute prurigo. Compared to oral PUVA therapy or other topical and systemic treatments, PUVA bath photochemotherapy with 8-MOP shows an excellent efficiency-side effect ratio. The clearance of chronic skin lesions refractive to other topical or systemic treatments by PUVA bath photochemotherapy with 8-MOP demonstrates that this therapy can be even superior to other common therapeutic modalities used for subacute prurigo.