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Human platelets (PLTs) are vulnerable to unfavorable conditions, and their adequate supply is limited by strict transportation conditions. We report here that PLTs preserved under three-dimensional (3D) conditions using novel biomimetic nanofiber peptides showed reduced apoptosis compared with classical PLTs stored at 22 °C and facilitated the storage and transportation of PLTs. The mechanism of PLT 3D preservation involves the formation of cross-links and a 3D nanofibrous network by a self-assembled peptide scaffold material at physiological conditions after initiation by triggers in plasma. PLTs adhere to the surface of the nanofibrous network to facilitate the 3D distribution of PLTs. The 3D microstructure, rheological properties, and effect on the inflammatory response and hemolysis were evaluated. Compared to traditional PLTs stored at 22 °C, PLTs subjected to 3D preservation showed similar morphology, number, aggregation activity, and reduced apoptosis. The detection of the reactive oxygen species (ROS) levels demonstrated that both reduced intracellular and mitochondrial ROS levels were correlated with reduced apoptosis. This study reveals a new 3D preservation method for PLTs based on the use of novel biomimetic nanofiber peptides that presents an attractive opportunity for various biomedical applications.

Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using in vitro transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the pol genes were defective at both the genome and transcript levels. We speculate that the defective PERV pol genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV pol genes as a source animal species for xenotransplantation.

Background The pathogenesis of transfusion-related acute lung injury (TRALI) progress is incompletely understood, and specific therapies for TRALI are lacking. Alveolar macrophages (AMs) are critical for initiation and resolution of lung inflammation. However, the role of AMs in the pathogenesis of TRALI-associated lung failure is poorly understood. Study design and methods Mouse model for in vivo imaging of interleukin (IL)-6 activation in AMs was established by intratracheal instillation of a lentiviral vector carrying the luciferase reporter gene. The TRALI mouse model was produced by intraperitoneal lipopolysaccharide plus intravenous major histocompatibility complex Class I monoclonal antibody treatment. We focused on the changes in AMs in the lung during TRALI and examined whether targeting AMs is an effective strategy to alleviate this condition. Measurements and main results We confirmed that TRALI progress is accompanied by IL-6 activation in AMs. Further study showed that AMs undergo M1 activation during TRALI progress. AM depletion protected mice from TRALI, and transfusion of M1-polarized AMs into 34-1-2 s-treated mice elevated acute lung injury, indicating that the severity of TRALI was able to be ameliorated by targeting AM polarization. Next, we showed that α1 -antitrypsin (AAT) expression improved lung injury by modulating the production of IL-6 in AMs and decreased polarization of AMs toward the M1 phenotype. Conclusions M1-polarized AMs are crucial in a mouse model of TRALI, and AAT may serve as a future treatment for TRALI by regulating the polarization of AMs.

The stromal vascular fraction (SVF) derived from adipose tissue contains heterogeneous cell populations and has enormous potential for clinical therapy. There are two main methods for SVF isolation: enzymatic isolation and mechanical isolation, both of which have shortcomings. In this study, optimized conditions for the isolation of high-quality SVF were established, and applications in fat grafting were evaluated.Adipose tissue was chopped into small pieces and then ground into an erosive shape using a syringe. The pieces were digested with 0.15% type II collagenase for 35 min at 37 °C. After centrifugation, the pellets were resuspended in DMEM and passed through a 100-μm strainer. The filtered cells were analyzed by flow cytometry. The fat graft was enriched with isolated SVF and subcutaneously transplanted into nude mice. Three weeks after transplantation, grafts were isolated, and HE staining, immunocytochemistry, and western blotting were conducted.The harvested SVF cells reached2 × 10Our study provides an efficient procedure for SVF isolation, its application in fat grafting, and possible underlying mechanisms.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Pasteurization is regularly used to inactivate viruses for the safety of plasma derivatives. Influence of pasteurization at 60 °C for 10 h on α2-Macroglobulin activity and virus inactivation were studied. With 40% sugar as stabilizers more than 70% α2-Macroglobulin activity was reserved after pasteurization compared with 20% in control. Glucose presented a better activity protection effect than sucrose and maltose. By pasteurization without stabilizer the virus titers of pseudorabies virus, Sindbis virus, porcine parvovirus and encephalomyocarditis virus were reduced more than 5.88 log10, 7.50 log10, 4.88 log10, and 5.63 log10 respectively within 2 h. By pasteurization with 40% glucose vesicular stomatitis virus was inactivated more than 5.88 log10 within 1 h. Only 2.71 log10 reduction was achieved for encephalomyocarditis virus after 10 h. 40% glucose protected α2-M activity and viruses simultaneously from pasteurization. Other viral inactivation methods need to be incorporated to ensure viral safety of this manufacturing process of α2-Macroglobulin.

Existence of porcine endogenous retrovirus (PERV) hinders pigs to be used in clinical xenotransplantation to alleviate the shortage of human transplants. Chinese miniature pigs are potential organ donors for xenotransplantation in China. However, so far, an adequate level of information on the molecular characteristics of PERV from Chinese miniature pigs has not been available. We described here the cloning and characterization of full-length proviral DNA of PERV from Chinese Wuzhishan miniature pigs inbred (WZSP). Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino-acid sequences of env. The results demonstrated that the full-length proviral DNA of PERV-WZSP belongs to gammaretrovirus and shares high similarity with other PERVs. Sequence analysis also suggested that different patterns of LTR existed in the same porcine germ line and partial PERV-C sequence may recombine with PERV-A sequence in LTR.

We conducted a large-scale survey on the existence and expression status of porcine endogenous retrovirus (PERV) in seven breeds of Chinese miniature pigs. Genotyping of PERV was examined by PCR using type-specific primers according to the env genotyping method. The presence and expression status of viral gag, pol and env genes were further analyzed in Wuzhishan pigs (WZSP) and Bama minipigs (BMP). The results showed that PERV existed in all 348 genomic DNA samples. The genotype distribution was subtype A-74.43%, subtype B-95.40% and subtype C-30.46%. No expression of subtype C was found in WZSP and BMP. This research obtained an adequate level of information on the molecular epidemiology of PERV in China. The results indicated that it is possible to monitor pig herds for individuals with the lowest PERV prevalence, especially lacking PERV-C.

Background α2-Macroglobulin (α2-M) has a curative effect on radiation injury. Virus transmission through plasma derivatives is still not risk-free. Effect of dry heat on α2-M activity and virus inactivation by dry heat in a new manufacturing process of α2-M were studied. Study design and methods Effects of 100°C for 30 minutes, 80°C for 72 hours, and lyophilization on α2-M activity were detected, and stabilizing agents were optimized. Effect of a treatment at 100°C for 30 minutes has been tested on a range of viruses and characteristics change of α2-M was investigated. Results More than 90 and 80% α2-M activity recovery were reserved after treatment at 100°C for 30 minutes and 80°C for 72 hours, respectively. A concentration of 0.05 mol/L histidine presented a better protecting effect for α-M activity. No substantial changes were observed in the characteristics of α2-M compared with the untreated. By lyophilization and dry-heat treatment at 100°C for 30 minutes, murine encephalomyocarditis virus and pseudorabies virus (PRV) were inactivated below detectable level within 5 minutes (virus titers reduction ≥ 5.75 log) and 30 minutes (virus titers reduction ≥ 6.00 log), respectively. Bovine viral diarrhea virus and porcine parvovirus were inactivated by 4.29 and 2.46 log reduction, respectively. Conclusion Treatment at 100°C for 30 minutes could improve the virus safety of α2-M with a slight activity loss.

BACKGROUND: A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77-79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection. METHODS: Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination. FINDINGS: Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation. CONCLUSIONS: These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.

Background Xenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs. Methods The proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptαSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis. Results The ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F. Conclusions Altogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced.