Genomic Analyses from Non-invasive Prenatal Testing Reveal Genetic Associations, Patterns of Viral Infections, and Chinese Population History.

We analyze whole-genome sequencing data from 141,431 Chinese women generated for non-invasive prenatal testing (NIPT). We use these data to characterize the population genetic structure and to investigate genetic associations with maternal and infectious traits. We show that the present day distribution of alleles is a function of both ancient migration and very recent population movements. We reveal novel phenotype-genotype associations, including several replicated associations with height and BMI, an association between maternal age and EMB, and between twin pregnancy and NRG1. Finally, we identify a unique pattern of circulating viral DNA in plasma with high prevalence of hepatitis B and other clinically relevant maternal infections. A GWAS for viral infections identifies an exceptionally strong association between integrated herpesvirus 6 and MOV10L1, which affects piwi-interacting RNA (piRNA) processing and PIWI protein function. These findings demonstrate the great value and potential of accumulating NIPT data for worldwide medical and genetic analyses.

Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and has very few mutations that are shared between different patients. To better understand the intratumoral genetics underlying mutations of ccRCC, we carried out single-cell exome sequencing on a ccRCC tumor and its adjacent kidney tissue. Our data indicate that this tumor was unlikely to have resulted from mutations in VHL and PBRM1. Quantitative population genetic analysis indicates that the tumor did not contain any significant clonal subpopulations and also showed that mutations that had different allele frequencies within the population also had different mutation spectrums. Analyses of these data allowed us to delineate a detailed intratumoral genetic landscape at a single-cell level. Our pilot study demonstrates that ccRCC may be more genetically complex than previously thought and provides information that can lead to new ways to investigate individual tumors, with the aim of developing more effective cellular targeted therapies.

Correlation Analysis Between Tumor Deposit and Clinicopathologic Characteristics and Prognosis of Gastric Cancer: A Multicenter Retrospective Study.

BACKGROUND: The mechanism underlying the formation of gastric tumor deposits (TDs) is unclear. We aimed to explore the risk factors for the formation and prognostic value of TDs. METHODS: This retrospective analysis included 781 locally advanced gastric cancer (LAGC) patients from four medical institutions in China, from June 2014 to June 2018. The risk factors for TD formation and prognostic value were determined through univariate and multivariate analyses. RESULTS: Univariate analysis revealed that TD positivity was closely related to tumor diameter, Borrmann classification, differentiation degree, pT stage, pN stage, pTNM stage, and nerve and vascular invasion (p < 0.05). Multivariate logistic regression revealed that tumor diameter ≥ 5 cm (odds ratio [OR] 1.836, 95% confidence interval [CI] 1.165-2.894, p = 0.009) and vascular invasion (OR 2.152, 95% CI 1.349-3.433, p = 0.001) were independent risk factors for TD positivity. Multivariate Cox analysis revealed that TD positivity (OR 1.533, 95% CI 1.101-2.134, p = 0.011), tumor diameter ≥ 5 cm (OR 1.831, 95% CI 1.319-2.541, p < 0.001), pT4a stage (OR 1.652, 95% CI 1.144-2.386, p = 0.007), and vascular invasion (OR 1.458, 95% CI 1.059-2.008, p = 0.021) were independent risk factors for GC prognosis. The 5-year overall and disease-free survival of the TD-positive group showed significant effects among patients in the pT4a and pN3b stages (p < 0.05). CONCLUSIONS: TDs are closely related to tumor diameter and vascular invasion in LAGC patients, and TD positivity is an independent prognostic factor for LAGC patients, especially those at pT4a and pN3b stages.

The short- and long-term outcomes of laparoscopic D2 lymphadenectomy plus complete mesogastrium excision for lymph node-negative gastric cancer.

BACKGROUND: Patients with T1-3N0M0 gastric cancer (GC) who undergo radical gastrectomy maintain a high recurrence rate. The free cancer cells in the mesogastric adipose connective tissue (Metastasis V) maybe the reason for recurrence in these individuals. We aimed to evaluate whether D2 lymphadenectomy plus complete mesogastrium excision (D2 + CME) was superior to D2 lymphadenectomy with regard to safety and oncological efficacy for T1-3N0M0 GC. METHODS: Patients with T1-3N0M0 GC who underwent radical resection from January 2014 to July 2018 were retrospectively analyzed; there were 323 patients, of whom 185 were in the D2 + CME group and 138 in the D2 group. The primary endpoint was 5-year disease-free survival (DFS). Secondary endpoints include the 5-year overall survival (OS), recurrence pattern, morbidity, mortality, and surgical outcomes. RESULTS: D2 + CME was associated with less intraoperative bleeding loss, a greater number of lymph nodes harvested, and less time to first postoperative flatus, but the postoperative morbidity was similar. The 5-year DFS was 95.6% (95% CI 92.7-98.5%) and 90.4% (95% CI 85.5-95.3%) in the D2 + CME group and the D2 group, respectively, with a hazard ratio (HR) of 0.455 (95% CI 0.188-1.097; p = 0.071). In terms of recurrence patterns, local recurrence was more prone to occur in the D2 group (p = 0.031). Subgroup analysis indicated that for patients with T1b-3N0M0 GC, the 5-year DFS in the D2 + CME group was considerably greater than that in the D2 group (95.3% [95% CI 91.6-99.0%] vs. 87.6% [95% CI 80.7-94.5%], HR 0.369, 95% CI 0.138-0.983; log-rank p = 0.043). CONCLUSION: Laparoscopic D2 + CME for T1-3N0M0 GC is safe and feasible. Furthermore, it not only reduces the local recurrence rate but also improves the 5-year DFS in cases of T1b-3N0M0 GC.

Construction of a nomogram based on clinicopathologic features to predict the likelihood of No. 253 lymph node metastasis in rectal cancer patients.

PURPOSE: To explore the high-risk factors for rectal cancer No.253 lymph node metastasis (LNM) and to construct a risk nomogram for the individualized prediction of No.253 LNM. METHODS: This was a retrospective analysis of 425 patients with rectal cancer who underwent laparoscopic-assisted radical surgery. Independent risk factors for rectal cancer No.253 LNM was identified using multivariate logistic regression analysis, and a risk prediction nomogram was constructed based on the independent risk factors. In addition, the performance of the model was evaluated by discrimination, calibration, and clinical benefit. RESULTS: Multivariate logistic regression analysis showed that No.253 lymphadenectasis on CT (OR 10.697, P < 0.001), preoperative T4-stage (OR 4.431, P = 0.001), undifferentiation (OR 3.753, P = 0.004), and preoperative Ca199 level > 27 U/ml (OR 2.628, P = 0.037) were independent risk factors for No.253 LNM. A nomogram was constructed based on the above four factors. The calibration curve of the nomogram was closer to the ideal diagonal, indicating that the nomogram had a better fitting ability. The area under the ROC curve (AUC) was 0.865, which indicated that the nomogram had high discriminative ability. In addition, decision curve analysis (DCA) showed that the model could show better clinical benefit when the threshold probability was between 1% and 50%. CONCLUSION: Preoperative No.253 lymphadenectasis on CT, preoperative T4-stage, undifferentiation, and elevated preoperative Ca199 level were found to be independent risk factors for the No.253 LNM. A predictive model based on these risk factors can help surgeons make rational clinical decisions.

The short- and long-term effect of membrane anatomy-guided laparoscopic D2 lymphadenectomy plus regional complete mesogastrium excision for locally advanced gastric cancer.

OBJECTIVE: Retrospectively analyzed the short- and long-term efficacy between laparoscopic D2 lymphadenectomy plus regional complete mesogastrium excision (D2 + rCME) and traditional laparoscopic D2 in the treatment of patients with locally advanced gastric cancer (LAGC), in order to obtain more evidence for D2 + rCME gastrectomy. METHODS: A total of 599 LAGC patients who underwent laparoscopy-assisted radical gastrectomy from January 2014 to December 2019, including 367 cases in the D2 + rCME group and 232 cases in the D2 group. Intraoperative and postoperative clinicopathological parameters, postoperative complications and long-term survival in the two groups were statistically analyzed. RESULTS: No significant differences in the positive rate of mesogastric tumor deposits, the number of positive lymph nodes and postoperative length of stay were found between the two groups (P > 0.05). In the D2 + rCME group, intraoperative blood loss was significantly reduced (84.20 ± 57.64 ml vs. 148.47 ± 76.97 ml, P < 0.001), the time to first postoperative flatus and first liquid diet intake were significantly shortened (3[2-3] days vs. 3[3-3] days, P < 0.001; 7[7-8] days vs. 8[7-8] days, P < 0.001), and the number of lymph nodes dissected was greater (43.57 ± 16.52 pieces vs. 36.72 ± 13.83 pieces, P < 0.001). The incidence of complications did not significantly differ between the D2 + rCME group (20.7%) and D2 group (19.4%) (P > 0.05). Although there was no statistically difference in 3-year OS and DFS between the two groups. However, the trend was better in D2 + rCME group. In subgroup analysis, patients with positive tumor deposits (TDs) in the D2 + rCME group had significantly better 3-year DFS compared With D2 group (P < 0.05). CONCLUSION: Laparoscopic D2 + rCME is safe and feasible for the treatment of LAGC and is characterized by less bleeding, greater lymph node dissection and rapid recovery, without increasing postoperative complications. D2 + rCME group showed a better trend of long-term efficacy, especially significant beneficial for LAGC patients who with positive TDs.

A prediction nomogram for postoperative gastroparesis syndrome in right colon cancer: a retrospective study.

PURPOSE: To investigate the high-risk factors for postoperative gastroparesis syndrome (PGS) in right colon cancer and to build a prediction nomogram for personalized prediction of PGS. METHODS: Our study retrospectively analyzed 361 patients with right colon cancer who underwent right hemicolectomy at The First Hospital of Putian City in Fujian Province, China and who were hospitalized between January 2012 and July 2022. Multivariate logistic regression was used to determine the independent risk factors for PGS and to establish a nomogram model. Furthermore, discrimination, calibration, and clinical benefits were used to evaluate the model. RESULTS: The multivariate logistic regression revealed that dissection of the subpyloric lymph nodes (No. 206 lymph node) (OR 5.242, P = 0.005), preoperative fasting blood glucose level (OR 3.708, P = 0.024), preoperative albumin level (OR 3.503, P = 0.020), and total operative time (OR 4.648, P = 0.014) were independent risk factors for PGS. Based on the above four factors, the area under the ROC curve (AUC) and C-index of the nomogram were 0.831. The prediction nomogram's calibration curve was closer to the ideal diagonal, and the Hosmer‒Lemeshow test indicated that the nomogram fit well (P = 0.399). Moreover, the decision curve analysis revealed that the model can present better clinical benefits when the threshold probability was between 1 and 28%, and the internal validation verified the dependability of the model (C-index = 0.948). CONCLUSIONS: A risk prediction nomogram based on perioperative factors provided the physician with a simple, visual, and efficient tool for the prediction and management of PGS in right colon cancer.

Comparison of ultrasound-guided serratus anterior plane block and thoracic paravertebral block in postoperative analgesia and inflammation control in patients undergoing upper abdominal surgery.

Objective: To compare the effects of ultrasound-guided serratus anterior plane block (SAPB) and thoracic paravertebral block (TPVB) on postoperative analgesia and inflammation control in patients undergoing upper abdominal surgery. Methods: This is a retrospective observational study. The records of patients who underwent upper abdominal surgery in our hospital from June 2019 to January 2021 were selected and retrospectively divided into two groups based on the analgesia method. Fifty-nine patients received ultrasound-guided SAPB analgesia (SAPB-group) and 55 patients received ultrasound-guided TPVB analgesia (TPVB-Group). Patients were matched for age, gender and body-mass index (BMI). The visual analogue scale (VAS) scores of pain at two hours(T1), six hours (T2), 12 hours (T3), 24 hours (T4) and 48 hours (T5) after the operation were compared between the two groups. The levels of interleukin-6 (IL-6), interleukin-10(IL-10) and tumor necrosis factor-α (TNF-α) at the completion of surgery (T0) and T4 were compared between the two groups. Results: The duration of block in SAPB-group was higher than that in TPVB-group (P<0.05). VAS scores of SAPB-groups were significantly lower than those of TPVB-group at all-time points (P<0.05) except at rest 48 hour after the procedure. The levels of IL-6, IL-10 and TNF-α at 24 hours after the operation in both groups were significantly higher than immediately at the end of the operation (P<0.05). Levels of IL-6 and TNF-α 24 hours after the operation were significantly lower in the SAPB-group than in the TPVB-group (P<0.05), while the levels of IL-10 24 hours after the operation were significantly higher in the SAPB-group (P<0.05). Conclusions: SAPB block under ultrasound guidance for patients undergoing upper abdominal surgery has good anesthetic and analgesic effect and can significantly improve the level of postoperative inflammation.

Impact of rapid on-site evaluation combined with endobronchial ultrasound and virtual bronchoscopic navigation in diagnosing peripheral lung lesions.

BACKGROUND: To investigate the value of endobronchial ultrasound (EBUS) and virtual bronchoscopic navigation (VBN) combined with rapid on-site evaluation (ROSE) in diagnosing peripheral pulmonary lesions (PPLs). METHODS: Between January 1st 2019 to September 1st 2021, EBUS and VBN examination were performed in expected consecutive patients with PPLs who were admitted to Zhangzhou Affiliated Hospital of Fujian Medical University (Fujian, China). Finally, based on the calculation of expected diagnostic yield of R-EBUS biopsy and drop out, 198 eligible patients were randomly divided into ROSE group (100 cases) and non-ROSE group (98 cases). The diagnostic yield of brushing and biopsy, the complications, the procedure time, the diagnosis time and expense during diagnosis were analyzed. RESULTS: In the ROSE group, the positive rate of EBUS brushing and biopsy were 68%, 84%, respectively. The average procedure time and diagnosis time were 18.6 ± 6.8 min, 3.84 ± 4.28 days, respectively, and the average expense was 643.44 ± 706.56 US.$ (4093.15 ± 4494.67 yuan ¥). In the controls, the positive rate of brushing and biopsy were 44%, 74%, respectively. The average procedure time and diagnosis time were 15.4 ± 5.7 min, 6.46 ± 3.66 days, respectively. And the average expense during diagnosis was 1009.27 ± 713.89 US.$ (6420.28 ± 4541.33 yuan ¥). There was significant difference in the positive rate of EBUS brushing and biopsy, diagnosis time and expense during diagnosis between both groups. And no significant difference was observed in the complications and the procedure time. Additionally, the impact of ROSE on diagnostic yield in right upper lobe and the size of lesion ≤ 2 cm in diameter was significant. CONCLUSION: In combination with ROSE, EBUS could significantly improve the positive rate of diagnosing PPLs, shorten diagnosis time and reduce expense during diagnosis. ROSE will be of great importance in the diagnosis of PPLs and medical resource.

Potential roles of mesenchymal stromal cells in islet allo- and xenotransplantation for type 1 diabetes mellitus.

Islet transplantation is poised to play an important role in the treatment of type 1 diabetes mellitus (T1DM). However, there are several challenges limiting its widespread use, including the instant blood-mediated inflammatory reaction, hypoxic/ischemic injury, and the immune response. Mesenchymal stem/stromal cells (MSCs) are known to exert regenerative, immunoregulatory, angiogenic, and metabolic properties. Here, we review recent reports on the application of MSCs in islet allo- and xenotransplantation. We also document the clinical trials that have been undertaken or are currently underway, relating to the co-transplantation of islets and MSCs. Increasing evidence indicates that co-transplantation of MSCs prolongs islet graft survival by locally secreted protective factors that reduce immune reactivity and promote vascularization, cell survival, and regeneration. MSC therapy may be a promising option for islet transplantation in patients with T1DM.

Modulation of SRSF2 expression reverses the exhaustion of TILs via the epigenetic regulation of immune checkpoint molecules.

The elevated expression of immune checkpoints by the tumor microenvironment is associated with poor prognosis in several cancers due to the exhaustion of tumor-infiltrating lymphocytes (TILs), and the effective suppression of the expression of these genes is key to reversing the exhaustion of TILs. Herein, we determined that serine/arginine-rich splicing factor 2 (SRSF2) is a target for blocking the tumor microenvironment-associated immunosuppressive effects. We found that the expression of SRSF2 was increased in exhausted T cells and that SRSF2 was involved in multiple immune checkpoint molecules mediating TILs' exhaustion. Furthermore, SRSF2 was revealed to regulate the transcription of these immune checkpoint genes by associating with an acyl-transferases P300/CBP complex and altering the H3K27Ac level near these genes, thereafter influencing the recruitment of signal transducer and activator of transcription 3 (STAT3) to these gene promoters. Collectively, our data indicated that SRSF2 functions as a modulator of the anti-tumor response of T cells and may be a therapeutic target for reversing the exhaustion of TILs.

CCL20-CCR6 axis directs sperm-oocyte interaction and its dysregulation correlates/associates with male infertility‡.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11 kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction.

Expression, purification and characterization of 5'-nucleotidase from caterpillar fungus by efficient genome-mining.

5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 µmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

Erratum: Association of 3 Common Polymorphisms of IL-27 Gene with Susceptibility to Cancer in Chinese People: Evidence from an Updated Meta-Analysis of 27 Studies.

The authors informed the journal that an error occurred in their manuscript.Figure 3C was mistakenly merged by the authors.The new version of the Figure 3C is as below.Reference:1. Meng Zhang, Xiuxiu Tan, Junjie Huang, Zekai Ke, Yukun Ge, Hu Xiong, Wei Lu, Lu Fang, Zhiming Cai, Song Wu: Association of 3 Common Polymorphisms of IL-27 Gene with Susceptibility to Cancer in Chinese: Evidence From an Updated Meta-Analysis of 27 Studies. Med Sci Monit 2015; 21: 2505-2513. DOI: 10.12659/MSM.895032.

Association between Aldose Reductase Gene C(-106)T Polymorphism and Diabetic Retinopathy: A Systematic Review and Meta-Analysis.

Controversial results regarding the associations between aldose reductase (AR) genetic polymorphisms and diabetic retinopathy (DR) have been reported for many years. The present meta-analysis was performed to clarify the effects of the AR gene C(-106)T polymorphism on DR risk. The PubMed, Web of Sciences, Cochrane library, EMBASE, Chinese National Knowledge Infrastructure, and Wan Fang databases were extensively searched in Chinese to select relevant studies with an updated date of April 25, 2018. The Newcastle-Ottawa Scale (NOS) was applied to assess quality. The random-effects model was applied to calculate the pooled OR and 95% CI. This meta-analysis identified 23 studies with an average score of 7.52 for NOS analysis, including 4,313 DR cases and 5,128 diabetes mellitus (DM) control cases. In the overall analysis, a significant association between the AR gene C(-106)T polymorphism and DR susceptibility was found. In subgroups stratified by DM type and ethnicity, significantly increased risks for DR were found in DM type 1, East Asian populations, and Middle Eastern populations. Compared with DR control cases, the following associations were found: T vs. C: OR 0.91, 95% CI 0.85-0.97, I2 = 72.9%; CT + TT vs. CC: OR 0.75, 95% CI 0.68-0.81, I2 = 86.7%; and CT vs. CC: OR 0.86, 95% CI 0.78-0.94, I2 = 70.5%. The results of this meta-analysis showed a significant association between the AR gene C(-106)T polymorphism and susceptibility to DR in DM patients. DM patients with allele T and CT+TT genotype of the AR gene may have a lower risk of DR.

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

Directing cellular information flow via CRISPR signal conductors.

The complex phenotypes of eukaryotic cells are controlled by decision-making circuits and signaling pathways. A key obstacle to implementing artificial connections in signaling networks has been the lack of synthetic devices for efficient sensing, processing and control of biological signals. By extending sgRNAs to include modified riboswitches that recognize specific signals, we can create CRISPR-Cas9-based 'signal conductors' that regulate transcription of endogenous genes in response to external or internal signals of interest. These devices can be used to construct all the basic types of Boolean logic gates that perform logical signal operations in mammalian cells without needing the layering of multiple genetic circuits. They can also be used to rewire cellular signaling events by constructing synthetic links that couple different signaling pathways. Moreover, this approach can be applied to redirect oncogenic signal transduction by controlling simultaneous bidirectional (ON-OFF) gene transcriptions, thus enabling reprogramming of the fate of cancer cells.

TNF-α promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression.

BACKGROUND: The major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated. METHODS: We assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus. RESULTS: Recombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs. CONCLUSION: Our data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.

A potential role of TLR2 in xenograft rejection of porcine iliac endothelial cells: An in vitro study.

BACKGROUND: Porcine vascular endothelial cells are a major participant in xenograft rejection. The Toll-like receptor 2 (TLR2) pathway plays an important role in both innate and adaptive immunity. The specific role of TLR2 in the response to a xenograft has not been reported. Whether the TLR2 pathway in pig vascular endothelial cells is involved in acute rejection needs to be investigated, and the mechanism is explored. METHODS: We used a modified antibody-dependent complement-mediated cytotoxicity (ADCC) assay to conduct in vitro experiments. In porcine iliac artery endothelial cells (PIECs), siRNA was used to knock down the expression of TLR2, CXCL8, and CCL2. The effect of human serum or inactivated human serum on the expression of TLR2 was analyzed by real-time PCR and Western blotting, and transwell assays were used to assess the chemotactic efficiency of PIECs on human monocyte-macrophages (THP-1 cells) and human neutrophils. The downstream signaling pathways activated by human serum were detected by Western blotting, and the regulation of proinflammatory chemokines and cytokines by TLR2 signaling was assessed by real-time PCR and ELISA. RESULTS: TLR2 was significantly upregulated in PIECs after exposure to human serum, and porcine proinflammatory chemokines, CXCL8 and CCL2, were induced, at least partially, in a TLR2-dependent pattern; the upregulated chemokines participated in the chemotaxis of human neutrophils and THP-1 cells across the species barrier. CONCLUSIONS: (i) TLR2 is significantly upregulated in PIECs by human serum, (ii) the elevated TLR2 participates in the chemotaxis of inflammatory cells through the secretion of chemokine CCL2 and CXCL8, and (iii) blockade of TLR2 would be beneficial for xenograft survival.

Potential pathological role of pro-inflammatory cytokines (IL-6, TNF-α, and IL-17) in xenotransplantation.

The major limitation of organ transplantation is the shortage of available organs from deceased human donors which leads to the deaths of thousands of patients each year. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection and coagulation dysfunction are two major hurdles for the successful survival of pig xenografts in primate recipients. Pro-inflammatory cytokines, such as IL-6, TNF-α, and IL-17, play important roles in many diseases and in allotransplantation. However, the pathological roles of these pro-inflammatory cytokines in xenotransplantation remain unclear. Here, we briefly review the signaling transduction and expression regulation of IL-6, TNF-α, and IL-17 and evaluate their potential pathological roles in in vitro and in vivo models of xenotransplantation. We found that IL-6, TNF-α, and IL-17 were induced in most in vitro or in vivo xenotransplantation model. Blockade of these cytokines using gene modification, antibody, or inhibitor had different effects in xenotransplantation. Inhibition of IL-6 signaling with tocilizumab decreased CRP but did not increase xenograft survival. The one possible reason is that tocilizumab can not suppress IL-6 signaling in porcine cells or organs. Other drugs which inhibit IL-6 signaling need to be investigated in xenotransplantation model. Inhibition of TNF-α was beneficial for the survival of xenografts in pig-to-mouse, rat, or NHP models. Blockade of IL-17 using a neutralizing antibody also increased xenograft survival in several animal models. However, the role of IL-17 in the pig-to-NHP xenotransplantation model remains unclear and needs to be further investigated. Moreover, blockade of TNF-α and IL-6 together has got a better effect in pig-to-baboon kidney xenotransplantation. Blockade two or even more cytokines together might get better effect in suppressing xenograft rejection. Better understanding the role of these cytokines in xenotransplantation will be beneficial for choosing better immunosuppressive strategy or producing genetic modification pig.