RESUMO
BACKGROUND: Breast implant infection and biofilm formation are major concerns in reconstructive and esthetic breast surgery, with significant medical and economic consequences. Staphylococcus is the common pathogen, with rapidly increasing rates of methicillin-resistant Staphylococcus aureus (MRSA). There is no consensus on prevention practices. This study compares the effect of several pocket irrigation and antibiotic prophylaxis regimens on implant colonization and biofilm formation in an established rat model of MRSA-infected silicone breast implants. METHODS: Silicone discs were inserted in a sub-pectoral pocket in 57 rats (114 implants). Implant infection was induced by injection of free planktonic MRSA into the surgical pocket. Rats were allocated to study groups treated by different antimicrobial protocols: pocket irrigation with vancomycin, povidone-iodine, or saline. Each group was divided into subgroups treated with or without additional peri-operative systemic vancomycin. Implant colonization or overt infection was assessed at post-operative day 14 both clinically and by cultures. RESULTS: Pocket irrigation with vancomycin prevented contamination in 87% of implants. Irrigation and systemic vancomycin prevented contamination in 100% of implants with no difference between a single preoperative dose and a 48-h regimen. Systemic vancomycin alone or irrigation with povidone-iodine alone resulted in 100% contamination rates. CONCLUSIONS: In this in vivo model, combination of systemic vancomycin with vancomycin pocket irrigation was the most effective regimen, preventing contamination in 100% of implants. Continuation of post-operative antibiotic treatment showed no added advantage.
Assuntos
Anti-Infecciosos , Implantes de Mama , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Ratos , Animais , Povidona-Iodo/farmacologia , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Implantes de Mama/efeitos adversos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Silicones/farmacologia , Silicones/uso terapêutico , Infecções Estafilocócicas/prevenção & controleRESUMO
Adipose tissue stromal vascular fraction (SVF) is increasingly used in the clinic. SVF separation from fat by enzymatic disruption is currently the gold standard for SVF isolation. However, enzymatic SVF isolation is time-consuming (~1.5 h), costly and significantly increases the regulatory burden of SVF isolation. Mechanical fat disruption is rapid, cheaper, and less regulatory challenging. However, its reported efficacy is insufficient for clinical use. The current study evaluated the efficacy of a novel rotating blades (RBs) mechanical SVF isolation system. Methods: SVF cells were isolated from the same lipoaspirate sample (n = 30) by enzymatic isolation, massive shaking (wash), or engine-induced RBs mechanical isolation. SVF cells were counted, characterized by flow cytometry and by their ability to form adipose-derived stromal cells (ASCs). Results: The RBs mechanical approach yielded 2 × 105 SVF nucleated cells/mL fat, inferior to enzymatic isolation (4.17 × 105) but superior to cells isolating from fat by the "wash" technique (0.67 × 105). Importantly, RBs SVF isolation yield was similar to reported yields achieved via clinical-grade enzymatic SVF isolation. RBs-isolated SVF cells were found to contain 22.7% CD45-CD31-CD34+ stem cell progenitor cells (n = 5) yielding quantities of multipotent ASCs similar to enzymatic controls. Conclusions: The RBs isolation technology provided for rapid (<15 min) isolation of high-quality SVF cells in quantities similar to those obtained by enzymatic digestion. Based on the RBs platform, a closed-system medical device for SVF extraction in a rapid, simple, safe, sterile, reproducible, and cost-effective manner was designed.
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Vascularized composite allotransplantation is the ultimate reconstructive tool when no other means of reconstruction are available. Despite its immense potential, the applicability of vascularized composite allotransplantation is hampered by high rejection rates and the requirement for high doses of immunosuppressive drugs that are associated with severe adverse effects and death. Because this is a non-life-saving procedure, widespread use of vascularized composite allotransplantation demands methods that will allow the reduction or elimination of immunosuppressive therapy. Efficient methods for the cryopreservation of biological cells and tissues have been sought for decades. The primary challenge in the preservation of viable tissue in a frozen state is the formation of intracellular and extracellular ice crystals during both freezing and thawing, which cause irreversible damage to the tissue. Recent proof-of-concept transplantations of a complete cryopreserved and thawed hindlimb in a rat model have demonstrated the potential of such methods. In the current review, the authors discuss how limb cryopreservation can attenuate or eliminate allograft rejection by either enabling better human leukocyte antigen matching or by adaptation of clinical tolerance protocols such as mixed chimerism induction. Also, the authors discuss the possible advantages of cryopreservation in autologous tissue salvage and cryopreservation following trauma. Clinical-grade cryopreservation may revolutionize the field of reconstruction, organ banking, and complex traumatic limb injury management.
Assuntos
Aloenxertos Compostos , Criopreservação/métodos , Extremidades/lesões , Preservação de Órgãos/métodos , Alotransplante de Tecidos Compostos Vascularizados/métodos , Animais , Extremidades/transplante , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Modelos Animais , Ratos , Bancos de Tecidos , Transplante Homólogo , Alotransplante de Tecidos Compostos Vascularizados/efeitos adversosRESUMO
BACKGROUND: The prevalence of peanut allergy (PA) is constantly on the rise. Atopic dermatitis (AD) is a major risk factor for developing food allergy. Some bath oils and skin creams used for treating AD contain peanut oil, and it has been suggested that exposure to peanut allergens through a disrupted skin barrier is a potential cause of PA. Our aim was to investigate whether application of peanut oil to irritated skin causes a systemic or respiratory allergic response to peanuts in an animal model. METHODS: BALB/c mice underwent epicutaneous sensitization with either peanut oil (PM, n = 9) or phosphate buffered solution (controls, n = 9) daily for 5 consecutive days. Ten days after the last exposure the mice were challenged with intranasal peanut protein for 5 consecutive days. Bronchial alveolar lavage fluid was collected for cellular studies and measurement of cytokine levels. Sera were collected for immunoglobulin E (IgE) measurement. RESULTS: Epicutaneous peanut oil sensitization increased leukocyte and eosinophil counts and interleukin-13 levels (p = 0.003, p = 0.0006 and p = 0.03, respectively), in addition to increasing total serum IgE (p = 0.03). CONCLUSIONS: The results suggest that topical application of peanut oil may play a role in the etiology of PA.
Assuntos
Alérgenos/administração & dosagem , Antígenos de Plantas/administração & dosagem , Hipersensibilidade a Amendoim , Óleo de Amendoim/administração & dosagem , Hipersensibilidade Respiratória , Administração Cutânea , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Feminino , Imunoglobulina E/sangue , Interleucina-13/imunologia , Interleucina-4/imunologia , Contagem de Leucócitos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos Endogâmicos BALB C , Hipersensibilidade a Amendoim/sangue , Hipersensibilidade a Amendoim/imunologia , Hipersensibilidade Respiratória/sangue , Hipersensibilidade Respiratória/imunologiaRESUMO
Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation. METHODS: SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at -196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone. RESULTS: While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45-CD34+CD31- stem cell progenitor cells compared to fresh SVF cells. CONCLUSIONS: Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.
RESUMO
Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.
Assuntos
Tecido Adiposo/citologia , Proteína Ligante Fas/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Osso e Ossos/citologia , Inibidores de Caspase/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ASCs) can be isolated from subcutaneous fat harvested by tissue resection or liposuction. OBJECTIVES: The authors compared ASCs isolated by tissue resection or power-assisted liposuction (PAL) to determine whether either surgical procedure yielded ASCs with improved purity and competence that was preserved for several passages. METHODS: For this experimental study, ASCs were isolated from fat harvested by tissue resection or PAL from six patients who underwent abdominoplasty. ASCs were counted to determine cell yields, and viabilities were assessed with an amine-reactive dye and by fluorescence-activated cell sorting (FACS). Cell phenotypes were determined by immunostaining and FACS, and doubling times were calculated. Senescence ratios of the cells were detected by gene profiling and by assaying ß-galactosidase activity. Multipotency was evaluated by induced differentiation analyses. RESULTS: No significant differences were observed in cell numbers or viabilities of ASCs isolated following either surgical method of fat harvesting. Both populations of cultured ASCs expressed markers of mesenchymal stem cells and preserved this expression pattern through the third passage. PAL and tissue resection yielded ASCs with similar division rates, similar senescence ratios into the fourth passage, and similar capacities to differentiate into osteocytes or adipocytes. CONCLUSIONS: Fat harvested by PAL or tissue resection yielded uniform cultures of ASCs with high division rates, low senescence ratios, and multipotency preserved into passages 3 and 4. Because PAL is less invasive, it may be preferable for the isolation of ASCs.
Assuntos
Gordura Abdominal/citologia , Adipócitos/citologia , Lipectomia , Células-Tronco Mesenquimais , Coleta de Tecidos e Órgãos/métodos , Abdominoplastia , Adipócitos/metabolismo , Adulto , Contagem de Células , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Senescência Celular , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state. METHODS: MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA). RESULTS: We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs. CONCLUSIONS: The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.
Assuntos
Tecido Adiposo/citologia , Instabilidade Genômica , Células-Tronco Mesenquimais/metabolismo , RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Ploidias , Proteína Supressora de Tumor p53/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.
Assuntos
Transformação Celular Neoplásica/genética , Inativação Gênica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Poliploidia , RNA Longo não Codificante/genética , Animais , Transformação Celular Neoplásica/patologia , Células Cultivadas , Instabilidade Genômica , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/genética , RNA Longo não Codificante/antagonistas & inibidoresRESUMO
The existence of incomplete T cell receptor (TCR) mRNA forms, including germline transcripts and products of unfruitful TCR rearrangements, has long been known. However, it is unclear whether these molecules are functional. We have previously shown that T cells also contain truncated TCRß peptides that lack the N-terminal part and contain C-terminus sequences. These partial forms of TCRß, target the mitochondrion and induce apoptosis, exhibiting a novel mode of TCR mediated cell death. Here we aimed at analyzing the minimal TCR sequences that direct the peptide to the mitochondrion. It is shown that truncated TCRß, targets mitochondria and induces mitochondrial perinuclear clustering, in both monkey COS-7 and human 293 cells. These phenomena are mediated by the C-terminus of the molecule. Whereas the positively charged amino acids flanking the transmembrane domain (TMD) of TCRß are beneficial for this process, they are not essential. Indeed, the isolated TMD of TCRß serves as a sufficient mitochondrial targeting sequence. These results indicate that any given partial form of TCRß, that contains the TMD, is bound to be sequestered by the mitochondrion. This may assure that incomplete TCR forms would not interfere with correct TCR complex formation.
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Apoptose/fisiologia , Mitocôndrias/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (ß-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.
Assuntos
Alérgenos/metabolismo , Metabolismo dos Carboidratos/imunologia , Celulose/metabolismo , Pólen/metabolismo , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes , Celulose/administração & dosagem , Celulose/imunologia , Cisteína Endopeptidases , Feminino , Lolium/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Pólen/imunologia , Ligação Proteica/imunologia , Pyroglyphidae/imunologia , Pyroglyphidae/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Wistar , Hipersensibilidade Respiratória/patologiaRESUMO
In vitro and in vivo studies implicate a series of cytokines in regulation of lymphohematopoiesis. However, direct indications for a local role of most of these cytokines within the bone marrow is lacking. In the present study, we aimed to test the contribution of a specific cytokine, activin A, a member of the transforming growth factor-beta (TGF-beta) family, to lymphohematopoiesis in mouse bone marrow. We show that mouse embryonic fibroblasts (MEFs) are indistinguishable from multipotent stromal cells (MSCs). Such MEFs overexpressing activin A, supported in vitro myelopoiesis in long-term bone marrow cultures as effectively as control MEFs. In contrast, activin A-overexpressing MEFs interfered with the in vitro generation of B lineage cells in such cultures. Thus, excessive expression in vitro of activin A, by supportive stromal cells, causes preferential maturation of myeloid rather than lymphoid cells. Moreover, the activin A-overexpressing MEFs caused an increased incidence in vivo of relatively immature B lineage cells; upon transplantation through the spleen route, MEFs engrafted the bone marrow specifically. Activin A-overexpressing MEFs accumulated in the bone marrow compartment and slowed down the progression of B cell precursors along the differentiation pathway, while sparing the myeloid population. The assay system described in this paper provides a means to assess the contribution of a wide range of molecules to hematopoiesis without perturbing the constitution of other organs.