ABSTRACT
Abstract Background: In the past 20 years, hematopoietic stem cell transplantation (HSCT) has been investigated as treatment for systemic sclerosis (SSc). The goal of HSCT is to eradicate the autoreactive immune system, which is replaced by a new immune repertoire with long-lasting regulation and tolerance to autoantigens. Here, we describe the clinical outcomes of severe and refractory SSc patients that underwent HSCT at a single Brazilian center. Patients and methods: This is a longitudinal and retrospective study, including 70 adult SSc patients, with an established diagnosis of SSc, and who underwent autologous HSCT from 2009 to 2016. The procedure included harvesting and cryopreservation of autologous hematopoietic progenitor cells, followed by administration of an immunoablative regimen and subsequent infusion of the previously collected cells. Patients were evaluated immediately before transplantation, at 6 months and then yearly until at least 5-years of post-transplantation follow-up. At each evaluation time point, patients underwent clinical examination, including modified Rodnan's skin score (mRSS) assessment, echocardiography, high-resolution computed tomography of the lungs and pulmonary function. Results: Median (range) age was 35.9 (19-59), with 57 (81.4%) female and median (range) non-Raynaud's disease duration of 2 (1-7) years. Before transplantation, 96% of the patients had diffuse skin involvement, 84.2%, interstitial lung disease and 67%, positive anti-topoisomerase I antibodies. Skin involvement significantly improved, with a decline in mRSS at all post-transplantation time points until at least 5-years of follow-up. When patients with pre-HSCT interstitial lung disease were analyzed, there was an improvement in pulmonary function (forced vital capacity and diffusing capacity of lung for carbon monoxide) over the 5-year follow-up. Overall survival was 81% and progression-free survival was 70.5% at 8-years after HSCT. Three patients died due to transplant-related toxicity, 9 patients died over follow-up due to disease reactivation and one patient died due to thrombotic thrombocytopenic purpura. Conclusions: Autologous hematopoietic progenitor cell transplantation improves skin and interstitial lung involvement. These results are in line with the international experience and support HSCT as a viable therapeutic alternative for patients with severe and progressive systemic sclerosis.(AU)
Subject(s)
Humans , Adult , Scleroderma, Systemic/surgery , Hematopoietic Stem Cells , Cryopreservation/instrumentation , Hematopoietic Stem Cell Transplantation/instrumentation , Disease Progression , Retrospective Studies , Longitudinal StudiesABSTRACT
Abstract A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 h gave significantly higher survival (75 %) compared to 24 h preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana. Rev. Biol. Trop. 66(3): 1314-1323. Epub 2018 September 01.
Resumen Se desarrolló un protocolo de crioconservación in vitro para ápices caulinares de Garcinia hombroniana mediante la técnica de vitrificación, con cuatro etapas críticas: precultivo, carga de crioprotector, deshidratación con solución de vitrificación vegetal 2 (PVS2), y descarga. Ápices precultivados por 48 h sobrevivieron más (75 %) que los de 24 h (50 %) después de la crioconservación. El tratamiento con glicerol 1 M más sacarosa 0.4 M como solución de carga permitió mayor sobrevivencia (45.83 %) que los otros tratamientos (sacarosa 0.4 M + glicerol 2 M; sacarosa 0.4 M). Los ápices deshidratados con PVS2 por 25 min registraron la mayor sobrevivencia tras inmersión en nitrógeno líquido. El tratamiento gradual por 15 min con solución de PVS2 al 50 %, seguido por 10 min al 100 %, mejoró la sobrevivencia de ápices tras la crioconservación (41.67 %). El medio Murashige-Skoog (MS) con sacarosa 0.4 M produjo una sobrevivencia significativamente mayor (66.67 %) que MS con sacarosa 1.2 M (25 %) como solución de descarga. El contenido de agua disminuyó a lo largo del proceso de vitrificación desde 6.83 ± 1.66 g g-1 peso seco, en ápices frescos, hasta 2.93 ± 0.28 g g-1 peso seco después del tratamiento con PVS2. Se requiere de más investigación sobre cada etapa, incluyendo el medio de recuperación, para mejorar la tasa de sobrevivencia. Sin embargo, este estudio muestra el potencial de la vitrificación para la crioconservación de G. hombroniana.
Subject(s)
Cryopreservation/instrumentation , Garcinia , Vitrification , Plants , Sucrose/therapeutic use , Glycerol/therapeutic use , Malaysia , Nitrogen/therapeutic useABSTRACT
After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.
A recuperação de espermatozoides da cauda do epidídimo pode ser a última chance para preservação do germoplasma quando ocorre morte súbita ou lesão grave em garanhões de alto valor genético. O presente trabalho comparou a viabilidade após refrigeração dos espermatozoides do ejaculado (G1), recuperados da cauda do epidídimo imediatamente após a orquiectomia (G2) e recuperados após armazenamento do epidídimo por 24 horas a 5ºC (G3). No G1 foram colhidos dois ejaculados. Uma semana após a colheita dos ejaculados os garanhões foram submetidos à orquiectomia bilateral e realizada a colheita dos espermatozoides da cauda do epidídimo de um testículo de cada garanhão (G2). O testículo contralateral permaneceu a 5°C por 24 horas, antes da recuperação espermática (G3). A análise das amostras foi realizada imediatamente após a adição do meio de refrigeração, e após 24 e 48 horas de armazenamento a 5°C. Após 24 e 48 horas de armazenamento, os espermatozoides do epidídimo demonstraram características de cinética maiores que os do ejaculado (P<0.05). Estes resultados indicam que espermatozoides recuperados da cauda do epidídimo foram mais resistentes ao processo de refrigeração, com maiores parâmetros de cinética espermática e integridade da membrana plasmática quando comparados aos espermatozoides do ejaculado.
Subject(s)
Animals , Cryopreservation/instrumentation , Epididymis/anatomy & histology , Semen Preservation/methods , Spermatozoa , Horses/classification , Orchiectomy/methodsABSTRACT
Foram utilizados ejaculados (n=25) de garanhões para avaliar o efeito de glutationa peroxidase (GPx) e cisteína na viabilidade de espermatozoides congelados. O sêmen foi diluído em Botu Crio, com antioxidantes, e foram formados os grupos: G1, Controle; G2, 1U GPx ; G3, 5U GPx; G4, 0,5mM cisteína; G5, 1mM cisteína. Depois foi envasado em palhetas (0,5mL) e congelado. Após descongelação, 37°C por 30 segundos, alíquotas foram analisadas quanto à integridade de membrana plasmática (IMP) e acrossoma (IAc), potencial de membrana mitocondrial (PMM) e cinética, nos tempos zero (T0) e 60 minutos (T60). GPx 5U e cisteína 0,5mM determinaram maior (P<0,05) IAc em T0 do que em T60. Cisteína 1mM resultou em maior (P<0,05) IAc em T60 do que GPx 1 e 5U e cisteína 0,5mM. O PMM de um garanhão no T60 foi mais alto (P<0,05) do que o de dois garanhões. VCL e VAP foram maiores (P<0,05) no T0 do que no T60 do grupo controle, e um garanhão apresentou, em geral, valores cinéticos mais altos (P<0,05) do que os demais. Conclui-se que a adição de glutationa peroxidase, nas concentrações de 1U e 5U, e de cisteína, nas concentrações de 0,5mM e 1mM, não interferem na integridade de espermatozoides criopreservados de equinos, mas preservam os parâmetros cinéticos de VCL e VAP após 60 minutos de incubação. Ressalta-se, ainda, que o garanhão tem uma forte influência nas características espermáticas pós-congelação.
Ejaculates (n=25) of horses were used to assess the effect of glutathione peroxidase (GPx) and cysteine on the viability of frozen sperm cells. Semen was extended at Botu Crio with antioxidants, and divided in groups: G1, control; G2, 1 U GPx; G3, 5U GPx; G4, 0.5mM cysteine and G5, 1mM cysteine, packed in 0.5mL straws, and frozen. After thawing (37° C for 30 seconds) samples were analyzed for plasma membrane (IMP) and acrosome integrity (IAc), mitochondrial membrane potential (MMP) and kinematic, at zero (T0) and 60 minutes after (T60). GPx 5U and cysteine 0.5mM increased (P<0.05) IAc at T0, when compared to T60. Cysteine 1mM resulted in a higher (P<0.05) IAc on T60, than GPx 1 and 5U, and cysteine 0.5mM. The PMM from a stallion on T60 was higher (P<0.05) than those of two stallions. In sperm kinematic, VCL and VAP were higher (P<0.05) at T0 compared to T60 for the control group, and one stallion showed larger (P<0.05) kinematic values than other animals. It is concluded that the addition of glutathione peroxidase at concentrations 1U and 5U, and cysteine, at concentrations of 0.5mM and 1mM, does not interfere with the integrity of cryopreserved equine sperm, but preserves the kinetic parameters VCL and VAP after 60 minutes of incubation. It should be noted also that the stallion has a strong influence on sperm characteristics post-freezing.
Subject(s)
Animals , Semen Analysis/methods , Cysteine/chemistry , Glutathione Peroxidase , Horses/classification , Cryopreservation/instrumentationABSTRACT
OBJECTIVE: To compare the survival rate of mouse oocytes and fertilization rate between using open pulled straws (OPS) and needles for vitrification. MATERIAL AND METHOD: Meiosis II oocytes from female C57B/6J mice aged 7-8 weeks were collected and allocated to two groups for vitrification by using OPS or needles. Vitrified oocytes were thawed, morphological survival and fertilization rate were examined. RESULTS: There was no obvious difference between the morphological survival rates of vitrified mouse oocytes using OPS and needles (66.7% vs 64.8%). Proportions Difference 1.9% (95% CI -7.1, 10.7). The vitrified oocytes from the needle had significantly higher percentages of fertilization rate than OPS (76.8% vs 62.5%). Proportions Difference -14.3% (95% CI -24.5, -3.6). CONCLUSION: Vitrification method of mouse oocytes using needles when compared to OPS provides a similar morphological survival rate and higher fertilization rate.
Subject(s)
Animals , Cryopreservation/instrumentation , Female , Meiosis , Mice , Mice, Inbred C57BL , Needles , Oocytes/ultrastructureABSTRACT
This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5M EG + 1.0M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p=0.024; 90.0% vs. 78.9%, p=0.033; 56.7% vs. 38.7%, p=0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes, compared to conventional EM grid vitrification and slow freezing methods.