Comparative Analysis of Post-Translational Modifications of Human Serum Albumin Derived from Human Plasma and Recombinant Sources in China.

BACKGROUND: The variations in sequence, three-dimensional structure, and post-translational modifications (PTMs) of human serum albumin (HSA) are crucial for its physiological functions. This study aims to analyze and compare the disparities in PTMs between HSA derived from human plasma and genetically recombinant sources for clinical treatments in China. METHODS: Six distinct PTMs, namely acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, were identified using pan-specific antibodies via Western blot analysis. The samples, comprising human plasma-derived HSA (pHSA) from six different manufacturers and recombinant HSA (rHSA) expressed in yeast and Oryza sativa, underwent detection for various types of PTMs. Additionally, a 4D label-free quantitative proteomic analysis was performed to identify N-glycosylation and the aforementioned PTMs in both pHSA and rHSA samples. This analysis aimed to discern disparities in modification sites and levels. RESULTS: Through Western blot analysis, all six pHSA and two rHSA samples displayed positive bands for albumin (66.5 kDa) across the six PTMs. Subsequent analysis using 4D label-free quantitative proteomics revealed 25 (29) acetylated, 30 (32) succinylated, 41 (50) malonylated, 15 (23) phosphorylated, 36 (30) beta-hydroxybutyrylated, and 27 (34) lactylated modification sites in pHSA and rHSA samples, with no N-glycosylation modification sites detected. The analysis identified 1 acetylation (ALB_K160), 2 beta-hydroxybutyrylation (ALB_K569, ALB_K426), and 3 crotonylation (ALB_K264, ALB_K581, ALB_K560) specific modification sites in pHSA, as well as 3 crotonylation (ALB_K560, ALB_K562, ALB_K75), 1 succinylation (ALB_K490), and 23 phosphorylation specific modification sites in rHSA. In pHSA (rHSA), 2 (6) acetylation, 10 (12) succinylation, 0 (9) crotonylation, 1 (9) phosphorylation, 6 (0) beta-hydroxybutyrylation, and 0 (7) lactylation specific modification sites were found. Moreover, in the shared modification sites between pHSA and rHSA, pHSA exhibited up-regulation of amberylation (16:1) and beta-hydroxybutyrylation (12:2) in more sites, and up-regulation of acetylation (7:11), crotonylation (2:11), phosphorylation (1:8), and lactylation (1:14) in fewer sites compared to rHSA. CONCLUSION: In clinical practice, both pHSA and rHSA utilized in China commonly display acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation. Notably, there exist distinctions in the site characteristics and modification levels of these alterations between pHSA and rHSA. Further experimental inquiries are imperative to delve into the implications of these disparities in PTMs on the biological functionality, effectiveness, and safety of pHSA and rHSA.

Effect of apheresis plasma donation on plasma uric acid levels, the lipid profile, and major plasma proteins in plasma donors in China: A multicenter, prospective cohort study.

Abnormal plasma uric acid (UA) levels, the lipid profile, and plasma proteins in blood are associated with a range of adverse health outcomes. This multicenter, prospective cohort study aimed to determine the possible effects of multiple apheresis plasma donations on plasma UA levels, the lipid profile, and major proteins in plasma donors. Participants were enrolled from 1 April 2021 to 31 August 2022. When their plasma UA (men: >420 µmol/L, women: >360 µmol/L) and/or lipid levels (total cholesterol [TC]: ≥6.2 mmol/L, triglycerides [TGs]: ≥2.3 mmol/L, low-density lipoprotein cholesterol: ≥4.1 mmol/L, or high-density lipoprotein cholesterol [HDL-C]: <1.0 mmol/L) were abnormal at their first plasma donation, the enrolled participants were followed up until they had completed 10 plasma donations. A total of 11485 participants were enrolled, of whom 1861 met the inclusion criteria. During the study period, 320 donors completed 10 plasma donations. None of the participants took any corrective medicine for their abnormal index. The measured parameters were significantly different from the first to the tenth plasma donations (donors with asymptomatic hyperuricemia: UA, P < 0.001; donors with asymptomatic hyperlipidemia: HDL-C, P < 0.001; TC, P = 0.025; TGs, P < 0.001; apolipoprotein B, P = 0.025; all of the plasma donors, immunoglobulin G, P < 0.001). The levels of HDL-C, TC, and apolipoprotein B were increased, and the levels of UA, TGs, and immunoglobulin G were decreased over this time. However, immunoglobulin G levels were still in the normal range. Moreover, the changes in these parameters were closely associated with the frequency of plasma donation during the study period. Repeated apheresis plasma donations can reduce plasma UA and TG levels and increase HDL-C levels; and further evaluation of the clinical significance with a larger sample size is required.

Intravenous immunoglobulin protects the integrity of the intestinal epithelial barrier and inhibits ferroptosis induced by radiation exposure by activating the mTOR pathway.

Radiation exposure often leads to serious health problems in humans. The intestinal epithelium is sensitive to radiation damage, and radiation causes destruction of the intestinal epithelial barrier, which leads to radiation enteritis (RE), the loss of fluids, and the translocation of intestinal bacteria and toxins; radiation can even threaten survival. In this study, we aimed to explore the influence of IVIg on the integrity of the intestinal epithelial barrier after RE. Using a RE mouse model, we investigated the protective effects of intravenous immunoglobulin (IVIg) on the epithelial junctions of RE mice and validated these findings with intestinal organoids cultured in vitro. In addition, transmission electron microscopy (TEM), western blotting (WB) and immunostaining were used to further investigate changes in intestinal epithelial ferroptosis and related signaling pathways. When RE occurs, the intestinal epithelial barrier is severely damaged. IVIg treatment significantly ameliorated this damage to epithelial tight junctions both in vivo and in vitro. Notably, IVIg alleviated RE by inhibiting intestinal epithelial ferroptosis in RE mice. Mechanistically, IVIg promoted activation of the mTOR pathway and inhibited ferroptosis in the intestinal epithelium of mice. Rapamycin, which is a potent inhibitor of the mTOR protein, significantly abolished the protective effect of IVIg against radiation-induced damage to intestinal epithelial tight junctions. Overall, IVIg can prevent RE-induced damage to the intestinal epithelial barrier and inhibit ferroptosis by activating the mTOR pathway; this study provides a new treatment strategy for patients with RE caused by radiotherapy or accidental nuclear exposure.

Study on the Treatment of ITP Mice with IVIG Sourced from Distinct Sex-Special Plasma (DSP-IVIG).

Intravenous immunoglobulin (IVIG) is a first-line drug prepared from human plasma for the treatment of autoimmune diseases (AIDs), especially immune thrombocytopenia (ITP). Significant differences exist in protein types and expression levels between male and female plasma, and the prevalence of autoimmune diseases varies between sexes. The present study seeks to explore potential variations in IVIG sourced from distinct sex-specific plasma (DSP-IVIG), including IVIG sourced from female plasma (F-IVIG), IVIG sourced from male plasma (M-IVIG), and IVIG sourced from a blend of male and female plasma (Mix-IVIG). To address this question, we used an ITP mouse model and a monocyte-macrophage inflammation model treated with DSP IVIG. The analysis of proteomics in mice suggested that the pathogenesis and treatment of ITP may involve FcγRs mediated phagocytosis, apoptosis, Th17, cytokines, chemokines, and more. Key indicators, including the mouse spleen index, CD16+ macrophages, M1, M2, IL-6, IL-27, and IL-13, all indicated that the efficacy in improving ITP was highest for M-IVIG. Subsequent cell experiments revealed that M-IVIG exhibited a more potent ability to inhibit monocyte phagocytosis. It induced more necrotic M2 cells and fewer viable M2, resulting in weaker M2 phagocytosis. M-IVIG also demonstrated superiority in the downregulation of surface makers CD36, CD68, and CD16 on M1 macrophages, a weaker capacity to activate complement, and a stronger binding ability to FcγRs on the THP-1 surface. In summary, DSP-IVIG effectively mitigated inflammation in ITP mice and monocytes and macrophages. However, M-IVIG exhibited advantages in improving the spleen index, regulating the number and typing of M1 and M2 macrophages, and inhibiting macrophage-mediated inflammation compared to F-IVIG and Mix-IVIG.

Parvovirus B19 DNA and antibodies in Chinese plasma donors, plasma pools and plasma derivatives.

Background: Human parvovirus B19 (B19V) is a common contaminant found in plasma pools and plasma derivatives. Previous studies were mainly focused on limited aspects, further assessment of prevalence of B19V DNA and antibodies in plasma donors, the contamination of B19V in pooled plasma and plasma derivatives should be performed in China. Study Design and Methods: Individual plasma donors' samples from four provinces and pooled plasma from four Chinese blood product manufacturers were collected and screened using B19V DNA diagnostic kits between October 2018 and May 2020. The positive samples were investigated for the seroprevalence of B19V antibodies and subjected to sequence analysis and alignment for phylogenetic studies. Moreover, 11 plasma donors who were B19V DNA-positive at their first testing were also followed during the later donation period. Additionally, 400 plasma pools and 20 batches of plasma derivatives produced by pooled plasma with a viral load of B19V DNA exceeding 104IU/mL were also collected and tested for B19V DNA and antibodies. Objectives: To comprehensively and systematically determine the frequency and viral load of B19V DNA in plasma donors, pooled plasma, and plasma derivatives from four Chinese blood product manufacturers. Results: A total of 17,187 plasma donors were analyzed and 44 (0.26%) specimens were found positive for B19V DNA. The quantitative DNA levels ranged from 1.01 × 101 to 5.09 × 1012 IU/mL. Forty-four DNA-positive specimens were also investigated for the seroprevalence of B19V antibodies, 75.0% and 2.3% of which were seropositive for B19V IgG and IgM antibodies, respectively. The phylogenic analyses showed that the prevalent genotypes in the four provinces' plasma donors belonged to B19V Genotype 1. Eleven individual plasma donors who were B19V DNA-positive at the first donation were then followed for a period, and in general, the DNA levels of B19V gradually decreased. Moreover, 64.8% (259/400) of the pooled plasma was contaminated by B19V, with concentrations of 1.05 × 100-3.36 × 109IU/mL. Approximately 72.6% of the DNA-positive plasma pools were only moderately contaminated (<104 IU/mL), while 27.4% contained >104 IU/mL. Twenty batches of plasma derivatives produced by pooled plasma with a viral load of B19V DNA exceeding 104IU/mL were also tested. B19V was detected in 5/5 PCC samples and 5/5 factor VIII samples but was not found in the intravenous immune globulin and albumin samples. Conclusion: The contamination of B19V in pooled plasma and plasma-derived clotting factor concentrates is serious. Whether B19V nucleic acid testing (NAT) screening of plasma and plasma derivatives is launched in China, blood product manufacturers should spontaneously perform B19V NAT screening in plasma donors and mini-pool plasma. These measures can ensure that samples with high titer B19V DNA are discarded in order to prevent and control this transfusion transmitted virus.

Blood Service in a Region of China's Qinghai-Tibetan Plateau.

Objectives: The purpose of this paper is to describe blood services in the Aba Tibetan and Qiang Regions, (hereinafter referred to as Aba Prefecture), a region of China's Qinghai-Tibetan Plateau, the third largest area of Tibet and the main inhabited area of the Qiang people. Design: We present a comprehensive investigation into blood donations, donors, screening and supply in the 13 counties of Aba Prefecture based on data from 2013 to 2018. Geography and population were also used to analyze the differences in blood services among different regions. Participants: The number of blood donors totaled 19,047. Results: Over the past 6 years, blood donations have increased by 29 and clinical blood usage by 45%. The blood donation rate was 3.4‱ and per capita blood use was 1.04 mL, both of which were significantly lower than the national average, and blood donation decreased with altitude. It should be noted that the donation rate of the Tibetan and Qiang peoples is much lower than that of the Han population. Moreover, the rejection rate of blood in laboratory testing was found to be higher than the national average, especially in counties located at higher altitudes. Conclusions: Blood donations and usage increased every year in Aba Prefecture, but blood shortage is still an important issue. In addition, the prevalence of transfusion-transmitted diseases is relatively high, which may be linked to lower-education and unfavorable geographical and medical conditions.

Antibody Assay and Anti-Inflammatory Function Evaluation of Therapeutic Potential of Different Intravenous Immunoglobulins for Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease that currently has no known cure. Intravenous immunoglobulin (IVIG), which contains AD-related antibodies and has anti-inflammatory properties, has shown potential as a treatment for AD. However, the efficacy of clinical trials involving AD patients treated with IVIG has been inconsistent. Our previous study found that different IVIGs had significantly varied therapeutic effects on 3xTg-AD mice. In order to investigate the relationship between the composition and function of IVIG and its efficacy in treating AD, we selected three IVIGs that showed notable differences in therapeutic effects. Then, the concentrations of specific antibodies against ß-amyloid (Aß)42, tau, and hyperphosphorylated tau (p-tau) in three IVIGs, as well as their effects on systemic inflammation induced by lipopolysaccharide (LPS) in Balb/c mice, were analyzed and compared in this study. The results indicated that these IVIGs differed greatly in anti-Aß42/tau antibody concentration and anti-p-tau ratio, and improved LPS-stimulated peripheral inflammation, liver and kidney injury, and neuroinflammation in Balb/c mice to varying degrees. Combined with our previous results, the efficacy of IVIG against AD may be positively correlated with its level of AD-related antibodies and anti-inflammatory ability. AD-related antibody analysis and functional evaluation of IVIG should be given sufficient attention before clinical trials, as this may greatly affect the therapeutic effect of AD treatment.

Sexual dimorphism in gut microbiota dictates therapeutic efficacy of intravenous immunoglobulin on radiotherapy complications.

INTRODUCTION: With the mounting number of cancer survivors, the complications following cancer treatment become novel conundrums and starve for countermeasures. Intravenous immunoglobulin (IVIg) is a purified preparation for immune-deficient and autoimmune conditions. OBJECTIVES: Here, we investigated whether IVIg could be employed to fight against radiation injuries and explored the underlying mechanism. METHODS: Hematopoietic or gastrointestinal (GI) tract toxicity was induced by total body or abdominal local irradiation. High-throughput sequencing was performed to analyze the gut microbiota configurations and gene expression profile of small intestine. The untargeted metabolomics of gut microbiome was assessed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. Hydrodynamic-based gene delivery was used to knockdown the target genes in vivo. RESULTS: Intravenous injection of IVIg protected against radiation-induced hematopoietic and GI tract toxicity in female mice but not in males. IVIg structured sex-characteristic gut microbiota configurations in abdominal irradiated mice. The irradiation enriched gut Lachnospiraceae in female mice but reduced those in males. IVIg injection combined with oral gavage of Lachnospiraceae or its metabolite hypoxanthine, alleviated radiation toxicity in male mice however, Lachnospiraceae or hypoxanthine alone failed to ameliorate the injuries. Abdominal local irradiation drove sex-distinct gene expression signatures in small intestine. Mechanistic investigation showed that replenishment of Lachnospiraceae or hypoxanthine offset abdominal radiation-reduced PLD1 expression in male mice. In females, irradiation elevated PLD1 expression. Deletion of PLD1 in GI tract of female mice erased the radioprotective effects of IVIg. CONCLUSION: IVIg battles against radiation injuries in a sex-specific, gut microbiome-dependent way through Lachnospiraceae/hypoxanthine/PLD1 axis. Our findings provide a sex-precise therapeutic avenue to improve the prognosis of cancer patients with radiotherapy in pre-clinical settings.

Analysis of influencing factors of serum total protein and serum calcium content in plasma donors.

Background and objectives: The adverse effects of plasma donation on the body has lowered the odds of donation. The aim of this study was to investigate the prevalence of abnormal serum calcium and total serum protein related to plasma donation, identify the influencing factors, and come up with suggestions to make plasma donation safer. Methods: Donors from 10 plasmapheresis centers in five provinces of China participated in this study. Serum samples were collected before donation. Serum calcium was measured by arsenazo III colorimetry, and the biuret method was used for total serum protein assay. An automatic biochemical analyzer was used to conduct serum calcium and total serum protein tests. Results: The mean serum calcium was 2.3 ± 0.15 mmol/L and total serum protein was 67.75 ± 6.02 g/L. The proportions of plasma donors whose serum calcium and total serum protein were lower than normal were 20.55% (815/3,966) and 27.99% (1,111/3,969), respectively. There were significant differences in mean serum calcium and total serum protein of plasma donors with different plasma donation frequencies, gender, age, regions, and body mass index (BMI), (all p < 0.05). Logistic regression analysis revealed that donation frequencies, age, BMI and regions were significantly associated with a higher risk of low serum calcium level, and donation frequencies, gender, age and regions were significant determinants factors of odds of abnormal total serum protein. Conclusions: Donation frequencies, gender, age, regions, and BMI showed different effects on serum calcium and total serum protein. More attention should be paid to the age, donation frequency and region of plasma donors to reduce the probability of low serum calcium and low total serum protein.

Proteomics profiles of blood glucose-related proteins involved in a Chinese longevity cohort.

BACKGROUND: High blood glucose level is one of the main characteristics of diabetes mellitus. Based on previous studies, it is speculated longevity families may have certain advantages in blood glucose regulation. However, limited information on these items has been reported. The purpose of this study was to profile differences of plasma proteomics between longevity subjects (with normal fructosamine (FUN) level) and non-longevity area participants (with exceeding standard FUN level). METHODS: In this study, a TMT-based proteomics analysis was used to profile differences of plasma proteomics between longevity subjects (with normal FUN level) and non-longevity area participants (with exceeding standard FUN level). Results were validated by Luminex detection. RESULTS: A total of 155 differentially expressed proteins (DEPs) were identified between these two groups. The DEPs related to blood glucose regulation were mainly involved in glycolysis/gluconeogenesis, pyruvate metabolism and propanoate metabolism, and most of the DEPs were contained in carbohydrate metabolism, PI3K-Akt pathway, glucagon signaling pathway and inflammatory response. Validation by Luminex detection confirmed that CD163 was down-regulated, and SPARC, PARK 7 and IGFBP-1 were up-regulated in longevity participants. CONCLUSIONS: This study not only highlighted carbohydrate metabolism, PI3K-Akt pathway, glucagon signaling pathway and inflammatory response may play important roles in blood glucose regulation, but also indicated that YWHAZ, YWHAB, YWHAG, YWHAE, CALM3, CRP, SAA2, PARK 7, IGFBP1 and VNN1 may serve as potential biomarkers for predicting abnormal blood glucose levels.

GDF11 Regulates PC12 Neural Stem Cells via ALK5-Dependent PI3K-Akt Signaling Pathway.

Growth differentiation factor 11 (GDF11), belonging to the transforming factor-ß superfamily, regulates anterior-posterior patterning and inhibits neurogenesis during embryonic development. However, recent studies recognized GDF11 as a rejuvenating (or anti-ageing) factor to reverse age-related cardiac hypertrophy, repair injured skeletal muscle, promote cognitive function, etc. The effects of GDF11 are contradictory and the mechanism of action is still not well clarified. The objective of the present study was to investigate effects of GDF11 on PC12 neural stem cells in vitro and to reveal the underlying mechanism. We systematically assessed the effects of GDF11 on the life activities of PC12 cells. GDF11 significantly suppressed cell proliferation and migration, promoted differentiation and apoptosis, and arrested cell cycle at G2/M phase. Both TMT-based proteomic analysis and phospho-antibody microarray revealed PI3K-Akt pathway was enriched when treated with GDF11. Inhibition of ALK5 or PI3K obviously attenuated the effects of GDF11 on PC12 neural stem cells, which exerted that GDF11 regulated neural stem cells through ALK5-dependent PI3K-Akt signaling pathway. In summary, these results demonstrated GDF11 could be a negative regulator for neurogenesis via ALK5 activating PI3K-Akt pathway when it directly acted on neural stem cells.

The proteome profiling of EVs originating from senescent cell model using quantitative proteomics and parallel reaction monitoring.

Senescence is the inevitable biological processes and is also considered as the biggest risk factor for the development of age - related diseases (ARDs) and geriatric syndrome (GS). Senescence is also known as inflammaging because it is characterized by persistent, long-term, low-grade inflammation named senescence-associated secretory phenotype (SASP). However, the mechanism for the persistence of inflammaging remains largely unclear. To explore the role of extracellular vesicles (EVs) in senescence/inflammaging, we established the cellular senescence model and performed TMT-based comparative quantitative proteomics and parallel reaction monitoring (PRM) to reveal the changes of EVs between young cells and senescent cells. A total of 3966 proteins were quantifiable, of which 132 were up-regulated, 144 were down-regulated, compared with the young cells. Subsequently, we chose 19 proteins involved in inflammation or proliferation to carry out PRM validation analysis. The result indicated that proteins promoting NF-κB signal pathway were up-regulated, and proteins promoting cell proliferation were down-regulated. The study provided a comprehensive altered proteomics profiles of EVs from senescent cells, and the result showed that EVs could serve as information carrier for further research on the pathogenesis and progression of senescence/inflammaging. SIGNIFICANCE: The mechanism of inflammaging occurrence and development has yet been clear. Therefore, this study attempts to provide an improved understanding of inflammaging from the perspective of EVs. The proteomics analysis revealed that the most changed proteins were connected to inflammation signaling pathways, cell growth and cell death, and PRM analysis results showed that proteins involved in NF-κB signal pathway and cell proliferation were more changed. The research systematically analyzed the profiles of proteins in senescence cell model, and the result indicated that further research should focus on the relationship between EVs and senescence/inflammaging.

In vitro Aggregation Ability of Five Commercially Available Aß42 peptide.

BACKGROUND: As the most basic material, synthetic human Amyloid-ß (1-42) (Aß42) peptide from different manufacturers have been widely used. Their aggregation ability is vital to the reliability, repeatability and comparability of studies on Aß42 physiology and pathology. However, it has not been evaluated and compared. OBJECTIVE: To analyze the consistency of the aggregation ability of 5 commercially available Aß42 peptide. METHODS: 5 Aß42 peptide represented as A, B, C, D and E were pretreated by HFIP. The pretreated Aß42 peptide were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptide were aggregated in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to SH-SY5Y cells was further evaluated using Cell Counting Kit-8. RESULTS: For aggregation kinetics, peptide A, C and E remained low level curves, while peptide B and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of peptide B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37 mdeg, while the height of peptide A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence intensity of the aggregates of peptide B and D were 7.79 and 8.82, higher than 1.19, 1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers remained in all aggregates. Furthermore, peptide B and D aggregated to dimers and trimers, peptide A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils were observed only in peptide B, while substantial spherical aggregates were formed in other peptide. Additionally, peptide B, D and E exhibited higher cytotoxicity after aggregated for 8 h, whereas peptide A, B and D presented relatively high cytotoxicity after 12-hour aggregation. CONCLUSION: Commercially available Aß42 peptide showed obvious differences in aggregation ability, which should arouse enough attention in the field of basic study related to Aß42. The aggregation ability evaluation with the various assay methods has some discrepancies, and it is highly urgent to establish a reasonable and uniform measurement strategy.

Effects of long-term high-altitude exposure on fibrinolytic system.

OBJECTIVE: High altitude (HA), with the main feature of hypobaric hypoxia, is an independent risk factor for thrombosis. However, little is known on the alterations of fibrinolytic system in adaptation to HA. In this study, we investigated changes of fibrinolytic system parameters between individuals permanently living at HA and low altitude (LA) regions, and provided data for further studies on HA-induced thrombotic disease. MATERIAL AND METHODS: A total of 226 eligible participants, including 103 LA participants, 100 healthy HA subjects and 23 high altitude polycythemia (HAPC) patients, were recruited in this study. Six fibrinolytic parameters, i.e. fibrinogen (Fbg), D-dimer (DDi), antithrombin III (AT-III), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and plasminogen (PLG) were analyzed respectively. PAI-1 and tPA were performed by using bio-immuno-assays and an automated coagulation analyzer was used to conduct Fbg, DDi, AT-III and PLG tests. RESULTS: Plasma levels of Fbg, DDi, PAI-1 and PLG were significantly higher in healthy HA group than in LA group (all p < 0.05), whereas tPA was significantly lower in healthy HA group. No significant difference in AT-III was observed between healthy HA and LA groups (p > 0.05). All these fibrinolytic parameters showed no significant distinctions between healthy HA subjects and HAPC patients (all p > 0.05). HGB showed no relationship with fibrinolytic parameters in HA cohort. CONCLUSION: This study demonstrates that HA environment has a significant effect on fibrinolytic system and provides a foundation for further studies on HA hypobaric hypoxia-induced thrombotic disease.

Content of anti-ß-amyloid42 oligomers antibodies in multiple batches from different immunoglobulin preparations.

Immunoglobulin preparations are one of the promising drugs for Alzheimer's disease (AD). Anti-ß-amyloid (Aß) oligomers antibodies in immunoglobulin preparations are considered to be critical for the therapeutic effect against Alzheimer's disease. However, the antibodies content in immunoglobulin preparations varies greatly. In order to determine which factor contributes to the difference of the antibodies content, the content of anti-Aß oligomers antibodies in multiple batches of immunoglobulin preparations from two manufacturers were measured by enzyme-linked immunosorbent assay. The results showed that no significant difference was found in the antibodies content among different bathes of normal immunoglobulin preparations prepared by the same process from the same manufacturer, whereas significant difference was found in the antibodies content between normal immunoglobulin preparations prepared by ethanol fractionation and those by chromatography process from the same manufacturer. In addition, significant variation existed in the antibodies content between normal immunoglobulin preparations and specific immunoglobulin preparations that are produced by plasma pool of immunized donors. Based on analysis of these results, the preparation process and raw plasma could be the main contributing factors affecting the content of anti-Aß oligomers antibodies in immunoglobulin preparations. This finding might help to develop AD-specific immunoglobulin preparation containing higher content of anti-Aß oligomers antibodies.

Antibodies against polyethylene glycol in human blood: A literature review.

Polyethylene glycol (PEG) conjugation, i.e. PEGylation, is a successful strategy to improve the pharmacokinetics and pharmacodynamics of biopharmaceuticals. In the past few decades, PEGylation technology has developed tremendously, and >15 PEGylated therapeutics have been brought to market, with more in development. However, the widely accepted assumption that PEG would have no antigenicity or immunogenicity is increasingly challenged with popularization of PEGylation technique. Although PEGylation indeed reduces the immunogenicities of the modified molecules, and even appears to completely eliminate their immunogenicities, yet emerging clinical evidence of anti-PEG antibodies (including both pre-existing and PEGylated therapeutics-treatment induced anti-PEG antibodies) have been attracted more and more attention. Anti-PEG antibodies were detected in not only patients treated with PEGylated therapeutics but also PEGylated drugs treatment-naïve individuals with a prevalence from <1% to 72%. In patients, the existing anti-PEG antibodies may attenuate therapeutic efficacy of PEGylated drugs and increase adverse effects. Although there is no golden standard avenue, several types of methods, including passive hemagglutination, Western Blot, enzyme linked immunosorbent assay, flow cytometry, Meso Scale Discovery technology, Acoustic Membrane Microparticle assay, and surface plasmon resonace technique, were established and used to screen, confirm and quantitatively detect anti-PEG antibodies. Herein, we focused on reviewing the prevalence of anti-PEG antibodies in healthy and PEGylated therapeutics-treated patients, and highlighting the detection methods for pre-screening and quantitative detection of anti-PEG antibodies.

Plasma proteomic and autoantibody profiles reveal the proteomic characteristics involved in longevity families in Bama, China.

BACKGROUND: Chinese Bama Yao Autonomous County is a well-known longevity region in the world. In the past 30 years, population and genome studies were undertaken to investigate the secret of longevity and showed that longevity is the result of a combination of multiple factors, such as genetic, environmental and other causes. In this study, characteristics of the blood plasma proteomic and autoantibody profiles of people from Bama longevity family were investigated. METHODS: Sixty-six plasma donors from Chinese Bama longevity area were recruited in this study. Thirty-three offsprings of longevous families were selected as case studies (Longevous group) and 33 ABO (blood type), age, and gender-matched subjects from non-longevous families were selected as controls (Normal group). Each group contains 3 biological replicates. Tandem mass tag-based proteomic technique was used to investigate the differentially expressed plasma proteins between the two groups. The auto-reactive IgG antibody profiles of the 3 pooled samples in each group were revealed by human proteome microarrays with 17,000 recombinant human proteins. RESULTS: Firstly, 525 plasma proteins were quantified and 12 proteins were discovered differentially expressed between the two groups. Secondly, more than 500 proteins were recognized by plasma antibodies, 14 proteins ware differentially reacted with the autoantibodies in the two groups. Bioinformatics analysis showed some of the differential proteins and targeted autoantigens were involved in cancer, cardiovascular disease and immunity. CONCLUSIONS: Proteomic and autoantibody profiles varied between the offspring of longevous and normal families which are from the same area and shared the same environmental factors. The identified differences were reported to be involved in several physiological and pathological pathways. The identified proteins will contribute to a better understanding of the proteomic characteristics of people from Bama longevous area and a revelation of the molecular mechanisms of longevity.

Alterations of Human Plasma Proteome Profile on Adaptation to High-Altitude Hypobaric Hypoxia.

For individuals migrating to or residing permanently in high-altitude regions, environmental hypobaric hypoxia is a primary challenge that induces several physiological or pathological responses. It is well documented that human beings adapt to hypobaric hypoxia via some protective mechanisms, such as erythropoiesis and overproduction of hemoglobin; however, little is known on the alterations of plasma proteome profiles in accommodation to high-altitude hypobaric hypoxia. In the present study, we investigated differential plasma proteomes of high altitude natives and lowland normal controls by a TMT-based proteomic approach. A total of 818 proteins were identified, of which 137 were differentially altered. Bioinformatics (including GO, KEGG, protein-protein interactions, etc.) analysis showed that the differentially altered proteins were basically involved in complement and coagulation cascades, antioxidative stress, and glycolysis. Validation results demonstrated that CCL18, C9, PF4, MPO, and S100A9 were notably up-regulated, and HRG and F11 were down-regulated in high altitude natives, which were consistent with TMT-based proteomic results. Our findings highlight the contributions of complement and coagulation cascades, antioxidative stress, and glycolysis in acclimatization to hypobaric hypoxia and provide a foundation for developing potential diagnostic or/and therapeutic biomarkers for high altitude hypobaric hypoxia-induced diseases.

Correlation between RBC changes and coagulation parameters in high altitude population.

OBJECTIVE: To explore the correlations between RBCs indexes and the basic coagulation parameters, and provide data for further studies on high altitude-induced thrombotic disease. METHODS: A total of eligible 433 volunteers were divided into different groups according to HGB concentration and HCT, respectively. PT, APTT, TT and Fbg were measured by clotting assays. HGB content, HCT and PLT count were assessed by automated hematology analyzer. RESULTS: APTT and PT were significantly higher in group 4 (high HGB or HCT groups) (p < 0.05 for all comparison) and PLT count was significantly lower in group 4 than in other groups (p < 0.01 for all comparison). APTT and PT showed negative correlations with HGB concentration (r = -0.168 and -0.165 resp.; both p < 0.01), whereas positive correlations were found between APTT and HCT, PT and HCT (r = 0.225 and 0.258, resp.; both p < 0.01). PLT, TT and Fbg showed no correlation with HGB and HCT. CONCLUSIONS: HGB and HCT may not correlate with basic coagulation parameters in high altitude population, their predictive value for high altitude-induced thrombotic disease may relatively independent and this remain to be determined in further studies.

Plasma proteome profiling of high-altitude polycythemia using TMT-based quantitative proteomics approach.

High-altitude polycythemia (HAPC) is one of the classic chronic mountain sicknesses and has been a serious public health problem in high-altitude regions. Despite numerous studies on HAPC via genomics or transcriptomics approaches, the pathogenesis of HAPC is still unclear. Here, we performed a TMT- based comparative quantitative proteomics analysis to reveal the changes of plasma proteomics profiles between HAPC subjects and healthy controls. Of identified 818 proteins, 7 and 12 proteins were up-regulated and down-accumulated, respectively, compared HAPC patients with healthy controls. GO and KEGG pathway analyses revealed the dysregulated proteins were primarily involved in complement and coagulation cascades, inflammation and immune response. ELISA validation demonstrated that C4A, C6 and CALR were down-regulated, and MASP1 and CNDP1 were up-regulated in HAPC patients. By ROC analysis, combinations of these five proteins (i.e., C4A, C6, CALR, MASP1 and CNDP1) resulted in a high AUC value (0.919; 95% CI, 0.817-961; p < .0001) to diagnose HAPC patients. Moreover, CNDP1 seems to be a robust biomarker for HAPC. This study not only provided a comprehensive dataset on overall proteomics changes in HAPC patients compared with healthy controls, but also indicated that CNDP1 can serve as a strong plasma biomarker of HAPC for the diagnostic and therapeutic potential. SIGNIFICANCE: HAPC, one of the classic chronic mountain sicknesses, has been a serious public health problem in high-altitude regions. Despite numerous studies on HAPC via genomics or transcriptomics approaches, the pathogenesis of HAPC is still largely unknown to date. In this study, we addressed this issue by performing TMT-based quantitative analyses of the plasma proteome profiles of HAPC patients and healthy controls. We identified 818 proteins, of which 19 were differentially expressed. Bioinformatics analysis revealed the differentially expressed proteins were mainly involved in complement and coagulation cascades, inflammation and immune response. By ROC analysis, combinations of C4A, C6, CALR, MASP1 and CNDP1 resulted in a high AUC value (0.919, p < .0001) to distinguish HAPC patients from healthy controls. Collectively, the current study provided a comprehensive dataset on overall proteomic changes in HAPC patients for the first time, and it also revealed C4A, C6, CALR, MASP1 and CNDP1 can be served as candidate plasma biomarkers of HAPC for their diagnostic and therapeutic potential.