Evidence of key role of Cdk2 overexpression in pemphigus vulgaris

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy.     

A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG-/-) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG-/- cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG-/- keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.     

Immune modulation in pemphigus vulgaris: role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28-/- mice keratinocytes. No alteration in in vitro adhesion was detected in CD28-/--type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28-/- mice. Lower levels of IL-10 mRNA were found in lesions from CD28-/- mice. To determine whether pemphigus susceptibility in CD28-/- was related to IL-10 deficiency, we performed passive transfer experiments in IL-10-/- mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.     

Cytodiagnosis of oral pemphigus vulgaris

A study was undertaken to verify the reliability of the Tzanck test, performed both by traditional cytomorphology and by a direct immunofluorescence technique, for the diagnosis of oral pemphigus vulgaris. Cytologic smears were obtained from oral erosions of 129 patients with various bullous diseases of the oral mucosa, clinically suspected of being oral pemphigus, as well as from 30 healthy subjects. The 40 cases with subsequent histologic proof of oral pemphigus were cytologically diagnosed as such, based on the significant cytomorphologic findings of acantholytic cells or on the pericellular deposition of IgG (which persisted after cytocentrifugation) in epithelial cells, as studied by direct immunofluorescence. Cytomorphology gave positive results in 37 patients with pemphigus and in one patient with a final diagnosis of herpetic stomatitis and gave negative results in all other cases. Immunocytology gave positive results in all patients with pemphigus and negative results in all other cases. The findings indicate that cytomorphologic studies may be useful in screening suspected cases of oral pemphigus vulgaris while the immunocytologic test may provide a reliable definitive diagnosis.     

Modeling of pemphigus vulgaris in guinea pigs]

In 146 guinea pigs with body weight of 150-200 g pemphigus vulgaris (PV) was induced using screening techniques. The animals were injected with PV patients' IgG (IgGPV) and blister fluid (PVBF) with the PV patient mononuclears. Intraperitoneal administration of IgGPV caused dystrophy in epidermis of experimental animals while intracutaneous injection of PVBF resulted in balloon dystrophy. Clinical manifestations of -PV were obtained after two-day intraperitoneal IgGPV administration in a total dose of 1.5 g (6 mg/g) following a cycle of intracutaneous injections of 2.0 ml PVBF in 10-15 sites of guinea pig back. The presence of acantholysis, intraepidermal localization of blisters, pemphigus antibodies fixation on spinous cell surface, and almost 100% mortality of experimental animals were the major criteria for the developed model of PV in guinea pigs. Experimental PV in guinea pigs was abolished due to treatment of PVBF with dexamethasone, contrykal, heating (56 degrees C, 30 min), preincubation with human skin samples, and PVBF cell-free supernatant application. The conclusion was made that for occurrence of pemphigus in laboratory animals a combined effect of pemphigus antibodies, sensitized mononuclears, complement and endogenous proteinases is required.     

Intermittent cyclophosphamide in pemphigus vulgaris and bullous pemphigoid

Cyclophosphamide, given in widely spaced doses, was used in the treatment of a patient with pemphigus vulgaris and a patient with bullous pemphigoid. To our knowledge, this form of therapy has not previously been reported in these two diseases. The distinct advantages of the larger intermittent dose method of cyclophosphamide therapy over the more conventional daily dose regimen are discussed.     

Expression of human endogenous retroviruses in pemphigus vulgaris patients

Molecular Biology Reports - Pemphigus is a severe, potentially life-threatening autoimmune blistering mucocutaneous disease which establishes with autoreactive IgG antibodies that target cellular...     

Apoptosis is not required for acantholysis in pemphigus vulgaris

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory proteins FLIP(L) and FLIP(S) to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.     

Dissecting the anti-desmoglein autoreactive B cell repertoire in pemphigus vulgaris patients

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. V(H) gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two V(H) genes. V(L) gene segment use was diverse even among IgG hybridomas suggesting that the V(L) is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in V(H) use, is somatically mutated, and is Ag selected.     

Defining the role of astrocytes in neuromodulation

Astrocytes undergo elevations in intracellular calcium following activation of metabotropic receptors, which may trigger glutamate secretion and excitation of surrounding neurons. In this issue of Neuron, Fiacco et al. use transgenic mice that express a foreign G(q)-coupled receptor in astrocytes to show that selective stimulation of astrocytes is not sufficient to induce the release of glutamate.     

Experimental Toxoplasma gondii infection in mice: the role of the fifth component of complement.

Experiments performed to determine the influence of the C5 component of complement in experimental Toxoplasma infection revealed that mice deficient in C5 had reduced mortality due to acute toxoplasmosis. Similar results were noted when inbred congenic mice of known complement type, as well as random-bred mice selected for complement type, were used. In both, mice with high complement activity were less resistant to Toxoplasma than were mice deficient in C5. However, many factors must interact in susceptibility to infection with T. gondii. Thus, lower resistance to Toxoplasma was noted in C5-deficient DBA/2J mice, whereas a high degree of resistance was noted in DBA/1J mice, which are not related to DBA/2J mice and which possess a normal sequence of complement. This accentuates the importance of using both random-bred and where possible cogenic lines in assessing the importance of individual factors in infectious immunity.     

Desmoglein versus non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.     

Defining the role of laminin-332 in carcinoma

The deadly feature of cancer, metastasis, requires invasion of cells through basement membranes (BM), which normally act as barriers between tissue compartments. In the case of many epithelially-derived cancers (carcinomas), laminin-332 (Ln-332) is a key component of the BM barrier. This review provides a historical examination of Ln-332 from its discovery through identification of its functions in BM and possible role in carcinomas. Current understanding points to distinct roles for the three Ln-332 subunits (α3, β3, γ2) in cell adhesion, extracellular matrix stability, and cell signaling processes in cancer. Given the large number of studies linking Ln-332 γ2 subunit with cancer prognosis, particular attention is given to the crucial role of this subunit in cancer invasion and to the unanswered questions in this area.