Structure and Concentration Capacity of the Kidneys in Brattleboro Rats under Conditions of Long-Term Vasopressin Treatment

Endoprostheses made from knitted fabric of 3 loop types were used for hernioplasty in rats. Biocompatibility of implanted textile constructions was evaluated after 6 months. The intensity of inflammatory reaction and types of complications in animals depended on the loop type and method of finishing of the mesh endoprosthesis tissue. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 582–587, May, 2008     

Allergic FcεRI‐ and pseudo‐allergic MRGPRX2‐triggered mast cell activation routes are independent and inversely regulated by SCF

While allergic mast cell (MC) degranulation occurs by FcεRI aggregation and varies in strength among subjects, the analogous pseudo‐allergic route was recently uncovered to proceed via MRGPRX2. Here, we examine interindividual variability in skin MC responses to FcεRI triggering vs those evoked by MRGPRX2. While population‐based variability is comparable between the routes, FcεRI‐ and MRGPRX2‐stimulated pathways are completely independent from each other, and responsiveness to one has therefore no predictive value for the other. Conversely, degranulation triggered by compound 48/80 is highly correlated to the process elicited by substance P. MRGPRX2 mRNA shows pronounced population‐based variability (coefficient of variation 102.9%). Surprisingly, stem cell factor (SCF) as the MC‐supportive mediator par excellence potently inhibits pseudo‐allergic degranulation, while it simultaneously promotes allergic stimulation via FcεRI . We conclude that SCF can have selective MC‐dampening functions. Clinically, the data imply that subjects highly reactive in one pathway are not automatically hyper‐responsive in terms of the alternative route.     

Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.The membrane protein CD44 is a multifaceted molecule involved in many different cellular processes, including organ development, neuronal axon guidance, immune functions, hematopoiesis, and migration.^1–4 It acts as a receptor for the extracellular matrix component hyaluronic acid (HA)^5,6 and for the secreted extracellular protein osteopontin.⁷ CD44 is an important mediator of cellular adhesion and migration due to its active influence on the organization of the actin cytoskeleton. This occurs through direct interactions between CD44 and different actin-binding partners, of which the most common are proteins belonging to the ezrin-moesin-radixin (ERM) family. The ERM proteins form a bridge between CD44 and the actin cytoskeleton, mediating cell morphology changes that are important for cell migration. Ezrin interacts with CD44 and F-actin, respectively, through its conserved N-terminal band four-point-one, ezrin, radixin, moesin domain and C-terminal ERM Association Domain domain. In the inactive configuration of ezrin, both domains interact with each other and block the binding sites for CD44 and F-actin. Ezrin activation is mediated by phosphorylation-induced conformational changes,⁸ with phosphorylation on threonine-567 being necessary for binding to the F-actin cytoskeleton.⁹Phosphorylation of CD44 has also been shown to be important for its activation, particularly on serine residues in the C-terminal domain.^8,10 CD44 has been described to be enriched in cholesterol- and sphingomyelin-enriched membrane microdomains termed lipid rafts.¹¹ Much evidence has suggested the involvement of lipid rafts in regulating different cellular events, including migration.¹² Because some of these cellular events are frequently altered in cancer, it has been hypothesized that lipid rafts play a crucial role in regulating cancer progression.¹³ However, although alterations in CD44 expression have been associated with many cancers,¹⁴ how lipid rafts influence the subcellular localization (and thus migratory functions) of CD44 and its contribution toward cancer progression is not well understood.Whether or not CD44 and its binding partners localize to lipid rafts may in fact regulate several signaling cascades. CD44 is usually directed toward lipid rafts via posttranslational lipid modifications called acylation reactions, the most common of which is palmitoylation. Due to its dynamic and reversible nature, palmitoylation can have important functions in dictating protein fate such as protein trafficking, lateral segregation, and cellular localization. Palmitoylation plays an important role in CD44-HA turnover, with palmitoylated CD44 promoting CD44-HA endocytosis. Accordingly, lipid rafts have been described to play an important role in cellular endocytosis.¹⁵Ezrin localization to lipid rafts is controversial¹⁶ and the mechanisms regulating its affiliation with lipid rafts incompletely understood. Ezrin interactions with phosphatidylinositol 4,5-biphosphate (PIP2) may be important for its activation, causing the four-point-one and C-terminal ERM domains to open¹⁷ and permitting ezrin localization at the plasma membrane.¹⁸ Because PIP2 has been described to be enriched in lipid rafts,¹⁹ it is possible that ezrin localizes to lipid rafts through an interaction with PIP2.In this paper, we set out to investigate the role of lipid rafts in regulating CD44-dependent breast cancer cell migration. Our initial findings revealed that CD44 and ezrin localized to different membrane fractions in nonmigrating cells, biochemically characterized as lipid raft and nonraft domains, respectively. In response to migratory stimuli (either random or CD44-specific), the proportion of raft-affiliated CD44 decreased whereas that of ezrin did not change. Moreover, under migrating compared to nonmigrating conditions, immunofluorescence confocal microscopy revealed increased colocalization of CD44 with the nonraft marker transferrin receptor. Altogether, we present novel evidence that physical interactions between CD44 and ezrin occur in nonraft fractions of migrating cells. In support of our observations, pharmacological disruption of lipid rafts increased CD44-ezrin coprecipitation, whereas enhanced retention of CD44 within rafts abolished CD44-ezrin coprecipitation. Surprisingly, flotillin-1 or caveolin-1 transient knockdown alone did not affect cell migration in these cells, suggesting compensatory mechanisms that make up for the presumed loss of one or other raft compartment. In support of this assumption, coincident knockdown of flotillin-1 and caveolin-1 significantly impaired cell migration. Nonetheless, our data are consistent with a novel regulatory mechanism in which CD44 translocates outside lipid rafts to bind ERM binding partners such as ezrin and drive cell migration. Future exploration of the precise mechanisms regulating this translocation may reveal future targets for interfering with breast cancer cell migration during the early stages of metastasis.     

Opposing effects on immune function and skin barrier regulation by the proteasome inhibitor bortezomib in an allergen‐induced eczema model

Proteasome inhibition (PI) has been reported to interfere with antibody‐driven autoimmune diseases. The impact of PI on the allergic immune response and on skin diseases like atopic dermatitis (AD) has not been thoroughly explored, however. Here, we examined whether the PI bortezomib interferes with the allergic immune response and the severity of AD by using an established mouse model of allergen‐driven dermatitis, to which bortezomib was applied after the establishment of systemic sensitization to ovalbumin. The treatment indeed resulted in a remarkable decrease in total and allergen‐specific plasma cells/antibody‐secreting cells, as evidenced by flow cytometry and ELISpot, respectively. This was accompanied by rapid reductions in serum antibody titres, including a prominent reduction of the IgE isotype. CD4+ and CD8+ cells were greatly diminished in lesional skin on immunohistological staining. The impressive effects at the level of immune modulation did not result in any improvement in the eczema, however. Following up on this unexpected result, we found that the skin itself was susceptible to bortezomib, by which it was instructed to lower the expression of critical skin barrier genes, especially transglutaminase‐1 and filaggrin. Together, bortezomib eliminates plasma cells and decreases immunoglobulin responses, including allergenic IgE. Although anti‐inflammatory effects are detectable in the skin, counter‐regulatory effects from PI on resident skin cells likely undermine improvement in the eczema. These results caution against the therapeutic use of bortezomib for inflammatory skin disorders, which are characterized by inherently impaired barrier function, especially AD.