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1.
Cryobiology ; 2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35413361

RESUMO

Dimethyl sulphoxide (DMSO) used in haematopoietic stem cell (HSC) cryopreservation has been linked to an increased incidence of adverse reactions following transplantation. In the interest of reducing the required DMSO concentrations, we have evaluated the use of unilamellar liposomes to internalize the non-toxic, cell-impermeable disaccharide, trehalose into HSCs and characterized the cryoprotective efficacy of this strategy. A fluorescent marker, 5(6)-carboxyfluorescein (200 µmol/L), was used for trehalose internalization following a 5 h incubation at 37 °C with liposome concentrations ranging from 0.5 mM to 4 mM. Cells were frozen (1 °C/min to -80 °C) following treatment with either 3 mM or 4 mM of liposomes (5 h, 37 °C) containing 0.2 mol/L trehalose either in the presence or absence of 0.2 mol/L extracellular trehalose. Increasing the liposome concentration from 3 mM to 4 mM corresponded to a significant (p = 0.046) increase in the mean fluorescent intensity (MFI) (3 mM 512 ± 7.07; 4 mM: 916 ± 28.3). Post-thaw membrane integrity indicated that the presence of trehalose both inside and outside when internalized using a liposome concentration of 4 mM significantly improved survival relative to the sole presence of extracellular trehalose (p = 0.02). However, viability was diminished relative to a standard DMSO control (trehalose: 32.5% ± 1.7%; DMSO: 85.0% ± 4.6%). This study confirms that the protective efficacy of trehalose is enhanced when it is present on both sides of the membrane; however, it reinforces concerns surrounding the efficiency of using liposomes as a vehicle to transfer trehalose into cells.

2.
Clin Chim Acta ; 530: 119-125, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35378092

RESUMO

BACKGROUND AND AIMS: Subpopulations of immature red blood cells (RBCs) named CD71+ erythroid cells (CECs) with different properties may contribute to RBC transfusion outcomes. However, it is challenging to quantify CECs in leukoreduced RCCs and whole blood due to their rarity and fragility. Current flow cytometry methods are not applicable to leukoreduced RCCs as there is limited peripheral blood mononuclear cells (PBMCs) to use for determination of CECs. We have developed and validated a flow cytometry method for quantifying CECs in WB. METHODS: We determined optimal PE-Cy7-CD235a, BV711-CD71, APC-CD45 concentrations and instrument setting by titration. Linearity and level of detection was determined by spiking labelled PBMCs from cord blood units into unlabeled WB. Low, medium, high levels of CECs were used to determine intra- and inter -run precision. RESULTS: Detection of CECs was linear (R2 = 0.98) over the range of concentrations assessed with a limit of detection of 0.005%. The overall CVs for the intra- and inter-run precision were 6.9% and 9.7%. CONCLUSION: We developed a simple, sensitive, and cost-effective flow cytometric method for quantifying the proportion of CECs in non-manipulated WB, which could help understand the impact of RBC products on recipient transfusion outcomes.


Assuntos
Eritrócitos , Leucócitos Mononucleares , Contagem de Células , Contagem de Eritrócitos , Citometria de Fluxo , Humanos
3.
Blood Adv ; 2022 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-35286383

RESUMO

Transfusion of red blood cell (RBC) from female donors has been associated with increased risk of mortality. This study aims to investigate the associations between donor-recipient sex and post-transfusion mortality and morbidity in critically ill patients who received RBC transfusions from either male only donors or from female only donors (unisex-transfusion cases). Survival analysis was used to compare 4 groups: female-to-female, female-to-male, male-to-female, and male-to-male transfusion. Multivariate logistic model was used to evaluate the association between donor sex and ICU mortality. Associations between transfusion and acute kidney injury (AKI), acute respiratory distress syndrome (ARDS) and nosocomial infections were assessed. Of the 6992 patients included in the original cohort study, 403 patients received unisex-transfusion. Survival analysis and the logistic model showed that transfusion of female RBCs to male patients was associated with an increased ICU mortality compared to transfusion of female RBCs to female patients (OR 2.43; 95% CI 1.02- 5.77; p-value < 0.05). There was a trend towards increased ARDS in patients receiving RBC from female donors compared to those receiving blood from males (p-value = 0.06) while AKI was higher in donor-recipient sex-matched transfusion groups compared to sex-mismatched groups (p-value = 0.05). This was an exploratory study with potential uncontrolled confounders that limits broad generalization of the findings. Results warrant further studies investigating biological mechanisms underlying the association between donor sex with adverse outcomes as well as studies on the benefit of matching of blood between donor and recipient.

4.
Vox Sang ; 2022 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-35157317

RESUMO

BACKGROUND AND OBJECTIVES: Di(2-ethylhexyl) phthalate (DEHP) must be removed from blood bag sets in Europe by 27 May 2025. DEHP is known to interact with the red blood cell (RBC) membrane, resulting in reduced haemolysis and thus prolonging shelf-life. Current non-DEHP alternatives result in increased haemolysis requiring reconsideration of the RBC shelf-life. Although the immediate impact of eliminating DEHP is to the European community, the non-DEHP movement could affect blood bag set availability globally. The purpose of this survey is to understand blood centre readiness regarding the transition to non-DEHP blood collection and storage systems. MATERIALS AND METHODS: A 24-question on-line survey was completed by members of the Biomedical Excellence for Safer Transfusion Collaborative research network. RESULTS: Responses were obtained from 16 blood collection or processing institutions. A majority of respondents (12/16) indicated that both shelf-life and haemolysis were equally important in selecting non-DEHP blood bag sets. Six respondents would accept a lower RBC product shelf-life compared to current practice. Respondents were not clear on the best non-DEHP vinyl material or RBC storage solution. Three European blood centres indicated they have developed non-DEHP transition plans. One challenge identified regarding the transition to non-DEHP is the extensive validation testing that will be required. CONCLUSION: Blood centres in Europe are concerned with meeting the sunset date for DEHP, considering that limited non-DEHP blood bag and RBC storage solutions are currently available. Banning DEHP in Europe, which may have global ramifications, represents a major challenge not yet fully understood by the transfusion medicine community.

5.
Transfusion ; 62(4): 751-757, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35098538

RESUMO

BACKGROUND: Gamma irradiation of red cell concentrates (RCCs) is regularly used to prevent transfusion-associated graft-versus-host disease (TA-GvHD) in at-risk patients. While studies have indicated that irradiated RCCs exhibit increased hemolysis, there have been no efforts to differentiate between free- and microvesicle (MV)-bound hemoglobin (Hb). As an increase in the proportion of free-Hb in irradiated RCCs could alter vascular function, we sought to characterize differences in the state of extracellular Hb based on the timing of irradiation. STUDY DESIGN AND METHODS: Four separate pools of seven CPD/SAGM leukoreduced RCCs were produced and split into four sets of seven identical units. The units from each set were subject to irradiation (25 Gy) at six different points during storage, with one unit serving as a nonirradiated control. All testing was performed immediately following unit expiry on day 43. RESULTS: The earlier in storage that units were irradiated, the higher the hemolysis and the lower the proportion of MV-bound Hb. Units irradiated earlier in storage (1-8 days post collection) additionally had lower membrane rigidity (KEI ), lower mean corpuscular Hb concentrations (MCHC), and higher mean corpuscular fragility (MCF). Morphology indices, mean cell volume (MCV), mean corpuscular Hb (MCH), phosphatidylserine (PS) expression, as well as MV production and size did not however differ significantly between groups based on the timing of irradiation. CONCLUSIONS: Our findings indicate that irradiation timing can alter the state of extracellular Hb, with "early" irradiation promoting an increased proportion of cell-free Hb as well as mechanical damage to the RBC membrane.


Assuntos
Preservação de Sangue , Potássio , Eritrócitos/metabolismo , Raios gama , Hemoglobinas/metabolismo , Hemólise , Humanos
8.
Cryobiology ; 102: 15-26, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33905707

RESUMO

In recent years there have been several advancements in organ preservation that have yet to see widespread clinical translation. While static cold storage (SCS) at 2 °C-4 °C continues to be the state-of-the-art strategy, it contributes to the current shortage of transplantable organs due to the limited preservation times it affords combined with the limited ability of marginal grafts to tolerate SCS. The era of optimizing storage solutions to minimize SCS-induced hypothermic injury has largely plateaued in its improvements, resulting in a shift towards the use of machine perfusion systems to provide continuous metabolic support, or the use of sub-zero storage temperatures to leverage the protection brought forth by a reduction in metabolic demand. Many of the rigors that organs are subjected to at low sub-zero temperatures (-80 °C to -196 °C) commonly used for mammalian cell preservation have yet to be surmounted, and therefore the focus of this article lies on an intermediate range of storage temperatures (0 °C to -20 °C) where much success has been seen in the past two decades. Numerous mechanisms leveraged by organisms capable of withstanding prolonged periods at these temperatures through either avoiding or tolerating the formation of ice has provided a foundation for some of the more promising efforts, and thus we aim to contextualize the translation of these nature-derived strategies to mammalian organ preservation.


Assuntos
Soluções para Preservação de Órgãos , Preservação de Órgãos , Animais , Temperatura Baixa , Criopreservação/métodos , Perfusão
9.
Vet Parasitol ; 292: 109400, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33713884

RESUMO

Prompt and reliable diagnostic tests for taeniid infection in canids are important due to the risk of zoonoses like Echinococcus spp. Current diagnostic methods relying on fecal flotation lack sensitivity and specificity, but this has rarely been quantified due to the challenges in performing adult cestode recovery (the gold standard) in domestic dogs (Canis familiaris). Therefore, we recovered adult Taenia and Echinococcus spp. from intestines, as well as fecal/intestinal material from 484 wild canids trapped for fur in two Canadian provinces (276 foxes - primarily Vulpes vulpes, coyotes - Canis latrans, and wolves - Canis lupus in Québec and 208 coyotes in Saskatchewan). The performances of a newly developed coproPCR for tapeworm DNA detection in dogs, and centrifugal fecal flotation using Sheather's solution, were evaluated against adult cestode recovery. Overall, adult taeniid cestode prevalence (Taenia and/or Echinococcus) was 28 % (95 % CI: 23-33 %) in Québec (62 % (CI: 51-73%) of 74 coyotes, 65 % (CI: 44-82) of 23 wolves, and 11 % (CI: 7-16%) of 179 foxes) and 79 % (CI: 73-84%) of 208 coyotes in Saskatchewan. In Québec, E. canadensis and Taenia spp. were detected in coyotes and wolves, and foxes were only infected with Taenia spp., whereas Saskatchewan coyotes were predominantly infected with E. multilocularis (at significantly higher prevalence, but not intensity, than coyotes in Québec). Compared with centrifugal fecal flotation, the new coproPCR had at least double the sensitivity (58 % vs 23 % in QC coyotes, 57 % vs 23 % in QC wolves, 24 % vs 0% in QC foxes, and 80 % vs 25 % in SK coyotes). Notably, no taeniid eggs were detected on flotations from foxes infected with Taenia spp., and the new coproPCR had highest sensitivity in Saskatchewan coyotes, which were predominantly infected with E. multilocularis. CoproPCR has promising prospects for use in Veterinary clinics and diagnostic laboratories to detect taeniid cestode infections because of its higher sensitivity than faecal flotation methods. This is particularly important for zoonotic Echinococcus spp. where, from a public health perspective, false negatives are a much greater concern than false positives.


Assuntos
Canidae/parasitologia , Equinococose/veterinária , Echinococcus/isolamento & purificação , Fezes/parasitologia , Reação em Cadeia da Polimerase/veterinária , Animais , Animais Selvagens , DNA de Helmintos/genética , Equinococose/diagnóstico , Equinococose/parasitologia , Contagem de Ovos de Parasitas/veterinária , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
10.
BMJ Open ; 11(2): e049598, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622960

RESUMO

INTRODUCTION: With over 1 million units of blood transfused each year in Canada, their use has a significant clinical and economic impact on our health system. Adequate screening of blood donors is important to ensure the safety and clinical benefit of blood products. Some adverse transfusion reactions have been shown to be related to donor factors (eg, lung injury), whereas other adverse outcomes have been theoretically related to donor factors (mortality and infection). Our clinical trial will test whether male donor blood leads to a greater benefit for transfusion recipients compared with female donor blood. METHODS AND ANALYSIS: We have designed a pragmatic, double-blind, randomised trial that will allocate transfusion recipients to receive either male-only or female-only donor transfusions. We will enrol 8850 adult patients requiring at least one transfusion at four sites over an approximate 2-year period. Randomisation and allocation will occur in the blood bank prior to release of the units of blood for transfusion. Our primary outcome is mortality. An intent-to-treat analysis will be applied using all randomised and transfused patients. The principal analysis will be a survival analysis comparing the time from randomisation to death between patients allocated to male donor red blood cells (RBCs) and female donor RBCs. ETHICS AND DISSEMINATION: Approval has been obtained from research ethics boards of all involved institutions, as well as from privacy offices of Canadian Blood Services, Institute for Clinical Evaluative Science and The Ottawa Hospital Data Warehouse. Our findings will be published in peer-reviewed journals and presented at relevant stakeholder conferences and meetings. TRIAL REGISTRATION NUMBER: NCT03344887; Pre-results.


Assuntos
Transfusão de Eritrócitos , Eritrócitos , Adulto , Canadá , Método Duplo-Cego , Feminino , Hospitais , Humanos , Masculino , Ensaios Clínicos Controlados Aleatórios como Assunto
11.
Transfusion ; 61(4): 1247-1257, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33481275

RESUMO

BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.


Assuntos
Criobiologia/métodos , Eritrócitos/fisiologia , Procedimentos de Redução de Leucócitos/métodos , Reação Transfusional/prevenção & controle , Preservação de Sangue/métodos , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos/efeitos da radiação , Filtração , Citometria de Fluxo/métodos , Humanos , Incidência , Procedimentos de Redução de Leucócitos/estatística & dados numéricos , Leucócitos/imunologia , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Fatores de Tempo , Reação Transfusional/epidemiologia , Reação Transfusional/imunologia
12.
World J Plast Surg ; 9(3): 313-320, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33330009

RESUMO

BACKGROUND: Feijoa is widely used in medicine due to their anti-inflammatory, antioxidant, antimicrobial and antitumor properties. The current investigation studied the proliferative and regenerative effect of acetonic extract of Feijoa sellowiana on stem cells. METHODS: Acetone extract of Feijoa was prepared using percolator and rotary machines. Human bone marrow stem cells (hBMSCs) were used as experimental in vitro model and characterized morphologically, by flowcytometry, and differentiation properties. The toxicity of the extract on hBMSCs was determined by MTT assay. The viability and growth kinetics of hBMSCs treated to Feijoa was determined. Real time PCR was used for changes in expression of proliferative and apoptotic genes on day 7th. RESULTS: MTT assay demondtrated that Feijoa at doses less than 200 ng/ml did not show any cytotoxic effect on hBMSCs and increased the cell proliferation until day 3rd followed by a non-significant slow decreasing trend until day 7th. Population doubling time (PDT) showed a decline until day 3rd followed by an increase until day 7th. A significant rise in expression of Bax and decline in Bcl-2 expression were noted on day 7th. CONCLUSION: The modulatory activity of Feijoa may be responsible for its increasing effect on cell proliferation till day 3rd. Therefore, when faster proliferation during a shorter time period is targeted, Feijoa can be safely added to the culture media in the first three days.

13.
Transfus Apher Sci ; 59(6): 103020, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33246838

RESUMO

Improving blood product quality and patient outcomes is an accepted goal in transfusion medicine research. Thus, there is an urgent need to understand the potential adverse effects on red blood cells (RBCs) during pre-transfusion storage. Current assessment techniques of these degradation events, termed "storage lesions", are subjective, labor-intensive, and complex. Here we describe emerging technologies that assess the biochemical, biophysical, and morphological characteristics of RBC storage lesions. Of these emerging techniques, machine learning (ML) has shown potential to overcome the limitations of conventional RBC assessment methods. Our previous work has shown that neural networks can extract chronological progressions of morphological changes in RBCs during storage without human input. We hypothesize that, with broader training and testing of multivariate data (e.g., varying donor factors and manufacturing methods), ML can further our understanding of clinical transfusion outcomes in multiple patient groups.


Assuntos
Inteligência Artificial/normas , Eritrócitos/metabolismo , Citometria de Fluxo/métodos , Aprendizado de Máquina/normas , Humanos
14.
Cryobiology ; 97: 93-100, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33031822

RESUMO

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth. Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of -5 °C/min to -20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of -5 °C/min to -20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin. The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034). The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.


Assuntos
Criopreservação , Crioprotetores , Animais , Criopreservação/métodos , Crioprotetores/farmacologia , Humanos , Gelo , Pulmão , Perfusão , Ratos , Ratos Sprague-Dawley
15.
Cryobiology ; 97: 217-221, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33031823

RESUMO

Immediate post-thaw evaluation of membrane integrity has proven to yield overestimates of cell survival under conditions that preclude intracellular ice formation (IIF). However, prominent theories on the mechanisms of intracellular nucleation suggest a damaged membrane can reseal, prompting us to evaluate whether immediate post-thaw assessments of membrane integrity can in fact underestimate cell survival under conditions that promote IIF. HUVEC and HepG2 monolayers were treated with 1.4 M DMSO and frozen to -25 °C under conditions that formed either 0% or 100% IIF. Membrane integrity was evaluated both immediately and 24 h post-thaw, with metabolic activity assessments performed 24 h post-thaw as a secondary measure of survival. Treatment with 1.4 M DMSO and nucleation of 100% IIF resulted in a drastic increase in the relative percent of membrane intact cells following a 24 h culture period (HUVEC: 90.2% ± 0.7%; HepG2: 70.4% ± 4.0%), which correlated with 24 h post-thaw metabolic activity. These differences between the immediate and 24 h post-thaw membrane integrity assessments were significantly more than those seen in the absence of either IIF or DMSO treatment. Therefore, a high incidence of IIF in DMSO-treated monolayers may lead to erroneous underestimates of cell survival when conducting immediate post-thaw assessments of membrane integrity.


Assuntos
Dimetil Sulfóxido , Gelo , Membrana Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células Endoteliais , Congelamento , Células Hep G2 , Hepatócitos , Células Endoteliais da Veia Umbilical Humana , Humanos
16.
Cryobiology ; 97: 123-130, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33007287

RESUMO

To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that the recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most mammalian cells, and thus we assessed the ability of novel small molecule carbohydrate-based ice recrystallization inhibitors (IRIs) to permeate and function within hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-ß-D-glycoside (IRI 3) and fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3. However, trehalose did increase the activity of IRI 1 while decreasing that of IRI 2. While this study suggests that each of these compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.


Assuntos
Criopreservação , Hepatócitos , Gelo , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido , Etilenoglicol , Células Hep G2 , Humanos
18.
Cryobiology ; 97: 250-253, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32986987

RESUMO

Minimizing ice recrystallization injury in tissues and organs has historically been sought using biological antifreeze proteins. However, the size of these compounds can limit permeation and their potential immunogenicity disqualifies them from use in several cryopreservation applications. Novel small molecule carbohydrate-derived ice recrystallization inhibitors (IRIs) are not subject to these constraints, and thus we sought to evaluate the ability of a highly active IRI to permeate liver tissue and control recrystallization. Rat liver tissue blocks (0.5 mm2) were incubated with the IRI for 6 h at 22 °C and subsequently plunged in liquid nitrogen. Ice crystals within the tissue were fixed using a formal acetic alcohol fixative as it was rewarmed from -80 °C to 22 °C over the course of 48 h. The untreated control demonstrated a gradient of increasing crystal size from the exterior to the interior region of the tissue; however, the IRI-treated condition had no such gradient and exhibited small crystals throughout. Threshold segmentation confirmed a significant reduction in the ice crystal size within the interior region of the IRI-treated condition, suggesting the IRI permeated throughout and effectively controlled recrystallization within the tissue.


Assuntos
Criopreservação , Gelo , Animais , Proteínas Anticongelantes , Carboidratos , Criopreservação/métodos , Cristalização , Fígado , Ratos
19.
Transfus Apher Sci ; 59(5): 102933, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32919879

RESUMO

COVID-19 convalescent plasma (CCP) therapy involves the use of circulating antibodies administration from recovered COVID 19 patients as a practical strategy to provide immediate passive immunity in susceptible recipients in need. Global concern over the potential for "second" or "third" waves of infection to occur before effective vaccines or drug therapies are available has many looking at other biological sources for large-scale production of neutralizing SARS-CoV-2 antibodies. This report summarizes some of the novel strategies for developing alternative safe sources of therapeutic autologous antibodies from COVID -19 infected patients, and provides some original thoughts on how to rapidly implement a safe passive immunity in those COVID-19 patients who are most in need of intervention. COVID-19 antibodies can be isolated or delivered using a number of other techniques including: plasmapheresis, plasma cryoprecipitate reduced (cryosupernatant), antibody hyperconcentrates and advanced cell-based delivery systems. While these proposed technological options may, in some cases, be theoretical, the growing concern over the rapid spread of the SARS-CoV-2 virus has prompted many to pursue innovative and creative solutions to reduce the mortality and morbidity resulting from the current global pandemic. A comparative analysis of various strategies currently in use deserved exploring and this highlighted separately as the essential part of this concise theme.


Assuntos
COVID-19/terapia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Sistemas de Liberação de Medicamentos , Humanos , Plasmaferese , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia
20.
Cell Commun Signal ; 18(1): 155, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948210

RESUMO

BACKGROUND: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.


Assuntos
Antígeno CD47/metabolismo , Deformação Eritrocítica , Eritrócitos/citologia , Transdução de Sinais , Trombospondina 1/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Sobrevivência Celular , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos
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