Evaluation of neoadjuvant immunotherapy in resectable gastric/gastroesophageal junction tumors: a meta-analysis and systematic review.

Background: Neoadjuvant therapy for resectable gastric cancer/gastroesophageal junction tumors is progressing slowly. Although immunotherapy for advanced gastric cancer/gastroesophageal junction tumors has made great progress, the efficacy and safety of neoadjuvant immunotherapy for locally resectable gastric cancer/gastroesophageal junction tumors have not been clearly demonstrated. Here, we conducted a systematic review and meta-analysis to assess the efficacy and safety of neoadjuvant immunotherapy and advance the current research. Methods: Original articles describing the safety and efficacy of neoadjuvant immunotherapy for resectable gastric cancer/gastroesophageal junction tumors published up until October 15, 2023 were retrieved from PubMed, Embase, the Cochrane Library, and other major databases. The odds ratios (OR) and 95% confidence intervals (CIs) were calculated for heterogeneity and subgroup analysis. Results: A total of 1074 patients from 33 studies were included. The effectiveness of neoadjuvant immunotherapy was mainly evaluated using pathological complete remission (PCR), major pathological remission (MPR), and tumor regression grade (TRG). Among the included patients, 1015 underwent surgical treatment and 847 achieved R0 resection. Of the patients treated with neoadjuvant immunotherapy, 24% (95% CI: 19%-28%) achieved PCR and 49% (95% CI: 38%-61%) achieved MPR. Safety was assessed by a surgical resection rate of 0.89 (95% CI: 85%-93%), incidence of ≥ 3 treatment-related adverse events (TRAEs) of 28% (95% CI: 17%-40%), and incidence of ≥ 3 immune-related adverse events (irAEs) of 19% (95% CI: 11%-27%). Conclusion: Neoadjuvant immunotherapy, especially neoadjuvant dual-immunotherapy combinations, is effective and safe for resectable gastric/gastroesophageal junction tumors in the short term. Nevertheless, further multicenter randomized trials are required to demonstrate which combination model is more beneficial. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=358752, identifier CRD42022358752.

Clinical study of thoracoscopic assisted different surgical approaches for early thymoma: a meta-analysis.

OBJECTIVE: The efficacy and safety of subxiphoid thoracoscopic thymectomy (SVATS) for early thymoma are unknown. The purposes of this meta-analysis were to evaluate the effectiveness and safety of SVATS for early thymoma, to compare it with unilateral intercostal approach video thoracoscopic surgery (IVATS) thymectomy, and to investigate the clinical efficacy of modified subxiphoid thoracoscopic thymectomy (MSVATS) for early anterior mediastinal thymoma. METHODS: Original articles describing subxiphoid and unilateral intercostal approaches for thoracoscopic thymectomy to treat early thymoma published up to March 2023 were searched from PubMed, Embase, and the Cochrane Library. Standardized mean differences (SMDs) and 95% confidence intervals (CIs) were calculated and analyzed for heterogeneity. Clinical data were retrospectively collected from all Masaoka stage I and II thymoma patients who underwent modified subxiphoid and unilateral intercostal approach thoracoscopic thymectomies between September 2020 and March 2023. The operative time, intraoperative bleeding, postoperative drainage, extubation time, postoperative hospital stay, postoperative visual analog pain score (VAS), and postoperative complications were compared, and the clinical advantages of the modified subxiphoid approach for early-stage anterior mediastinal thymoma were analyzed. RESULTS: A total of 1607 cases were included in the seven studies in this paper. Of these, 591 cases underwent SVATS thymectomies, and 1016 cases underwent IVATS thymectomies. SVATS thymectomy was compared with IVATS thymectomy in terms of age (SMD = - 0.09, 95% CI: -0.20 to - 0.03, I2 = 20%, p = 0.13), body mass index (BMI; SMD = - 0.10, 95% CI: -0.21 to - 0.01, I2 = 0%, p = 0.08), thymoma size (SMD = - 0.01, 95% CI: -0.01, I2 = 0%, p = 0.08), operative time (SMD = - 0.70, 95% CI: -1.43-0.03, I2 = 97%, p = 0.06), intraoperative bleeding (SMD = - 0.30. 95% CI: -0.66-0.06, I2 = 89%, p = 0.10), time to extubation (SMD = - 0.34, 95%CI: -0.73-0.05, I2 = 91%, p = 0.09), postoperative hospital stay (SMD = - 0.40, 95% CI: -0.93-0.12, I2 = 93%, p = 0.13), and postoperative complications (odds ratio [OR] = 0.94, 95% CI: 0.42-2.12, I2 = 57%, p = 0.88), which were not statistically significantly different between the SVATS and IVATS groups. However, the postoperative drainage in the SVATS group was less than that in the IVATS group (SMD = - 0.43, 95%CI: -0.84 to - 0.02, I2 = 88%, p = 0.04), and the difference was statistically significant. More importantly, the postoperative VAS was lower in the SVATS group on days 1 (SMD = - 1.73, 95%CI: -2.27 to - 1.19, I2 = 93%, p < 0.00001), 3 (SMD = - 1.88, 95%CI: -2.84 to - 0.81, I2 = 97%, p = 0.0005), and 7 (SMD = - 1.18, 95%CI: -2.28 to - 0.08, I2 = 97%, p = 0.04) than in the IVATS group, and these differences were statistically significant. A total of 117 patients undergoing thoracoscopic thymectomy for early thymoma in the Department of Thoracic Surgery of the Second Hospital of Jilin University were retrospectively collected and included in the analysis, for which a modified subxiphoid approach was used in 42 cases and a unilateral intercostal approach was used in 75 cases. The differences between the two groups (MSVATS vs. IVATS) in general clinical characteristics such as age, sex, tumor diameter, Masaoka stage, Word Health Organization (WHO) stage, and intraoperative and postoperative conditions, including operative time, postoperative drainage, extubation time, postoperative hospital stay, and postoperative complication rates, were not statistically significant (p > 0.05), while BMI, intraoperative bleeding, and VAS on postoperative days 1, 3, and 7 were all statistically significant (p < 0.05) in the MSVATS group compared with the IVATS group. CONCLUSION: The meta-analysis showed that the conventional subxiphoid approach was superior in terms of postoperative drainage and postoperative VAS pain scores compared with the unilateral intercostal approach. Moreover, the modified subxiphoid approach had significant advantages in intraoperative bleeding and postoperative VAS pain scores compared with the unilateral intercostal approach. These results indicate that MSVATS can provide more convenient operation conditions, a better pleural cavity view, and a more complete thymectomy in the treatment of early thymoma, indicating that is a safe and feasible minimally invasive surgical method.

Transmission and resistome of extremely drug-resistant tuberculosis in Beijing, China: A retrospective population-based epidemiological study.

BACKGROUND: In this study, we utilized whole genome sequencing (WGS) of clinical extremely drug-resistant tuberculosis (EDR-TB) strains collected during 2014-2020 in Beijing to detect clustered strains. METHODS: A retrospective cohort study was conducted by inclusion of EDR-TB patients with positive cultures in Beijing between 2014 and 2020. RESULTS: A total of 95 EDR-TB patients were included in our analysis. Up on the WGS based genotyping, 94 (94/95, 98.9%) out of 95 were identified as lineage 2 (East Asia). The pairwise genomic distance analysis identified 7 clusters, ranging in size from 2 to 5 isolates. The clustering rate of EDR-TB was 21.1%; while no patients had significantly higher odds of clustering. All isolates harbor rpoB RRDR mutations that confer RIF resistance and katG or inhA promoter mutations that confer INH resistance. Of 95 EDR-TB isolates, a total of 15 mutation types were recorded in the transcriptional regulator mmpR5. In vitro susceptibility testing results revealed that 14 (14/15, 93.3%) out of 15 mutation types were resistant to CFZ; whereas only 3 (3/15, 20.0%) showed resistance to BDQ. Interestingly, 12 isolates harbored mutations within rrl locus, whereas only mutations at positions 2294 and 2296 conferred CLA resistance. Favorable outcomes of EDR-TB patients were positively associated with more effective drugs in the regimes. CONCLUSION: WGS data demonstrate limited transmission of EDR-TB in this metropolis city. WGS-based drug susceptibility predictions will bring benefits to EDR-TB patients to formulate optimal therapeutic regimens.

Neoadjuvant chemotherapy plus nivolumab with or without ipilimumab in operable non-small cell lung cancer: the phase 2 platform NEOSTAR trial.

Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7-43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6-61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129 .).

Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations.

BACKGROUND & OBJECTIVES: Accurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings. METHODS: A total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant. RESULTS: Overall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 µg/ml, and the resistotype population were no lower than 4 µg/ml, indicating that 4 µg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 µg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 µg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity. CONCLUSIONS: We propose 4 µg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Significant difference in Th1/Th2 paradigm induced by tuberculosis-specific antigens between IGRA-positive and IGRA-negative patients.

False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1ß, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals [271 TB patients and 36 individuals with latent TB infection (LTBI)], demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting Mycobacterium tuberculosis (MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.

A Peptidomic Approach to Identify Novel Antigen Biomarkers for the Diagnosis of Tuberculosis.

Background: Here, we conducted a peptidomic study in murine model to identify novel antigen biomarkers for the diagnosis of tuberculosis (TB) with improved performance. Methods: Four recombinant proteins, including Mycobacterium tuberculosis protein 32 (MPT32), Mycobacterium tuberculosis protein 64 (MPT64), culture filtrate protein 10 (CFP10), and phosphate ABC transporter substrate-binding lipoprotein (PstS1) were expressed and intravenously injected into BALB/c mice. The serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of candidate peptides in serum of suspected TB patients were determined using competitive enzyme-linked immunosorbent assay. Results: A total of 65 peptides from 4 MTB precursor recombinant proteins were identified in mouse serum by LC-MS/MS, of which 5 peptides were selected as candidates for serological analysis. The concentrations of peptides MPT64-2, CFP10-2 and PstS1-2 in TB patients were significantly higher than those in non-TB patients. MPT64-2 exhibited the most promising sensitivity (81.4%), followed by PstS1-2 and CFP10-2. In addition, PstS1-2 had the highest specificity (93.3%), followed by CFP10-2 and MPT64-2. According to the area under the curve (AUC), MPT64-2 (AUC = 0.863), PstS1-2 (AUC = 0.812) and CFP10-2 (AUC = 0.809) exhibited better diagnostic validity. Conclusion: We develop an effective approach to identify new antigen biomarkers via LC-MS/MS-based peptidomics. Multiple peptides exhibit promising efficacy in diagnosis of active TB patients.

Prevalence of extensively drug-resistant tuberculosis in a Chinese multidrug-resistant TB cohort after redefinition.

OBJECTIVES: Recently, the definition of extensively drug-resistant TB (XDR-TB) has been revised. In this study, we conducted a descriptive and retrospective study to determine the prevalence of XDR-TB in a Chinese multidrug-resistant TB (MDR-TB) cohort. METHODS: Broth microdilution method was performed to determine in vitro susceptibilities of Mycobacterium tuberculosis (MTB) isolates to (FQs), bedaquiline (BDQ) and linezolid (LZD). The putative drug target genes conferring drug resistance were screened by DNA sequencing. RESULTS: A total of 425 MDR-TB isolates were included from 13 pilots in China. LZD and BDQ resistance were noted in 30 (7.1%) and 10 (2.4%) isolates. On the basis of latest definitions, 114 (26.8%) were MDR-TB, 282 (66.4%) were pre-XDR-TB, and 29 (6.8%) were XDR-TB. Among 311 FQ-resistant isolates, 265 harbored genetic mutations within QRDRs. The most common mutations were observed at codon 94 of gyrA, accounting for 47.2% of FQ-resistant MTB isolates. Only mutations within the Rv0678 gene were found to confer BDQ resistance in our cohort, conferring 40.0% of BDQ resistance. For LZD resistance, 53.3% of LZD-resistant isolates carried genetic mutations in rplC or 23S rRNA. The most frequent mutation was Cys154Arg in the rplC gene. In addition, we recorded two MDR-TB patients with resistance to both BDQ and LZD, of which one patient experienced continuous positive culture of MTB despite inclusion of efficacious moxifloxacin. CONCLUSION: Our results demonstrate that the low prevalence of XDR-TB holds great promise for MDR-TB treatment with WHO-endorsed regimens containing BDQ-LZD combination, whereas the high prevalence of FQ-resistance in MDR-TB patients warrants national attention.

Comparative in vitro susceptibility of a novel fluoroquinolone antibiotic candidate WFQ-228, levofloxacin, and moxifloxacin against Mycobacterium tuberculosis.

OBJECTIVE: WFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. METHODS: We selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. RESULTS: MFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. CONCLUSION: WFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.

Comparison of clinical effectiveness of nurse led care among Chinese patients with cancer: A prospective study evaluating effective patient care compared to consultant oncologist.

OBJECTIVE: The NLC model in oncology setting was not well established in China, and there was no study evaluating the clinical effectiveness of NLC versus oncologist-led care (OLC) in Chinese patients with cancer. We therefore designed a pilot study to evaluate the clinical usefulness after NLC versus OLC in Chinese patients with cancer. METHODS: This pilot, single center, prospective study was designed to evaluate clinical effectiveness of NLC versus oncologist-led care (OLC) in Chinese patients with cancer. Adult patients of either gender (aged between 20 and 65 years) who were diagnosed with any cancer at China were included. The patients' with stage IV cancer or who were not willing to give written consent to participate in this study were excluded during screening phase of our study. We assessed the symptoms from each enrolled patients, the most common symptoms associated with any cancer patients are pain, dyspnea and constipation. Also distress symptoms (caused by pain and dyspnea) and low quality of life are seen in patients with advanced stage of cancer. RESULTS: A total of 220 patients who were visited in our hospital for consultation were enrolled and assigned to nurse led care and oncologist led care group (110 patients in each group). Pain intensity, dyspnea intensity and constipation intensity was recorded for patients of nurse led care and oncologist led care group. Pain intensity, dyspnea intensity and constipation intensity on numerical rating scale was numerically lesser in Nurse led care group as compared to oncologist led care group. However, the difference was not statistically significant (p>0.05). The mean QoL score of each key domain of QoL was higher in nurse led care group when compared to oncologist led care group. Overall, significant improvement in quality of life was observed in individuals underwent in nurse led care group than oncologist led care group. CONCLUSION: The results of this preliminary study showed that NLC results in better clinical outcome in terms of improvement in pain intensity, dyspnea intensity, symptom distress, constipation score, and QOL in comparison to oncologist led care among Chinese cancer patients. This study results can aid as a base for steering outsized clinical study to form the model of NLC among Chinese cancer patients.

Effect of carbodiimide cross-linking of decellularized porcine pulmonary artery valvular leaflets.

Decellularization provides low immunogenicity and is only slightly subject to calcification in tissue engineering. However, the mechanical properties of the tissues are weakened after decellularization. We adopted cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to treat decellularized porcine pulmonary artery valvular leaflets to improve their mechanical properties. Twenty porcine pulmonary artery valvular leaflets were divided into three groups: the fresh control group A, group B treated with trypsin and Triton X-100 to remove cells, and group C cross-linked with EDC after decellularization. All samples were evaluated the physical and mechanical properties and were then subcutaneously embedded in rabbits. These valvular leaflets were removed after 1, 2, or 4 weeks and checked for pathological changes. The cells of the valvular leaflets were completely removed. The thickness of the valvular leaflets was thinner in group B than in group A (P<0.01). In the subcutaneous embedding of the group B samples, there was mild immunological response after 1-2 weeks, and parts of the scaffolds were degraded. After 4 weeks, fibroblasts had grown into the scaffolds. In group C, there was an increase in the tensile strength and thermal shrinkage temperature in group C compared with group B (P<0.01). In subcutaneous embedding of the group C samples, there was a mild immunological response after 1-2 weeks. The fibroblasts had grown into the samples. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary artery valvular leaflets and both decrease the valvular leaflets' rejection and promote tissue regeneration in vivo.

Intracellular ATP concentration contributes to the cytotoxic and cytoprotective effects of adenosine.

Extracellular adenosine (ADE) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.

A novel surgical procedure: scaffold-pulmonary autograft transplantation.

Mitral valve-related operations are easy to perform and show good results, but to prevent severe thromboembolism or a high ratio of prosthetic valve destruction by tissue, lifetime anticoagulant therapy is essential after the operation. Thus, identifying a new type of surgical procedure and prosthetic valve to cure mitral valve diseases is necessary. Pulmonary valve autograft transplantation (Ross II) with the "top hat" transplantation technique was first reported by Ross DN to cure mitral disease. Because the "top hat" procedure has some shortcomings, we designed the scaffold-pulmonary autograft transplantation procedure and performed animal experiments to confirm the feasibility and effectiveness of the procedure. A total of 13 minipigs, weighing 20-25 kg, were employed as experimental animals to undergo scaffold-pulmonary autograft valve transplantation in our surgical animal lab. The surgical procedure was performed under hypothermic general anaesthesia and extracorporeal circulation (or cardiopulmonary bypass, CPB). Briefly, the chest cave was opened through the left intercostal, the pulmonary valve autograft was harvested during on-pump beating heart, and the pulmonary valve autograft was mounted in a self-made pulmonary valve scaffold and transferred to the mitral valve annulus without removing the mitral instruments. Finally, the outflow tract of the right ventricle was re-established with a pig pulmonary homograft. After finishing data collection, all animals were executed 1 hour after removal from the CPB. For the 13 minipigs that underwent the operation, the CPB time was 182.4 ± 23.4 min. Two of the thirteen cases died of bleeding during the operation and of a post-operative pulmonary embolism, and the remaining eleven survived for one hour. The pressure of the left atrium did not increase significantly (P = 1.00), and the ultrasonic cardiograph (UCG) showed good function of the new mitral valves, with mean ejection fraction (EF) values of 63.6%. The mitral valve orifice areas were 1.10 ± 0.13 cm(2) (pre-operation) and 1.01 ± 0.08 cm(2) (post-operation) (P = 0.013). The function and structure of the new mitral valves were normal. We preliminarily consider scaffold-pulmonary autograft valve transplantation to be a new alternative to cure mitral valve disease, but advanced chronic animal experiments will be needed to confirm the long-term results of the operation. The results showed it could be a new alternative to cure mitral valve disease.

Decellularized porcine pulmonary arteries cross-linked by carbodiimide.

The physical properties of the tissues are weakened after decellularization, and the exposed collagen fibers are prone to thrombogenesis. Several studies have proven that the use of carbodiimide (EDC) as a cross-linking agent can improve the properties of decellularized xenogeneic scaffold materials. We adopted EDC for the treatment of porcine pulmonary arteries in an effort to improve the physical properties of these arteries following decellularization. Twenty porcine pulmonary arteries were randomly divided into 3 groups. The control group (group A) consisted of fresh porcine pulmonary arteries with no further processing; group B was treated with trypsin and the detergent Triton X-100 to remove cells; and group C was cross-linked with EDC after trypsin and Triton X-100 treatment, as in group B. The pulmonary arteries were assessed based on water content, thickness, tensile strength, and thermal shrinkage temperature, to evaluate the physical properties of all of the samples. The scaffolds were then subcutaneously embedded in rabbits. These constructs were removed after 4 weeks and checked. The cells and matrix components of the arterial walls were removed and the fibrous scaffolds were retained. In group B, the moisture content of the pulmonary arterial walls was increased; and the thickness of the walls and the tensile strength of the pulmonary arteries were decreased in comparison with group A. In subcutaneous embedding of the group B samples in rabbits, after 4 weeks, fibroblasts had grown into the scaffolds and regenerated the tissue. The water content was decreased in the pulmonary arterial walls, there was an increase in the tensile strength and the thermal shrinkage temperature in group C compared with group B. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary arteries and decrease scaffold rejection and degradation and promote tissue regeneration in vivo.

Gambogic acid is a tissue-specific proteasome inhibitor in vitro and in vivo.

Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1) gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1) expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

Sanggenon C decreases tumor cell viability associated with proteasome inhibition.

Several flavonoids have been reported to be proteasome inhibitors, but whether prenylated flavonoids are able to inhibit proteasome function remains unknown. We report for the first time that Sanggenon C, a natural prenylated flavonoid, inhibits tumor cellular proteasomal activity and cell viability. We found that (1) Sanggenon C inhibited tumor cell viability and induced cell cycle arrest at G0/G1 phase; (2) Sanggenon C inhibited the chymotrypsin-like activity of purified human 20S proteasome and 26S proteasome in H22 cell lysate, and Sanggenon C was able to dose-dependently accumulate ubiquitinated proteins and proteasome substrate protein p27; (3) Sanggenon C-induced proteasome inhibition occurred prior to cell death in murine H22 and P388 cell lines; (4) Sanggenon C induced death of human K562 cancer cells and primary cells isolated from leukemic patients. We conclude that Sanggenon C inhibits tumor cell viability via induction of cell cycle arrest and cell death, which is associated with its ability to inhibit the proteasome function and that proteasome inhibition by Sanggenon C at least partially contributes to the observed tumor cell growth-inhibitory activity.

Gambogic acid enhances proteasome inhibitor-induced anticancer activity.

Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy.

Physiological levels of ATP negatively regulate proteasome function.

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is ∼100 µM. Intracellular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50 and 100 µM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.