Human Serum Albumin Treatment Research Articles

Coexpression of Tim-3 and PD-1 of T Lymphocytes Prevents Acute Graft-Versus-Host Disease

T lymphocytes are critical players in acute graft-versus-host disease (aGVHD). TIM3expression on the T cell surface was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, their clinical potential is greatly limited by their low cell number in peripheral blood (PB). We already introduced a novel method to obtain a high number of PD1 +TIM3 + lymphocytes that culturing CD3 + cells isolated from G-CSF mobilized peripheral blood stem cells (CD3 +G-PBSC) with human serum albumin (HSA). We also confirmed that cultured PD1 +TIM3 + lymphocytes are the most potent subset of the CD3 + cells that inhibit T cell proliferation. In this study, we investigate the immunosuppressive effects of PD1 +TIM3 + lymphocytes after a 2-day culture with HSA (D2-CD3 +G-PBSC) or after a 4-day culture with HSA (D4-CD3 +G-PBSC). Also, we evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes in the xenograft mouse aGVHD model. Methods. The healthy donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3 + cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. CD3 + G-PBSC were cultured with 5% HSA for 2 days or 4 days (1x10 5 cells/mL). PD1 and TIM3 expressions were analyzed using flow cytometry (FACS). Sorted T lymphocytes were cultured with irradiated allo-MNCs for 3 days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. aGVHD was induced by intraperitoneal injection of human PB MNCs to the NOD-SCID IL-2Rγ null mouse after exposure of 3Gy irradiation. The treatment efficacy of PD1 +TIM3 + lymphocytes was analyzed by determining the survival rate after tail vein injection of PD1 +TIM3 + lymphocytes (1x10 7/kg). Results. Normally, very few cells (0.2±0.2%) were PD1 +TIM3 + lymphocytesin PB. The numbers of PD1 +TIM3 + lymphocytes markedly increased by 74-fold in D2-CD3 +G-PBSC and 370-fold in D4-CD3 +G-PBSC, respectively. CD3 + G-PBSC were composed of three types of cells such as PD1 -TIM3 -, PD1 +TIM3 -, and PD1 +TIM3 + lymphocytes. To check the origin of PD1 +TIM3 + lymphocytes after HSA treatment, PD1 -Tim3 - and PD1 +Tim3 - lymphocytes from CD3 +G-PBSC were sorted and cultured with HSA. It was confirmed that PD1 +TIM3 + lymphocytes originated from PD1 -TIM3 - lymphocytes. To assess the immunosuppressive effects of cultured T lymphocytes, we used CSFE-MLR. D2-CD3 +G-PBSC significantly increased the levels of unstimulated T cells compared to D4-CD3 +G-PBSC (% of unstimulated T cells in D2-CD3 +G-PBSC vs. in D4-CD3 +G-PBSC; 38.9±3.0% vs. 13.9±6.6%). Also, both PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated significantly enhanced levels of immunosuppression (% of unstimulated T cells PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 31.6±5.5 and 36.6±13.3, respectively). Lastly, we tested the effects of anti-PD1 or anti-TIM3 blocking mAb on the PD1 +TIM3 + lymphocytes. The immunosuppressive effects of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC were significantly decreased by anti-PD1 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 21.1±4.5 from 31.6±5.5 and 18.4±7.8 from 36.6±13.3, respectively). However, the immunosuppression of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC was not decrease by anti-TIM3 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 28.7±3.3 from 31.6±5.5 and 22.7±5.6 from 36.6±13.3, respectively). These data indicated that PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC have the same phenotype as PD1 +TIM3 + lymphocytes from D4-CD3 +G-PBSC, however, their functions are different. Next, we used the xenograft mouse aGVHD model to evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes for preventing aGVHD. In the control group, 14% of the mice survived at 100 days. Mice that were treated with PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated 100% and 71% survival rate at 100 days, respectively. Conclusion. Ex vivo HSA treatment of CD3 + G-PBSCs for 2 days shows promising therapeutic potential for the treating patients with aGVHD.

Human serum albumin promotes self-renewal and expansion of umbilical cord blood CD34+ hematopoietic stem/progenitor cells.

We investigated the effect of human serum albumin (HSA) on human umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells (HSPCs) cultured in vitro and transplanted in vivo. Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation. CD34+ cells were then sorted by CD34 conjugated magnetic microbeads. The sorted cells were cultured with or without HSA for 8 days in vitro. After 8 days, all cells were harvested for flow phenotyping and colony formation cell (CFC) experiments. The cells were injected into immunodeficient mice (NOD/Shi-scid/IL2Rγnull, NOG) via intravenous injections. From 4 weeks post-transplantation, flow cytometry was used to calculate human cell chimerism in the peripheral blood (PB) every 2 weeks. Flow phenotyping of human cell chimerism in bone marrow and spleen was calculated 16 weeks post-transplantation. Compared to the control group, CD34+ cells cultured with HSA increased significantly in vitro. The long-term engraftment of HSPCs and the hematopoietic multilineage reconstruction capacity were preserved by HSA. Normal engraftment of human cells could be maintained via HSA treatment could maintain normal engraftment of human cells in recipient PB. Here, we found that HSA was beneficial to maintaining CD34+ cell expansion and short-term colony formation in vitro and optimizing multilineage reconstitution in immunodeficient mice in vivo.

Ex Vivo Culture with Albumin Greatly Increases the Immunosuppressive Subset of T Lymphocytes

Emerging studies suggest that the expression of co-inhibitory receptors such as PD1, TIM3, TIGIT, and LAG3 on the T cell surface is important in regulating T cell response. However, the low number of cells expressing such co-inhibitory receptors in peripheral blood severely limits their clinical potential in treating GVHD. In this study, we introduce a novel method to culture and obtain a high number of CD3+PD1+TIM3+ lymphocytes that also exert strong immunosuppression. We demonstrate that culturing CD3+ cells isolated from G-CSF mobilized peripheral blood stem cells (G-PBSCs) with human serum albumin (HSA) greatly increases the number of immunosuppressive CD3+ cells. Also, we confirm that the CD3+PD1+TIM3+ lymphocytes are the most potent subset of the CD3+ cells that induce immunosuppression and inhibit T cell proliferation. Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3+ cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. Isolated CD3+ cells were cultured with 5% HSA for 4 days (1x105 cells/mL). PD1, TIM3, LAG3, and TIGIT expressions were analyzed using flow cytometry (FACS). CD3+, CD3+PD1+, and CD3+PD1+TIM3+ lymphocytes were sorted and cultured with irradiated allo-MNCs for three days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. Results. We discovered that treating G-PBSC cells with 5% HSA resulted in a dramatic increase in the number of immunosuppressive lymphocytes after the HSA treatment. In untreated G-PBSCs, there were very low numbers of CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ lymphocytes (0.9±0.3x103, 0.1±0.1x103, and 0.3±0.2x103, respectively). In G-PBSCs, the HSA treatment markedly increased the cell numbers (47.6±1.9x103, 7.8±3.0x103, and 0.7±0.3x103, respectively) while resulting in a mild increase in the total number of CD3+ cells (1.2-fold increase, 1x105 to 1.2±0.4x105). Our data also demonstrated significant percent changes in subpopulations of CD3+ lymphocytes. In untreated G-PBSCs, very low percentages of cells were CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ (9.0±3.1%, 1.2±0.1% and 3.2±2.8% in G-PBSC, respectively). After the HSA treatment, the percentages of the immunosuppressive lymphocytes in G-PBSCs markedly increased to 39.7±9.1%, 6.5±1.3%, and 0.6±0.4%, respectively. In particular, CD3+PD1+TIM3+ lymphocytes demonstrated the greatest increase in the cell number and the percentage after the HSA treatment, increasing by ~52 fold. Next, we tested whether the HSA-treatment induced proliferation of immunosuppressive T cells translated into increased immunosuppression. To do this, we used CSFE-MLR, co-cultured the CD3+ G-PBSCs with allo-MNCs, and analyzed the number of unstimulated T cells. The HSA-treated CD3+ G-PBSCs significantly increased the levels of unstimulated T cells compared to the untreated G-PBSCs (% of unstimulated T cells in untreated cells vs. in HSA-treated cells; 0.5±0.1 vs. 15.4±11.7), indicating that the HSA treatment increased CD3+ cell-mediated immunosuppression. Lastly, we tested the immunosuppressive effects of CD3+PD1+ and CD3+PD1-lymphocytes in HSA-treated CD3+ G-PBSCs. Both CD3+PD1+ and CD3+PD1+TIM3+ lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3+PD1-lymphocytes and CD3+PD1+TIM3- lymphocytes, respectively (% of unstimulated T cells in CD3+PD1+ vs. in CD3+PD1-lymphocytes, 27.9±5.5 vs. 4.3±7.5, and % of unstimulated T cells in CD3+PD1+TIM3+ vs. in CD3+PD1+TIM3- lymphocytes, 46.1±5.5 vs. 17.4±7.4). Collectively, these data suggest that PD1 and TIM3 are key markers in the HSA-dependent increase in immunosuppression. Conclusion. The ex vivo culture of CD3+ G-PBSCs with HSA markedly increases both the number and the effect of immunosuppressive CD3+ lymphocytes. Our analysis also indicates that the increased immunosuppression is likely due to the proliferation of CD3+PD1+TIM3+ lymphocytes. Taken together, our data suggest that the ex vivo HSA treatment of CD3+ G-PBSCs shows promising therapeutic potential for treating patients with GVHD.

Pathological Significance and Prognostic Roles of Indirect Bilirubin/Albumin Ratio in Hepatic Encephalopathy

Background and Aim: Hepatic encephalopathy (HE) is a neurological disease caused by severe liver disease. Early identification of the risk factor is beneficial to the prevention and treatment of HE. Free bilirubin has always been considered to be the culprit of neonatal kernicterus, but there is no research to explore its role in HE. In this study, we aim to study the clinical significance of the indirect bilirubin-albumin ratio in HE.Methods: A retrospective case-control study of 204 patients with liver failure was conducted. Human serum albumin (HSA) or heme oxygenase-1 (HO-1) inhibitor SnPP (Tin protoporphyrin IX dichloride) was injected intraperitoneally into Ugt1−/− mice to establish a treatment model for endogenous hyperbilirubinemia.Results: IBil/albumin ratio (OR = 1.626, 95% CI1.323–2.000, P < 0.001), white blood cell (WBC) (OR = 1.128, 95% CI 1.009–1.262, P = 0.035), ammonia (OR = 1.010, 95% CI 1.001–1.019, P = 0.027), platelet (OR=1.008, 95% CI 1.001–1.016, P = 0.022), Hb (OR = 0.977, 95% CI 0.961–0.994, P = 0.007), and PTA (OR = 0.960, 95% CI 0.933–0.987, P = 0.005) were independent factors of HE. Patients with a history of liver cirrhosis and severe HE (OR = 12.323, 95% CI 3.278–47.076, P < 0.001) were more likely to die during hospitalization. HSA or SnPP treatment improved cerebellum development and reduced apoptosis of cerebellum cells.Conclusion: The IBil/albumin ratio constitutes the most powerful risk factor in the occurrence of HE, and reducing free bilirubin may be a new strategy for HE treatment.

Expert consensus on the use of human serum albumin in critically ill patients.

Expert consensus on the use of human serum albumin in critically ill patients.

Human serum albumin attenuates global cerebral ischemia/reperfusion-induced brain injury in a Wnt/β-Catenin/ROS signaling-dependent manner in rats

This study sought to clarify the role and underlying mechanisms of human serum albumin (HSA) therapy in global cerebral ischemia/reperfusion (GCI/R)-induced brain damage in rats. Five groups of adult male Wistar rats (n = 12 per group) were created as follows: sham operation (Sham), global cerebral ischemia/reperfusion (GCI/R), HSA treatment (GCI/R + HSA), Dickkopf-1 (DDK1) treatment (GCI/R + DDK1), and DDK1 plus HSA treatment (GCI/R + DKK1 + HSA). The GCI/R injury model was created using the modified Pusinelli four-vessel occlusion method. After 24 h, rats were evaluated using neurological scoring, Nissl staining, and brain tissue water content. The mRNA expression of Wnt, GSK3β, and β-Catenin in the brain were detected by quantitative real time polymerase chain reaction. The protein expression of β-Catenin and GSK-3β were investigated by western blot and immunohistochemical analysis in the presence and absence of the Wnt/β-Catenin antagonist, DKK-1. Complex I activity and ROS content were also measured. After 24 h of reperfusion, the behavior score and brain tissue water content in the GCI/R + HSA group were lower than that in the GCI/R group. In addition, the degree of neuronal injury was significantly reduced in the GCI/R + HSA group (P < 0.05). The ROS content was significantly decreased and Complex I activity was markedly raised in the GCI/R + HSA group compared to the GCI/R group (P < 0.05). Further, GSK-3β expression in the GCI/R + HSA group was lower than that in the GCI/R group, while the Wnt and β-catenin expression were increased. These effects were reversed by DKK1. Taken together, we showed that HSA attenuates GCI/R-induced brain damage and may be neuroprotective via regulation of the Wnt/β-catenin/ROS signaling pathway.

Neuroprotective effects of high-dose human albumin against traumatic spinal cord injury in rats.

Human serum albumin (HA) is a unique multifunctional protein with neuroprotective properties. We aimed to delineate the mechanisms of HA-induced neuroprotection, supresses inflammatory response and lipid peroxidation after spinal cord injury (SCI). Adult female Wistar rats weighing 210-250 g were used for the study. The rats were randomly and blindly allocated into five groups. The one-way analysis of variance (ANOVA) for parametric data and Shapiro-Wilk test was used for evaluating the normal distribution of the variables. Kruskal-Wallis for nonparametric data was used to compare groups. Electron and light microscopies were used to demonstrate ultrastructural changes in spinal cord. The HA group was significantly different from all the other groups (p < 0.05). Both MPSS and HA treatments decreased the MPO significantly. HA treatment decreased the lipid peroxidation. HA treatment prevented the worsening of clinical results. In the HA treatment group, the ultrastructure was protected significantly. The neuronal bodies and axonal structures were normal except for some limited edematous spaces. HA improves early clinical results, protects spinal cord ultrastructure, and decreases MPO and LPO levels after spinal cord contusion injury (Tab. 3, Fig. 3, Ref. 39).

A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies.

Recently, a quasi-lipid-enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated. Cell culture lysates and supernatants of two cytopathic HAV variants, HM175/18f and HM175/24a, were characterized for their LE and NLE particle content by isopycnic gradient centrifugation. The obtained fractions were characterized for relative infectivity and then subjected to heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) in 12.5% human serum albumin to investigate their respective heat sensitivity. Preparations of the two HAV variants contained either LE particles (HM175/24a) or LE and NLE particles (HM175/18f) with equivalent specific infectivity. For HM175/18f, heat sensitivity of LE and NLE fractions did not differ significantly, and inactivation of the whole virus stock was identical to the NLE particle inactivation profile, whereas the HM175/24a variant was more heat sensitive. The results indicate that, in heat-treatment studies, the LE or NLE HAV phenotype is less important than the choice of HAV variant, and the most heat-resistant HM175/18f should be used.

Zika virus is not thermostable: very effective virus inactivation during heat treatment (pasteurization) of human serum albumin.

The ongoing Zika virus (ZIKV) outbreak in the Americas has also raised concerns around the potential for ZIKV transmission via blood products. Plasma-derived products are considered safe, because effective viral-inactivation and removal methods are implemented in their manufacturing processes. However, a recent study has indicated that ZIKV is "thermally stable" compared with the closely related Dengue virus, thus raising the question of whether heat treatments, as embedded in the manufacturing of plasma-derived products, are as effective against ZIKV as was previously shown for other Flaviviruses. Therefore, the sensitivity of ZIKV to heat inactivation was investigated using the pasteurization of human serum albumin (HSA) as an example. Heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) of HSA was investigated for the capacity to reduce ZIKV in two different protein concentrations (5% and 25% HSA). The results were compared with data obtained in identical set-ups for the closely related West Nile virus, tick-borne encephalitis virus, and bovine viral diarrhea virus. Heat treatment of HSA inactivated ZIKV to below the limit of detection already during the heating phase to 57.0°C, that is, even before the 10-hour incubation at 58.0 ± 1.0°C commenced. For West Nile virus, bovine viral diarrhea virus, and tick-borne encephalitis virus, incubations up to 180 minutes were required to achieve inactivation to below the limit of detection. ZIKV was more sensitive to heat treatment than other members of the Flaviviridae and thus does not pose a concern for plasma products that include a heat treatment in their manufacturing process.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

Multifunctional Effect of Human Serum Albumin Reduces Alzheimer's Disease Related Pathologies in the 3xTg Mouse Model.

Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifests simultaneously, eventually leading to cognitive impairment and death. No treatment is currently available; however, an agent addressing several key pathologies simultaneously has a better therapeutic potential. Human serum albumin (HSA) is a highly versatile protein, harboring multifunctional properties that are relevant to key pathologies underlying AD. This study provides insight into the mechanism for HSA's therapeutic effect. In vivo, a myriad of beneficial effects were observed by pumps infusing HSA intracerebroventricularly, for the first time in an AD 3xTg mice model. A significant effect on amyloid-β (Aβ) pathology was observed. Aβ1-42, soluble oligomers, and total plaque area were reduced. Neuroblastoma SHSY5Y cell line confirmed that the reduction in Aβ1-42 toxicity was due to direct binding rather than other properties of HSA. Total and hyperphosphorylated tau were reduced along with an increase in tubulin, suggesting increased microtubule stability. HSA treatment also reduced brain inflammation, affecting both astrocytes and microglia markers. Finally, evidence for blood-brain barrier and myelin integrity repair was observed. These multidimensional beneficial effects of intracranial administrated HSA, together or individually, contributed to an improvement in cognitive tests, suggesting a non-immune or Aβ efflux dependent means for treating AD.

Role of homeobox A13 in epithelial-to-mesenchymal transition induced by serum albumin overload in human renal tubular epithelial cells

Objective To explore the role of nuclear translational factor homeobox A13(HOXA13) gene in epithelial-to-mesenchymal transition (EMT) induced by human serum albumin(HSA) overload in human renal tubular epithelial(HKC) cells. Methods HKC cells were treated with different concentrations of HSA (ranging from 0-30 g/L) for 48 h or 20 g/L HSA for different times (ranging from 0-72 h) in vitro.The protein expressions of cytokeratin (CK), Vimentin, and HOXA13 protein in HKC cells were detected by using Western blot respectively.Meanwhile, liposome-mediated DNA transfection was used to transfect the HOXA13 gene into HKC cells before HSA treatment, and the expressions of CK, Vimentin and HOXA13 protein in HKC cells were also detected by using Western blot. Results (1)The protein expression of CK decreased but Vimentin increased after HKC cells were exposed to HSA, which was in a concentration-and time-dependent manner.(2)Expression of HOXA13 was down-regulated by HSA in a dose-and time-dependent manner, and the expressions of HOXA13 protein in HKC in 5 g/L, 10 g/L, 20 g/L, 30 g/L group were 58.24%(P=0.005), 44.73%(P=0.003), 38.40%(P=0.033)and 24.83%(P=0.011)respectively as compared with 0 g/L group.Likewise, the protein expressions of HOXA13 in 24 h, 48 h, 72 h group were 52.00%(P=0.023), 46.83%(P=0.008)and 35.10%(P=0.034)respectively as compared with 0 h group.(3)There was a positive correlation between the levels of HOXA13 protein expression and CK protein expression(r=0.86, P=0.005), while the relationship between the levels of HOXA13 protein expression and Vimentin protein expression was negative(r=-0.94, P=0.002). (4)Up-regulated expression of HOXA13 in HKC cells by lipofectamine transfection alleviated the degree of EMT induced by HSA significantly.The expression of Vimentin decreased by 35.34% (P=0.005) while the expression of CK increased 360.00%-fold (P=0.005), compared with that of untransfected HKC cells. Conclusion EMT induced by HSA in HKC cells may play a role through HOXA13. Key words: Albumin overload; Renal tubular epithelial cell; Epithelial-to-mesenchymal transition; Homeobox A13

Albumin overload induces expression of hypoxia-inducible factor 1α and its target genes in HK-2 human renal proximal tubular cell line

The aim of this study was to investigate the effect of human serum albumin (HSA) overload on the expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in human renal proximal tubular cell line HK-2. First, the cell viability and cytotoxic activity were examined to assess the cellular conditions in HK-2 cells with HSA treatment employed in this study. HSA treatment for 48h decreased the cell viability and increased the leakage of lactate dehydrogenase (LDH) into the medium in a concentration-dependent manner, but the toxicity was relatively mild. Western Blot analysis revealed that HSA treatment induced the expression of HIF-1α protein in a concentration-dependent manner without a change in β-actin protein expression. Confocal microscopy analysis revealed that HIF-1α protein was predominantly localized in the nucleus but was also observed in the cytoplasm. The HIF-1 target gene mRNAs, glucose transporter 1 and glyceraldehyde 3-phosphate dehydrogenase, were up-regulated by HSA treatment, leading to the increases in the protein expression levels. In addition, the mRNA of HIF-1α was increased by HSA treatment. In conclusion, albumin loading induces HIF-1α in HK-2 cells, resulting in the increases in the expression of proteins of its target genes.

Beyond plasma bilirubin: The effects of phototherapy and albumin on brain bilirubin levels in Gunn rats

Severe unconjugated hyperbilirubinemia, as occurs in Crigler-Najjar disease and neonatal jaundice, carries the risk of neurotoxicity. This neurotoxicity is related to the increased passage of free bilirubin (UCB(free)), the fraction of bilirubin that is not bound to plasma proteins, into the brain. We hypothesized that albumin treatment would lower the UCB(free) fraction, and thus decrease bilirubin accumulation in the brain. We treated chronic (e.g., as a model for Crigler-Najjar disease) and acute hemolytic (e.g., as a model for neonatal jaundice) moderate hyperbilirubinemic Gunn rats with phototherapy, human serum albumin (HSA) or phototherapy+HSA. In the chronic model, adjunct HSA increased the efficacy of phototherapy; it decreased plasma UCB(free) and brain bilirubin by 88% and 67%, respectively (p<0.001). In the acute model, adjunct HSA also increased the efficacy of phototherapy; it decreased plasma UCB(free) by 76% (p<0.001) and completely prevented the hemolysis-induced deposition of bilirubin in the brain. Phototherapy alone failed to prevent the deposition of bilirubin in the brain during acute hemolytic jaundice. We showed that adjunct HSA treatment decreases brain bilirubin levels in phototherapy-treated Gunn rats. We hypothesize that HSA decreases these levels by lowering UCB(free) in the plasma. Our results support the feasibility of adjunct albumin treatment in patients with Crigler-Najjar disease or neonatal jaundice.

Abstract 17487: S-Nitroso Human Serum Albumin (S-NO-HSA) Attenuates Pulmonary Hypertension, Improves Right Ventricular-Arterial Coupling and Reduces Oxidative Stress in a Chronic Right Ventricle Volume Overload Model

BACKGROUND The study was aimed to examine the acute overall hemodynamic and oxidative stress effects to intravenous S-NO-HSA infusion in a model of chronic left-to-right shunt-induced pulmonary arterial hypertension and right ventricle failure. METHODS AND RESULTS Fifty male Wistar rats underwent surgical creation of aorto-caval fistula (Qp/Qs&gt; 2.0) After 10 weeks they were randomly treated with S-NO-HSA (0.5 υmol/kg/h) (n. 20) or human serum albumin (HSA) (n. 25) infusion for 60 minutes. Right ventricular contractility, right ventriculo-vascular coupling and ventricular interdependence were assessed in vivo ad different preloads by biventricular conductance catheters prior and after 60 minutes infusion. Heart and lung biopsies were obtained to determine oxidative stress by reduced to oxidized glutathione (GSH/GSSG) ratio and high energy phosphates content. S-NO-HSA compared to HSA infusion resulted in a significant reduction in right ventricular afterload expressed by effective pulmonary arterial elastance (Ea) (0.49 +/- 0.3 versus 1.2 +/- 0.2 mmHg/ml; P = 0.0005). S-NO-HSA significantly improve right ventricular diastolic function (slope of end-diastolic pressure-volume relations) but not contractility (slope of preload recruitable stroke work), whereas a significant increase in the efficiency of ventricular-vascular coupling (ratio of end-systolic to pulmonary arterial elastances: Ees/Ea) occurred after S-NO-HSA but not HSA infusion (from 0.35 +/- 0.17 to 0.94 +/- 0.21; P = 0.005 and from 0.37 +/- 0.11 to 0.43 +/- 0.19; P = 0.1 respectively) with significant increase in left ventricular stroke volume (56 +/- 4 vs 9 +/- 5 %; P = 0.0013). S-NO-HSA compared to HSA treatment significantly improved ATP and PCr right ventricle myocardial content (13.9 +/- 1.1 vs. 7.0 +/- 1.8 and 5.9 +/- 3.3 vs. 1.9 +/- 0.6 micromol/g protein; P = 0.01) and increase tissue GSH/GSSG ratio (P = 0.001). CONCLUSIONS: S-NO-HSA reduces pulmonary hypertension and improves right ventricular diastolic function and right ventricular-arterial coupling with a positive effect on ventricular interdependence. This results in an increased energetic reserve of the heart and reduction of oxidative stress.

CD20, CD22, CD23, but Not CD37 Expression on CD19+ B-Cells Is Altered by Exogenous Factors in a Sub-Population of Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia (CLL/SLL) Patients,

Abstract 3685 Introduction:Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia (CLL/SLL) is a lymphoproliferative disorder that is characterized by the slow accumulation of malignant B cells. Patients follow heterogeneous clinical courses. The effectiveness of Rituximab, an anti-CD20 monoclonal antibody (mAb), is limited because target B cells from CLL patients express low levels of CD20. We previously reported that human serum suppresses CD20 expression ex vivo. Therefore, understanding mechanisms of low expression of CD20 as well as other target surface receptors would benefit CLL patients. New therapies such as epratuzumab (anti-CD22 mAb), lumiliximab (anti-CD23 mAb), and TRU-016 (anti-CD37 SMIP protein) are being investigated as alternative means to treat CLL. We therefore investigated CD20, CD22, CD23, and CD37 expression during ex vivo culture of CLL B cells from thirteen patients with and without human serum albumin (HSA), normal donor serum (NS), autologous CLL serum (CS), fetal bovine serum (FBS), IL2, IL4, IL13, IL15, IL21, TNFα, IFNα, IFNγ, G-CSF, or GM-CSF. Methods: Peripheral blood (PB) from thirteen CLL patients and five healthy normal donors was obtained with IRB approval. Nine CLL patients were Rai 0/1, one Rai 4, and three unknown. Prognosis, as determined by cytogenetics and/or FISH, was: four good, three intermediate, one poor, and five unknown. Beta-2 Microglobulin (B2M) ranged from 1.4–7.1 mg/L. CD19+ cells, isolated by positive magnetic selection, were cultured in AIM-V serum-free media at 37°C, 5% CO2, 100% humidity for 0, 24, 48, and 96 hours. Cells were either untreated, treated with 5% serum, or 5ng/mL cytokine. Receptor expression was measured by multivariate flow cytometry and calculated as percent loss or increase in response to treatment. Data was presented as mean ± SEM and analyzed using the Mann-Whitney U test as compared to HSA treatment. Results: Prior to culture, CD19+ CLL cells had 11.7±1.7% CD20, 40.3±7.9% CD22, 60.6±6.8% CD23, and 81.6±2.7% CD37 positive expression (N=13). Analysis of untreated CLL cells cultured for 96 hours showed that the CLL patients subdivided into two groups based on changes in CD20 expression: variable (72.5±7.4% CD20+, N=9) and stable (27.2±4.2% CD20+, N=4). Within the variable group, losses in CD20 expression resulted from treatment as compared to HSA were: NS (40.2±3.6%, p<0.05), CS (43.6±8.8%, p<0.05), FBS (37.2±5.0%, p=0.064), IL2 (11.5±5.1%, p=0.860), IL4 (33.3±8.9%, p=0.185), and IL13 (24.2±9.1%, p=0.596). The percent loss in CD20 expression due to IL4 was significantly greater than IL2 (p<0.05). Percent losses in CD22 expression were: NS (62.4±9.4%, p<0.05), CS (63.2±8.1%, p<0.05), and FBS (66.6±6.8%, p<0.05). No loss in CD37 expression was significant. Percent increases in CD23 expression were: IL4 (253.7±79.4%, p=0.930) and IL13 (86.7±77.9%, p=0.377). Within the stable group after treatment, percent losses in CD22 were: IL2 (40.0±10.1%, p=1.000) and IL4 (29.1±6.5%, p=0.114). Percent increase in CD37 expression was: FBS (53.3±18.3%, p<0.05). Conclusions: Our findings show that cells from CLL patients form groups based on the ability of cells to maintain low CD20 expression after serum-free culture for 96 hours: variable and stable. Only CLL cells within the variable group have a high upregulation of CD20. These patient samples respond to serum through a reduction of CD20. In addition, suppression of CD20 expression due to IL4 or IL13 treatment was similar to that of serum. Both IL4 and IL13 share a common receptor that signals through the JAK/STAT pathway, suggesting downstream signals may be responsible for suppression of CD20 expression in CLL. Similarly, CLL patients belonging to the variable group also have reduced expression of CD22 in response to serum. Loss of CD23 expression in CLL cells cultured over time, in the absence of IL4 or IL13 treatment, may be predictive of poor clinical outcomes in patients treated with the anti-CD23 monoclonal antibody. CD20, CD22, and CD23 are susceptible to environmental cues, whereas expression of CD37 remained high throughout culture and independent of treatment. As a result, we predict CD37 to be a good target for treating CLL due to its stability. Ongoing clinical trials with TRU-016 or other agents that specifically target CD37 may therefore be of potential therapeutic benefit. Disclosures:No relevant conflicts of interest to declare.

Human serum albumin improves endothelial dysfunction and survival during experimental endotoxemia: Concentration-dependent properties*

The mechanisms by which human serum albumin might protect against sepsis-induced organ dysfunction and improve survival are not elucidated. The present study was designed to assess the effects of two concentrations of human serum albumin on endotoxin-induced mortality as well as on endothelial and organ dysfunctions in both mouse and cell models. Prospective, randomized, controlled experimental study. University research laboratories. Swiss mice (n = 10-15/group) were injected with either lipopolysaccharide or vehicle. Four and 12 hrs later, mice were infused or not with human serum albumin HSA (4% or 20%, 10 mL/kg) or normal saline (0.9% NaCl, 30 mL/kg). Human uterine vein endothelial cells were exposed to both lipopolysaccharide and tumor necrosis factor-α during 8 hrs in the presence or absence of human serum albumin (4% or 20%). Mice survival, reactivity of mesenteric arteries, and Western blot protein analysis were assessed. Circulating endothelin-1, gluthatione, gluthatione disulfide, and creatinine plasma levels were measured. Nitric oxide production, oxidative, and nitrosative stresses were also measured in situ in endothelial cells. Human serum albumin 4%, but not human serum albumin 20% or normal saline solution, improved survival time of endotoxemic mice. Furthermore, human serum albumin 4% activated endothelial nitric oxide synthase and restored lipopolysaccharide-impaired flow-dependent endothelial dilation in mesenteric arteries. This was associated with a downregulation of nuclear factor κB and an upregulation of nuclear respiratory factor-2 and heme oxygenase-1. Human serum albumin 4% reduced lipopolysaccharide-induced renal dysfunction, enhanced endothelin-1 production and glutathione plasmatic levels, whereas human serum albumin 20% increased gluthatione disulfide. Furthermore, human serum albumin 4% but not 20% blunted lipopolysaccharide-tumor necrosis factor-α-induced oxidative and nitrosative stresses in endothelial cells and increased their gluthatione levels. The present data confirm a protective effect of 4% human serum albumin treatment both on mice survival and endothelial dysfunction by inhibiting inflammatory and oxidative stress pathways induced by endotoxins. Conversely, higher concentrations of human serum albumin were detrimental suggesting a dose-dependent effect.

Unique cyanide adduct in human serum albumin: potential as a surrogate exposure marker.

Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.

Presence of Soluble CD26 Peptidase Activity in Human Serum Albumin (FlexBumin): Implications for Cell Freezing and Thawing Protocols during Transplantation

Hematopoietic cells are commonly exposed to human serum albumin (HSA) in the clinical setting and fetal bovine serum (FBS) in the research setting. Serum is utilized as a standard reagent across multiple research disciplines for tissue culture (10–20% FBS). HSA is a common reagent in the clinical setting and is routinely added to hematopoietic cells (0.5–4% HSA) in preparation for cryopreservation. Many labs also wash cryopreserved hematopoietic stem cells with HSA (1–5%) prior to autologous or allogenic transplantation to rid the cells of the DMSO used during cryopreservation. Lastly, HSA is sometimes infused directly into patients in the clinic for such indications as the short-term treatment of nephrotic syndrome. We have generated data suggesting that the peptidase CD26 (dipeptidylpeptidase IV/DPPIV) regulates HSC/HPC function, release of HPC out of the bone marrow (BM) during G-CSF induced mobilization, and engraftment of HSC/HPC into the BM during transplantation. It is within this context that we evaluated the presence of CD26/DPPIV activity in HSA. HSA of 20%, 10%, 2.5%, and 1% were prepared by diluting Flexbumin 25% (Baxter Healthcare) with 0.9% NaCl. FBS (Hyclone) concentrations were prepared likewise. Bovine Serum Albumin Fraction V (BSA) concentrations were prepared weight by volume. CD26/DPPIV activity was assayed utilizing the Gly-Pro-p-nitroanilide substrate in a 96-well format. Standard p-nitroanilide (pNA) curves were created by serially diluting pNA with 5mM Tris-HCl pH 7.5. 4mM Gly-Pro-pNA substrate was then combined with each test solution in 5mM Tris-HCl. Absorbance readings (A405) were then taken at 37° C every 5 minutes for 60 minutes. CD26 inhibition was achieved by treating with Diprotin-A (Ile-Pro-Ile). Prior to measurement of CD26 activity, HSA, BSA, or FBS were combined with Diprotin-A incubated at 37° C for 15 minutes. Final Diprotin-A concentrations during treatment were 5000μM, 500μM, 50μM, 5μM, 0.5μM, 0.05μM, 0.005μM, and 0 μM. Evaluation of CD26 activity in 1%, 2.5%, 10%, and 20% HSA solutions revealed a statistically significant and dose dependent level of CD26 activity within the serum (p<0.05). CD26 activity of 10% HSA was quantitated at 49.62±1.5 pmoles/min and activity of 10% FBS was quantitated at 53.42±1.44 pmoles/min. Evaluation of CD26 activity in 2.5%, 10%, or 20% FBS also showed a quantifiable and dose dependent CD26 activity (p< 0.05). 1%, 2.5%, 10%, and 20% BSA Fraction V had no detectable levels of CD26 activity as compared to saline alone. Furthermore, treatment with the CD26 inhibitor, Diprotin A, actively inhibits CD26 activity ex vivo in a dose dependent manner (p< 0.05). These results suggest that significant levels of CD26 activity are present in HSA (Flexbumin) that is utilized routinely for clinical purposes. Additional evaluations are indicated to test whether this is an industry wide phenomenon across multiple preparation procedures and manufactures. Additionally, these results suggest that the CD26 activity that is present is specific and can be inhibited utilizing CD26 inhibitors. No clinical protocol methodology currently exists for the treatment of HSA in the clinical or research setting. Lastly, these results suggest that the use of HSA that may contain CD26 activity during clinical freezing and thawing procedures of transplant units should be reconsidered, especially if the HSA remains with the unit during infusion. Future studies will be needed to evaluate if the results these pre-clinical experiments necessitate changes to clinical methodologies.

Human Serum Albumin Improves Arterial Dysfunction during Early Resuscitation in Mouse Endotoxic Model via Reduced Oxidative and Nitrosative Stresses

Human serum albumin (HSA) is used as a resuscitation fluid in sepsis. This study investigated the potential protective properties of HSA on vascular function in a mouse endotoxic model in terms of oxidative and nitrosative stresses. Swiss mice were treated with either lipopolysaccharide (LPS) (50 mg/kg i.p.) or vehicle. One and five hours later, mice were infused with HSA (4%, 10 ml/kg), normal saline (0.9% NaCl, 30 ml/kg), or no fluid. Six hours after treatment, vascular reactivity was assessed on aortae and small mesenteric arteries. Measurements of NO and superoxide anion (O2(-)) by spin trapping and nuclear factor (NF)-kappaB, inducible NO synthase (iNOS), and peroxynitrite by Western blotting and immunohistochemical studies were conducted. HSA partially prevented the reduction of blood pressure induced by LPS and completely prevented both vascular hyporeactivity to phenylephrine and myogenic tone as well as endothelial dysfunction induced by the endotoxin. This was associated with a decreased up-regulation of NF-kappa B, iNOS, and peroxynitrite in the vascular wall. LPS-induced tissue increases in both NO and O2(-) production was decreased by HSA. These data demonstrate the protective effect of HSA treatment in experimental endotoxic shock by reducing the inflammatory process leading to oxidative and nitrosative stresses and vascular hyporeactivity.