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Lymphedema is a common complication following breast cancer treatment with axillary lymphadenectomy and radiotherapy. Currently, there is no curative treatment for this disease, hence there is a need for new therapeutic suggestions. The aim of this study was to investigate the effect of hyaluronidase (HYAL) injections after inducing hindlimb lymphedema in 36 female C57BL/6 mice. HYAL injections were administered every second day for 14 days in three groups: (1) HYAL for 1 week followed by saline for 1 week, (2) HYAL for 2 weeks, and (3) saline injections for 2 weeks. Volume of the lymphedema limb was weekly assessed with micro-computed tomography (µ-CT) scans for a total course of 6 weeks. Lymph vessel morphometry was assessed in the end of the study after staining cross-sections of the hindlimb for anti-LYVE-1 blindly. Lymphatic function was assessed by lymphoscintigraphy to assess lymphatic clearance. There was a significant reduction of the volume of lymphedema in mice treated with HYAL-7 compared with mice treated with HYAL-14 (p < 0.05) and saline (p < 0.05). No differences were detected in lymph vessel morphometry and the lymphoscintigraphy between groups. Short-term treatment with HYAL-7 might be a potential therapeutic suggestion for secondary lymphedema induced in mouse hindlimbs. In the future, clinical studies are needed to investigate the potential of HYAL treatment in human beings.
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Hialuronoglucosaminidase , Linfedema , Camundongos , Feminino , Humanos , Animais , Hialuronoglucosaminidase/farmacologia , Hialuronoglucosaminidase/uso terapêutico , Microtomografia por Raio-X/efeitos adversos , Camundongos Endogâmicos C57BL , Linfedema/diagnóstico por imagem , Linfedema/tratamento farmacológico , Linfedema/etiologia , Membro Posterior , Extremidade Inferior , Linfocintigrafia/efeitos adversos , Doença CrônicaRESUMO
OBJECTIVES: To investigate Stone Heart Syndrome (SHS) as consequence of prolonged ischemic arrest in an experimental study on pigs in regards to onset of SHS and pathological changes. Outcomes defined as aortic cross clamp (ACC) time until onset of SHS and cellular changes characterized by SHS. METHODS: Eight pigs were included to undergo normothermic cardioplegia induced cardiac arrest ranging from 80 to 240 minutes of ACC. Duration of ACC was defined as time from initiation of aortic cross clamping until cessation. Normothermic, cardioplegic solution administered directly into the arterial system, though in a reduced dose compared to clinical practice. Myocardial contracture evaluated by palpation of the myocardium. Biopsies were collected from the left ventricle just after the induction of cardiac arrest and after reperfusion. Biopsies were evaluated for pathological changes indicative of SHS by electron microscopy. RESULTS: Six pigs completed the full trial, while two were lost to bleeding. Pigs undergoing 80 to 120 minutes of ACC regained heart rhythm either spontaneously or after defibrillation. Pigs undergoing more than 180 minutes of ACC had contracted hearts with no electrocardiographic response indicating the development of SHS. Electron microscopy findings after ACC of 80 to 120 minutes showed no or low degrees of cellular changes, whereas pig hearts with more than 180 minutes of ACC showed severe mitochondrial changes, endothelial damage, and shortening of sarcomeres consistent with SHS. CONCLUSION: Development of SHS in pigs was ACC time dependent and solely avoided when ACC was limited to a maximum of 120 minutes.
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Parada Cardíaca Induzida , Isquemia Miocárdica , Animais , Soluções Cardioplégicas/efeitos adversos , Parada Cardíaca/induzido quimicamente , Parada Cardíaca Induzida/efeitos adversos , Isquemia Miocárdica/etiologia , Miocárdio/patologia , Projetos Piloto , SuínosRESUMO
BACKGROUND: Rotator cuff (RC) tendon tear leads to impaired shoulder function and pain. The supraspinatus (SS) tendon is most often affected, but the biological response of the SS muscle to SS tendon tear is largely unknown. This study aimed to investigate time-dependent muscle inflammation, degeneration, fatty infiltration, and regeneration in experimental SS tear conditions. METHODS: Forty-five C57BL/6 mice were subjected to SS tendon tear and allowed to recover for 1, 3, 5, 7, 14, or 28 days. The extent of muscle damage was examined using histologic, flow cytometric, proteomic, and chemiluminescence analyses. RESULTS: We found that muscle inflammation peaked around day 5 with increased monocyte infiltration and increased cytokine levels in the ipsilateral compared to the contralateral SS muscle. Bioinformatics analysis of proteomics on mice that survived 5 days after RC tendon tear revealed upregulated proteins involved in "neutrophil activation involved in immune response" and "extracellular matrix organization," whereas "skeletal muscle tissue development and contraction" and "respiratory electron transport chain" were among the most downregulated. Histologic analysis of collagen showed increased collagen accumulation and fatty infiltration of the ipsilateral SS over time. Finally, we observed time- and lesion-dependent changes in satellite cell and fibro-adipogenic progenitor populations. CONCLUSION: Altogether, we demonstrate that the SS muscle shows severe signs of acute inflammation, early degeneration, and fatty infiltration, as well as reduced regenerative potential following SS tendon tear.
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Lesões do Manguito Rotador , Manguito Rotador , Tecido Adiposo/patologia , Animais , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/patologia , Proteômica , Manguito Rotador/patologiaRESUMO
The need for a substitute for allograft and autograft is rising as bone graft surgeries exceed available supplies. We investigated the efficacy of the low-molecular weight marine bioactive compound fucoidan (FUC) on bone regeneration and implant fixation in seven female sheep, as FUC has shown great promise as a bone substitute. Titanium implants were inserted bilaterally in the distal femurs to test three hydroxyapatite/fucoidan (HA/FUC) groups and compared to allograft. The HA was coated with either 500 or 1500 µg of FUC, obtained by microwave-assisted chemical extraction, or 500 µg of FUC obtained by an enzyme-assisted extraction method. The concentric 2-mm gap around the implant was filled with either one of the HA/FUCs or allograft from the donor sheep. After 12 weeks, implant-bone blocks were harvested and divided into three parts for mechanical push-out testing, immunohistochemistry, and micro-CT and histomorphometry. Pronounced bone formations were observed by micro-CT and histomorphometry in all groups, but higher bone volume fractions were seen in the allograft group compared to the three HA/FUC groups. The trabecular thickness, trabecular separation, and architectural anisotropy were all significantly higher in the allograft group compared to the three HA/FUC groups. In conclusion, adequate bone formation was observed in all groups, although the bone formation was significantly greater in the allograft group. Also, no significant differences existed in the shear mechanical properties between groups, suggesting that the combination of HA and FUC can achieve a similar fixation strength to allograft in this model.
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Substitutos Ósseos , Animais , Regeneração Óssea , Substitutos Ósseos/química , Durapatita/química , Feminino , Osseointegração , Polissacarídeos , Próteses e Implantes , Ovinos , TitânioRESUMO
BACKGROUND: Esophageal atresia (EA) is a congenital malformation affecting 1:3000-4500 newborns. Approximately 15% have a long-gap EA (LGEA), in which case a primary anastomosis is often impossible to achieve. To create continuity of the esophagus patients instead have to undergo lengthening procedures or organ interpositions; methods associated with high morbidity and poor functional outcomes. Esophageal injections of Botulinum Toxin Type A (BTX-A) could enable primary anastomosis and mitigate stricture formation through decreased tissue tension. METHODS AND ANALYSIS: In this randomized controlled blinded animal trial, 24 pigs are divided into a long- or short-gap EA group (LGEA and SGEA, respectively) and randomized to receive BTX-A or isotonic saline injections. In the LGEA group, injections are given endoscopically in the esophageal musculature. After seven days, a 3 cm esophageal resection and primary anastomosis is performed. In the SGEA group, a 1 cm esophageal resection and primary anastomosis is performed, followed by intraoperative injections of BTX-A or isotonic saline. After 14 days, stricture formation, presence of leakage, and esophageal compliance is assessed using endoscopic and manometric techniques, and in vivo and ex vivo contrast radiography. Tissue elongation is evaluated in a stretch-tension test, and the esophagus is assessed histologically to evaluate anastomotic healing. ETHICS AND DISSEMINATION: The study complies with the ARRIVE guidelines for animal studies and has been approved by the Danish Animal Experimentation Council. Results will be published in peer-reviewed journals and presented at national and international conferences. HIGHLIGHTS: The optimal management of long-gap esophageal atresia remains controversialPrimary anastomosis could improve functional outcomes and reduce complicationsBotulinum Toxin Type A decreases tissue tension and could facilitate anastomosisReduced tension could further abate the risk for anastomotic stricture and leakageWe present a model to evaluate the method in long- and short-gap esophageal atresia.
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AIMS: In vivo experiments were performed to establish and validate a rat model of urethral sphincter injury and to develop a method for leak point pressure (LPP) measurements performed repeatedly in the same animal. METHODS: Twenty-four Sprague-Dawley female rats underwent bladder and epidural catheter implantation. Five days later, cystometry was performed using continuous infusion. Anesthesia with isoflurane, ketamine-xylazine (KX) or fentanyl-fluanisone-midazolam (FFM) was used. After three micturition cycles, intrathecal bupivacaine was administered leading to the suppression of reflex bladder contractions. LPP measurements were performed using vertical tilt. After the initial LPP measurement, animals underwent partial resection of the striated urethral sphincter. The effect was evaluated 6 weeks after surgery, by repeating the LPP measurement in the same animal. RESULTS: Ten out of 19 animals showed full micturition cycles under isoflurane, and all 9 animals under KX anesthesia. No significant difference in micturition pressures (Mean ± SEM; 30.1 ± 2.3 vs. 26.8 ± 1.6 mmHg) and LPP (31.0 ± 2.4 vs. 28.0 ± 0.9 mmHg) was observed between isoflurane and KX groups, respectively. Reflex micturition was suppressed with FFM. Bupivacaine led to overflow incontinence in all cases. Sphincter injury caused fibrotic changes and a significant increase in LPP (26.4 ± 2.3 before vs. 46.9 ± 4.6 mmHg after injury, p < 0.05). CONCLUSIONS: KX anesthesia preserves bladder contractions. Intrathecal bupivacaine eliminates reflex micturition, allowing for repeated LPP measurements in the same animal. Resection of striated sphincter resulted in increased LPP 6 weeks post injury. The site of urethral sphincter resection healed with fibrosis.
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Uretra , Doenças Uretrais , Animais , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Bexiga Urinária , MicçãoRESUMO
OBJECTIVES: To correlate the level of fibrocytes in peripheral blood, synovial tissue and in vitro culture in rheumatoid arthritis (RA) with changes in disease activity, imaging and pulmonary function. METHODS: Twenty patients with early RA (ERA) and 20 patients with long-standing RA (LRA) were enrolled in a 6-month prospective study. Sixteen patients undergoing wrist arthroscopy were healthy controls. Patients with RA underwent pulmonary function tests, ultrasound and synovial ultrasound-guided needle biopsy of the same wrist at baseline and 6 months. Wrist MRI was performed at baseline (all) and 6 months (ERA). Circulating fibrocytes were measured by flow cytometry, in vitro by the number of monocytes that were differentiated to fibrocytes and in synovial biopsies by counting in histological sections. RESULTS: Fibrocytes were primarily located around vessels and in the subintimal area in the synovium. Fibrocyte levels did not decline during the trial despite effective RA treatment. In the ERA group, increased synovitis assessed by ultrasound was moderate and strongly correlated with an increase in circulating and synovial fibrocyte levels, respectively. Increased synovitis assessed by MRI during the trial in the ERA group was moderately correlated with both increased numbers of circulating and cultured fibrocytes. Absolute diffusion capacity level was overall weakly negatively correlated with the level of circulating and synovial fibrocytes. The decline in diffusion capacity during the trial was moderately correlated with increased levels of synovial fibrocytes. CONCLUSION: Our findings suggest that fibrocytes are involved in RA pathogenesis, both in the synovium and the reduction in lung function seen in a part of patients with RA. TRIAL REGISTRATION NUMBER: NCT02652299.
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Artrite Reumatoide , Artrite Reumatoide/diagnóstico por imagem , Humanos , Biópsia Guiada por Imagem , Estudos Prospectivos , Testes de Função Respiratória , Articulação do Punho/diagnóstico por imagemRESUMO
BACKGROUND: Rotator cuff (RC) disorders involve a spectrum of shoulder conditions from early tendinopathy to full-thickness tears leading to impaired shoulder function and pain. The pathology of RC disorder is, nonetheless, still largely unknown. Our hypothesis is that a supraspinatus (SS) tendon tear leads to sustained inflammatory changes of the SS muscle along with fatty infiltration and muscle degeneration, which are threshold markers for poor RC muscle function. The aim of this study was to determine the extent of this muscle inflammation in conjunction with lipid accumulation and fibrosis in RC tear conditions. METHODS: We used proteomics, histology, electrochemiluminescence immunoassay, and quantitative polymerase chain reaction analyses to evaluate inflammatory and degenerative markers and fatty infiltration in biopsies from 22 patients undergoing surgery with repair of a full-thickness SS tendon tear. RESULTS: Bioinformatic analysis showed that proteins involved in innate immunity, extracellular matrix organization, and lipid metabolism were among the most upregulated, whereas mitochondrial electronic transport chain along with muscle fiber function was among the most downregulated. Histologic analysis confirmed changes in muscle fiber organization and the presence of inflammation and fatty infiltration. Inflammation appeared to be driven by a high number of infiltrating macrophages, accompanied by elevated matrix metalloprotease levels and changes in transforming growth factor-ß and cytokine levels in the SS compared with the deltoid muscle. CONCLUSIONS: We demonstrated massive SS muscle inflammation after the tendon tear combined with fatty infiltration and degeneration. The regulation of tissue repair is thus extremely complex, and it may have opposite effects at different time points of healing. Inhibition or stimulation of muscle inflammation may be a potential target to enhance the outcome of the repaired torn RC.
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Lesões do Manguito Rotador , Tendinopatia , Humanos , Atrofia Muscular/patologia , Manguito Rotador/patologia , Lesões do Manguito Rotador/patologia , Lesões do Manguito Rotador/cirurgia , Ruptura/patologiaRESUMO
C57BL/6J-related mouse strains are widely used animal models for diet-induced obesity (DIO). Multiple vendors breed C57BL/6J-related substrains which may introduce genetic drift and environmental confounders such as microbiome differences. To address potential vendor/substrain specific effects, we compared DIO of C57BL/6J-related substrains from three different vendors: C57BL/6J (Charles Rivers), C57BL/6JBomTac (Taconic Bioscience) and C57BL/6JRj (Janvier). After local acclimatization, DIO was induced by either a high-fat diet (HFD, 60% energy from fat) or western diet (WD, 42% energy from fat supplemented with fructose in the drinking water). All three groups on HFD gained a similar amount of total body weight, yet the relative amount of fat percentage and mass of inguinal- and epididymal white adipose tissue (iWAT and eWAT) was lower in C57BL/6JBomTac compared to the two other C57BL/6J-releated substrains. In contrast to HFD, the three groups on WD responded differently in terms of body weight gain, where C57BL/6J was particularly prone to WD. This was associated with a relative higher amount of eWAT, iWAT, and liver triglycerides. Although the HFD and WD had significant impact on the microbiota, we did not observe any major differences between the three groups of mice. Together, these data demonstrate significant differences in HFD- and WD-induced adiposity in C57BL/6J-related substrains, which should be considered in the design of animal DIO studies.
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Dieta Hiperlipídica , Absorciometria de Fóton , Animais , Peso Corporal , Glucose/administração & dosagem , Insulina/sangue , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Tamanho do Órgão , Especificidade da Espécie , Triglicerídeos/metabolismo , Aumento de PesoRESUMO
Background: Lymphedema is one of the most common complications following breast cancer. Axillary lymph node dissection and radiotherapy are two well-known risk factors resulting in either removal or damage to the lymph nodes. As stem cells are known for their regenerative capabilities, they could theoretically repair/restore the damaged lymph vessels leading to a decrease in lymphedema.Methods: We evaluated the treatment of SVF and ASC on a mouse lymphedema model. Forty-five mice were allocated into three groups containing 15 mice each. The SVF group was injected with 100 µl containing 1 × 106 SVF, the ASC group with 100 µl ml containing 1 × 106 ASC and the NS with 100 µl ml of NS. Volumes of the mice were assessed weekly by µCT hindlimb volumetry for a total of 8 weeks. Lymph vessel morphometry was assessed by cross-sections of both hindlimbs stained for anti-LYVE1. Lymphatic function was assessed by lymphatic clearance.Results: The volume change between the groups was non-significant throughout all 8 weeks. The immunohistochemistry showed a statistically significant difference between the hindlimbs in ASC vs. NS group p = 0.032, 95% CI [-2121, -103].Conclusion: The volume of the hindlimbs showed that treatment with SVF or ASC yielded very similar results compared to the control group when assessed after 8 weeks. In week two the biggest difference between ASC and NS was seen but the difference diminished during the 8 weeks. The secondary outcomes showed that the lymph vessel lumen decreased when treated with ASC compared to the control group. Lymphoscintigraphy yielded non-significant results.
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Tecido Adiposo/citologia , Linfedema/terapia , Transplante de Células-Tronco , Células Estromais/transplante , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Membro Posterior/diagnóstico por imagem , Linfedema/diagnóstico por imagem , Linfocintigrafia , Camundongos Endogâmicos C57BL , Tomografia Computadorizada de Emissão de Fóton Único , Microtomografia por Raio-XRESUMO
Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.
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Antígenos CD/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Adolescente , Ataxia/genética , Ataxia/fisiopatologia , Atrofia , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Colina/farmacologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Transtornos de Deglutição/genética , Transtornos de Deglutição/fisiopatologia , Disartria/genética , Disartria/fisiopatologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Incontinência Fecal/genética , Incontinência Fecal/fisiopatologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Mutação da Fase de Leitura , Globo Pálido/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Homozigoto , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Leucoencefalopatias/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Nootrópicos/farmacologia , Atrofia Óptica/genética , Atrofia Óptica/fisiopatologia , Linhagem , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestrutura , Substância Negra/diagnóstico por imagem , Síndrome , Tremor/genética , Tremor/fisiopatologia , Incontinência Urinária/genética , Incontinência Urinária/fisiopatologiaRESUMO
BACKGROUND: Lymphedema is a common and debilitating complication following cancer treatment with surgical lymph node excision and radiotherapy. Currently there are no curative treatments for lymphedema. Animal models that intended to replicate the disease have been inadequate, making a troublesome transition from experimental therapeutic studies into the clinic. It is therefore imperative to establish an experimental animal model that can reliably replicate clinical lymphedema. METHODS: To discover the optimal method of lymphedema induction, surgical lymph ablation and irradiation or silicone splint emplacement were combined in 8 experimental groups (n = 4). In total, 32 mice served in this study and were followed for 8 weeks after surgery. Outcomes included micro-computed tomography hind limb volumetry, lymphatic clearance measured with technetium Tc 99m (Tc) human serum albumin lymphoscintigraphy and lymph vessel ectasia quantified with LYVE-1 immunohistochemistry. RESULTS: All trialed models but one resulted in only transient lymphedema or lasting lymphedema with adverse morbidity. Combined surgical lymph obstruction with 2 fractions of 10-Gy irradiation successfully induced lasting lymphedema without adverse events. Over the 8 weeks' follow-up, limb volumes were significantly increased at all time points (P < 0.001), lymph drainage was impaired (P < 0.001), and lymph vessels were ectatic (P < 0.001), when compared with the unoperated limbs. CONCLUSIONS: The presented model of acquired lymphedema is a reduction and refinement of previous works and can transpose to future observational and interventional studies. In addition, it is shown how Tc-HSA lymphoscintigraphy can quantify lymphatic clearance, which can prove insightful in therapeutic studies aiming to enhance lymphatic drainage.
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Modelos Animais de Doenças , Linfedema , Complicações Pós-Operatórias , Animais , Doença Crônica , Dinamarca , Membro Posterior , Linfedema/diagnóstico por imagem , Linfocintigrafia , Camundongos , Complicações Pós-Operatórias/diagnóstico por imagemRESUMO
BACKGROUND: Interstitial lung disease (ILD) can be a severe extra-articular disease manifestation in Rheumatoid Arthritis (RA). A potential role of fibrocytes in RA associated ILD (RA-ILD) has not previously been described. We present a modified faster method for measuring circulating fibrocytes, without intracellular staining. The results are compared to the traditional culture method, where the number of monocytes that differentiate into mature fibrocytes in vitro are counted. The results are following compared to disease activity in patients with severe asthma, ILD, RA (without diagnosed ILD) and RA with verified ILD (RA-ILD). METHOD: CD45+ CD34+ CD11b+ (7-AAD- CD3- CD19- CD294-) cells were isolated by cell sorting and stained for pro-collagen type 1. Thirty-nine patients (10 RA, 9 ILD and 10 with severe asthma, 10 with RA-ILD) and 10 healthy controls (HC) were included. Current medication, disease activity, pulmonary function test and radiographic data were collected. Circulating fibrocytes were quantified by flow cytometry. Peripheral blood mononuclear cells were isolated and cultured for 5 days and the numbers of mature fibrocytes were counted. RESULTS: 90.2% (mean, SD = 1.5%) of the sorted cells were pro-collagen type 1 positive and thereby fulfilled the criteria for being circulating fibrocytes. The ILD and RA-ILD groups had increased levels of circulating fibrocytes compared to HC (p < 0.05). Levels of circulating fibrocytes correlated overall to number of monocytes that subsequently in vitro differentiated to mature fibrocytes (r = 0.81, p < 0.001). RA patients with pathologically reduced diffusion capacity for carbon monoxide adjusted for hemoglobin (DLCOc) in both the RA and in the combined RA + RA-ILD group, had significantly higher levels of both circulating and number of cultured mature fibrocytes (both p < 0.05). In both groups, the level of circulating fibrocytes and number of mature fibrocytes in culture also correlated to a reduction in DLCOc (r = -0.61 an r = -0.58 both p < 0.05). CONCLUSIONS: We presented a fast and valid method for measuring circulating fibrocytes using flow cytometry on lysed peripheral blood. Further, we showed for the first time, that the level of circulating fibrocytes correlated with the number of peripheral blood mononuclear cells, that differentiated into mature fibrocytes in vitro. Reduced DLCOc was correlated with high levels of circulating and mature fibrocytes in RA, which have not been reported previously. In such, this study suggests that fibrocytes may exhibit an important role in the pathogenesis of RA-ILD, which requires further clarification in future studies. TRIAL REGISTRATION: ClinicalTrials.gov : NCT02711657 , registered 13/3-2016, retrospectively registered.
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Artrite Reumatoide/complicações , Diferenciação Celular , Separação Celular/métodos , Citometria de Fluxo , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/patologia , Monócitos/patologia , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Asma/sangue , Asma/patologia , Biomarcadores/sangue , Estudos de Casos e Controles , Células Cultivadas , Colágeno Tipo I/metabolismo , Estudos Transversais , Feminino , Humanos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/sangue , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Fenótipo , Valor Preditivo dos Testes , Pró-Colágeno/metabolismo , Capacidade de Difusão Pulmonar , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid)-based microparticles supported prolonged proliferation. Myoblasts released from the alginate and fibrin gels were studied, and cells released from these scaffolds had retained the ability to proliferate and differentiate. Thus, the study shows that human myogenic cells combined with injectable scaffold materials are guided into different states depending on the choice of scaffold. This opens for in vivo experiments, including testing of the significance of the cell state on regeneration potential of primary human myoblasts.
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OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFß, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and ß-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.
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Pontos de Checagem do Ciclo Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Oxigênio/farmacologia , Fase de Repouso do Ciclo Celular/genética , Adolescente , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Proliferação de Células/efeitos dos fármacos , Separação Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Receptores Notch/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Adulto JovemRESUMO
Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms. Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50% of the families reported worldwide. Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.