Protein Permeability Research Articles

Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes.

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.

Fas activation alters tight junction proteins in acute lung injury

Background:The acute respiratory distress syndrome (ARDS) is characterized by protein-rich oedema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved.Objective:To determine whether...

Puerarin inhibits hyperglycemia-induced inter-endothelial junction through suppressing endothelial Nlrp3 inflammasome activation via ROS-dependent oxidative pathway

Background: Recent studies indicate that vascular complications are closely related to diabetes mellitus; in particular, inflammatory-mediated endothelial dysfunction plays a crucial role in diabetes-induced cardiovascular diseases. Therefore, exploring effective methods to suppress endothelial dysfunction via inhibition of inflammatory responses is imperative. Puerarin (Pu), a flavonoid common in Pueraria, has been widely and successfully used to treat cardiovascular diseases in China for many years. However, information on its protective properties in hyperglycemia-induced vascular complications is insufficient.Hypothesis/Purpose: In this study, we investigate the protective effects of puerarin against high glucose-induced endothelial dysfunction and the underlying mechanism of the flavonoid.Methods: we investigated the protective effects of Pu against hyperglycemia-induced inter-endothelial junction by permeability and transendothelial electrical resistance (TEER) assay. In addition, changes in the Nlrp3 inflammasome activation via reactive oxygen species (ROS)-dependent oxidative pathway were investigated using western blot, immunofluorescence microscopy analyses and flow cytometry. ROS scavenger and Nlrp3 gene silencing were used to determine the roles of the ROS-Nlrp3 pathway involved in the molecular mechanism of Pu.Results: Our findings demonstrate that puerarin inhibits high glucose-induced Nlrp3 inflammasome formation and activation, as shown by fluorescence confocal microscopy and Western blot. Puerarin decreases Nlrp3 protein, which is a critical factor necessary to form an inflammasome complex. We demonstrate that puerarin exerts anti-oxidation and ROS scavenged effects, similar to apocynin (APO). Interestingly, thioredoxin-interacting protein (TXNIP) protein and TXNIP binding to Nlrp3 markedly decreased with puerarin treatment. Together with these changes, puerarin could decrease high mobility group box 1 (HMGB1) release from mouse vascular endothelial cell (mMVECs). We also demonstrate the decreased expression of the tight junction proteins ZO-1/ZO-2, which are related to endothelial permeability after stimulation by high glucose in endothelial cells. Puerarin could recover the gap junction protein and decrease monolayer cell permeability in endothelial cells. In conclusion, we reveal a new protection mechanism of puerarin that inhibits Nlrp3 inflammasome activation and decreases subsequent caspase-1 activation, triggering the release of HMGB1 by reducing ROS generation.Conclusions: Our findings indicate that puerarin exhibits immense potential and specific therapeutic value in hyperglycemia-related cardiovascular disease and the development of innovative drugs.

Therapeutic effects of human mesenchymal stem cell microvesicles in an ex vivo perfused human lung injured with severe E. coli pneumonia

BackgroundWe previously reported that microvesicles (MVs) released by human mesenchymal stem cells (MSC) were as effective as the cells themselves in both Escherichia coli lipopolysaccharide and live bacteria-induced acute lung...

Therapeutic Implications of Human Umbilical Cord Mesenchymal Stromal Cells in Attenuating Influenza A(H5N1) Virus-Associated Acute Lung Injury.

Highly pathogenic avian influenza viruses can cause severe forms of acute lung injury (ALI) in humans, where pulmonary flooding leads to respiratory failure. The therapeutic benefits of bone marrow mesenchymal stromal cells (MSCs) have been demonstrated in a model of ALI due to influenza A(H5N1) virus. However, clinical translation is impractical and limited by a decline in efficacy as the age of the donor increases. Umbilical cord MSCs (UC-MSCs) are easier to obtain by comparison, and their primitive source may offer more-potent therapeutic effects. Here we investigate the therapeutic efficacy of UC-MSCs on the mechanisms of pulmonary edema formation and alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs were also tested in a mouse model of influenza ALI. We found that UC-MSCs were effective in restoring impaired alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs consistently outperformed bone marrow MSCs, partly because of greater growth factor secretion of angiopoietin 1 and hepatocyte growth factor. Conditioned UC-MSC medium and UC-MSC exosomes were also able to recapitulate these effects. However, UC-MSCs only slightly improved survival of A(H5N1)-infected mice. Our results suggest that UC-MSCs are effective in restoring alveolar fluid clearance and protein permeability in A(H5N1)-associated ALI and confer functional in addition to practical advantages over conventional bone marrow MSCs.

Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation in Caco-2 cells by regulation of TLR4 expression.

Purpose. Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis. In inflammatory conditions, commensal bacteria exploit transcytotic pathways to cross the intestinal epithelium in a TLR4-dependent manner. The aim of this study was to test the hypothesis that Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation by regulation of Toll-like receptor-4 expression.Methodology. L. plantarum strains were investigated to determine their capacity to inhibit the initial adhesion of Escherichia coli B5 to Caco-2 cells. The inhibitory effects of L. plantarum on TNF-α-induced E. coli B5 translocation across Caco-2 cells were studied. Barrier function and integrity were simultaneously assessed by transepithelial electrical resistance, HRP permeability, LDH release and distribution of tight junctional proteins. Expression of TLR4 was assessed by RT-PCR.Results/Key findings. Pretreatment of monolayers with L. plantarum L2 led to a significant decrease in E. coli B5 adhesion and cell internalization (P<0.01). Exposure to TNF-α for six hours caused a significant increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability and disruption of tight junction proteins. Manipulations that induced bacterial translocation were associated with a marked increase in TLR4 mRNA expression and IL-8 secretion. L. plantarum L2 significantly abrogated TNF-α-induced bacterial translocation of E. coli B5, and also downregulated expression of TLR4 and IL-8 in intestinal epithelial cells.Conclusion. Live L. plantarum L2 can inhibit TNF-α-induced transcellular bacterial translocation via regulation of TLR4 expression.

α4 Coordinates Small Intestinal Epithelium Homeostasis by Regulating Stability of HuR.

The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis depends on a dynamic balance among proliferation, migration, apoptosis, and differentiation of intestinal epithelial cells (IECs). The protein phosphatase 2A (PP2A)-associated protein α4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. Here, using a genetic approach, we investigated the mechanisms whereby α4 controls the homeostasis of the intestinal epithelium. In mice with ablated α4, the small intestinal mucosa exhibited crypt hyperplasia, villus shrinkage, defective differentiation of Paneth cells, and reduced IEC migration along the crypt-villus axis. The α4-deficient intestinal epithelium also displayed decreased expression of different intercellular junction proteins and abnormal epithelial permeability. In addition, α4 deficiency decreased the levels of the RNA-binding protein HuR in the mucosal tissue. In cultured IECs, ectopic overexpression of HuR in α4-deficient cells rescued the production of these intercellular junction proteins and restored the epithelial barrier function to a nearly normal level. Mechanistically, α4 silencing destabilized HuR through a process involving HuR phosphorylation by IκB kinase α, leading to ubiquitin-mediated proteolysis of HuR. These findings indicate that the critical impact of α4 upon the barrier function and homeostasis of the intestinal epithelium depends largely on its ability to regulate the stability of HuR.

Mesenchymal Stem Cell Microvesicles Restore Protein Permeability Across Primary Cultures of Injured Human Lung Microvascular Endothelial Cells.

Our previous study demonstrated that mesenchymal stem cell (MSC) microvesicles (MV) reduced lung inflammation, protein permeability, and pulmonary edema in endotoxin‐induced acute lung injury in mice. However, the underlying mechanisms for restoring lung protein permeability were not fully understood. In this current study, we hypothesized that MSC MV would restore protein permeability across injured human lung microvascular endothelial cells (HLMVEC) in part through the transfer of angiopoietin‐1 (Ang1) mRNA to the injured endothelium. A transwell coculture system was used to study the effect of MSC MV on protein permeability across HLMVECs injured by cytomix, a mixture of IL‐1β, TNF‐α, and IFN‐γ (50 ng/ml). Our result showed that cytomix significantly increased permeability to FITC‐dextran (70 kDa) across HLMVECs over 24 hours. Administration of MSC MVs restored this permeability in a dose dependent manner, which was associated with an increase in Ang1 mRNA and protein secretion in the injured endothelium. This beneficial effect was diminished when MSC MV was pretreated with an anti‐CD44 antibody, suggesting that internalization of MV into the HLMVEC was required for the therapeutic effect. Fluorescent microscopy showed that MSC MV largely prevented the reorganization of cytoskeleton protein F‐actin into “actin stress fiber” and restored the location of the tight junction protein ZO‐1 and adherens junction protein VE‐cadherin in injured HLMVECs. Ang1 siRNA pretreatment of MSC MV prior to administration to injured HLMVECs eliminated the therapeutic effect of MV. In summary, MSC MVs restored protein permeability across HLMVEC in part by increasing Ang1 secretion by injured HLMVEC. Stem Cells Translational Medicine 2018;7:615–624

Dexamethasone Prevents Lipopolysaccharide-Induced Epithelial Barrier Dysfunction in Rat Ileum.

Inflammatory mediators have been postulated as elementary inducing factors to the disruption of the intestinal tight junctions (TJ) and consequently, gut permeability and bacterial translocation. Corticosteroids are considered the mainstay in the treatment of septic shock; however, the impact of this therapy on the intestinal epithelial barrier dysfunction during septic shock remains unknown. Our aims were to demonstrate the role of low dexamethasone (DEX) doses in modulation of the inflammatory response, as well as the expression and the arrangement of TJ proteins in endotoxemic rats. One hour before the endotoxemia induction by lipopolysaccharide (LPS) administration, rats were pretreated with DEX at two low-doses (0.1 and 1.0 mg/kg). The parameters assessed included intestinal permeability, bacterial translocation, cytokines production, histology injury, localization, and expression of TJ proteins. Endotoxemic rats displayed intestinal epithelial barrier dysfunction, characterized by increased permeability and bacterial translocation, TJ disruption (opening and changes to its constituent proteins expression) and hyperactivation of the inflammatory response. On the other hand, the pretreatment with DEX attenuated the systemic and mucosal production of inflammatory mediators and also reverted the LPS-induced ileal injuries, increasing the expression of occludin and claudin-1, but also reducing claudin-2. Moreover, the histological damages and the morphology of the TJ were preserved by the DEX administration, therefore reducing their LPS-induced opening. The present study sheds light on the fact that early DEX treatment breaks the vicious cycle of local gut inflammation and barrier dysfunction in endotoxemia, especially preserving an essential structure of this monolayer epithelium, the TJ.

Characterization of uniaxial high-speed stretch as an in vitro model of mild traumatic brain injury on the blood-brain barrier

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.

Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine.

Antibiotic resistant features of Acinetobacter baumannii is partly due to the decreased outer membrane proteins (OMPs) permeability. The OmpA is one of the most conserved proteins among A. baumannii with a considerable antigenic potential to stimulate the multidimensional immune system responses. The present study was aimed to clone the ompA gene into the eukaryotic expression vector with potential as DNA vaccine. The ompA gene of A. baumannii was amplified using polymerase chain reaction (PCR). The target DNA was cloned and sub-cloned into the pTZ57R/T and pBudCE4.1 vectors, respectively. The recombinant vectors containing ompA were then validated using colony PCR, vector sequencing and double-digestion strategies. The pBudCE4.1-ompA recombinant plasmid was transfected into the human dermal fibroblast cells (HDF) and presence of ompA transcript and protein was evaluated using reverse transcribed-PCR (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Our finding from colony PCR, sequencing and enzyme double digestion result confirmed that target gene has been successfully inserted into the pTZ57RT and pBudCE4.1. The presence of an expected band (1112bp) in RT-PCR as wells as a~38kDa band during SDS-PAGE showed that the recombinant pBudCE4.1-ompA construct was efficiently transfected into the HDF cells and expressed. Altogether, our observation demonstrated that the recombinant pBudCE4.1-ompA construct was successfully produced although further experiments are needed.

Physicochemical Characterization of Mitragyna speciosa Alkaloid Extract and Mitragynine using In Vitro High Throughput Assays.

Mitragynine, a major active alkaloid of Mitragyna speciosa, acts as an agonist on µ-opioid receptors, producing effects similar to morphine and other opioids. It has been traditionally utilized to alleviate opiate withdrawal symptoms. Besides consideration about potency and selectivity, a good drug must possess a suitable pharmacokinetic profile, with suitable absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) profile, in order to have a high chance of success in clinical trials. The purity of mitragynine in a Mitragyna speciosa alkaloid extract (MSAE) was determined using Ultra-Fast Liquid Chromatography (UFLC). In vitro high throughput ADMETox studies such as aqueous solubility, plasma protein binding, metabolic stability, permeability and cytotoxicity tests were carried out to analyze the physicochemical properties of MSAE and mitragynine. The UFLC quantification revealed that the purity of mitragynine in the MSAE was 40.9%. MSAE and mitragynine are highly soluble in aqueous solution at pH 4.0 but less soluble at pH 7.4. A parallel artificial membrane permeability assay demonstrated that it is extensively absorbed through the semi-permeable membrane at pH 7.4 but very poorly at pH 4.0. Both are relatively highly bound to plasma proteins (> 85 % bound) and are metabolically stable to liver microsomes (> 84 % remained unchanged). In comparison to MSAE, mitragynine showed higher cytotoxicity against WRL 68, HepG2 and Clone 9 hepatocytes after 72 h treatment. The obtained ADME and cytotoxicity data demonstrated that both MSAE and mitragynine have poor bioavailability and have the potential to be significantly cytotoxic.

Peritoneal Protein Losses Depend on More Than Just Peritoneal Dialysis Modality and Peritoneal Membrane Transporter Status.

Peritoneal protein clearance (PPCl) depends upon vascular supply and size selective permeability. Some previous reports suggested PPCl can distinguish fast peritoneal membrane transport due to local or systemic inflammation. However, as studies have been discordant, we wished to determine factors associated with an increased PPCl. Consecutive patients starting peritoneal dialysis (PD) who were peritonitis-free were studied. Data included a baseline peritoneal equilibration test (PET), measurement of dialysis adequacy, 24-h dialysate PPCl and body composition measured by multifrequency bioimpedance. 411 patients, mean age 57.2 ± 16.6 years, 60.8% male, 39.4% diabetic, 20.2% treated by continuous ambulatory peritoneal dialysis (CAPD) were studied. Mean PET 4-h Dialysate/Serum creatinine was 0.73 ± 0.13, with daily peritoneal protein loss 4.6 (3.3-6.4) g, and median PPCl 69.6 (49.1-99.6) mL/day. On multivariate analysis, PPCl was most strongly associated with CAPD (β 0.25, P < 0.001), extracellular water (ECW)/total body water (TBW) ratio (β 0.21, P < 0.001), skeletal muscle mass index (β 0.21, P < 0.001), log N-terminal brain natriuretic peptide (NT-proBNP) (β 0.17, P = 0.001), faster PET transport (β 0.15, P = 0.005), and normalized nitrogen appearance rate (β 0.13, P = 0.008). In addition to the longer dwell times of CAPD, greater peritoneal creatinine clearance and faster PET transporter status, we observed an association between increased PPCl and ECW expansion, increased NT-proBNP, estimated dietary protein intake and muscle mass, suggesting a link to sodium intake and sodium balance, increasing both ECW and conduit artery hydrostatic pressure resulting in greater vascular protein permeability. This latter association may explain reports linking PPCl to patient mortality.

Rejection of trace organic water contaminants by an Aquaporin-based biomimetic hollow fiber membrane

Abstract Forward osmosis (FO) is a promising key strategy for energy efficient water purification. Different draw solutions, that create an osmotic gradient, and water selective FO membranes exist that allow for a significant concentration of waste streams while water is being filtered and absorbed into the draw solution. Such technologies are often limited by the solute rejection and water flux characteristics of the membrane used. A new approach is the utilization of aquaporin-based membranes (ABMs). These biomimetic membranes make use of the high water permeability and selectivity of aquaporin proteins, which are embedded into the active layer of the membrane. In this study, a 0.6 m2 hollow fiber ABM module was tested for its rejection of three different trace organic contaminants: the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), the plastic component bisphenol A (BPA) and the preservative methyl paraben. Over 95% of methyl paraben was rejected by the membrane while for 2,4-D and BPA rejection rates of over 99% were attained. However, it could also be observed that BPA and methyl paraben adsorbed on the aquaporin membrane and were flushed out again during subsequent experiments. This phenomenon may have to be addressed in future studies by developing specific cleaning protocols for ABMs.

Measurement of Protein Permeability and Fluid Transport of Human Alveolar Epithelial Type II Cells Under Pathological Conditions.

Alveolar epithelial barrier dysfunction contributes to the influx of protein-rich edema fluid and the accumulation of inflammatory cells in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). To study the alveolar epithelial barrier function under pathological conditions, we developed an in vitro model of acute lung injury using cultured human alveolar epithelial type II (ATII) cells. Here we describe the methods that we use to measure protein permeability and fluid transport across human ATII cell monolayers under stimulated conditions. Both proinflammatory cytokines and edema fluid from ALI/ARDS patients can increase protein permeability and decrease fluid transport across the human ATII cells monolayer.

Blood-brain barrier on a chip.

The blood-brain barrier (BBB) plays a vital role in the maintenance of brain homeostasis. It strictly restricts the passage of molecules from the brain vasculature into the brain via its high transendothelial electrical resistance and low paracellular and transcellular permeability. Specialized brain endothelial cells, astrocytes, pericytes, neurons, and microglia contribute synergistically to the functional properties of the BBB. Because of its complexity and relative inaccessibility, BBB research is fraught with difficulties. Most studies rely on animal or cell culture models, which are not able to fully recapitulate the properties of the human BBB. The recent development of three-dimensional (3D) microfluidic models of the BBB could address this issue. This chapter aims to provide an overview of the recent advances in modeling the BBB on microdevices, and illustrate important considerations for the design of such models. In addition, protocols for the fabrication of a 3D BBB microfluidic chip and BBB assessment experiments, including immunocytochemistry for analyzing cell morphology and protein marker expression, permeability assay, and calcium imaging for studying neuronal function as a measure of BBB integrity, are presented here. It is envisioned that continued advancements in microtechnology can lead to the creation of realistic in vivo-like BBB-on-chip models.

Branched BBB-shuttle peptides: chemoselective modification of proteins to enhance blood-brain barrier transport.

The blood-brain barrier (BBB) hampers the delivery of therapeutic proteins into the brain. BBB-shuttle peptides have been conjugated to therapeutic payloads to increase the permeability of these molecules. However, most BBB-shuttles have several limitations, such as a lack of resistance to proteases and low effectiveness in transporting large biomolecules. We have previously reported on the THRre peptide as a protease-resistant BBB-shuttle that is able to increase the transport of fluorophores and quantum dots in vivo. In this work, we have evaluated the capacity of linear and branched THRre to increase the permeability of proteins in cellular models of the BBB. With this purpose, we have covalently attached peptides with one or two copies of the BBB-shuttle to proteins in order to develop chemically well-defined peptide-protein conjugates. While THRre does not enhance the uptake and transport of a model protein in BBB cellular models, branched THRre peptides displaying two copies of the BBB-shuttle result in a 2.6-fold increase.

Separation of metabolic products by electroosmotic dialysis in the plasma model

Hemodialytic membrane separation of low molecular weight solutes e.g. urea and creatinine bases on diffusion processes generated by the respective concentration gradient between dialysate and blood plasma. Electroosmotic drag of liquid through membranes offers a new transport concept for separation of metabolic products in dialysis. In this work electroosmotic drag of membranes made of textile reinforced agar, collagen, bovine pericardium, acrylonitrile/sodium methallylsulfonate copolymer as well as cation exchange membranes was studied using a five chamber electroosmotic cell in continuous operation mode. The textile reinforced agar membrane, the copolymer membrane and the bovine pericard membrane were selected for electroosmotic dialysis experiments using plasma as biological model. Vitamin B12, myoglobin, albumin and beta-2-microglobulin plasma concentrations were monitored to investigate both, membrane permeation of these markers in direction of the electroosmotic flow and possible negative effects of the electroosmotic treatment on protein stability. Transport of low molecular weight substances urea and creatinine into the recipient electrolyte was achieved without permeation of Vitamin B12 and plasma proteins. Coagulation of proteins due to the electrical field applied was not on a significant level. Monitoring of biologically relevant ions Na+, K+, Ca2+, PO43−, Cl− demonstrated that the ion movement in electroosmosis also will permit control of ion concentrations during dialysis. The transported volume of up to 14 ml Ah−1 demonstrates the potential for a significant reduction in dimensions of devices for dialysis, thus offering new aspects for miniaturisation and home dialysis.

Biocompatibility and small protein permeability of hydrophilic-coated membrane dialyzer (NV) in hemodialysis patients: a pilot study

BackgroundWe evaluated the biocompatibility and small protein permeability of a newly developed hydrophilic-coated membrane dialyzer (NV) compared with conventional polysulfone dialyzer (APS) for hemodialysis (HD) therapy.MethodsIn a prospective crossover study, 11 maintenance HD patients (7 males; mean age 67.0 ± 10.2 years) received HD three times a week for 4 weeks with the NV membrane and then for another 4 weeks with the APS membrane. We evaluated the variation in several parameters including white blood cell (WBC) count and fibrinogen as indexes for biocompatibility. The plasma and dialysate concentrations of β2-microglobulin (β2-M), α1-microglobulin (α1-M), and albumin were measured at baseline and after 4 h of each study treatment in order to assess the removal of small proteins.ResultsReductions in the WBC count were seen with APS compared with NV at 60 min (NV 5.65 ± 1.60, APS 5.17 ± 1.65 × 103/μL, p < 0.05) and 240 min (NV 5.28 ± 1.38, APS 4.63 ± 1.2 × 103/μL, p < 0.005) after the start of HD. With NV, we found significantly greater rates of variation of β2-M (NV 45.5 ± 1.2, APS 40.1 ± 1.2%, p < 0.0001), α1-M (NV 41.2 ± 9.9, APS 34.2 ± 18.5%, p < 0.05), and albumin (NV 31.6 ± 7.8, APS 18.1 ± 6.5%, p < 0.0001) during HD than with APS. However, there were no significant differences in the removal of β2-M between the two dialyzers.ConclusionsThe clinical characteristics of NV may reveal an improved biocompatibility and a comparable efficiency in small protein removal as compared to those of APS.Trial registrationClinical effects of polysulfone membrane, NV-13U UMIN000011764

Abstract 121: Development of Enzyme Replacement Therapy in Mammalian Models of Barth Syndrome

Barth Syndrome (BTHS) is caused by a single gene mutation in the mitochondrial transacylase, tafazzin (TAZ), which results in impaired lipid metabolism leading to dysfunction in highly energetic tissues such as the heart and skeletal muscle. TAZ remodels the signature mitochondrial phospholipid, cardiolipin (CL), which is responsible for providing support to the electron transport chain. BTHS patients suffer from growth deficiencies, cardiomyopathy, hypotonia and neutropenia. Currently, treatment for patients with BTHS is supportive, seeking to ameliorate rather than prevent heart problems, skeletal muscle problems and recurring infections. Protein therapy, on the other hand, might treat and even prevent cardiac, skeletal muscle as well as infection-related morbidities. We designed a recombinant TAZ protein containing a cell penetrating peptide in its C-terminus, which enables the recombinant TAZ to penetrate cells and then treated TAZ-deficient cells with it. We tested the permeability of the recombinant protein by direct delivery to H9C2 cardiomyoblasts and found that the protein is successfully taken up by the cells. We have generated a CRISPR-mediated TAZ knock out cardiomyoblast cell line and we found that TAZ knock out cells show a decrease in oxygen consumption as compared to the wild type cells; this is consistent with data from BTHS patient-derived cells. We are using this cell line to assess the enzymatic activity of the delivered protein by conducting mitochondrial respiration measurements. We have also acquired a mouse model of BTHS and are testing the recombinant TAZ in vivo. Preliminary data shows an augmentation in oxygen consumption following treatment with TAZ. These results indicate that the protein is able to reach the mitochondria, where it is enzymatically active and able to enhance respiration. As the protein is able to rescue respiration in cells in which tafazzin was absent, this suggests that our approach should not only be able to prevent onset of symptoms, but also rescue the phenotype in already affected tissues.