[Show abstract][Hide abstract] ABSTRACT: In pemphigus vulgaris (PV), IgG autoantibodies against the ectodomain of desmoglein 3 (Dsg3) have been shown to be directly responsible for the loss of keratinocyteadhesion. The aim of the present study was to study the effect of the B cell depleting anti-CD20 monoclonal antibody, rituximab, on the profile of pathogenic IgG against distinct regions of the Dsg3 ectodomain in 22 PV patients who were followed up clinically and serologically by Dsg3 ELISA over 12-24 months. Prior to rituximab, all the 22 PV patients showed IgG against Dsg3 (Dsc3EC1-5). Specifically, 14/22 showed IgG reactivity against the Dsg3EC1 subdomain, 5/22 patients against Dsg3EC2, 7/22 against Dsg3EC3, 11/22 against Dsg3EC4, and 2/22 against Dsg3EC5. Within 6 months after rituximab, all the patients showed significant clinical improvement and reduced IgG against Dsg3 (5/22) and the various subdomains, that is, Dsg3EC1 (7/22), Dsg3EC2 (3/22), Dsg3EC3 (2/22), sg3EC4 (2/22), and Dsg3EC5 (0/22). During the entire observation period, 6/22 PV patients experienced a clinical relapse which was associated with the reappearance of IgG against previously recognized Dsg3 subdomains, particularly against the Dsg3EC1. Thus, in PV, rituximab only temporarily depletes pathogenic B cell responses against distinct subdomains of Dsg3 which reappear upon clinical relapse.
Dermatology Research and Practice 06/2010; 2010(1687-6105):321950. DOI:10.1155/2010/321950
[Show abstract][Hide abstract] ABSTRACT: Epidermolysis bullosa acquisita (EBA) is a severe immunobullous disease and is caused by IgG against type VII collagen (Col VII) of anchoring fibrils. In this study, utilizing ELISA and immunoblot, 13/15 EBA sera but 0/20 bullous pemphigoid sera and 0/30 healthy control sera showed IgG reactivity with distinct recombinant subregions of the non-collagenous domain 1 (NC1) of Col VII. In two EBA patients, IgG titers against Col VII-NC1 were grossly correlated to clinical disease activity. Moreover, Col VII-reactive T cells were identified in a representative EBA patient which recognized identical subdomains of Col VII-NC1. These findings strongly suggest that (1) the Col VII-NC1 ELISA is a powerful tool for making the diagnosis of EBA, (2) Col VII-specific IgG grossly relates to disease activity and (3) IgG reactivity is associated with T cell recognition of identical subdomains of Col VII-NC1.
[Show abstract][Hide abstract] ABSTRACT: Pemphigus vulgaris (PV) is associated with autoantibodies against desmoglein (Dsg) 3 inducing epidermal loss of adhesion. The major pathogenic epitopes of Dsg3 are presumably dependent of their conformation. The aim of this study was to characterize the IgG reactivity of sera from a cohort of clinically well-characterized PV patients against presumably non-conformational subdomains of the Dsg3 ectodomain including recently described NH2-terminal immunodominant epitopes. By ELISA, IgG reactivity against distinct subdomains of Dsg3 was related to disease activity and the clinical phenotype of PV patients. Our findings suggest that (i) autoantibody from PV sera react with non-conformational epitopes of Dsg3; (ii) IgG reactivity against the NH2-terminus and the extracellular domains (EC) 2-4 of Dsg3 was associated with active PV, while IgG titres were not strictly correlated with disease activity and (iii) IgG reactivity against the EC1-4 was associated with mucosal dominant PV and was decreased in cutaneous dominant PV. The findings may help to define more refined serological disease markers of PV.
[Show abstract][Hide abstract] ABSTRACT: Pemphigus vulgaris (PV) is an autoimmune disease caused by immunoglobulin G (IgG) autoantibodies against the desmosomal adhesion molecules, desmoglein (Dsg)3 and Dsg1. The aim of the study was to relate IgG reactivity of 123 PV sera and 40 control sera against NH(2)-terminal non-conformational epitopes of Dsg3 and Dsg1 with disease activity and clinical phenotype by enzyme-linked immunosorbent assay. The results show that (i) the overall reactivity and the titres of IgG reactive with the Dsg3 ectodomain, Dsg3(1-566), significantly correlated with the disease activity of the PV patients; (ii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with active PV while there was no direct correlation between the IgG titres and the disease activity; (iii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with mucosal and mucocutaneous PV; (iv) IgG titres against a small stretch of the NH(2)-terminus of Dsg3, Dsg3(25-88), were associated with active PV; and (v) IgG in the PV sera detected non-conformational epitopes in addition to the previously identified conformation-dependent epitopes of the Dsg3 and Dsg1 ectodomains.
[Show abstract][Hide abstract] ABSTRACT: Alopecia areata (AA) is a T cell-mediated autoimmune disease that can be treated with the contact sensitizer diphenylcyclopropenone (DCP). Peripheral blood leukocytes from AA patients are relatively resistant to apoptosis which might be due to decreased Fas Ligand (FasL) expression, or to an increase in CD44v7 expression. Moreover it has been suggested in a murine model of AA that contact allergen treatment might interfere with the emigration of Langerhans cells into the draining lymph node, thus hampering autoreactive T-cell activation. To assess whether and which of these mechanisms is of clinical relevance, immunohistochemistry was performed in scalp biopsies of successfully DCP-treated AA patients in the early phase of hair regrowth. In line with recent studies in a murine model of AA, there was no evidence that DCP treatment would interfere with extravasation and skin homing of activated leukocytes. Perifollicular infiltrates of DCP-treated as compared to untreated AA patients actually showed an increased number of perifollicular CD8(+) and CD1a(+) cells. Furthermore, the expression of CD44 and CD49d, which are of major importance in leukocyte extravasation, was even increased in DCP-treated as compared to AA patient infiltrates. The same accounted for the skin homing receptor CD44v10. When we evaluated the leukocyte subpopulations in DCP-treated as compared to untreated AA patients' skin biopsies, there was an undue increase in CD1a(+) cells, that could well be indicative of hampering of the emigration of antigen presenting cells (APC) by allergen treatment. Most importantly, the number of perifollicular TUNEL- and FasL-positive cells was strikingly increased, whereas the number of CD44v7(+) cells remained unaltered. Taken together, this study provides strong evidence that long term treatment with a contact sensitizer allows for the recovery of hair follicle by driving autoreactive T cells into activation-induced cell death. In addition the replacement with newly activated autoreactive T-cells might be impaired due to a DCP-mediated hindrance of APC emigration.
European journal of dermatology: EJD 11/2005; 16(5):537-42. DOI:10.1684/ejd.2006.0033 · 1.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the relationship between clinical phenotype, clotting activity (FVIIc) and FVII genotype, a multi-center study of factor VII (FVII) congenital deficiency with centralized genotyping and specific functional assays was carried out. FVII mutations characterized in patients (n=313) were extremely heterogeneous (103 different, 22 novel). Clinical phenotypes ranged from asymptomatic condition, including 15 homozygotes and 14 double heterozygotes, to patients with a severe disease characterized by life-threatening and disabling symptoms (CNS, GI bleeding and hemarthrosis) strongly associated with an early age of presentation. Based on type and number of symptoms we classified 90 'severe' (median FVIIc 1.4%, IQR [Interquartile Range] 0.9-3.8), 83 'moderate' (FVIIc 3%, IQR 1-21.7), and 140 'mild' bleeders (FVIIc 14%, IQR 3-31). The significantly different FVIIc levels, and the decreasing prevalence of homozygotes or double heterozygotes among severe (98%), moderate (84%) and mild (56%) bleeders, further support our classification. The excess of females among moderate bleeders (female/male ratio =2.6) is attributable to menorrhagia. There was no evidence for modulation of clinical features by frequent functional polymorphisms. Homozygotes for the same mutation (Ala294Val; 11125delC) with similar FVIIc and FXa generation levels, showed striking differences in clinical phenotypes. Our study depicts the ample clinical picture of this rare disorder, proposes a severity classification and provides arguments for the early management of the disease in the severe cases. Genotype-phenotype relationships indicate the presence of major environmental and/or extragenic components modulating expressivity of FVII deficiency.
[Show abstract][Hide abstract] ABSTRACT: Green fluorescent protein (GFP)-expressing wild-type, and nontransgenic mouse vibrissa follicle cells were cultured and implanted to mouse ears and footpads. Dermal papiller (DP)-derived cells and cells from the peribulbar dermal sheath "cup" (DSC) induced new hair follicles in both implanted ears and footpads, while nonbulbar dermal sheath cells did not. Confocal microscopy revealed that GFP-expressing DP and DSC cells induced hair growth associated with the formation of DP exclusively comprised of fluorescent cells. In mouse ears, but not footpads, fluorescent DP and DSC cells could also be identified in DP along with nonfluorescent cells. DSC cells were characterized in vivo and in vitro by low alkaline phosphatase activity in contrast to high alkaline phosphatase in DP cells. The results indicate transplanted DP and DSC cells were equally capable of DP formation and hair follicle induction. This suggests the DP and peribulbar DSC may be functionally similar. In addition to observing papillae exclusively composed of GFP-expressing cells, DP and DSC cells may also have combined with resident cells to form papillae composed of implanted GFP-expressing cells and host-derived non-GFP-expressing cells. Alkaline phosphatase expression may be utilized as a simple marker to identify hair follicle mesenchyme derived cells with hair follicle inductive abilities.
[Show abstract][Hide abstract] ABSTRACT: Alopecia areata is suspected to be a T cell-mediated autoimmune disease of the hair follicle, where Fas is expressed on hair follicles and Fas ligand on perifollicular infiltrates. To elucidate whether the Fas/Fas ligand pathway is of pathogenetic significance in alopecia areata, we investigated whether alopecia areata can be induced in Fas-deficient and Fas ligand-deficient mice and whether alopecia areata develops in Fas-deficient and Fas ligand-deficient skin. Therefore, we induced alopecia areata by grafting alopecia areata-affected C3H/HeJ mouse skin on to C3H/HeJ mice (control), on to Fas ligand-deficient C3H/HeJ-Tnfsf6(gld) mice or Fas-deficient C3.MRL-Tnfrsf6(lpr) mice. All control mice developed alopecia areata, whereas no Fas-deficient mice showed hair loss and two of seven Fas ligand-deficient mice developed only transitory, limited alopecia areata. Moreover, skin from C3H/HeJ mice (control), C3H/HeJ-Tnfsf6(gld) mice, and C3.MRL-Tnfrsf6(lpr) mice was grafted on to C3H/HeJ mice with extensive alopecia areata. Skin grafts from control mice developed hair loss, whereas Fas-deficient and Fas ligand-deficient skin grafts were spared from alopecia areata. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and immunofluorescence studies revealed an increased number of apoptotic cells and expression of Fas on hair follicles as well as expression of Fas ligand on cells of the perifollicular infiltrate in C3H/HeJ mice with alopecia areata, whereas in Fas-deficient and Fas ligand-deficient mice apoptotic cells were virtually absent in hair follicles. The results suggest that the Fas/Fas ligand pathway plays an important pathogenetic role in alopecia areata.
[Show abstract][Hide abstract] ABSTRACT: Alopecia areata (AA) is a complex, multi-factorial disease where genes and the environment may affect susceptibility and severity. Diet is an environmental factor with the potential to influence disease susceptibility. We considered dietary soy (soya) oil content and the soy-derived phytoestrogen genistein as potential modifying agents for C3H/HeJ mouse AA. Normal haired C3H/HeJ mice were grafted with skin from spontaneous AA affected mice, a method previously shown to induce AA. Grafted mice were given one of three diets containing 1%, 5% or 20% soy oil and observed for AA development. In a separate study, mice on a 1% soy oil diet were injected with 1 mg of genistein three times per week for 10 weeks or received the vehicle as a control. Of mice on 1%, 5%, and 20% soy oil diets, 43 of 50 mice (86%), 11 of 28 mice (39%), and 2 of 11 mice (18%) developed AA, respectively. Four of 10 mice injected with genistein and 9 of 10 controls developed AA. Mice with AA had hair follicle inflammation consistent with observations for spontaneous mouse AA, but no significant association was observed between the extent of hair loss and diet or genistein injection. Mice that failed to develop AA typically experience white hair regrowth from their skin grafts associated with a moderate macrophage and dendritic cell infiltration. Soy oil and derivatives have previously been reported to modify inflammatory conditions. Hypothetically, soy oil compounds may act on C3H/HeJ mice through modulating estrogen-dependent mechanisms and/or inflammatory activity to modify AA susceptibility.
[Show abstract][Hide abstract] ABSTRACT: Grafting alopecia areata affected C3H/HeJ mouse skin to littermates induces alopecia areata, but high dietary soy oil reduces alopecia areata susceptibility. Alopecia areata affected and resistant mice were characterized to evaluate possible mechanisms involved in alopecia areata resistance. Of 44 mice that received alopecia areata affected skin grafts but failed to develop alopecia areata, only two of 22 receiving further alopecia areata affected skin grafts developed alopecia areata, whereas 39 of 44 controls developed alopecia areata. Alopecia areata affected skin contained increased numbers of CD4+ and CD8+ cells, increases in pro inflammatory T helper 1 and T helper 2 type cytokines, and upregulation of CD28, CD40L, and their ligands. In draining lymph nodes, a relatively high number of antigen-presenting cells was recovered, whereas several CD44v variants were downregulated. In contrast, alopecia areata resistant mouse skin did not display increased numbers of CD4+ and CD8+ cells, whereas counter-regulatory cytokines interleukins 4 and 10 were upregulated. High expression of CD28, CD80, CD86, CD40, CTLA4, CD44v variants, and FasL occurred in alopecia areata resistant mouse spleens. In vitro, lymph node cells of susceptible and resistant mice responded equally to a mitogenic stimulus, but only lymph node cells from alopecia areata affected mice displayed an increased response with T cell receptor stimulation via anti-CD3 cross-linking. These results suggest alopecia areata is a cell-mediated autoimmune disease, but alopecia areata affected skin graft hosts may resist alopecia areata onset through active counter-regulatory mechanisms. Because alopecia areata resistant mice showed unimpaired responsiveness and a transient inflammatory response towards the graft, it is suggested that alopecia areata develops as a consequence of an inappropriate immune response regulation.
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-1 has been shown to be a potent inhibitor of hair growth in vitro. We hypothesized that this cytokine might be a decisive factor causing hair loss during the lymphocytic attack in alopecia areata. Neither the intracellular pathways involved in hair growth inhibition mediated by IL-1beta nor the signal transduction processes within hair follicles in general are known. We therefore investigated the intracellular signals involved in human hair growth in vitro. Hair follicles were isolated from scalp biopsies by microdissection, and hair growth was measured daily by image analysis. We assessed intracellular signal transducing elements using specific inhibitors or activators either alone or in combination with IL-1beta. The calcium ionophore A 23187 induced a rapid and complete arrest of hair growth, and phorbol-12-myristate-13-acetate (PMA), genistein, or IL-1beta decreased hair growth by approximately 60%-80%. IL-1beta-elicited hair growth arrest was not antagonized by calphostin C, a specific inhibitor of protein kinase C. In contrast, coincubation of IL-1beta with pertussis toxin or H 1004 neutralized the effect of IL-1beta, and dibutyryl-cAMP and cholera toxin, an activator of adenylate cyclase, inhibited hair growth. These data suggest that cAMP acts as a second messenger for IL-1beta-induced inhibition of hair growth. Moreover, our data indicate that in vitro hair growth is dependent on intracellular Ca2+ levels and activation of tyrosine kinase as well as protein kinase C. We were unable to detect a signal transducing element responsible for enhanced hair growth in vitro.
Journal of Investigative Dermatology 02/1997; 108(1):40-2. · 7.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-1 has been shown to be a potent inhibitor of hair growth in vitro. We hypothesized that this cytokine might be a decisive factor causing hair loss during the lymphocytic attack in alopecia areata. Neither the intracellular pathways involved in hair growth inhibition mediated by IL-1β nor the signal transduction processes within hair follicles in general are known. We therefore investigated the intracellular signals involved in human hair growth in vitro. Hair follicles were isolated from scalp biopsies by microdissection and hair growth was measured daily by image analysis. We assessed intracellular signal transducing elements using specific inhibitors or activators either alone or in combination with IL-1β. The calcium ionophore A 23187 induced a rapid and complete arrest of hair growth and phorbol-12-myristate-13-acetate (PMA), genistein, or IL-1β decreased hair growth by approximately 60%- 80%. IL-1β- elicited hair growth arrest was not antagonized by calphostin C, a specific inhibitor of protein kinase C. In contrast, coincubation of IL-1β with pertussis toxin or H 1004 neutralized the effect of IL-1β and dibutyryl-cAMP and cholera toxin, an activator of adenylate cyclase, inhibited hair growth. These data suggest that cAMP acts as a second messenger for IL-1β-induced inhibition of hair growth. Moreover, our data indicate that in vitro hair growth is dependent on intracellular Ca22+ levels and activation of tyrosine kinase as well as protein kinase C. We were unable to detect a signal transducing element responsible for enhanced hair growth in vitro.
[Show abstract][Hide abstract] ABSTRACT: Factors that influence the growth of the anagen hair follicle or initiate the switch to a catagen growth pattern have so far not been definitely determined, but there is increasing evidence that cytokines and growth factors play an important role during these processes. Recently we detected an aberrant in situ expression pattern of cytokines of the Th1 type (IFN gamma, IL-2) plus IL-1 beta expression in untreated alopecia areata (AA), and a switch to high levels of IL-10 TGF-beta 1 expression after successful treatment with the contact allergen diphenylcyclopropenone (DCP). Hence the question arose as to whether cytokines are able to arrest hair growth and whether IL-10 or TGF beta 1 have the capacity to antagonize this process. Using whole-organ cultures of microdissected human hair follicles we studied the effect of a panel of cytokines and growth factors on hair growth and on the gross morphology of the hair follicles in vitro. IL-2, IL-10 and IFN-gamma had no effect in this regard, whereas TGF beta 1 partially inhibited hair growth and EGF, TNF alpha and IL-1 beta completely abrogated it. EGF and TNF alpha induced the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb, whereas hair follicles grown in the presence of IL-1 beta or TGF beta 1 showed no particular morphological changes. We conclude that cytokines and growth factors are pivotal regulators of hair growth at least in vitro. IL-1 is suggested as playing an important role during the pathogenesis of AA. Possible mediators of therapeutic contact dermatitis (IL-10, TGF beta 1, TNF alpha, PGE2) are, at least in vitro, not able to antagonize the IL-1 beta-triggered hair growth inhibition. Therefore, we infer that these mediators rather "modulate' the immune response in AA.
Archives for Dermatological Research 04/1996; 288(3):153-6. DOI:10.1007/BF02505825 · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The early immune response in alopecia areata is characterized by a Th1 T helper cell cytokine pattern and an aberrant expression of ICAM-1 and HLA-DR molecules on lesional hair bulbs. A counteracting cytokine pattern induced by a therapeutic contact dermatitis is supposed to mediate the hair regrowth. In addition to cytokines, growth factors have been shown to influence immune responses, and we therefore investigated the expression levels for a panel of growth factors in untreated versus alopecia areata after treatment with the contact sensitizer diphenylcyclopropenone. Using semiquantitative reverse transcriptase polymerase chain reaction we detected a striking overexpression of transforming growth factor beta 1 mRNA in successfully treated patients. This cytokine has been shown to be a potent immune response modifier, which can suppress Th1 immune responses. The way in which topical immunotherapy induces hair regrowth in alopecia areata is unknown, but a lesional increased expression of transforming growth factor beta 1 may be a possible mechanism.
[Show abstract][Hide abstract] ABSTRACT: Although the nature of the noxious signal and the anatomical target in alopecia areas (AA) are still unknown, it has been assumed that CD4+ T lymphocytes surrounding and infiltrating the hair bulb might trigger the hair loss. As these T lymphocytes do not promote cytotoxic activity we hypothesize that AA is triggered by cytokines. Topical immunotherapy with diphenylcyclopropenone (DCP) is at present the most effective approach. If it is true that AA results from a distinct cytokine pattern, we can hypothesize that the beneficial effect of DCP should be mediated by locally secreted cytokines during the contact allergy. Using semiquantitative reverse transcription-polymerase chain reaction with RNA extracted from scalp biopsies from patients with AA before and after successful treatment with DCP, and from healthy controls we detected a T-cell response with increased steady state mRNA levels for interferon (IFN)-, interleukin (IL)-1, and IL-2 in untreated AA of the totalis type. After DCP treatment, the IFN- expression was reduced but still above the constitutive level found in controls, whereas mRNA expression of IL-2, IL-8, IL- 10, and tumor necrosis factor- was increased. Our results point towards cytokines involved in the pathogenesis in AA. A TH1 type cytokine pattern is present in untreated AA, and this is modified by cytokines secreted during DCP treatment. IL-10 has recently been described as an immunomodulator of the TH1 response and, therefore, we hypothesize that basal keratinocytes or lesional T cells secrete bioactive IL-10 after DCP application, resulting in an inhibitory effect on lesional T lymphocytes. This hypothesis would explain the effectiveness of DCP and implies the theoretical possibility of a response to topical or intralesional application of recombinant IL-10.Keywords: alopecia areata, diphenylcyclopropenone
[Show abstract][Hide abstract] ABSTRACT: Serum-free conditioned media (CM) of acute myelogenous leukemia cell lines induce growth stimulation of other leukemic cell lines. A transferrin (Tf)-like iron binding kD 50 protein was found to be the main growth factor of these CM's. Purification and further characterization of this protein was performed by chromatographic methods, SDS-PAGE, peptide mapping and amino acid analysis. In addition m-RNA of leukemic cell lines was analysed by Northern blot analysis with oligonucleotide probes specific for human serum Tf and melanotransferrin (p97) respectively. No hybridization signals for these probes have been detected. Biochemical results show significant differences between human serum Tf and kD 50 growth factor with respect to retention time on reversed phase FPLC, pattern of digestion peptides in SDS-PAGE and amino acid composition, which suggests, that this protein is an additional tumor associated member of the transferrin superfamily.
Leukemia and Lymphoma 12/1993; 11(5-6):435-41. DOI:10.3109/10428199309067937 · 2.89 Impact Factor