Utilidad del apósito liofilizado de piel de cerdo en el manejo de cicatrización de úlcera de pie diabético / Usefulness of lyophilized pig skin dressings in wound healing as treatment for diabetic foot ulcer

Resumen Objetivo: Mostrar la utilidad del apósito liofilizado de piel de cerdo comparado con el manejo conservador con sulfadiazina de plata en el proceso de cicatrización de la úlcera de pie diabético. Materiales y Método: Estudio cuasiexperimental en pacientes con diagnóstico de pie diabético, se establecieron 2 grupos de estudio utilizando una relación 2:1, el grupo de exposición (10 pacientes) tratado con apósito liofilizado de piel de cerdo y el grupo de control (5 pacientes) manejado con sulfadiazina de plata. La utilidad se midió con la cicatrización en semanas de tratamiento. El análisis estadístico incluyó prueba de t, prueba de z, regresión logística simple y cálculo de la probabilidad del evento. Resultados: El tiempo de cicatrización fue más corto en el grupo manejado con apósito liofilizado de piel de cerdo (10,20 semanas) que en el grupo con manejo a base de sulfadiazina de plata (13,8 semanas). A las 9 semanas de iniciado el tratamiento, la mitad de las pacientes con apósito de piel de cerdo ya habían cicatrizado comparado con la cicatrización en el grupo manejado con sulfadiazina de plata (20%). La probabilidad de cicatrización a las 11 semanas en paciente manejados con sulfadiazina de plata es 20% y con apósito liofilizado de piel de cerdo 80%. Conclusión: El apósito liofilizado de piel de cerdo tuvo mejores resultados en el estudio, comparado con el manejo estándar con sulfadiazina de plata. Es necesario realizar un estudio aleatorizado para determinar la efectividad de este material como herramienta terapéutica.

Aim: To demonstrate the usefulness of lyophilized pig skin dressings versus usual management with silver sulfadiazine in wound healing treatment for diabetic foot ulcers. Materials and Method: In this quasi-experimental study, we included patients diagnosed with diabetic foot. We established two groups with a distribution (2:1), the exposure group treated with lyophilized pig skin dressings (10 patients) and the control group (5 patients), the standard of care with silver sulfadiazine. Usefulness was measured with wound healing in treatment weeks. Statistical analysis included t-test, z-test, simple logistic regression, and calculation of probability of an event. Results: Wound healing time was shorter in the group treated with lyophilized pig skin dressing (10.20 weeks) than in the group treated with silver sulfadiazine (13.8 weeks). At 9 weeks after treatment started, 50% of patients treated with lyophilized pig skin dressings had complete wound healing compared with the patients in the group managed with silver sulfadiazine. (20%). The probability of wound healing been completed at 11 weeks in a patient managed with silver sulfadiazine is 20%, compared to lyophilized pig skin dressings is 80%. Conclusion: Lyophilized pig skin dressings had better outcomes than silver sulfadiazine in wound healing treatment for diabetic foot ulcers inside the study. Is mandatory develop another study with a randomized design to determinate the effectiveness as a therapeutic alternative.

Analysis of Mrgprb2 Receptor-Evoked Ca 2+ Signaling in Bone Marrow Derived (BMMC) and Peritoneal (PMC) Mast Cells of TRPC-Deficient Mice.

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca2+ concentration ([Ca2+]i) in both BMMC and PMC, robust [Ca2+]i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca2+]i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca2+ oscillations in BMMC and robust [Ca2+]i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca2+]i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca2+]i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca2+]i rise was reduced by ~20% in PMC from Trpc1/4/6-/- mice compared to Trpc1/4-/- littermatched control mice, whereas FcεRI-evoked [Ca2+]i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca2+]i rise in Trpc1/4/6-/- PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (FcεRI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a ß-hexosaminidase release assay. The amount of ß-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. ß-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis.

All three members of the Orai family of cation channels-Orai1, Orai2 and Orai3-are integral membrane proteins that can form store-operated Ca2+ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca2+ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca2+-dependent exocytosis of inflammatory mediators. We show that the Ca2+ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca2+ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2-/- PMCs at high (2mM) extracellular Ca2+ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1mM) Ca2+ concentration. Likewise, the density of CRAC currents, Ca2+-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca2+ transients, store-operated Ca2+ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca2+ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.

Measuring intracellular Ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging.

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca(2+)-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca(2+) dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.