Effect of water calcium, copper, and silver on branchial Na+ permeability in a characid and cichlid fish.

We tested the hypothesis that water Ca2+ is involved in control of branchial Na+ permeability in low pH tolerant convict cichlids and black neon tetras. We measured Na+ efflux in water with different Ca2+ concentrations during exposure to low pH, silver, and copper, at levels which are known to stimulate Na+ efflux. For convict cichlids at pH 7.5 exposure to 0 µmol L-1 Ca2+caused Na+ efflux to rise 2.5 times above controls at 100 µmol L-1 Ca2+. However, raising [Ca2+] to 500 µmol L-1 had no effect. Upon exposure to pH 3.5 (control [Ca2+]) Na+ efflux rose almost 5× and increasing the [Ca2+] 5-fold did not reduce the magnitude of stimulation. Exposure to 1 µmol L-1 silver and 25 µmol L-1 copper stimulated Na+ efflux 7×, and 2×, respectively. Raising [Ca2+] concentration during metal exposure halved the stimulation of Na+ efflux caused by silver, and eliminated the stimulation elicited by copper. For black neon tetras raising or lowering water [Ca2+] had no effect on Na+ efflux at pH 7.5. Exposure to pH 3.5 caused Na+ efflux to rise 2.5× but changing [Ca2+] had no effect. Exposure to 1 µmol L-1 silver, or 25 µmol L-1 copper caused Na+ efflux of tetras to rise 4-fold and 3-fold, respectively. Raising [Ca2+] during silver exposure reduced the stimulation of Na+ efflux by about 50%, but during copper exposure increased [Ca2+] had no effect on stimulation of Na+ efflux. These results suggest water Ca2+ plays a role in control of branchial Na+ permeability in cichlids, but perhaps not tetras. In addition, the silver and copper concentrations required to inhibit Na+ uptake and stimulate Na+ efflux were higher than the concentrations used on non-characids and non-cichlids, which indicates that our fish are much more tolerant of these metals.

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.

Late-onset inversa recessive dystrophic epidermolysis bullosa caused by glycine substitutions in collagen type VII.

Dystrophic epidermolysis bullosa (DEB) is a rare hereditary skin disorder caused by mutations in COL7A1, encoding collagen type VII.1 Clinical manifestations of COL7A1 mutations range from generalized skin blistering to mild localized blistering or nail dystrophy.2 The investigation of the molecular basis of DEB has revealed more than 540 different mutations that cannot entirely explain phenotypic variations (HGMD Professional 2010.3, https://portal.biobase-international. com/hgmd/). Inversa recessive DEB (RDEB-I) is a subtype characterized by generalized blistering in the neonatal period. The condition improves with age, and in adults blistering is restricted to intertriginous areas, and severe lesions of the oral and genital mucosa and nail changes occur in the majority of described patients.2 Recent data suggested that amino-acid substitutions affecting arginines or glycines at borders of collagenic subdomains might cause this phenotype.3 We report a German patient with an unusually mild RDEB-I harbouring compound heterozygous mutations in COL7A1.

Characterization of coated vesicles that participate in endocytic recycling.

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.

Perforin granule release from cytotoxic lymphocytes ex vivo is inhibited by ciclosporin but not by methotrexate.

The 70-kD plasma membrane pore-forming protein perforin is a key component of lymphocyte cytotoxicity mediated by lytic granules. It represents a major player in the regulation of various immune reactions like immunoglobulin synthesis, T-cell activation and homeostasis, and in the elimination of virus-infected and tumor cells. Dysregulation of the perforin-granule system, i.e. an increase of perforin-containing lymphocytes, was recently demonstrated in exacerbated psoriasis and generalized drug reactions. In contrast, in patients with exacerbated atopic dermatitis or unsymptomatic rhinitis allergica, a severe perforin depletion in cytotoxic T cells was demonstrated. In addition, these cells displayed a remarkable transport defect of lytic granules, i.e. a perforin hyperreleasability. Thus, the process of perforin-granule release may represent an attractive target for therapeutic immune modulation in various dermatological diseases. Ficoll isolated peripheral blood mononuclear cells (PBMCs) of healthy volunteers were preincubated with different concentrations of ciclosporin or methotrexate (MTX) for 1 h. A newly developed flow cytometry based perforin release assay was used to quantify the velocity of ionomycin/phorbol 12-myristate 13-acetate stimulated perforin-granule release in the presence or absence of pharmacological agents. The immunosuppressant MTX did not influence perforin-granule release. Ciclosporin, in contrast, was found to inhibit perforin-granule release significantly and dose dependently: whereas release from CD8(+) lymphocytes was almost maximal for the untreated control after 60 min (41% of CD8(+) perforin(+) cells at time zero), ciclosporin at 20, 4 and 2 microg/ml elevated the aforementioned parameter up to 73, 65 and 53%, respectively. Our data demonstrate that (i) perforin-granule release can be targeted efficiently by pharmacological agents which can be monitored directly in a newly developed perforin-granule release assay, and (ii) suppression of perforin-granule based cytotoxicity by ciclosporin might contribute to the beneficial therapeutic effects of this drug as an immunomodulating and immunosuppressant target.

Dithranol and dimethylfumarate suppress the interferon-gamma-induced up-regulation of cytokeratin 17 as a putative psoriasis autoantigen in vitro.

In psoriasis an etiopathogenetic vicious circle has been hypothesized in which disease manifestation is triggered by skin-specific autoantigen structures. Autoreactive T cells are supposed to mediate inflammation and hyperproliferation in the epidermopapillary compartment, positively feeding back the expression and accessibility of decisive antigen structures. Recently an epitope within cytokeratin 17 (K17) has been described as such a putative psoriasis autoantigen, which is moreover known to be up-regulated under the influence of proinflammatory interferon-gamma (IFN-gamma), which is abundantly detected in psoriatic plaques. The present study proposes an in vitro model for this presumptive IFN-gamma/K17 autoimmune loop, i.e. the incubation of hyperproliferative human HaCaT keratinocytes with 25 U IFN-gamma/ml for 72 h. This treatment led to a significant up-regulation of K17 protein expression (> or =300%) measured by flow immunocytometry as compared to the untreated control (100%, p < or = 0.05). Preincubation with a subcytotoxic and antiproliferative dithranol concentration as low as 0.3 microM for 2 h prior to the IFN-gamma exposure resulted in a K17 expression that was significantly lower than the IFN-gamma-induced K17 expression reference level. The IFN-gamma-induced K17 expression was also significantly lowered by coincubation with a subcytotoxic and nonantiproliferative concentration of 3 microM dimethylfumarate. The data indicate for dithranol and dimethylfumarate that a part of their antipsoriatic mode of action may be related to a direct down-regulation of putative psoriasis autoantigen structures.

Perforin hyperreleasability and depletion in cytotoxic T cells from patients with exacerbated atopic dermatitis and asymptomatic rhinoconjunctivitis allergica.

BACKGROUND: As a plasma membrane pore-forming protein, perforin is essential for T-cell cytotoxicity mediated by lytic granules. Recent studies on the immune system of perforin knockout mice demonstrated striking similarities to the immunopathology of atopic diseases. OBJECTIVE: We sought to investigate the perforin system of atopic patients. METHODS: Monoclonal antibodies were used to characterize perforin-positive PBMCs of patients with exacerbated atopic dermatitis (AD) and asymptomatic rhinoconjunctivitis allergica (RCA) by means of immunoflow cytometry. In addition, a perforin release assay was developed to quantify the velocity of ionomycin and phorbol 12-myristate 13-acetate-induced secretion of lytic granules. RESULTS: In atopic patients significantly fewer lymphocytes contained perforin-positive lytic granules compared with those of healthy control subjects (patients with AD: 14% +/- 5%, n = 13, P <.0001; patients with RCA: 24% +/- 5%, n = 9, P <.01; healthy control subjects: 33% +/- 11%, n = 13). Of all CD8(hi+) cytotoxic T lymphocytes (CTLs), only 18% +/- 9% and 17% +/- 12% were perforin-positive in patients with AD and RCA, respectively, compared with 44% +/- 13% in control subjects (P <.0001). In addition, perforin-positive CD8(hi+) CTLs of atopic patients released their perforin twice as fast and more completely than control CTLs. This means that 50% of initially perforin-positive CD8(hi+) CTLs from patients with AD and RCA released their perforin completely within 32 +/- 16 and 36 +/- 19 minutes, respectively, and an over 85% release was reached within 113 +/- 41 and 118 +/- 60 minutes, respectively. In CTLs of healthy control subjects, however, it took 64 +/- 40 minutes to achieve a 50% release of lytic granules, and an 85% depletion was not reached in 60% of healthy control subjects, even after 180 minutes. CONCLUSION: The perforin hyperreleasability explains, at least in part, the decreased percentage of perforin-positive CD8(hi+) CTLs in atopic patients. These distortions in the system of lytic granules of atopic patients may contribute to the functional defects observed in T-cell cytotoxicity in vivo and in vitro in patients with AD and RCA.

The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes.

Cofilin, an actin-depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co-stimulation through accessory receptors (e.g. CD2 or CD28) - however, not following TCR/CD3 stimulation alone - cofilin undergoes dephosphorylation. The subcellular localization as well as the actin-binding activity of cofilin are regulated by the phosphorylation state of serine-3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM-kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM-kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19-kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506-resistant co-stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co-stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).

Inhibition of constitutive serine phosphatase activity in T lymphoma cells results in phosphorylation of pp19/cofilin and induces apoptosis.

In untransformed T lymphocytes, pp19/cofilin, a cytoplasmic actin-binding protein, undergoes dephosphorylation and nuclear translocation in response to costimulation through accessory receptors (e.g., CD2), but not following TCR/CD3 triggering. In malignant T lymphoma cells, dephosphorylation and nuclear translocation of pp19/cofilin occur spontaneously through constitutive activation of a serine phosphatase. Blockade of these processes by the serine phosphatase inhibitor okadaic acid leads to apoptosis. Moreover, lowering the intracellular pp19/cofilin concentrations by antisense-cofilin transfection results in reduced cloning efficiencies. These findings provide support for the view that pp19/cofilin plays a critical role in the growth and survival of both untransformed and malignant T lymphocytes.

Evidence for cell-mediated immune mechanisms in the pathology of pemphigus.

Pemphigus is an autoimmune blistering disease in which autoantibodies have clearly been shown to be pathogenic. Because autoantibodies are also found in uninvolved skin, further mechanisms may be important in the development of pemphigus lesions. In addition to granulocytes, mononuclear cells are commonly found in pemphigus lesions. To elucidate the role of mononuclear cells in the pathology of this disease, we determined the levels of soluble interleukin-2 receptor in blister fluid and serum samples from pemphigus patients prior to treatment. The interleukin-2 receptor (IL-2R) is expressed on activated mononuclear cells. Depending on its rate of synthesis, a portion is released from the cell surface in a soluble form (sIL-2R). In blister fluid, sIL-2R levels were 2186 +/- 288 U/ml (+/- SD), which was significantly higher than levels in concurrently obtained serum samples (1299 +/- 165 U/ml; P < 0.001). In suction blisters in volunteers, and in patients with second-degree burns or friction-induced bullae, sIL-2R levels were normal in both blister fluid and serum. In pemphigus patients, sIL-2R serum levels continuously declined during systemic therapy, correlating with disease activity. Immunohistological studies demonstrated a marked increase in IL-2R+ cells in both the epidermis and dermis of lesional skin compared with perilesional skin. In the dermis, CD3+ T cells predominated, whereas monocytes/macrophages were most frequent in the epidermis. In pemphigus vulgaris, monocytes/macrophages were restricted to the basal keratinocytes, whereas in pemphigus foliaceus, they were found throughout the lesional epidermis. Our data indicate that activated mononuclear cells are present in lesional skin of pemphigus patients, and may contribute to the pathology of this disease.

[Immune phenotyping of mononuclear infiltrate in bullous pemphigoid]. / Immunphänotypisierung des mononukleären Infiltrats beim bullösen Pemphigoid.

In bullous pemphigoid, autoantibodies alone are not capable of blister formation. In addition, they need complement and various inflammatory cells. The role of mononuclear cells, however, is poorly understood. Therefore, we studied the inflammatory infiltrate in 21 patients with bullous pemphigoid and 11 controls (normal skin, n = 5; eczematous skin, n = 6) using a panel of monoclonal antibodies. In bullous pemphigoid, 60% of the mononuclear infiltrate consists of CD3+ T-lymphocytes, while 25% represents monocytes/macrophages (MOMA); B-lymphocytes are absent. In the area of the basement zone (BMZ), which was studied separately, MOMA predominate. In 30% of bullous pemphigoid biopsies we found a close, almost linear attachment of MOMA to the BMZ. Along the BMZ, lymphocytes belong to the CD3+, CD4+ T-helper subset. CD8+ T-cytotoxic-suppressor cells, however, are rarely encountered there, and other cytotoxic lymphocytes are not found at all. All lymphocytes along the BMZ are memory cells. In small, early bullous pemphigoid lesions, MOMA are frequently the only effector cells. Eosinophilic granulocytes are rare in these lesions. Our findings suggest that mononuclear cells, and particularly MOMA, may be more important than believed hitherto in the inflammatory process resulting in separation of the BMZ.

The interleukin-2 receptor in lesions and serum of bullous pemphigoid.

The interleukin-2 receptor (IL-2R) is mainly expressed on activated T cells. Depending on its rate of synthesis, a portion is released from the cell surface as soluble IL-2R (sIL-2R). Since the role of mononuclear cells in the pathology of bullous pemphigoid (BP) is not well understood, we determined the sIL-2R in both blister fluid and serum of 15 BP patients with generalized disease before initiating systemic treatment. In addition, we obtained both lesional and perilesional skin biopsies and examined the mononuclear infiltrate with a panel of monoclonal antibodies. In BP blisters, sIL-2R levels were significantly increased (2070 +/- 350 U/ml), (+/- SEM) compared with serum samples taken at the time of blister puncture (1340 +/- 290 U/ml). In six patients with blisters due to second-degree burns or friction and in five suction blister volunteers, sIL-2R levels were normal in both blisters and serum. In BP, elevated serum levels decreased to normal during therapy, correlating with disease activity. The immunohistology showed that 30% of mononuclear cells in the dermal infiltrate of lesional skin expressed the IL-2R, whereas only 15% were positive in perilesional skin. IL-2R-positive cells are the most likely source of the shed receptor in BP blisters. Our results indicate the presence of activated T cells in lesions and peripheral blood of BP and thus underline the importance of cell-mediated immune mechanisms in the pathology of this disease.