Treatment of severe autoimmune blistering skin diseases with combination of protein A immunoadsorption and rituximab: a protocol without initial high dose or pulse steroid medication.

BACKGROUND: Skin blistering diseases due to autoantibodies are typically treated with high dose systemic corticosteroids and other conventional immunosuppressants. However, in severe cases, this treatment may not be sufficient to achieve disease control or contraindicated because of comorbidity. METHODS: We describe 15 patients (pts.) with such diseases: 6 pts. with pemphigus vulgaris, 3 pts. with bullous pemphigoid, 3 pts. with mucous membrane pemphigoid (MMP), one being anti-laminin-332-MMP (AL332-MMP), 2 pts. with pemphigus foliaceus and 1 pt. with epidermolysis bullosa acquisita (EBA). Patients were treated with a combination of protein A immunoadsorption (PAIA, 3-21 treatments) and rituximab (3-6 treatments) in addition to low dose conventional immunosuppression. RESULTS: All patients showed rapid clinical improvement starting within the first 4 weeks and decline of circulating autoantibody levels. Complete/partial remission was 88%/12% in pemphigus and 71%/29% in subepidermal blistering diseases. Overall relapse rate was 13% with an average follow-up of 22 months. In the AL332-MMP pt. the PAIA/rituximab treatment was stopped because of an oesophagus cancer considered as the paraneoplastic cause of the skin disease. CONCLUSION: Combined treatment with PAIA and rituximab showed rapid and long-lasting response, thereby allowing substantial reduction of dosage of concomitant immunosuppressive medication. We hereby confirm data from other investigators that PAIA/rituximab treatment is a promising therapeutical modality for pemphigus, pemphigoids and EBA, characterized by a favourable ratio of beneficial efficacy and minimized long-term adverse effects.

Progressive lipo-lymphedema associated with increased activity of dermal fibroblasts in monoclonal gammopathy of undetermined significance: is there a causal relationship?

The pathophysiology of skin diseases associated with monoclonal gammopathies is generally unknown. Our aim was to investigate whether a monoclonal gammopathy could be a causal factor in progressive lymphedema. We describe a 75 year old patient with a rapidly progressive lipo-lymphedema and a monoclonal gammopathy of unknown significance (MGUS) suspected as a key etiological factor. Dermal fibroblasts were cultured from lesional lower leg skin and non-lesional abdominal skin and compared to healthy control fibroblasts. We found 10-fold elevated basic fibroblast growth factor 2 (FGF-2) in the patient's serum and significantly increased basal FGF-2 production of lesional and non-lesional fibroblasts compared to healthy controls. Upon restimulation with patient or healthy control serum, lesional fibroblasts showed significantly increased proliferation rates and FGF-2 production in vitro. Non-lesional abdominal fibroblasts showed an intermediate phenotype between lesional and control fibroblasts. Our findings provide the first evidence that lesional dermal fibroblasts from lipo-lymphedema with plasma cell infiltration show increased proliferation and FGF-2 production and that both local tissue factors and altered FGF-2 serum levels associated with monoclonal gammopathies might contribute to this phenotype. Thus we propose a possible pathophysiologic link between the gammopathy-associated factors and the generation of lymphedema with initial fibrogenesis aggravating pre-existing lipedema.

Selective regional perfusion of the bilateral external carotid arteries with pegylated liposomal doxorubicin and melphalan to treat metastatic malignant melanoma of the scalp.

We present the case of a 79-year-old patient with extensive metastatic malignant melanoma (MM) of the scalp. Cutaneous MM of the head and neck often presents a therapeutic challenge. Radical surgical procedures and conventional chemotherapy are often unfeasible and contraindicated because of the difficult anatomy, the extent of the tumour process, and systemic toxicity. In our patient, selective intra-arterial perfusion with pegylated liposomal doxorubicin (PLD) and melphalan was performed after catheterization of both bilateral external carotid arteries with an arterial port system. PLD 4.5 mg/m(2) and melphalan (1.35 mg/m(2), followed by 2.7 mg/m(2) after reaching tolerance) were given as short-term infusions at two-weekly intervals into the right and left external carotid arteries, respectively. After eight applications with tolerable side-effects, no MM cells were detected; however, infiltrates of lymphocytes and melanophages were seen. This case suggests that intra-arterial chemotherapy may be a useful treatment for metastatic melanoma of the scalp.

Altered immunohistochemical expression of small proteoglycans in the tumor tissue and stroma of basal cell carcinoma.

Small proteoglycans have been shown to act as receptors for matrix molecules or growth factors and to influence the attachment and the migration of cells. We therefore report here on the immunocytochemical expression of three small proteoglycans, i.e., decorin, biglycan, and the recently described PG-100, in normal human skin and in basal cell carcinoma. In normal human skin, staining for decorin revealed expression throughout the dermis with an increased signal in the papillary dermis, whereas no expression was observed in the epidermis. Biglycan and PG-100 were mainly detected in the epidermis, with biglycan being expressed only in suprabasal layers. In addition, biglycan could be detected in a narrow zone below the basement membrane. In tissue specimens obtained from 12 basal cell carcinomas, the expression of biglycan and PG-100 was absent or strongly down-regulated in the tumor tissue. Tumor cells thus displayed a staining pattern similar to that found on the basal cells of normal human skin. In the stroma surrounding the tumor, however, the expression of biglycan and to a lesser degree decorin was increased when compared with normal human dermis. The increased deposition appears to be due to an increased synthesis of these molecules, as total RNA extracted from basal cell carcinoma tissue revealed an induction of biglycan and decorin mRNA. This study indicates that the expression of proteoglycans in basal cell carcinoma tumor cells and in tumor stroma is altered from that in normal skin.

Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-gamma.

The immortalized human keratinocyte cell line HaCaT was used to assess the effect of interferon-gamma (IFN-gamma) on expression of keratin K17. Both IFN-gamma and K17 have been implicated in the pathophysiology of psoriasis. Western and quantitative enzyme-linked immunosorbent assay analyses demonstrated increasing induction of K17 protein by 48 h exposure to IFN-gamma at concentrations of 10, 50, and 250 U/ml. At 50 U/ml IFN-gamma, immunohistochemical analysis revealed numerous K17-positive foci, whereas in situ hybridization demonstrated K17 message in the majority of cells. In addition, at low (5 U/ml) concentrations of IFN-gamma, cell proliferation and protein synthesis decreased, as determined by 3H-thymidine labeling and 14C-amino acid uptake. These data suggest that aberrant K17 expression observed in psoriatic lesions may be a consequence of IFN-gamma overexpression, and that the HaCaT cell line may be a useful in vitro model system to elucidate the underlying mechanisms.

Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma.

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.

Ex vivo and in vivo adenovirus-mediated gene therapy strategies induce a systemic anti-tumor immune defence in the B16 melanoma model.

The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.

Inhibition of melanoma growth by adenoviral-mediated HSV thymidine kinase gene transfer in vivo.

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.

A delta opioid receptor lacking the third cytoplasmic loop is generated by atypical mRNA processing in human malignomas.

delta Opioid receptors were identified in human melanomas by RT-PCR and radioligand binding. In all tumors an additional PCR amplificate was detected in which 144 bp within the third exon were deleted. This fragment corresponded to the third cytoplasmic domain of the receptor protein. The short variant resulted from atypical mRNA processing. There were no common splice recognition sequences around the deleted fragment; instead its excision resembled the removal of a transposon. The deletion was not detected in normal human melanocytes nor in human or rat brain. However, it was present in a human neuroblastoma cell line (SH-SY5Y). Thus, it appears that the occurrence of the short delta opioid receptor is correlated to malignancy.

Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients.

This prospective study was performed to determine the prognostic value of tyrosinase mRNA detection in sentinel lymph nodes (SLN) of melanoma patients. About 847 SLNs from 322 consecutive patients were assessed by histopathology and immunohistochemistry as well as tyrosinase-reverse transcriptase-polymerase chain reaction (RT/PCR) for the presence of micrometastases. The results were correlated with the prognostic parameters employing a multivariate analysis after a median follow-up of 37 months. Histopathological analysis revealed metastases in 34/322 patients (10.6%). Among the 288 patients with histopathologically negative SLN, tyrosinase-mRNA was detected in 39 patients. A relapse of the tumour occurred in 44.1% of the patients with histopathologically positive SLN, in 25.6% with histopathologically negative, but tyrosinase-RT/PCR-positive SLN, and 8.0% with "double-negative" SLN. A multivariate analysis identified tumour thickness, the histopathological SLN status, and the ulceration of the primary tumour as independent prognostic factors. Thus, by assessing tyrosinase mRNA in the SLN of melanoma patients, we identified a subgroup with histopathologically negative, but Tyr-RT-PCR-positive SLN who have a high risk of disease relapse.

Vasoactive-intestinal-Peptide (vip) modulates the growth fraction of epithelial skin cells.

Using the human keratinocyte cell line HaCaT, modifications of the growth fraction due to vasoactive intestinal peptide (VIP) were determined by immunostaining with monoclonal antibody Ki67. In addition, the expression of VIP receptor and epidermal growth factor (EGF) receptor have been analysed. VIP (10-(7) to 10-(11) M) produced an almost doubling of the total number of Ki67-positive cells in cultures with 2% fetal calf serum (FCS), wheras it was ineffective in FCS-free and 10% FCS cultures. The nuclear Ki67-staining patterns were classified into four categories. In FCS-free cultures VIP induced a shift from type III (light nucleus, staining nuclei) to type II (multiple, intensely stained spots). In cultures with 2% FCS, VIP induced a shift from type II to type III. VIP receptor expression was facilitated by VIP, when cells were grown in a medium supplemented with 10% FCS. VIP increased EGF receptor expression in FCS-free cultures but decreased the number EGF receptor-positive cells in experiments with 2% FCS. In conclusion, VIP is capable to modulate the growth fraction and receptor expression of HaCaT cells in vitro. The effects are dependent on the concentration of FCS within the culture medium. The findings might be of interest for keratinocyte pathology in general and dermatooncology in particular.

Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.

Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.

Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action.

Complete remission of a primary cutaneous B-cell lymphoma of the lower leg by first-line monotherapy with the CD20-antibody rituximab.

BACKGROUND: Rituximab is a genetically engineered antibody recognizing the CD20 antigen known to be expressed by more than 95% of B-cell lymphomas. Recently the antibody has been approved for routine administration in primary extracutaneous, treatment-refractory or relapsed low-grade, follicular non-Hodgkin B-cell lymphomas. With regard to the pathogenetically related primary cutaneous lymphomas, the so-called large B-cell lymphoma of the leg represents a distinct, but rare subentity. In an 89-year-old, multimorbid patient who was affected by such a non-resectable CD20+ large B-cell lymphoma limited to the skin of both lower legs, rituximab was used as a first-line monotherapy in order to avoid local or systemic toxicities inevitably linked to conventional treatment modalities, i.e., radio- or chemotherapy. METHODS: Rituximab was administered at a dosage of 375 mg/m(2) i.v. eight times in weekly intervals. As a premedication we used prednisolone 150 mg i.v. as well as loratadine 10 mg p.o. 1 h before each rituximab infusion. RESULTS: The treatment was well tolerated without any adverse reactions, but was accompanied by a mild transient blood eosinophilia. The histologically proven, exophytic, multi-nodular lymphoma showed a substantial regression already at 2 weeks after the onset of the rituximab treatment. At 8 weeks we observed a complete clinical remission which is now stabile for a follow-up period of 6 months without any maintenance therapy. CONCLUSIONS: Our case observation demonstrates that an intensified, i.e. eightfold, rituximab application in weekly intervals may be a highly effective, tumor target cell-specific first-line monotherapy in the management of primary cutaneous large B-cell lymphoma of the leg. Given the rareness of the disease, the result as well as the possible contribution of the prednisolone premedication will have to be evaluated in a future, controlled, multi-centre study.

Antiproliferative and cytotoxic profiles of antipsoriatic fumaric acid derivatives in keratinocyte cultures.

Oral administration with complex mixtures of fumaric acid derivatives is known to have antipsoriatic efficacy. The present studies aimed to clarify the mode of action and toxicity of the individual compounds. Hyperproliferative HaCaT keratinocytes in monolayer cultures were exposed to fumaric acid, dimethylfumarate, zinc monoethylfumarate, calcium monoethylfumarate and magnesium monoethylfumarate at concentrations between 0.4 microM and 960 microM for 48 h. Cell proliferation was studied by [3H]thymidine incorporation. In addition 14C-labelled amino acid uptake and total protein content were measured. Direct cytotoxicity was determined by the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium. The corresponding 50% inhibition concentrations (IC50) were calculated for DNA/protein synthesis: 2.3/2.5 microM (dimethylfumarate), 133/145 microM (zinc monoethylfumarate), 215/230 microM (calcium monoethylfumarate), 275/270 microM (magnesium monoethylfumarate), > 960/> 960 microM (fumaric acid). The total protein content was less sensitive. Antiproliferative activity was found for dimethylfumarate and to a lesser degree for calcium monoethylfumarate already at the subtoxic concentrations of 1.3 and 4 microM, respectively. In the case of magnesium monoethylfumarate, zinc monoethylfumarate and fumaric acid there was no such dissociation between their cytotoxic and antiproliferative potential. These data indicate that most of the antipsoriatic potential of fumaric therapies is due to the dimethylfumarate compound.

Keratin pattern of acanthosis nigricans in syndromelike association with polythelia, polycystic kidneys, and syndactyly.

BACKGROUND: Acanthosis nigricans (AN) comprises a broad spectrum of etiologic subtypes. The underlying pathomechanisms have not yet been completely clarified. We present a patient affected with a syndromelike AN subtype including disturbed epidermopoiesis as evidenced by immunohistologic findings and in situ hybridization. OBSERVATIONS: A 54-year-old white man contracted AN during childhood. There were connate malformations consisting of webbed toes II/III on the right side and a supernumerary left mammilla. As an adult he developed psoriasis vulgaris, obesity, and latent diabetes mellitus, polycystic kidney and liver disease. With regard to keratin 6 mRNA, and the protein expression of keratin 6/16, KI-67, and proliferating cell nuclear antigen, the AN lesion showed moderate hyperproliferation. A much higher degree of hyperproliferation was evident in psoriatic areas of the patient's skin. In contrast to psoriatic tissue, basal keratinocytes of the AN showed an unusually high expression of keratin 18 and 19 protein. CONCLUSIONS: The observation thus deals with a unique, syndromelike constellation of AN characterized by a particular epidermal pattern of moderate hyperproliferation. A further dysregulation of protein expression in the epidermis is indicated by the demonstration of the rare keratins 18 and 19 in basal keratinocytes of the AN lesion.