In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Identifizierung und Charakterisierung cis regulatorischer Elemente des humanen Gens für Keratin 20 in vitro und im transgenen Mausmodell

ZusammenfassungKeratin 20 (K20) ist ein Intermediärfilament, das als Strukturelement in den Epithelien des Intestinaltrakts und den Merkelzellen der Haut exprimiert wird. Im Rahmen dieser Arbeit wurden verschiedene K20 Expressionsvektoren generiert, mit denen regulatorische Elemente des humanen Gens für K20 charakterisiert wurden. Analysiert wurde der Bereich von –4,8 kb bzw. –21,5 kb bis 12,9 kb vom Transkriptionsstart. Die Vektoren, welche die Sequenz von –21,5 kb bis 12,9 kb umfassten, konnten die Expression des EGFP Reportergens in HT-29 Zellen signifikant steigern. Zwei Enhancer-Elemente zwischen –21,5 und –18,4 kb bzw. –18,4 und –14,9 kb verstärkten die Expression der Reporterkonstrukte in vitro signifikant. Die Analyse der Vektoren in transgenen Mäusen zeigte, dass diese das Transgen gewebespezifisch exprimieren. Mit dem Bereich von –4,8 kb bis 12,9 kb ließ sich transgenspezifische mRNA Expression in intestinalen Geweben mittels RT-PCR nachweisen. Die Verwendung der Sequenz von –21,5 kb bis 21,8 kb steigerte die Expression in vivo nicht weiter.Für die gewebespezifische Expression des K20 Vektors reicht die 4,8 kb 5´upstream Sequenz aus, der Bereich bis –21,5 kb verstärkt die Expression in vitro, allerdings fehlen für die starke gewebespezifische Expression von Transgenen in vivo noch weitere Kontrollelemente.

In vitro diagnosis of T cell-mediated drug allergy

Diagnosis of drug hypersensitivity relies on history, skin tests, in vitro tests and provocation tests. In vitro tests are of great interest, due to possible reduction of drug provocation tests. In this review we focus on best investigated in vitro techniques for the diagnosis of T cell-mediated drug hypersensitivity reactions. As drug hypersensitivity relies on different pathomechanisms and as a single diagnostic test usually does not cover all possible reactions, it is advisable to combine different tests to increase the overall sensitivity. Recently, proliferation-based assays have been supplemented by a panel of novel in vitro tests including analysis of cytotoxic potential of effector cells (granzyme B, granulysin, CD107a), evaluation of cytokine secretion (IL-2, IL-5, IL-13, and IFN-γ) and up-regulation of cell surface activation markers (CD69). We discuss the latest findings and readout systems to identify causative drugs by detecting functional and phenotypic markers of drug-reacting cells, and their ability to enable a more conclusive diagnosis of drug allergy.

Analgesic activity of a non-peptide imidazolidinedione somatostatin agonist: in vitro and in vivo studies in rat

Several lines of evidence support an important role for somatostatin receptors (SSTRs) in pain modulation. The therapeutic use of established SSTR peptide agonists for this indication is limited by their broad range of effects, need for intrathecal delivery, and short half-life. Therefore, the goal of the present study was to investigate the analgesic effect of SCR007, a new, highly selective SSTR2 non-peptide agonist. Behavioral studies demonstrated that paw withdrawal latencies to heat were significantly increased following intraplantar SCR007. Furthermore, both intraperitoneal and intraplantar injection of SCR007 significantly reduced formalin- and capsaicin-induced flinching and lifting/licking nociceptive behaviors. Recordings from nociceptors using an in vitro glabrous skin-nerve preparation showed that SCR007 reduced heat responses in a dose-dependent fashion, bradykinin-induced excitation, heat sensitization and capsaicin-induced excitation. In both the behavioral and single fiber studies, the SCR007 effects were reversed by the SSTR antagonist cyclo-somatostatin, demonstrating receptor specificity. In the single fiber studies, the opioid antagonist naloxone did not reverse SCR007-induced anti-nociception suggesting that SCR007 did not exert its effects through activation of opioid receptors. Analysis of cAMP/protein kinase A (PKA) involvement demonstrated that SCR007 prevented forskolin- and Sp-8-Br-cAMPS (a PKA activator)-induced heat sensitization, supporting the hypothesis that SCR007-induced inhibition could involve a down-regulation of the cAMP/PKA pathway. These data provide several lines of evidence that the non-peptide imidazolidinedione SSTR2 agonist SCR007 is a promising anti-nociceptive and analgesic agent for the treatment of pain of peripheral and/or central origin.

In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test

BACKGROUND Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

In vitro and in vivo activity of human interleukin-8 in dogs

Interleukin-8 (IL-8), a proinflammatory cytokine produced by human monocytes, fibroblasts, and endothelial and epithelial cells, is effective not only on cells and tissues of human beings but also on those of several animal species. We investigated the importance of recombinant human IL-8 for the activation of canine neutrophils in vitro and its potential for inducing inflammation in vivo. Shape change (10(-9)-10(-7) M IL-8) and chemotaxis (10(-10)-10(-6) M IL-8) assays were used to determine the activation of canine neutrophils in vitro. Chemotaxis was induced by IL-8 at doses > 10(-8) M with a maximum response at 10(-6) M. A rapid shape change of comparable intensity was elicited by 10(-9)-10(-7) M IL-8. Thirty minutes after intradermal injection of 10(-9) moles of IL-8, emigration of neutrophils could be observed and became more intense at 60 minutes and 240 minutes, respectively. Zymosan-activated canine plasma, which served as a positive control, induced a rapid, massive, and more diffuse neutrophil accumulation, whereas the reaction after IL-8 was weaker but still significant. The neutrophil accumulation after IL-8 was preferentially located in perivenular areas of the deep dermis. Recombinant human IL-8 is capable of activating canine neutrophils in vitro and is able to generate significant neutrophil accumulation in dog skin. Its activity is lower than that in human, rabbit, and rat systems.

Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.

Dendritic cell modulation by 1α,25 dihydroxyvitamin D3 and its analogs: A vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo

Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1α,25 dihydroxyvitamin D3 [1α,25(OH)2D3] and a related analog, 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D3 (D3 analog). Conditioning of bone marrow cultures with 10−10 M D3 analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor β1. These DCs retained an immature phenotype after withdrawal of D3 analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1α,25(OH)2D3 receptor (VDR). In an in vivo model of DC-mediated antigen-specific sensitization, D3 analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1α,25(OH)2D3/VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1α,25(OH)2D3/VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responses in vivo.

Interazione di nanoparticelle metalliche con menbrane biologiche: risultati di studi in vitro su cute, mucosa del cavo orale e meningi ed implicazioni per la salute

L’utilizzo di nanomateriali, ovvero una nuova classe di sostanze composte da particelle ultrafini con dimensioni comprese fra 1 e 100 nm (American Society for Testing Materials - ASTM), è in costante aumento a livello globale. La particolarità di tali sostanze è rappresentata da un alto rapporto tra la superficie e il volume delle particelle, che determina caratteristiche chimico-fisiche completamente differenti rispetto alle omologhe macrosostanze di riferimento. Tali caratteristiche sono tali da imporre una loro classificazione come nuovi agenti chimici (Royal Society & Royal Academy of Engineering report 2004). Gli impieghi attuali dei nanomateriali risultano in continua evoluzione, spaziando in diversi ambiti, dall’industria farmaceutica e cosmetica, all’industria tessile, elettronica, aerospaziale ed informatica. Diversi sono anche gli impieghi in campo biomedico; tra questi la diagnostica e la farmacoterapia. È quindi prevedibile che in futuro una quota sempre maggiore di lavoratori e consumatori risulteranno esposti a tali sostanze. Allo stato attuale non vi è una completa conoscenza degli effetti tossicologici ed ambientali di queste sostanze, pertanto, al fine di un loro utilizzo in totale sicurezza, risulta necessario capirne meglio l’impatto sulla salute, le vie di penetrazione nel corpo umano e il rischio per i lavoratori conseguente al loro utilizzo o lavorazione. La cute rappresenta la prima barriera nei confronti delle sostanze tossiche che possono entrare in contatto con l’organismo umano. Successivamente agli anni ‘60, quando si riteneva che la cute rappresentasse una barriera totalmente impermeabile, è stato dimostrato come essa presenti differenti gradi di permeabilità nei confronti di alcuni xenobiotici, dipendente dalle caratteristiche delle sostanze in esame, dal sito anatomico di penetrazione, dal grado di integrità della barriera stessa e dall’eventuale presenza di patologie della cute. La mucosa del cavo orale funge da primo filtro nei confronti delle sostanze che entrano in contatto con il tratto digestivo e può venir coinvolta in contaminazioni di superficie determinate da esposizioni occupazionali e/o ambientali. È noto che, rispetto alla cute, presenti una permeabilità all’acqua quattro volte maggiore, e, per tale motivo, è stata studiata come via di somministrazione di farmaci, ma, ad oggi, pochi sono gli studi che ne hanno valutato le caratteristiche di permeazione nei confronti delle nanoparticelle (NPs). Una terza importante barriera biologica è quella che ricopre il sistema nervoso centrale, essa è rappresentata da tre foglietti di tessuto connettivo, che assieme costituiscono le meningi. Questi tre foglietti rivestono completamente l’encefalo permettendone un isolamento, tradizionalmente ritenuto completo, nei confronti degli xenobiotici. L’unica via di assorbimento diretto, in questo contesto, è rappresentata dalla via intranasale. Essa permette un passaggio diretto di sostanze dall’epitelio olfattivo all’encefalo, eludendo la selettiva barriera emato-encefalica. Negli ultimi anni la letteratura scientifica si è arricchita di studi che hanno indagato le caratteristiche di assorbimento di farmaci attraverso questa via, ma pochissimi sono gli studi che hanno indagato la possibile penetrazione di nanoparticelle attraverso questa via, e nessuno, in particolar modo, ha indagato le caratteristiche di permeazione delle meningi. L’attività di ricerca svolta nell’ambito del presente dottorato ha avuto per finalità l’indagine delle caratteristiche di permeabilità e di assorbimento della cute, della mucosa del cavo orale e delle meningi nei confronti di alcune nanoparticelle, scelte fra quelle più rappresentative in relazione alla diffusione d’utilizzo a livello globale. I risultati degli esperimenti condotti hanno dimostrato, in vitro, che l’esposizione cutanea a Pt, Rh, Co3O4 e Ni NPs determinano permeazione in tracce dei medesimi metalli attraverso la cute, mentre per le TiO2 NPs tale permeazione non è stata dimostrata. È stato riscontrato, inoltre, che la mucosa del cavo orale e le meningi sono permeabili nei confronti dell’Ag in forma nanoparticellare.

An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream Silvazine (TM), 1% silver sulphadiazine cream Flamazine (TM) and a silver coated dressing Acticoat (TM)

Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped improve the survival in these patients. There are a number of anti-microbials available, one of which, Silvazine(TM) (1% silver sulphadiazine (SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared Silvazine(TM) with the new dressing Acticoat(TM). This study compared the anti-microbial activity of Silvazine(TM), Acticoa(TM) and 1% silver sulphadiazine (Flamazine(TM)) against eight common burn wound pathogens. Methods: Each organism was prepared as a suspension. A 10 mul inoculum of the chosen bacterial isolate (representing approximately between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were then incubated and 10 mul loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated for 18 hours and a colony count was performed. Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) results in the most effective killing of all bacteria. SSD and Acticoat(TM) had similar efficacies against a number of isolates, but Acticoat(TM) seemed only bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus mirabilis rei named at 24 h. Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) is a more effective anti-microbial against a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required. (C) 2003 Elsevier Ltd and ISBI. All rights reserved.

Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype. (c) 2005 Elsevier Inc. All rights reserved.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: An international multicenter study using quasi-standardized methods and materials

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. Standardized calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 mug cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 mug cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intra-laboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required. (C) 2005 Wiley-Liss, Inc.

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies-therm ally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 mu m. (C) 2005 Elsevier Ltd. All rights reserved.

Síntese verde de nanopartículas de prata a partir de extrato aquoso do tubérculo de Curcuma longa associadas à quitosana e avaliação da atividade antitumoral in vitro em câncer de pele não melanoma (linhagem A431)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação Nanociência e Nanobiotecnologia, 2016.