A phase II randomised controlled trial of adjuvant tumour-infiltrating lymphocytes for pretreatment Epstein-Barr virus DNA-selected high-risk nasopharyngeal carcinoma patients.

PURPOSE: The safety and objective clinical responses were observed in the phase I study using adjuvant autologous tumour-infiltrating lymphocytes (TILs) following concurrent chemoradiotherapy (CCRT) in nasopharyngeal carcinoma (NPC) patients. METHODS AND MATERIALS: One hundred fifty-six patients with stage III-IVb and pretreatment Epstein-Barr virus DNA levels of ≥4000 copies/ml were randomly assigned to receive CCRT combined with TIL infusion (n = 78) or CCRT alone (n = 78). All patients received CCRT and patients assigned to the TIL group received TIL infusion within 1 week after CCRT. The primary endpoint was investigator-assessed progression-free survival (PFS) at 3 years. RESULTS: After a median follow-up of 62.3 months, no significant difference was observed in the 3-year PFS rate between the CCRT plus TIL infusion group and CCRT alone group (75.6% versus 74.4%, hazard ratios, 1.08; 95% confidence intervals, 0.62-1.89). TIL infusion was safe without grade 3 or 4 adverse events and all the high-grade adverse effects were associated with myelosuppression caused by CCRT. Exploratory analysis showed that a potential survival benefit was observed with TILs in patients with lower levels of circulating CD8+TIM3+ cells, serum IL-8 or PD-L1. The infused TIL products in patients with favourable outcomes were associated with increased transcription of interferon-γ and a series of inflammatory related genes and a lower exhausted score. CONCLUSION: The primary objective of prolonging PFS with CCRT plus TILs in high-risk NPC patients was not met. These findings may provide evidence for the design of future trials investigating the combination of TILs plus immune checkpoint inhibitors based on CCRT in high-risk NPC patients. TRIAL REGISTRATION NUMBER: NCT02421640.

Phase I study of adjuvant immunotherapy with autologous tumor-infiltrating lymphocytes in locally advanced cervical cancer.

BACKGROUNDAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has achieved remarkable clinical efficacy in metastatic cancers such as melanoma and cervical cancer (CC). Here, we explored the safety, feasibility, and preliminary tumor response and performed translational investigations of adjuvant immunotherapy using infusion of autogenous TILs (auto-TILs) following concurrent chemoradiotherapy (CCRT) in patients with CC who had locally advanced disease.METHODSTwenty-seven patients with CC with stage III-IV disease were recruited in this single-center, phase I study. TILs were isolated from lesions in the uterine cervix and generated under good manufacturing practice (GMP) conditions and then infused after CCRT plus i.m. IL-2 injections.RESULTSTILs from 20 of the 27 patients were successfully expanded, with a feasibility of 74.1%. Twelve patients received TILs following CCRT. Adverse events (AEs) were primarily attributable to CCRT. Only 1 (8.3%) patient experienced severe toxicity with a grade 3 hypersensitivity reaction after TIL infusion. No autoimmune AEs, such as pneumonitis, hepatitis, or myocarditis, occurred, and there were no treatment-related mortalities. Nine of 12 patients (75.0%) attained a complete response, with a disease control duration of 9-22 months. Translational investigation showed that the transcriptomic characteristics of the infused TIL products and some immune biomarkers in the tumor microenvironment and serum of patients with CC at baseline were correlated with the clinical response.CONCLUSIONTIL-based ACT following CCRT was safe in an academic center setting, with potentially effective responses in patients with locally advanced CC. "Hot" inflammatory immune environments were beneficial to the clinical efficacy of TIL-based ACT as adjuvant therapy.TRIAL REGISTRATIONClinicalTrials.gov NCT04443296.FUNDINGNational Key R&D Program; Sci-Tech Key Program of the Guangzhou City Science Foundation; the Guangdong Province Sci-Tech International Key Program; the National Natural Science Foundation of China.

A rapid strategy for screening high-efficiency PCSK9 inhibitors from Ginkgo biloba leaves by ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an attractive target for new cholesterol-lowering drug development. Here, we developed a method integrating ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay, aiming to explore potential PCSK9 inhibitors from mixtures rapidly and accurately. PCSK9 was expressed and purified firstly, and then the recombined PCSK9 was coated on the surface of magnetic beads (MBs). The PCSK9-immobilized MBs (PCSK9-MBs) were used for ligand fishing combined with HPLC and Q-TOF-MS/MS. Ginkgo biloba leaves (GBL), an herbal medicine widely used in Asia and Europe with good efficacy in treatment of hypercholesterolemia, were chosen as an illustration for ligand fishing. Two PCSK9 ligands were discovered from GBL and identified as kaempferol-3-O-rutinoside (1) and kaempferol 3-O-26″-(6‴-p-coumaroyl) glucosylrhamnoside (KCGR) (2). In order to verify fishing results and pick out more powerful PCSK9 inhibitors, molecular docking assay was further performed and KCGR was optimized to be an excellent PCSK9 inhibitor by the confirmation of affinity and activity bioassay. These results suggested that the developed approach could be applied to screen and analysis potential bioactive constituents from mixtures, which may improve the efficiency of drug discovery. Moreover, KCGR separated from GBL was expected to be a potential candidate of PCSK9 inhibitors.

Type I neurofibromatosis with spindle cell sarcoma: A case report.

BACKGROUND: Neurofibromatosis type I (NF1) is the most frequent subtype of neurofibromatosis. Its related tumor-suppressor syndromes are characterized by a predisposition to multiple tumor types and other disorder presentations. In addition, the incidence of tumors is much higher in patients with neurofibromatosis type I. However, there are very few reports at home and abroad on this topic. Here, we present a case of NF1 with spindle cell sarcoma. CASE SUMMARY: A 50-year-old male was found to have a right axillary mass for 20 years. Specialist examination found cafe-au-lait spots on many parts of the skin, rounded nodules in the skin, a bulge in the right armpit, touching a lump (10 cm × 6 cm, hard, unclear boundary, poor mobility, local tenderness). The anterior side of the thigh felt weakened on the opposite side; in the right groin a swollen lymph node (hard, clear border, good mobility, local tenderness). According to the results of positron emission tomography/computed tomography, puncture pathology and immunohistochemistry, genetic testing, a diagnosis of NF1 with spindle cell sarcoma was confirmed. According to the genetic testing result, the patient was given a targeted treatment with crizotinib. CONCLUSION: Surgery, chemotherapy and radiotherapy are the main treatment methods of NF1. However, with the continuous progress of molecular biology research, molecular targeted therapy may bring benefits for patients.

The correlation between possible RBD and cognitive function in Parkinson's disease patients in China.

OBJECTIVE: Cognitive impairment is an important symptom of Parkinson's disease (PD) and seriously affects patients' quality of life and prognosis. However, its cause is still uncertain. In about one-third of patients, PD is associated with rapid eye movement sleep behavior disorder (RBD), which is an independent risk factor for PD-associated dementia; but whether or not it relates to the cognitive function of patients with nondemented PD is still controversial. METHODS: The data from 89 enrolled PD patients were retrospectively analyzed. The RBD Questionnaire Hong-Kong (RBD-HK) was used to diagnose possible RBD (pRBD). There are 46 patients with possible RBD (the PD-pRBD) and 43 without (the PD-npRBD). PD disease severity, neuropsychological function, overall cognitive function, and various cognitive functions were assessed. RESULTS: There were significant between-group differences in scores on the Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), Digit Symbol Test (DST), Trail Making Test-A (TMT-A)-Time, TMT-Trail Making Test-B (TMT-B)-Time, Stroop Color-word Test, Clock Drawing Test (CDT), Boston Naming Test (BNT), Verbal Fluency Test (fruit), etc. (P < 0.05). INTERPRETATION: Patients in the PD-pRBD group had more cognitive impairment.

Rapid identification of urokinase plasminogen activator inhibitors from Traditional Chinese Medicines based on ultrafiltration, LC-MS and in silico docking.

The urokinase plasminogen activator (uPA) is regarded as the crucial trigger for plasmin generation, which is involved in several diseases especially for neoplasm metastasis. In this study, an efficient approach integrating ultrafiltration, LC/MS, bioassay and in silico docking, was proposed for rapidly detecting uPA ligands from Traditional Chinese Medicines (TCMs). Forty-two TCMs were initially assessed, and as illustrative case studies, Galla Chinensis and Sanguisorbae Radix, which appeared significant inhibitory activities on uPA, were chosen to develpe and verify the strategy. A total of seven uPA ligands were successfully detected and identified. Two of them, pentagalloylglucose and 28-O-ß-d-glucopyranosyl pomolic acid, were demonstrated to be potential inhibitors, with IC50 at 1.639 µM and 37.82 µM repectively. Furthermore, a combinatorial compound library screening combined with in silico docking assay, was revealed that ursolic acid (IC50 = 2.623 µM) was also speculated to be a potent parent structure for inhibition of uPA. This approach offers a multidimensional perspective to discover uPA-binding leading compounds from TCMs or other complex mixtures, which would provide an efficient route for drug discovery.

Chinese consensus on management of tyrosine kinase inhibitor-associated side effects in gastrointestinal stromal tumors.

Tyrosine kinase inhibitors (TKIs) have improved the overall survival of patients with gastrointestinal stromal tumors (GISTs), but their side effects can impact dose intensity and, consequently, the clinical benefit. To date, no guideline or consensus has been published on the TKI-associated adverse reactions. Therefore, the Chinese Society of Surgeons for Gastrointestinal Stromal Tumor of the Chinese Medical Doctor Association organized an expert panel discussion involving representatives from gastrointestinal surgery, medical oncology, cardiology, dermatology, nephrology, endocrinology, and ophthalmology to consider the systemic clinical symptoms, molecular and cellular mechanisms, and treatment recommendations of GISTs. Here, we present the resultant evidence- and experience-based consensus to guide the management of TKI-associated side events in clinical practice.

A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro.

The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.

Screening for biomarkers reflecting the progression of Babesia microti infection.

BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.

Simultaneous inhibition of NMDA and mGlu1/5 receptors by levo-corydalmine in rat spinal cord attenuates bone cancer pain.

Bone cancer pain is a challenge for its not completely clarified mechanism and broad clinical morbidity. Therefore, novel and more effective drugs are urgent needed for improvement of patients' quality of life. Glutamate receptors have been associated with the development of the central sensitization of chronic pain. Inhibition of N-methyl-d-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors can effectively attenuate bone cancer pain, respectively. Herein, our results indicated that levo-Corydalmine (l-CDL), a compound from Corydalis yanhusuo W.T. Wang, which has been used in traditional Chinese medicine for pain relief could effectively attenuate bone cancer pain induced by tibia bone cavity tumor cell implantation (TCI) through simultaneously inhibiting the NMDA and mGlu1/5 receptors in rat spinal cord without notable side effects. Both intragastric and intrathecal administration of l-CDL significantly alleviated the mechanical hypersensitivity induced by TCI in rats, and the analgesic effect of l-CDL could be reversed by intrathecal administration of NMDA receptor agonist NMDA and mGlu1/5 receptor agonist DHPG but not AMPA receptor agonist AMPA. l-CDL could also selectively suppress NMDA and DHPG induced rapid rise in Ca2+ oscillations in primary cultures neurons of spinal cord in vitro. The antinociception of l-CDL were partially mediated by the reduced phosphorylation of PKC γ and ERK1/2 in spinal cord of TCI rats in a NMDA and mGlu1/5 dependent manner. In conclusion, these results suggested that l-CDL attenuates TCI induced bone cancer pain through simultaneously inhibiting the NMDA and mGlu1/5 receptors and the downstream PKC γ, ERK1/2 signaling pathways in the spinal cord.

The effect of process parameters and microstructural changes on a new convenience food - quick-frozen paste-coated mushrooms (Agaricus bisporus).

The technology of quick-freezing paste-coated mushrooms (Agaricus bisporus) was studied and optimized. The best microwave pretreatment condition for 1 cm slices, regarding color protection, was 5.4 W/g, for 55, 55-60 and 60 s for mushrooms with 3, 4 and 5 cm diameter caps respectively. For a batch of paste (668.2-1034.6 g), the process parameters considered were oil content (46.6-63.4 g), water content (381-562.6 g) and flour content (166-334 g) with a constant additional content of 30 g starch, 9 g baking powder, 2.6 g carrageenan, 30 g salt and 3 g pepper. These parameters were investigated using response surface methodology (RSM) with a central composite design. The optimal levels of the major paste components were 300 g flour, 432.5 g water and 50 g oil. The freezing time and sensory acceptability for paste-coated Agaricus bisporus(PCAB) under the optimized conditions were 7.49 min and 6.2 respectively. The freezing curves of PCAB were established at different temperatures and the freezing rates were calculated to find the freezing characteristics. In addition, the cell structure of PCAB, frozen at -75 °C, the lowest freezing temperature, and studied using transmission electron microscopy, was similar in quality to that of fresh Agaricus bisporus. The results suggested that Agaricus bisporus can be quick-frozen with a paste coating to produce an acceptable and nutritious convenience food.

[Prokaryotic expression and function analysis of Schistosoma japonicum calpain].

OBJECTIVE: To clone and express recombinant calpain of Schistosoma japonicum (Sjcalpain), observe the distribution of Sjcalpain in S. japonicum cercariae and analyze its role in skin invasion. METHODS: The primers were designed according to the full-length sequence of calpain (GenBank accession No. AB016726). The genes encoding catalytic domain and Ca2+ binding domain of Sjcalpain were amplified by PCR, and the target fragments were subcloned into pET-28a. The recombinant proteins were expressed in E. coli BL21 (DE3) and purified by Ni-NTA resin. The rabbit polyclonal antibodies were prepared with the two purified recombinant proteins by immunizing New Zealand white rabbits. ELISA was used to detect the titer of rabbit antiserum. Immunolocalization was used to investigate the distribution of Sjcalpain in S. japonicum cercariae. Cercariae were incubated with specific inhibitor before infection of mice and the worm reduction rate was calculated. RESULTS: The recombinant expression vector Sjcalpain catalytic domain/pET28a and Sjcalpain Ca2+ binding domain/pET28a were constructed and the recombinant proteins were successfully expressed in E. coli BL21 (DE3) (about M(r) 43 000 and M(r) 39 000, respectively). The two target proteins were expressed as inclusion bodies. The purified target proteins were obtained through Ni-NTA affinity purification. ELISA result showed that the titer of prepared rabbit polyclonal antibodies was higher than 1 : 80 000. Immunolocalization study demonstrated that Sjcalpain protein was mainly expressed in the head of cercariae. Inhibition assays suggested that the average number of adult worms in calpain inhibitor-incubation group and control group was 19 and 23, respectively, with a worm reduction rate of 17.4%. CONCLUSION: Sjcalpain is mainly expressed in the head of S. japonicum cercariae. Inhibition of Sjcalpain could reduce the number of invading cercariae in infected mice, which suggest that Sjcalpain may play a role in skin invasion by cercariae.

An ion-pair principle for enantioseparations of basic analytes by nonaqueous capillary electrophoresis using the di-n-butyl L-tartrate-boric acid complex as chiral selector.

A chiral recognition mechanism of ion-pair principle has been proposed in this study. It rationalized the enantioseparations of some basic analytes using the complex of di-n-butyl l-tartrate and boric acid as the chiral selector in methanolic background electrolytes (BGEs) by nonaqueous capillary electrophoresis (NACE). An approach of mass spectrometer (MS) directly confirmed that triethylamine promoted the formation of negatively charged di-n-butyl l-tartrate-boric acid complex chiral counter ion with a complex ratio of 2:1. And the negatively charged counter ion was the real chiral selector in the ion-pair principle enantioseparations. It was assumed that triethylamine should play its role by adjusting the apparent acidity (pH*) of the running buffer to a higher value. Consequently, the effects of various basic electrolytes including inorganic and organic ones on the enantioseparations in NACE were investigated. The results showed that most of the basic electrolytes tested were favorable for the enantioseparations of basic analytes using di-n-butyl l-tartrate-boric acid complex as the chiral ion-pair selector.

[Genetic variations of the elastase gene among eight populations of Schistosoma japonicum].

OBJECTIVE: To investigate genetic diversity in the elastase gene among eight Schistosoma japonicum populations, and whether natural selection occur. METHODS: S. japonicum populations were collected from the provinces of Anhui (Tongling and Guichi), Hunan (Yueyang), Hubei (Shashi), Sichuan (Xichang), Yunnan (Eryuan), Taiwan (Puye) in China, and the Philippines. The elastase gene from different populations was amplified by PCR and then sequenced. Watterson's theta, Tajima's pi, dN/dS ratio, Tajima's D and fixation index (F(st)) of each population were calculated. The phylogenetic networks based on the elastase gene were constructed by median-joining algorithm. RESULTS: A total of 73 elestase gene sequences (GenBank No. KF297654-KF297681) were obtained from 8 populations. The sequence analysis indicated that higher genetic diversity was found in the populations from the middle and lower reaches of the Yangtze River (i.e. Tongling City of Anhui, Yueyang City of Hunan), while there was no genetic variations in Hubei or Philippines populations. The value of Tajima's D was positive in Hunan population, while negative in the other populations. The dN/dS ratio was higher than 1 in Tongling population, whereas lower than 1 in Taiwan population. Significant genetic differentiations were observed between Taiwan population and other populations. CONCLUSION: The genetic diversity of the elastase gene among S. japonicum populations is very high, and a high level of gene flow has been detected among the populations from the middle and lower reaches of the Yangtze River. The S.japonicum elastase gene might have been under a positive selection. The level of genetic divergence is the highest between Taiwan population and others.

Prevalence and risk factors of organ failure in patients with severe acute pancreatitis.

BACKGROUND: This study was undertaken to determine the prevalence of organ failure and its risk factors in patients with severe acute pancreatitis (SAP). METHODS: A retrospective analysis was made of 186 patients with SAP who were had been hospitalized in the intensive care unit of Jinzhong First People's Hospital between March 2000 and October 2009. The patients met the diagnostic criteria of SAP set by the Surgical Society of the Chinese Medical Association in 2006. The variables collected included age, gender, etiology of SAP, the number of comorbidit, APACHEII score, contrast-enhanced CT (CECT) pancreatic necrosis, CT severity index (CTSI) , abdominal compartment syndrome (ACS) , the number of organ failure, and the number of death. The prevalence and mortality of organ failure were calculated. The variables were analyzed by unconditional multivariate logistic regression to determine the independent risk factors for organ failure in SAP. RESULTS: Of 186 patients, 96 had organ failure. In the 96 patients, 47 died. There was a significant association among the prevalence of organ failure and age, the number of comorbidity, APACHEII score, CECT pancreatic necrosis, CTSI, and ACS. An increase in age, the number of comorbidity, APACHEII score, CECT pancreatic necrosis were correlated with increased number of organ failure. Age, the number of comorbidity, APACHEII score, CECT pancreatic necrosis, CTSI and ACS were assessed by unconditional multivariate logistic regression. CONCLUSIONS: Organ failure occurred in 51.6% of the 186 patients with SAP. The mortality of SAP with organ failure was 49.0%. Age, the number of comorbidity, APACHEII score, CECT pancreatic necrosis, CTSI and ACS are independent risk factors of organ failure.

[Exploration of differential expressed genes involved in the development and progression of hepatocellular carcinoma using oligo microarray].

OBJECTIVES: To analyze the differential expression genes (DEGs) among hepatocellular carcinoma (HCC), para-cancerous tissue (PCT) and normal liver tissue (NLT) and explore the target genes related to the development and progression of HCC. METHODS: The total RNAs of matched HCC, PCT and NLT of HCC patients were isolated using one step Trizol method. Matched RNAs were qualified using 10 g/L agarose gel electrophoresis and lab-on-chip. cRNAs were synthesized, fluorescence labeled and purified after total RNAs were purified. The RNAs of HCC and NLT, HCC and PCT were hybridized with Agilent oligo microarray (21,074 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were confirmed by SYBR Green I stained real time RT-PCR. RESULTS: (1) The total RNA, reverse transcription product and fluorescence labeled cRNA were all of high quality; (2) There were 420 up-regulated genes and 552 down-regulated genes among 2-fold DEGs, including DKK1 (dickkopf homolog 1) which was 5-fold up-regulated; (3) The results of real time RT-PCR, using beta-actin as an internal control, showed that the 2-Delta Ct values of DKK1 in HCC, PCT and NLT were 0.089 504, 0.007,65 and 0.000,631 respectively. CONCLUSION: (1) The high throughput and effective Agilent oligo microarray can screen novel therapy targeted genes by analyzing the DEGs in development and progression of HCC; (2) The development and progression of HCC is a complicated process involving multigenes and multiprocedures; (3) DKK1, as a novel gene, is involved in the development and progression of HCC and may be a new therapy target.