Impact of hypoalbuminemia on the prognosis of relapsed/refractory B-cell lymphoma treated with axicabtagene ciloleucel.

INTRODUCTION: Hypoalbuminemia is a known adverse prognostic factor in lymphomas. Yet, it is unknown if axicabtagene ciloleucel (axi-cel) overcomes the adverse prognostic impact of hypoalbuminemia in relapsed/refractory large B-cell lymphoma. METHODS: We conducted a retrospective analysis across three Mayo Clinic centers to assess the relationship of hypoalbuminemia (defined as a serum albumin (SA) levels ≤ 3.5 g/dL) on outcomes of patients treated with axi-cel. RESULTS: This analysis included 81 patients. Two patients had no available SA levels preceding axi-cel infusion. Eighteen patients (22.8%) had hypoalbuminemia with a median SA of 3.3 g/dL. Patients with normal SA had a statistically higher ORR than those without hypoalbuminemia (P = .018). There was no difference in 1-year PFS and OS between the group with hypoalbuminemia and the group with normal SA levels (48% vs 49%, P = .81) and (74% vs 73%, P = .97), respectively. There was no difference in the severity or median duration of cytokine release syndrome or neurotoxicity between the two groups. CONCLUSION: Notwithstanding the limitations related to the relatively small sample size, axi-cel therapy appears to overcome the adverse effect of hypoalbuminemia on OS and PFS. Large multicenter clinical studies are certainly needed to validate these findings.

Comprehensive study of dexamethasone on albumin biogenesis during normal and pathological renal conditions.

CONTEXT: Dexamethasone (DXM) has an anti-immunoinflammatory effect, and is often used in acute kidney injury (AKI). However, the effects of DXM on albumin (ALB) have not been fully studied. OBJECTIVE: To investigate the effects of DXM on ALB production and renal function. MATERIALS AND METHODS: Male Wistar rats were divided into normal and DXM groups (0.25, 0.5, 1 mg/kg for 5 days) (n = 15) for a dose-dependent study. Rats were divided into normal group and DXM groups (0.5 mg/kg for 3, 5, 7 days) (n = 9) for a time-dependent study. In AKI experiment, rats were divided into normal (saline), cisplatin (CP, 5 mg/kg, i.v.), CP + DXM groups (0.25, 0.5 and 1 mg/kg, i.m.) (n = 16). The blood and the organs were isolated for analysis. RESULTS: In normal, serum ALB (sALB) and serum total protein (sTP) increased in DXM group with sALB increased 19.8-32.2% (from small to large dosages); and 30.2-32.5.6% (from 3 to 7 days of DXM); sTP 15.7-22.6% and 14.2-24.3%; urine ALB (uALB) 31.5-392.3%, and 1047.2-1390.8%; urine TP (uTP) 0.68-173.1% and 98.0-504.9%, compared with normal groups. DXM increased the mRNA expression of Cebp and Hnf, suppressing podocin. In AKI, DXM decreased serum BUN (53.7%), serum Cre (73.4%), sALB (30.0%), sTP (18.7%), uALB (74.5%), uTP (449.3%), rescuing the suppressed podocin in kidney. CONCLUSIONS: DXM acts on Cebp and Hnf and promotes ALB production. This finding helps to evaluate the rationale of DXM for kidney injury.

Thiol-Disulfide Homeostasis, Serum Ferroxidase Activity, and Serum Ischemia Modified Albumin Levels in Neonatal Jaundice.

AIM: Hyperbilirubinemia causes oxidative stress. METHOD: We evaluated three oxidative stress markers in hyperbilirubinemic neonates (native/total thiol levels, serum ferroxidase activity and ischemia modified albumin (IMA), comparing these levels to levels in a control group to determine which indicators were the most sensitive. RESULTS: Serum from 124 term infants (67 with pathologic jaundice and 57 controls) were evaluated. Native/total thiol ratio was significantly lower (p:0.021) while disulfide levels were significantly higher (p:0.001) in the jaundiced group. There was no significant difference in ferroxidase (p:0.603) or IMA (p:0.251) levels. CONCLUSION: Altered thiol/disulfide homeostasis in the favor of disulfide indicates augmented oxidative stress in jaundiced term infants. The lack of alteration in ferroxidase or IMA levels suggests these latter alterations take more time or more severe oxidative stress to become altered or are not as sensitive as the thiol/disulfide ratio to detect oxidative stress states.

Thiol-Disulfide Homeostasis, Serum Ferroxidase Activity, and Serum Ischemia Modified Albumin Levels in Childhood Iron Deficiency Anemia.

Aim: The aim is to compare the markers of oxidative stress in iron deficient children to that of non-anemic children. Method: Serum thiol-disulfide level, ferroxidase activity and ischemia-modified albumin (IMA) levels were compared between iron deficiency anemia (IDA) and non-anemic children. Results: A total of 117 children, 66 with IDA and 51 non-anemic children were included in the study. Disulfide, disulfide/native thiol, and disulfide/total thiol levels were significantly higher in the IDA group (p: 0.001). Serum ferroxidase levels were significantly lower in the IDA group (p: 0.04); but there was no significant difference between the two groups regarding serum IMA levels (p: 0.42). There was a weak negative correlation between disulfide and serum hemoglobin (p: 0.004), iron (p: 0.041), and ferritin (p: 0.023) levels while there was a weak positive correlation between ferroxidase activity and these parameters. Conclusion: There is an increased protein oxidation in children with IDA compared with non-anemic controls.

The fractional synthesis rates of plasma proteins as determined using deuterated water are sensitive to dietary intake of lysine in rats.

Traditionally, the effect of dietary lysine upon health is determined through the concentrations of plasma proteins, but sometimes they are not responsive to lysine intake. We hypothesized that the fractional synthesis rates (FSRs) of plasma proteins may be more sensitive to dietary intake of lysine than protein concentrations in plasma. Seventy-two male Sprague-Dawley rats were divided randomly into three groups based on their diets provided for 18 weeks: low lysine (LG), normal lysine (NG) and high lysine (HG). Rats underwent labeling with deuterated water, a more reliable tracer than amino-acid tracers. The FSRs of albumin and immunoglobulin (Ig) G in plasma increased with increasing dietary intake of lysine. However, the albumin concentration in plasma in rats in the LG did not decrease significantly compared with that in the NG, and a similar result was shown for the IgG concentration between the NG and HG. These results suggested that the FSRs of albumin and IgG in plasma were more sensitive to dietary intake of lysine than their concentrations, and could be useful as sensitive indicators of the effect of dietary lysine upon health.

The value of glycated albumin for the prediction of graft outcome in the non-human primate porcine islet transplantation model.

BACKGROUND: The development of a precise and easy-to-use tool for monitoring islet graft function is important in clarifying the causes of graft loss, identifying appropriate therapy, and ensuring graft survival in the nonhuman primate (NHP) model of porcine islet transplantation (PITx). Glycated albumin (GA) is an indicator of intermediate-term changes in blood glucose control and is useful in clinical diabetes management. The validity of GA for monitoring graft function in NHP recipients of PITx was evaluated using a retrospective analysis of cohort samples. METHODS: Data from a total of 23 PITxs performed in 20 recipients (3 were retransplanted) were included in this study. Islet clusters purified from adult wild-type pigs were transplanted via the intraportal route into streptozotocin-induced diabetic rhesus monkeys with immune suppression. Blood samples were obtained once per week from the recipients until they lost insulin-independence. Blood samples were also obtained from 69 non-diabetic monkeys that served as a control group. The levels of GA and albumin in stored plasma aliquots were measured using each enzymatic method, and the GA result was expressed as the percentage of GA level to the total albumin level. RESULTS: The median level of GA in the recipients on the day of PITx (median 18.6%, 95% confidence interval [CI] 16.7%-20.4%) was significantly higher than that of healthy controls (median 9.14%, 95% CI 9.0%-9.3%, P < .0001). However, the level decreased after PITx and remained low or increased depending on the extent of residual graft function. The GA level at a nadir (median 11.6%, 95% CI 10.8%-13.0%) and the time to reach a nadir (median 43 days, 95% CI 21.7-69.3 days) both correlated with the duration of insulin-independence (rho [ρ] = -.605, P = .0028 and ρ = .662, P = .0008, respectively). The GA level strongly correlated with KG , the glucose disappearance rate during intravenous glucose tolerance testing (ρ = -.76, P < .0001). At post-transplant week (PTW) 3 and at PTW 4, the GA levels in recipients with long-term insulin-independence (>90 days) were significantly lower than those with short-term insulin-independence, which revealed the excellent performance for the prediction of long-term insulin-independence that is comparable to that of porcine C-peptide (historic data). CONCLUSIONS: As a surrogate indicator for graft function, serial measurement of GA may provide Supporting Information to that obtained from conventional monitoring techniques of graft function for assessing porcine islet grafts in NHP models.

Simultaneous assessment of the synthesis rate and transcapillary escape rate of albumin in inflammation and surgery.

BACKGROUND: Better knowledge of albumin kinetics is needed to define the indications for albumin use in clinical practice. This study involved two approaches: the synthesis rate and transcapillary escape rate of albumin were measured simultaneously at different levels of plasma albumin concentration in relation to acute inflammation and surgery; and two different tracers were compared to determine plasma volume and the transcapillary escape rate. METHODS: Healthy volunteers (n = 10), patients with acute inflammatory abdominal disease (n = 10), and patients undergoing elective pancreatic resection (n = 10) were studied. The albumin synthesis rate was measured by the incorporation of deuterium-labeled phenylalanine. Plasma volume and the transcapillary escape rate were assessed using 123I-labeled and 125I-labeled albumin. RESULTS: A 50 % elevated de-novo albumin synthesis rate was seen in patients with acute inflammation and marked hypoalbuminemia, while patients with marginal hypoalbuminemia before the start of surgery had a normal albumin synthesis rate. The transcapillary escape rate was elevated intraoperatively during the reconstructive phase of pancreatic surgery, when plasma albumin was decreased but stable. In acute inflammation with marked hypoalbuminemia, the transcapillary escape rate was no different from normal. 123I-labeled and 125I-labeled albumin were found exchangeable for plasma volume determinations, but could be used only in groups of patients for the transcapillary escape rate. CONCLUSIONS: This observational study illustrates the limited information contained in albumin plasma concentrations to reflect albumin kinetics. On the contrary, single measurements of the synthesis rate and/or transcapillary escape rate of albumin obviously cannot explain the plasma level of albumin or the changes seen in plasma albumin concentration. TRIAL REGISTRATION: www.clinicaltrials.gov , study number NCT01686776 . Registered 13 September 2012.

Expression and bioactivity of recombinant human serum albumin and dTMP fusion proteins in CHO cells.

The 14-amino acid (IEGPTLRQWLAARA) thrombopoietin mimetic peptide (TMP) shares no sequence homology with native thrombopoietin (TPO). When dimerized, it displays a high-binding affinity for the TPO receptor and has equipotent bioactivity with recombinant human TPO (rhTPO) in stimulating proliferation and maturation of megakaryocytes in vitro. However, TMP is limited for clinical usage because of its short half-life in vivo. In this study, fusion proteins that composed of tandem dimer of TMP (dTMP) genetically fused at the C- or N-terminus of human serum albumin (HSA) were separately expressed in Chinese hamster ovary (CHO) cells. In vitro bioactivity assays showed that purified fusion proteins promoted the proliferation of megakaryocytes in a dose-dependent manner and activated signal transducer and activator of transcription (STAT) pathway in TPO receptor-dependent manner. Following subcutaneous administration, both HSA-dTMP and dTMP-HSA significantly elevated peripheral platelet counts in normal mice in a dose-dependent manner. In addition, fusion with HSA successfully prolonged dTMP half-life in mice. However, when HSA was fused at the C-terminus of dTMP, the bioactivity of dTMP-HSA was about half of that of HSA-dTMP. In conclusion, these results suggested that HSA/dTMP fusion proteins might be potential drugs for thrombocytopenia and, when HSA was fused at the N-terminus of dTMP, the fusion protein had a higher activity.

Impairment of liver synthetic function and the production of plasma proteins in primary breast cancer patients on doxorubicincyclophosphamide (AC) protocol.

Doxorubicin and Cyclophosphamide (AC protocol) combination is usually considered as a first line therapy in newly diagnosed breast cancer patients. Thus, a retrospective observational study was conducted to monitor the effect of AC protocol on liver synthetic functions and production of plasma proteins in breast cancer patients, reporting to specialized cancer care hospital of Lahore, Pakistan. A total of 75 patients (n=75) on AC protocol with breast cancer were observed in this study. The patient data including age, gender, body surface area, dosage, disease status and laboratory biochemical values were recorded by reviewing historical treatment records. Pre-treatment values were taken as baseline values for albumin, globulin, blood urea nitrogen (BUN), albumin/globulin (A/G) ratio and total proteins. The baseline values were compared after each cycle of by applying ANOVA using statistical tool SPSS® version 21. The plasma levels of blood urea nitrogen (BUN), total protein and globulin dropped significantly (p<0.05) in patients of all age groups. However, the albumin levels were not significantly changed (p>0.05). The A/G ratio level increased (p<0.05) as a result of reduction in globulin levels. Significant changes in plasma protein levels were observed in the elderly patients (50 to 65 years) than patients between 20 to 50 years of age. AC protocol impairs liver synthetic functions as observed by decreased blood urea nitrogen (BUN) and plasma protein levels.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Human serum albumin from recombinant DNA technology: challenges and strategies.

BACKGROUND: As the most abundant protein in the blood, human serum albumin (HSA) plays an important role in maintaining plasma oncotic pressure and fluid balance between the body's compartments. HSA is thus widely used in the clinic to treat diseases. However, the shortage of and safety issues arising from using plasma HSA (pHSA) underscore the importance of recombinant HSA (rHSA) as a promising substitute for pHSA. SCOPE OF REVIEW: Here, we review the production of rHSA, from expression to downstream processing, and highlight the scalability and cost-effectiveness of the two main expression platforms. We also discuss the biosafety of commercially available pharmaceutical rHSA with respect to impurities and contaminants, followed by an analysis of recent progress in preclinical and clinical trials. We emphasise the challenges of producing pharmaceutical-grade rHSA. MAJOR CONCLUSIONS: rHSA can be highly expressed in various hosts and seems to be identical to pHSA. rHSA generated from yeast appears to be as efficient and safe as pHSA in a series of preclinical and clinical trials, whereas rHSA from rice seeds exhibits great potential for more cost-effective production. Cost-effective products with no adverse effects will likely play a vital role in future human therapeutics. GENERAL SIGNIFICANCE: Our understanding of pharmaceutical-grade rHSA production has improved with respect to expression hosts, biochemical properties, downstream processing, and the detection and removal of impurities. However, due to the large dosages required for clinical applications, the production of sufficient quantities of rHSA still presents challenges. This article is part of a Special Issue entitled Serum Albumin.

Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model.

BACKGROUND: The liver has a crucial role in metabolic homeostasis as well as being the principal detoxification centre of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of detrimental health consequences of engineered NM exposure. It has been shown that exposure via inhalation, intratracheal instillation or ingestion can result in NM translocation to the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e. reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). METHODS: In order to rectify these issues and for the very first time we have utilised a 3D human liver microtissue model to investigate the toxicological effects associated with a single or multiple exposure of a panel of engineered NMs (Ag, ZnO, MWCNT and a positively charged TiO2). RESULTS: Here we demonstrate that the repeated exposure of the NMs is more damaging to the liver tissue as in comparison to a single exposure with the adverse effects more significant following treatment with the Ag and ZnO as compared with the TiO2 and MWCNT NMs (in terms of cytotoxicity, cytokine secretion, lipid peroxidation and genotoxicity). CONCLUSIONS: Overall, this study demonstrates that the human microtissue model utilised herein is an excellent candidate for replacement of traditional in vitro single cell hepatic models and further progression of liver nanotoxicology.

Large-scale production of functional human serum albumin from transgenic rice seeds.

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.

Acute-phase proteins and mortality in status epilepticus: a 5-year observational cohort study.

OBJECTIVE: Acute-phase proteins, such as C-reactive protein and albumin, may be related with course and outcome in status epilepticus, as changes of cytokine levels and blood-brain barrier breakdown during status epilepticus have been demonstrated. The aim of this study was to elucidate the association of C-reactive protein and albumin with course and outcome of status epilepticus. DESIGN: Observational cohort study. SETTING: This study was performed on the ICU of a university-affiliated tertiary care center. PATIENTS: All consecutive patients with status epilepticus from 2005 to 2009 were selected from a prospectively established electroencephalography database. INTERVENTION: None. MEASUREMENTS: Albumin was assessed at admission and status epilepticus onset, and C-reactive protein was assessed during the first 3 days of status epilepticus. Outcomes were defined as refractory status epilepticus and death. MAIN RESULTS: One hundred thirty-five consecutive status epilepticus patients were analyzed. Patients with higher levels of albumin at status epilepticus onset had significant lower odds for the development of refractory status epilepticus and death (with every 1g/L: odds ratio 0.91, 95% confidence interval 0.86-0.96, p = 0.001; odds ratio 0.88, 95% confidence interval 0.82-0.95, p < 0.0001, respectively). These associations remained significant after multiple adjustments for possible confounders and correction for multiple comparisons (with every 1g/L: odds ratio 0.92, 95% confidence interval 0.86-0.97, p = 0.004; odds ratio 0.87, 95% confidence interval 0.80-0.94, p = 0.001, respectively). Increased C-reactive protein levels at status epilepticus onset were associated with higher rates of refractory status epilepticus and death (with every 1mg/L: odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.021; odds ratio 1.01, 95% confidence interval 1.00-1.02, p < 0.007, respectively). These associations were inconsistent after adjustment for possible confounders and corrections for multiple comparisons (with every 1mg/L: odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.109; odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.043). CONCLUSIONS: Albumin levels measured early in status epilepticus are independently associated with refractory epileptic activity and death while C-reactive protein levels were inconsistent. Further studies are needed to assess the potential of acute-phase proteins for inclusion in prediction models allowing to identify patients with poor outcome of status epilepticus.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis.

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

Chicken hypersensitive site-4 insulator increases human serum albumin expression in bovine mammary epithelial cells modified with phiC31 integrase.

Achieving high expression levels of recombinant human serum albumin (HSA) for purification is a solution for the large amount of plasma-derived HSA needed in therapeutic applications. Here, we employed phiC31 integrase system and chicken hypersensitive site-4 (cHS4) insulators to construct a HSA expression vector for high-level HSA expression. The phiC31 integrase system mediated efficient transgene integration in bovine mammary epithelial cells (bMECs). A preferred pseudo attP site, which had 38 % identity with the 39 bp wild-type attP sequence, was detected in six out of 55 bMEC colonies. Addition of the cHS4 insulator to the phiC31 integrase system resulted in 8-20-fold increases of HSA expression compared with that of using integrase alone. Moreover, the reverse-oriented cHS4 insulator in the phiC31 integrase system provided the optimal level of HSA expression in bMECs.

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115.

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.

Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris.

Interleukin-2 (IL-2) plays important roles in variety of immune functions. Recombinant IL-2 has become an important therapeutic protein for therapy of melanoma and renal cell carcinoma. Previously, it was proved that the therapeutic efficacy of rIL-2 expressed in Saccharomyces cerevisiae was improved by prolonging its in vivo half-life through genetic fusion with albumin. In this study, a fusion protein composed of hIL-2 genetically fused to HSA was expressed as a secretory protein under AOX1 (alcohol oxidase 1) promoter in Pichia pastoris. An effective strategy was established to express rhIL-2-HSA fusion protein in 5L scale and the optimal purification procedure was investigated. The purity of rhIL-2-HSA in final product was about 95%. The purified rhIL-2-HSA fusion protein could be recognized by both anti-hIL-2 and anti-human serum albumin monoclonal antibody. Bioactivity analysis showed that the purified rhIL-2-HSA fusion protein displayed high level activity on proliferation in IL-2 dependent manner in CTLL2-cells. rhIL-2-HSA fusion protein also showed a extended half-life in plasma compared with IL-2 when tested in a BALB/c mouse model. This study provides an alternative strategy for large-scale production of bioactive rhIL-2-HSA fusion protein using P. pastoris as an expression host.

Fibrinogen and albumin synthesis rates in major upper abdominal surgery.

Plasma fibrinogen and albumin concentrations initially decrease after abdominal surgery. On postoperative days 3-5 fibrinogen concentration returns to the preoperative level or even higher, while albumin stays low. It is not known if these altered plasma concentrations reflect changes in synthesis rate, utilization, or both. In particular a low albumin plasma concentration has often been attributed to a low synthesis rate, which is not always the case. The objective of this study was to determine fibrinogen and albumin quantitative synthesis rates in patients undergoing major upper abdominal surgery with and without intact liver size. Patients undergoing liver or pancreatic resection (n = 9+6) were studied preoperatively, on postoperative days 1 and 3-5. De novo synthesis of fibrinogen and albumin was determined; in addition, several biomarkers indicative of fibrinogen utilization were monitored. After hemihepatectomy, fibrinogen synthesis was 2-3-fold higher on postoperative day 1 than preoperatively. On postoperative days 3-5 the synthesis level was still higher than preoperatively. Following major liver resections albumin synthesis was not altered postoperatively compared to preoperative values. After pancreatic resection, on postoperative day 1 fibrinogen synthesis was 5-6-fold higher than preoperatively and albumin synthesis 1.5-fold higher. On postoperative days 3-5, synthesis levels returned to preoperative levels. Despite decreases in plasma concentrations, de novo synthesis of fibrinogen was markedly stimulated on postoperative day 1 after both hemihepatectomies and pancreatectomies, while de novo albumin synthesis remained grossly unchanged. The less pronounced changes seen following hepatectomies were possibly related to the loss of liver tissue.

SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics.

The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.