Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG.Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin- and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin- and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.     

Digital glomus tumors: a 29-year experience with a lateral subperiosteal approach

The excision of distal digital glomus tumors has traditionally been performed directly over the involved nail bed. This can lead to nail deformities that are often unacceptable for the surgeon and the patient. The authors describe their experience with successful excision of digital glomus tumors using a lateral subperiosteal approach, which creates a dorsal flap. In 29 years, 19 patients were diagnosed with digital glomus tumors. All patients underwent excision using the lateral subperiosteal approach. The mean tumor size was 0.52 cm. The tumors were located on the pulp of the distal phalanx in two patients (10.5 percent) and subungually in 17 patients (89.5 percent). In all patients, preoperative clinical diagnosis was confirmed postoperatively with the biopsy result. Complications occurred in only two patients and included one paronychia and one temporary nail loss. The overall recurrence rate was 15.7 percent. All patients remained asymptomatic after surgery and regained full active and passive range of motion. There were no nail deformities by this approach. This technique represents a safe and effective approach to excising digital glomus tumors.     

Primary Localization and Tumor Thickness as Prognostic Factors of Survival in Patients with Mucosal Melanoma

Background

Data on survival with mucosal melanoma and on prognostic factors of are scarce. It is still unclear if the disease course allows for mucosal melanoma to be treated as primary cutaneous melanoma or if differences in overall survival patterns require adapted therapeutic approaches. Furthermore, this investigation is the first to present 10-year survival rates for mucosal melanomas of different anatomical localizations.

Methodology

116 cases from Sep 10 1984 until Feb 15 2011 retrieved from the Comprehensive Cancer Center and of the Central Register of the German Dermatologic Society databases in Tübingen were included in our analysis. We recorded anatomical location and tumor thickness, and estimated overall survival at 2, 5 and 10 years and the mean overall survival time. Survival times were analyzed with the Kaplan-Meier method. The log-rank test was used to compare survival times by localizations and by T-stages.

Principal Findings

We found a median overall survival time of 80.9 months, with an overall 2-year survival of 71.7%, 5-year survival of 55.8% and 10-year survival of 38.3%. The 10-year survival rates for patients with T₁, T₂, T₃ or T₄ stage tumors were 100.0%, 77.9%, 66.3% and 10.6% respectively. 10-year survival of patients with melanomas of the vulva was 64.5% in comparison to 22.3% of patients with non-vulva mucosal melanomas.

Conclusion

Survival times differed significantly between patients with melanomas of the vulva compared to the rest (p = 0.0006). It also depends on T-stage at the time of diagnosis (p<0.0001).     

A homogeneous enzyme immunoassay with avidin-ligand conjugate as the enzyme-modulator

A homogeneous enzyme immunoassay (EIA) based on the immunomodulation of an avidin-ligand conjugate and the inhibition of pyruvate carboxylase is described. The conjugation of the ligand, 5,5-diphenylhydantoin (DPH), to avidin does not affect avidin's capacity to bind biotin or inhibit pyruvate carboxylase. The DPH-avidin conjugate and free DPH were shown to compete for a limited number of antibody sites. The interaction of anti-DPH with the DPH-avidin conjugate sterically inhibited enzyme inactivation. Enzyme activity was correlated with DPH concentrations in the therapeutic range found in serum.     

Proteases from purulent sputum. Purification and properties of the elastase and chymotrypsin-like enzymes.

A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.     

The Desmosome

Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane. Desmogleins and desmocollins, members of the cadherin superfamily, mediate adhesion at desmosomes. Cytoplasmic components of the desmosome associate with the desmosomal cadherin tails through a series of protein interactions, which serve to recruit intermediate filaments to sites of desmosome assembly. These desmosomal plaque components include plakoglobin and the plakophilins, members of the armadillo gene family. Linkage to the cytoskeleton is mediated by the intermediate filament binding protein, desmoplakin, which associates with both plakoglobin and plakophilins. Although desmosomes are critical for maintaining stable cell–cell adhesion, emerging evidence indicates that they are also dynamic structures that contribute to cellular processes beyond that of cell adhesion. This article outlines the structure and function of the major desmosomal proteins, and explores the contributions of this protein complex to tissue architecture and morphogenesis.The desmosome is an adhesive intercellular junction that is crucial to tissues that experience mechanical stress, such as the myocardium, bladder, gastrointestinal mucosa, and skin (Getsios et al. 2004b; Holthofer et al. 2007). The desmosome was first observed in the spinous layer of epidermis by the Italian pathologist Giulio Bizzozero (1846–1901). Bizzozero''s observations of these small dense nodules, subsequently named “nodes of Bizzozero,” led him to the insightful interpretation of these structures as adhesive cell–cell contact points. The term desmosome was later coined by Josef Schaffer in 1920 and is derived from the Greek words “desmo,” meaning bond or fastening, and “soma,” meaning body (Wells 2005; Calkins and Setzer 2007). The introduction of electron microscopy yielded a series of advances by Porter, Odland, and Kelly in the 1950s and 1960s, which revealed desmosome organization at the ultrastructural level. These studies and others indicated that the desmosome can be divided into three morphologically identifiable zones: the extracellular core region (desmoglea), the outer dense plaque (ODP), and the inner dense plaque (IDP) (Fig. 1A) (Kowalczyk et al. 1994; Schmidt et al. 1994; Green and Jones 1996; North et al. 1999; Garrod and Chidgey 2008).Open in a separate windowFigure 1.A model for the structure of desmosomes. (A) Electron micrograph of a desmosome. (B) Schematic of desmosomal proteins and relative distance from the plasma membrane (PM). The desmosomal cadherins, the desmogleins and desmocollins, extend into extracellular core and outer dense plaque (ODP) to establish contact and adhere to neighboring cells in a Ca²⁺-dependent manner. The cadherin cytoplasmic tails associate linker proteins, plakoglobin (PG), the plakophilins (PKP), and desmoplakin (DP). DP binds to keratin intermediate filaments (KIF) within the inner dense plaque (IDP), serving to tether the intermediate filaments to the plasma membrane. (Adapted with permission from Kottke et al. 2006.)In the mid 1970s, Skerrow and Matoltsy (Skerrow and Matoltsy 1974a; Skerrow and Matoltsy 1974b) advanced the field by isolating desmosomes using biochemical approaches (Bass-Zubek and Green 2007).These landmark studies provided a foundation for the Franke and Steinberg laboratories to characterize the transmembrane glycoproteins and cytoplasmic plaque proteins that linked the structure to the intermediate filament cytoskeleton, and to develop immunological tools for localizing specific components (Franke et al. 1981; Kapprell et al. 1985; Steinberg et al. 1987). Collectively, these and other studies shaped our current view of how desmosomal components are organized.The transmembrane glycoproteins, termed desmogleins and desmocollins (Garrod and Chidgey 2008), represent separate subfamilies of the cadherin superfamily of calcium dependent adhesion molecules. The extracellular domains of the desmogleins and desmocollins mediate adhesion, whereas the cytoplasmic tails of these cadherins associate with the desmosomal plaque proteins. The outer dense plaque consists of the cytoplasmic tails of the desmosomal cadherins, which bind to members of the armadillo and plakin family of linker proteins (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008). Plakoglobin, a member of the armadillo family, binds directly to the cytoplasmic tails of both the desmogleins and the desmocollins (Wahl et al. 1996; Witcher et al. 1996). Desmoplakin, a member of the plakin family, interacts with both plakoglobin and another subgroup of armadillo family proteins, the plakophilins (Cowin and Burke 1996). Finally, the interaction between desmoplakin and the keratin filaments forms the inner dense plaque, tethering the cytoskeletal network to the adhesion complex (Fig. 1B) (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008).The following sections of this article describe the structural and functional characteristics of the major desmosomal proteins. In addition, we discuss differences in tissue expression patterns of desmosomal proteins and the role of desmosomes in human disease. A comprehensive review of additional proteins found to regulate or associate with desmosomes is provided elsewhere (Holthofer et al. 2007) and discussion of desmosome dynamics is provided in Green et al. 2009.     

Erythromelalgia mutation L823R shifts activation and inactivation of threshold sodium channel Nav1.7 to hyperpolarized potentials

Erythromelalgia (also termed erythermalgia) is a neuropathic pain syndrome, characterized by severe burning pain combined with redness in the extremities, triggered by mild warmth. The inherited form of erythromelalgia (IEM) has recently been linked to mutations in voltage-gated sodium channel Nav1.7, which is expressed in peripheral nociceptors. Here, we used whole-cell voltage-clamp recordings in HEK293 cells to characterize the IEM mutation L823R, which introduces an additional positive charge into the S4 voltage sensor of domain II. The L823R mutation produces a ∼15 mV hyperpolarizing shift in the midpoint of activation and also affects the activation slope factor. Closing of the channel from the open state (deactivation) is slowed, increasing the likelihood of the channel remaining in the open state. The L823R mutation induces a ∼10 mV hyperpolarizing shift in fast-inactivation. L823R is the only naturally-occurring IEM mutation studied thus far to shift fast-inactivation to more negative potentials. We conclude that introduction of an additional charge into the S4 segment of domain II of Nav1.7 leads to a pronounced hyperpolarizing shift of activation, a change that is expected to increase nociceptor excitability despite the hyperpolarizing shift in fast-inactivation, which is unique among the IEM mutations.     

Translating research findings into practice - the implementation of kangaroo mother care in Ghana

ABSTRACT: BACKGROUND: Kangaroo mother care (KMC) is a safe and effective method of caring for low birth weight infants and is promoted for its potential to improve newborn survival. Many countries find it difficult to take KMC to scale in healthcare facilities providing newborn care. KMC Ghana was an initiative to scale up KMC in four regions in Ghana. Research findings from two outreach trials in South Africa informed the design of the initiative. Two key points of departure were to equip healthcare facilities that conduct deliveries with the necessary skills for KMC practice and to single out KMC for special attention instead of embedding it in other newborn care initiatives. This paper describes the contextualisation and practical application of previous research findings and the results of monitoring the progress of the implementation of KMC in Ghana. METHODS: A three-phase outreach intervention was adapted from previous research findings to suit the local setting. A more structured system of KMC regional steering committees was introduced to drive the process and take the initiative forward. During Phase I, health workers in regions and districts were oriented in KMC and received basic support for the management of the outreach. Phase II entailed the strengthening of the regional steering committees. Phase III comprised a more formal assessment, utilising a previously validated KMC progressmonitoring instrument. RESULTS: Twenty-six out of 38 hospitals (68 %) scored over 10 out of 30 and had reached the level of 'evidence of practice' by the end of Phase III. Seven hospitals exceeded expected performance by scoring at the level of 'evidence of routine and institutionalised practice.' The collective mean score for all participating hospitals was 12.07. Hospitals that had attained baby-friendly status or had been re-accredited in the five years before the intervention scored significantly better than the rest, with a mean score of 14.64. CONCLUSION: The KMC Ghana initiative demonstrated how research findings regarding successful outreach for the implementation of KMC could be transferred to a different context by making context-appropriate adaptations to the model.