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Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fowl typhoid is an important disease of chickens and turkeys, which is caused by Salmonella Gallinarum (S. Gallinarum). Vaccines with high levels of protective effects against fowl typhoid need to be developed for the poultry industry. In this study, a S. Gallinarum strain, named SG01, was isolated from a poultry farm in Mashan region of Wuxi City, China, and identified through biochemical tests and specific PCR amplifications. Then, safety evaluations of the SG01 strain were performed in young chickens. No clinical symptom including depression and diarrhea and gross lesion involved in the cardiac nodule, hepatic necrotic lesion and splenic necrotic lesion, was determined on fifteen-day-old chickens after immunization with 1 × 10 10 CFU of the SG01 strain through the oral route. However, diarrhea symptoms and hepatic lesions were identified from chickens immunized with the commercial vaccine strain SG9R by the same dose and route. At 14 days post inoculation, SG01 strain was eliminated in the liver and spleen from SG01-immunized chickens, while the SG9R strain still could be identified from SG9R-immunized chickens. After challenge with the virulent S. Gallinarum strain, significant reduction of the morbidity rate was found in the SG01 immunized group (20 %) compared to the challenge group (100 %) according to signs scoring systems for clinical symptoms and gross lesions. Additionally, immunization with the SG01 strain could provide more than 8 weeks of protection periods against fowl typhoid. These results demonstrate the SG01 strain is avirulent to young chickens and might be safer compared to the SG9R strain. In addition, SG01 strain is a potential vaccine candidate against fowl typhoid in young chickens., Competing Interests: Conflict of interests The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heat shock protein 90 (Hsp90) is associated with resisting heat‑stress injury to the heart, particularly in myocardial mitochondria. However, the mechanism underlying this effect remains unclear. The present study was based on the high expression of Hsp90 during heat stress (HS) and involved inducing higher expression of Hsp90 using aspirin in mouse hearts. Higher Hsp90 levels inhibited HS‑induced myocardial damage and apoptosis, and mitochondrial dysfunction, by stimulating Akt (protein kinase B) activation and PKM2 (pyruvate kinase M2) signaling, and subsequently increasing mitochondrial Bcl‑2 (B‑cell lymphoma 2) levels and its phosphorylation. Functional inhibition of Hsp90 using geldanamycin verified that reducing the association of Hsp90 with Akt and PKM2 caused the functional decline of phosphorylated (p)‑Akt and PKM2 that initiate Bcl‑2 to move into mitochondria, where it is phosphorylated. Protection by Hsp90 was weakened by blocking Akt activation using Triciribine, which could not be recovered by normal initiation of the PKM2 pathway. Furthermore, increased Hsp70 levels induced by Akt activation in myocardial cells may flow into the blood to resist heat stress. The results provided in vivo mechanistic evidence that in myocardial cells, Hsp90 resists heat stress via separate activation of the Akt‑Bcl‑2 and PKM2‑Bcl‑2 signaling pathways, which contribute toward preserving cardiac function and mitochondrial homeostasis.

1. This study aimed to investigate the protective effects of Gingko biloba extract EGB761 on heat-stressed chicken heart in vivo and its underlying relevance to Hsp70.2. A total of 50 one-day-old female chicks were randomly divided into five groups: control (Con), heat-stress (HS), 0.1% EGB761 plus heat-stress (0.1%EGB+HS), 0.3%EGB761 plus heat-stress (0.3%EGB+HS) and 0.6%EGB761 plus heat-stress (0.6%EGB+HS) groups. After administration of EGB761 for 45 days, the chickens in each group were exposed to a single heat-stress event at 38 ± 1°C for 3 h.3. EGB761 attenuated the abnormal symptoms and pathological scores of myocardium of heat-stressed chickens. Despite a reduction in the transcription and translation of the Hsp70 gene in heat-stressed myocardium, EGB761 induced the expression of Hsp70 in endothelial cells of the microarteries and venules into the blood, and reduced heat-stress damage in vascular endothelial cells.4. Supplementation with EGB761 before heat-stress exposure protected chicken myocardium from damage by increasing serum Hsp70 protein from myocardial cells and cardiac microvascular endothelial cells and protected the microvascular system from adverse injury.

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken ( Gallus gallus ) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

Background: Stress has been proved to impair the porcine reproduction soundly. Endocrine disruption, which is closely related to the persistent follicles, is possibly one of the results of stress, although the mechanism is unclear. Since the expression of luteinizing hormone receptor (LHR) in ovarian follicular wall and concentrations of steroid hormone in follicular fluid are related to the development of persistent follicles, this study is designed to evaluate the effect of administered adrenocorticotrophic hormone (ACTH) to weaned pigs on their ovarian steroidogenesis capacity and LHR expression., Methods: Ten multiparous sows were weaned and randomly divided into two groups (n = 5 each). Sows received 1 IU/kg ACTH (ACTH group) or saline (control group) every 8 h from days 3-9 after jugular vein intubation. Blood samples were collected throughout the experiment, and ovaries were collected after slaughter on day 10. Follicular fluid (FF) was used to determine the steroid hormone concentrations. The ovarian follicle wall was obtained and stored in liquid nitrogen to detect mRNA levels., Results: The plasma cortisol concentration was significantly (P < 0.01) elevated after ACTH injection. The estradiol (E 2 ) and androstenedione (ASD) concentrations in FF were significantly lower (P < 0.05) in the ACTH group than in the control group. The LHR, 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (P450arom), and cytochrome P450 17a-hydroxylase (P450c17) mRNA levels were significantly (P < 0.05) reduced in the ACTH group. The steroidogenic acute regulatory protein (StAR) level and cytochrome P450 side-chain cleavage (P450scc) was lower in the ACTH group than in the control group, but the difference was not statistically significant (P > 0.05). Immunostaining results revealed 3β-HSD,P450c17, and LHR expression in theca cells, and P450arom expression in granulosa cells. Immunohistochemical staining showed significant differences in the distribution of 3β-HSD, P450c17, LHR, and P450arom between the two groups., Conclusions: These findings indicated that ACTH significantly diminished the LHR expression and steroidogenesis capacity of the ovaries of weaned sows.

In this study, the immune response induced by a mixture of polysaccharide and nucleic acid extracted from Bacillus Calmette-Guerin (BCG) was evaluated in chickens inoculated with infectious bursal disease virus (IBDV) vaccine. After the mixture was injected intramuscularly at a dose of 0.075, 0.15 or 0.3 mg·kg(-1)·day(-1) for 3 days, the 14-day-old chickens were inoculated with the attenuated IBDV vaccine via intranasal and ocular routes. The relative weight of bursa of Fabricius (BF) and thymus, the serum IBD antibody titer, the CD4+/CD8+ ratio, and the concentrations of IFN-γ, IL-2 and IL-6 in peripheral blood were investigated on days 5, 15 and 25. The IBD antibody titer in BCG-treated groups was higher than in the negative control and only IBD-vaccinated chickens, indicating that the mixture of BCG can significantly enhance chicken humoral response. CD4+/CD8+ and the secretions of IFN-γ, IL-2 and IL-6 were also clearly increased compared with that in the negative control and IBD-vaccinated chickens, indicating that the mixture can also enhance the cell-mediated immune response. The results also showed that the relative weights of BF and thymus increased after chickens were inoculated with BCG, indicating that the BCG mixture can clearly enhance the immunity of IBD-vaccine and can be expected to be viewed as a candidate for a new type of immune adjuvant.

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.

To investigate the mechanism of sudden death as a result of stress-induced damage to heart tissue and myocardial cells and to investigate the cardioprotective role of Hsp70 during heat stress, the distribution and expression of Hsp70 was evaluated in the heart cells of heat-stressed rats in vivo and heat-stressed H9c2 cells in vitro. After exposure to heat stress at 42°C for different durations, we observed a significant induction of CK, CK-MB, and LDH as well as pathologic lesions characterized by acute degeneration, suggesting that cell damage occurs from the onset of heat stress. Immunocytochemistry showed that Hsp70 was distributed mainly in the cytoplasm of myocardial cells in vivo and in vitro. Hsp70-positive signals in the cytoplasm were more prominent in intact areas than in degenerated areas after 60 min of heat stress. Hsp70 protein levels in myocardial cells in vitro decreased from the beginning to the end of heat stress. Hsp70 protein levels in rat heart tissues in vivo decreased gradually with prolonged heat stress, with a slight increase at the beginning of heat stress. These results indicate that Hsp70 plays a role in the response of cardiac cells to heat stress and that decreased Hsp70 levels are associated with damage to rat myocardial cells in vitro and in vivo. Significant differences were found in hsp70 mRNA, which began to increase after 20 min of heat stress in vitro and after 40 min in vivo. This indicates that hysteresis is involved in mRNA expression after heat stress in vivo.

The aim of the present study was to identify the correlation between expression of heat shock protein 47 (Hsp47) and stress injury in heat-stressed myocardial cells and to compare variations in Hsp47 expression in rat myocardial cells exposed to different heat stress for varying periods in vitro and in vivo. Exposure to heat stress at 42°C resulted in similar induction patterns of the heart damage-related enzyme aspartate aminotransferase in the supernatants of H9c2 cells and in the serum of rats. Histological analysis revealed that both H9c2 cells and heart tissues displayed cellular degeneration in response to different periods of heat stress. Hsp47 was constitutively expressed in the cytoplasm of H9c2 cells at all time points during heat stress, which was consistent with observations in heart fibers in vivo. Immunoblotting analysis revealed no significant difference between the expression of Hsp47 in H9c2 cells and heart tissue. However, the expression of hsp47 mRNA in response to heat stress was significantly increased in H9c2 cells at 60 min (P < 0.01) and 100 min (P < 0.01), which was comparable to that at 100 min (P < 0.01) in the rat heart. Thus, Hsp47 was elevated significantly after hyperthermia at the mRNA level but not at the protein level both in vitro and in vivo. The results suggest that Hsp47 turnover may increase during heat stress or that Hsp47 consumption exceeds its production.

To investigate the protective role of Hsp60 against stress damage and its role in the sudden death of stressed animals, changes in the levels of Hsp60 protein and hsp60 mRNA of myocardial cells in vivo and in vitro were studied. In addition, the relationship between Hsp60 expression and heat-induced damage was also studied. Rats were exposed to a temperature of 42° ± 1°C for 0, 20, 40, 60, 80, or 100 min. More than 50% of the rats died suddenly within 100 min. With increasing heat stress duration, hsp60 mRNA levels significantly increased in both in vivo and in vitro rat myocardial cells; however, a similar trend was not observed for Hsp60 protein levels. Although the changes observed in Hsp60 expression in myocardial cells in vitro were inconsistent with those of rat heart tissues in vivo, Hsp60 expression levels were consistent with the histopathological damage observed in myocardial cells both in vivo and in vitro. Differences in Hsp60 expression may reflect the degree of injury sustained by myocardial cells in vivo and in vitro. As a mitochondrial protein, Hsp60 represents a potential biomarker of heat stress, and may protect against heat stress induced myocardial cellular damage both in vivo and in vitro.

The objective of this study was to investigate the mechanism of heat shock protein 90 alpha (Hsp90α) protection against heart damage resulting from heat stress by detecting Hsp90α mRNA, Hsp90α protein, protein localization, and cell damage in primary myocardial cells of neonatal rats in response to heat stress in vitro. The cells were heat-stressed at 42°C in an incubator with 95% air and 5% CO2 for different periods. Levels of Hsp90α, protein localization, enzymes, and cytopathological lesions were detected using Western blot, immunocytochemistry enzymatic assays, and cytopathological techniques. Aspartate aminotransferase, lactate dehydrogenase, and creatine kinase enzyme levels were elevated during heat stress, and acute cellular lesions that were characterized by vacuolar degeneration and necrosis were observed. Hsp90α levels decreased between 10 and 60 min of heat stress and increased after 360 and 480 min, while Hsp90α mRNA decreased after 360 min. These results indicate that heat stress might induce irreversible damage in certain myocardial cells. The elevated Hsp90α level at the end of heat stress and its positive signal in the cytoplasm of myocardial cells after heat stress could be associated with its protective role. Additionally, the consumption of Hsp90α exceeded its production in the first period of treatment.

To understand the mechanism underlying the sudden animal death caused by acute heart failure during heat stress, the relationships among the heat-induced pathological changes and apoptosis and the variations in the levels of protective Hsp90α and its mRNA in the heat-stressed primary myocardial cells of neonatal rats in vitro were studied by cytopathological observation, immunoblotting, RT-PCR, and analysis of the related enzymes. After a period of adaptive cell culture, the myocardial cells were immediately exposed to heat stress at 42°C for 10, 20, 40, 60, 120, 240, 360, and 480 min. Levels of creatine kinase increased from the beginning of heat stress, and the cells exposed to heat stress showed acute cellular lesions characterized by vacuolar degeneration and necrosis after 40 min of heat stress, suggesting that the myocardial cells in vitro were obviously stressed and damaged by higher temperature. The levels of cleaved caspase-3 and cytochrome C, which were related to apoptosis, increased significantly after 40 min of heat stress while the Hsp90α protein level significantly decreased. In contrast, after 6 h of exposure to heat stress, the levels of cleaved caspase-3 and cytochrome C decreased while those of Hsp90α significantly increased, suggesting that early depletion of Hsp90α coincides with a high rate of necrosis and apoptosis in heat-stressed myocardial cells, while the Hsp90α level in surviving cells increases again with significantly less apoptosis after 6 h of heat stress. These findings also indicate that apoptosis of myocardial cells occurs through the activation of the cytochrome C and caspase-3 pathway. The cell repair capacity of Hsp90α is overstrained in the early phase of heat treatment and needs some hours to stabilize. As a result, in the primary myocardial cells in vitro, Hsp90α shows protective activity against damage at the end period of the heat exposure.

The mechanisms involved in sudden animal death due to acute heart failure during heat stress are not well understood. We examined the relationship between heat stress-induced variations of protective Hsp60 and expression of its regulatory factor, HSF-1, in heat-stressed primary myocardial cells of neonatal rats in vitro through cardiac enzyme detection, immunoblotting, immunocytochemistry, and qPCR. Increases in cardiac damage-related enzyme levels demonstrated injury to myocardial cells after heat exposure at 42°C. Hsp60 expression levels fluctuated during heat stress; they decreased significantly after 20 min, then increased at 120 min and decreased again at 360 min after initiation of heat stress. The highest levels of Hsp60 were observed at 240 min, while the lowest were at 60 min. Damage to myocardial cells was characterized by increases in cardiac enzyme levels and low levels of Hsp60 due to functional disorder of myocardial cells at early stages of heat stress. However, the significant induction of hsp60 mRNA levels from the beginning up to 240 min of heat stress was not consistent with the classic regulatory mechanisms that link transcription and translation, suggesting that Hsp60 expression is delayed due to loss of Hsp60 during the early stages of heat stress. hsf-1 mRNA levels were significantly increased from 10 min of heat stress; however, HSF-1 protein levels did not simultaneously increase, indicating that HSF-1 is not the sole regulator of Hsp60 expression.

Previous studies investigating the association between TP53 Arg72Pro polymorphism and bladder cancer risk reported controversial results. To quantify the strength of association between TP53 Arg72Pro polymorphism and bladder cancer risk, we performed this meta-analysis. We searched PubMed, Embase and Wangfang databases for studies relating the association between TP53 Arg72Pro polymorphism and bladder cancer risk. We used the pooled odds ratios (ORs) with their 95 % confidence intervals (95 % CIs) to assess the association. Finally, data were available from a total of 16 case-control studies including a total of 5, 545 subjects (2,345 cases and 3,200 controls). Meta-analysis of all 16 studies showed TP53 Arg72Pro polymorphism was not associated with bladder cancer risk (All P values were more than 0.10). Subgroup analyses by ethnicity showed that TP53 Arg72Pro polymorphism contributed to bladder cancer risk in East Asians in three genetic models (For Pro vs. Arg, Fixed-effects OR 1.18, 95 % CI 1.05-1.32; For ProPro vs. ArgArg, Fixed-effects OR 1.40, 95 % CI 1.11-1.77; For ProPro vs. ArgPro/ArgArg, Fixed-effects OR 1.32, 95 % CI 1.07-1.62). However, there was no significant association in Caucasians and the others (All P values were more than 0.05). Heterogeneity analyses suggested ethnicity was the major sources of heterogeneity. Thus, meta-analyses of available data suggest the Pro variant of TP53 Arg72Pro contributes to bladder cancer risk in East Asians. Besides, TP53 Arg72Pro polymorphism may have race-specific effects on bladder cancer risk and further studies are needed to elucidate this possible effect.

Background: SUMO-specific protease 2 (SENP2) is a de-SUMOylation protease family member which has an indispensable role in the regulation of NF-κB transcriptional activation and Wnt signaling. However, whether SENP2 plays a role in tumor metastasis is completely unknown., Methods: Real-time PCR and Western blot was used to detect the expression of SENP2 in human bladder cancer samples and cell lines. Small interfering RNA (siRNA) was used to silencing the expression of SENP2. Matrigel-coated invasion chambers were used to detect the invasion ability of SENP2 in bladder cancer cells., Results: SENP2 was down-regulated in bladder cancer samples. SENP2 inhibited bladder cancer cells migration and invasion in vitro. Transcriptional analysis of several genes associated with tumor metastasis and invasion demonstrated that SENP2 selectively down-regulated MMP13 in bladder cancer cells. Further analysis indicated that silencing of MMP13 rescued the invasive phenotype in SENP2 expressing T24 cells., Conclusion: SENP2 functions as a tumor metastasis suppressor in bladder cancer. The effects of SENP2 on bladder cancer invasion are partially mediated by inhibiting the expression of MMP13., (© 2013 S. Karger AG, Basel.)

We investigated and described the kinetics of heat shock protein (Hsp) 110 expression and distribution in rat primary myocardial cells exposed to heat stress in vitro. After incubation at 37°C for 72 h, myocardial cells were heat stressed at 42°C for 0, 10, 20, 40, 60, 120, 240, 360, and 480 min. Significant increases in aspartate transaminase, lactate dehydrogenase, and creatine kinase enzymatic activities in the myocardial cell culture media were observed during heat stress, suggesting that the integrity of the myocardial cells was altered. Immunocytochemical analysis revealed that the expressed Hsp110 was constitutively localized in the cytoplasm and in the nuclei in small amounts characterized by a granular pattern. Nuclear Hsp110 levels increased significantly after 240 min of heat stress compared with levels in the control. The overall levels of Hsp110 expression increased significantly after 20 min. After 240 min, Hsp110 levels were approximately 1.2-fold higher than those in the control. Increasing levels of hsp110 messenger RNA detected using real-time quantitative polymerase chain reaction were observed after 20 min of heat stress, and the levels peaked with a 10-fold increase after 240 min of heat stress. These results indicate that the expression of Hsp110 in primary myocardial cells in vitro is sensitive to hyperthermic stress and that Hsp110 is involved in the potential acquisition of thermotolerance after heat stress. Therefore, Hsp110 might play a fundamental role in opposing and alleviating heat-induced damage caused by hyperthermic stress in primary myocardial cells.

Gene polymorphisms of cytotoxic T lymphocyte associated antigen 4 (CTLA4) play an influential role in the graft rejection and long-term clinical outcome of organ transplantation. We investigated the associations of five CTLA4 single nucleotide polymorphisms (SNPs) (rs733618T/C, rs4553808A/G, rs5742909C/T, rs231775G/A, rs3087243G/A) on the early acute rejection (AR) of Chinese deceased donor renal transplantation recipients. Genotyping of the CTLA4 SNPs was performed in 167 deceased donor renal transplantation recipients. Each patient underwent a 6-month follow-up observation for AR. The incidence of AR during the 6 months post-transplantation was 26.9% (45 out of 167 patients). Patients experiencing AR were found to have a higher frequency of the rs733618TT genotype and T allele (p=0.000 and p=0.002, respectively). While the haplotype CACAG was merely observed in non-AR group (corrected p=0.000), the frequency of haplotype TACGG was significantly higher in AR group than in non-AR group even after 50,000 permutation tests (corrected p=0.018). In conclusion, these polymorphisms statistically significantly associated with acute renal allograft rejection may be considered as a risk factor of AR in Chinese renal transplantation recipients except for haplotype CACAG as a protective one., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs., Animals: 24 pigs (mean weight, 20 +/- 1 kg)., Procedures: Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA., Results: No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs., Conclusions and Clinical Relevance: Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein (P) and matrix protein (M) in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts (CEF) cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDV-GFP) expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive ailantoic fluid were used for further egg passages. All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1 x 10(4) EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers (10(9) EID50/mL). In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1 x 10(6) EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.