Characterization of microbial community and antibiotic resistome in intra urban water, Wenzhou China.

The present study investigated the water quality index, microbial composition and antimicrobial resistance genes in urban water habitats. Combined chemicals testing, metagenomic analyses and qualitative PCR (qPCR) were conducted on 20 locations, including rivers from hospital surrounds (n = 7), community surrounds (n = 7), and natural wetlands (n = 6). Results showed that the indexes of total nitrogen, phosphorus, and ammonia nitrogen of hospital waters were 2-3 folds high than that of water from wetlands. Bioinformatics analysis revealed a total of 1,594 bacterial species from 479 genera from the three groups of water samples. The hospital-related samples had the greatest number of unique genera, followed by those from wetlands and communities. The hospital-related samples contained a large number of bacteria associated with the gut microbiome, including Alistipes, Prevotella, Klebsiella, Escherichia, Bacteroides, and Faecalibacterium, which were all significantly enriched compared to samples from the wetlands. Nevertheless, the wetland waters enriched bacteria from Nanopelagicus, Mycolicibacterium and Gemmatimonas, which are typically associated with aquatic environments. The presence of antimicrobial resistance genes (ARGs) that were associated with different species origins in each water sample was observed. The majority of ARGs from hospital-related samples were carried by bacteria from Acinetobacter, Aeromonas and various genera from Enterobacteriaceae, which each was associated with multiple ARGs. In contrast, the ARGs that were exclusively in samples from communities and wetlands were carried by species that encoded only 1 to 2 ARGs each and were not normally associated with human infections. The qPCR showed that water samples of hospital surrounds had higher concentrations of intI1 and antimicrobial resistance genes such as tetA, ermA, ermB, qnrB, sul1, sul2 and other beta-lactam genes. Further genes of functional metabolism reported that the enrichment of genes associated with the degradation/utilization of nitrate and organic phosphodiester were detected in water samples around hospitals and communities compared to those from wetlands. Finally, correlations between the water quality indicators and the number of ARGs were evaluated. The presence of total nitrogen, phosphorus, and ammonia nitrogen were significantly correlated with the presence of ermA and sul1. Furthermore, intI1 exhibited a significant correlation with ermB, sul1, and blaSHV, indicating a prevalence of ARGs in urban water environments might be due to the integron intI1's diffusion-promoting effect. However, the high abundance of ARGs was limited to the waters around the hospital, and we did not observe the geographical transfer of ARGs along with the river flow. This may be related to water purifying capacity of natural riverine wetlands. Taken together, continued surveillance is required to assess the risk of bacterial horizontal transmission and its potential impact on public health in the current region.

Effects of Lactobacillus on hyperlipidemia in high-fat diet-induced mouse model.

Introduction: Hyperlipidemia refers to a group of lipid metabolism disorders characterized by increased levels of total cholesterol, triglyceride, and/or low-density lipoprotein cholesterol and/or decreased levels of high-density lipoprotein cholesterol. This study aims to investigate the effects of Lactobacillus on lipid metabolism and hepatic steatosis in male mice fed with a high-fat diet by measuring blood lipid, hepatic function and hepatocyte morphology. Material and methods: Eighty male Institute of Cancer Research (ICR) mice were fed with a high-fat diet for 6 weeks to establish hyperlipidemic models. Then, mice were treated with a high or low concentration of Lactobacillus of human source, mouse source, or plant source, respectively. Results: After 3 weeks of therapy, except for the human Lactobacillus treatment group, the blood cholesterol, triglyceride and low-density lipoprotein cholesterol in mice treated with Lactobacillus of mouse and plant source were lower than those in the hyperlipidemic model group. After 4 weeks of treatment, the levels of blood biochemical indexes in mice in all treatment groups were significantly different, when compared to those in the hyperlipidemic model group. Conclusions: Lactobacillus may regulate blood lipid in mice fed with a high-fat diet. Lactobacillus can improve the high cholesterol, high blood lipid, and injury of hepatic function, and prevent further development of atherosclerosis caused by a high-fat diet to some extent. Correct dietary structure is the basis for the treatment of dietary hyperlipidemia and its complications.

Albuca Bracteata Polysaccharides Attenuate AOM/DSS Induced Colon Tumorigenesis via Regulating Oxidative Stress, Inflammation and Gut Microbiota in Mice.

Inflammation is an important risk factor in the development of inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Accumulating evidence indicates that some phytochemicals have anti-cancer properties. Polysaccharides extracted from Albuca bracteata (AB) have been reported to possess anti-neoplastic activities on colorectal cancer (CRC) models. However, it is still unclear whether they exert therapeutic effects on colorectal cancer. In this study, we investigate the properties of polysaccharides of A. bracteate, named ABP. The average molecular weight of ABP was 18.3 kDa and ABP consisted of glucose, mannose, galactose, xylose, galacturonic acid, glucuronic acid at a molar ratio of 37.8:8:2.5:1.7:1:1. An Azoxymethane/Dextran sodium sulfate (AOM/DSS) induced CAC mouse model was established. The CAC mice treated with ABP showed smaller tumor size and lower tumor incidence than untreated ones. ABP increased anti-inflammatory cytokine IL-10, inhibited secretion of pro-inflammatory cytokines (IL-6, IFN-γ, and TNF-α), mitigated oxidative stress by increasing GSH and decreasing MDA levels, suppressed the activation of STAT3 and expressions of its related genes c-Myc and cyclin D1. Moreover, ABP treatment increased the relative abundance of beneficial bacteria (f_Ruminococcaceae, g_Roseburia, g_Odoribacter, g_Oscillospira, and g_Akkermansia) and the levels of fecal short-chain fatty acid (SCFA) in CAC model mice. In summary, our data suggest that ABP could be a potential therapeutic agent for treating CAC.

Albuca Bracteate Polysaccharides Synergistically Enhance the Anti-Tumor Efficacy of 5-Fluorouracil Against Colorectal Cancer by Modulating ß-Catenin Signaling and Intestinal Flora.

The first-line treatment for colorectal cancer (CRC) is 5-fluorouracil (5-FU). However, the efficacy of this treatment is sometimes limited owing to chemoresistance as well as treatment-associated intestinal mucositis and other adverse events. Growing evidence suggests that certain phytochemicals have therapeutic and cancer-preventing properties. Further, the synergistic interactions between many such plant-derived products and chemotherapeutic drugs have been linked to improved therapeutic efficacy. Polysaccharides extracted from Albuca bracteata (Thunb.) J.C.Manning and Goldblatt (ABP) have been reported to exhibit anti-oxidant, anti-inflammatory, and anti-tumor properties. In this study, murine CRC cells (CT26) and a murine model of CRC were used to examine the anti-tumor properties of ABP and explore the mechanism underlying the synergistic interactions between ABP and 5-FU. Our results revealed that ABP could inhibit tumor cell proliferation, invasion, and migratory activity in vitro and inhibited tumor progression in vivo by suppressing ß-catenin signaling. Additionally, treatment with a combination of ABP and 5-FU resulted in better outcomes than treatment with either agent alone. Moreover, this combination therapy resulted in the specific enrichment of Ruminococcus, Anaerostipes, and Oscillospira in the intestinal microbiota and increased fecal short-chain fatty acid (SCFA) levels (acetic acid, propionic acid, and butyric acid). The improvement in the intestinal microbiota and the increase in beneficial SCFAs contributed to enhanced therapeutic outcomes and reduced the adverse effects of 5-FU. Together, these data suggest that ABP exhibits anti-neoplastic activity and can effectively enhance the efficacy of 5-FU in CRC treatment. Therefore, further research on the application of ABP in the development of novel anti-tumor drugs and adjuvant compounds is warranted and could improve the outcomes of CRC patients.

Corrigendum: Role of HK2 in the Enzootic Cycle of Borrelia burgdorferi.

[This corrects the article DOI: 10.3389/fmed.2020.573648.].

Role of HK2 in the Enzootic Cycle of Borrelia burgdorferi.

The two-component response regulator Rrp2 is a key activator controlling the production of numerous virulence factors of Borrelia burgdorferi, the Lyme disease pathogen. Previously it was shown that the cognate histidine kinase HK2 is not required for Rrp2 activation in vitro, nor for mammalian infection upon needle inoculation, raising the question whether HK2 has any role in the enzootic cycle of B. burgdorferi. In this study, we demonstrated that HK2 is not required for spirochetal survival in the tick vector. When fed on naive mice, the hk2 mutant had reduced infectivity through the route of tick bite, suggesting that the spirochetes lacking HK2 had a disadvantage in the enzootic cycle. Furthermore, overexpression of hk2 reduced the level of Rrp2 phosphorylation, suggesting that HK2 can function as a phosphatase to dephosphorylate Rrp2. Strains overexpressing hk2 impaired the expression of RpoN regulon whose activation is dependent on Rrp2 phosphorylation and activation, and had reduced infectivity in mice. Taken together, these results demonstrate that although HK2 does not play an essential role in Rrp2 activation, it is important for the optimal fitness of B. burgdorferi in the enzootic cycle.

Sodium Butyrate Selectively Kills Cancer Cells and Inhibits Migration in Colorectal Cancer by Targeting Thioredoxin-1.

BACKGROUND: Sodium butyrate (NaB) is a short-chain fatty acid which is produced by bacterial fermentation of nondigestible dietary fiber and has been reported to exert anti-tumor effects in many tumors including colorectal cancer (CRC). However, the role of thioredoxin-1 (Trx-1) in NaB-induced anti-tumor effect has not been completely clarified. MATERIALS AND METHODS: Effects of NaB on the growth of CRC cell lines HT29 and SW480 were detected by the Cell Counting Kit-8 (CCK-8) and colony formation assays. The apoptotic cells were determined by flow cytometry, and cell migration was assessed by a Transwell assay. Western blot analysis was used to test the Trx-1 and epithelial-to-mesenchymal transition (EMT)-related proteins level. Reactive oxygen species (ROS) level was determined and N-acetylcysteine (NAC) recovery experiment was performed in CRC cells. In addition, mice xenograft model was established to test the effect of NaB on CRC growth in vivo. Further, the effects of NaB on CRC cells with overexpression or knockdown were tested by the CCK-8 and Transwell assays. RESULTS: NaB treatment significantly inhibited cell growth and decreased Trx-1 protein expression in CRC cells but not in normal colon epithelial cells. NaB also induced apoptosis, inhibited colony formation, migration and EMT in CRC cells. Besides, NaB increased ROS level in CRC cells and NAC reversed NaB-induced inhibition of cell proliferation. Moreover, downregulation of Trx-1 significantly enhanced NaB-induced inhibitory effects on cell growth and migration, whereas overexpression of Trx-1 attenuated NaB-induced inhibitory effects on growth and migration in CRC cells. CONCLUSION: These findings indicate that the NaB-mediated anti-tumor effects on CRC cells are related to downregulation of Trx-1.

Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, encounters two disparate host environments during its enzootic life cycle, Ixodes ticks and mammalian hosts. B. burgdorferi has a small genome that encodes a streamlined cyclic dimeric GMP (c-di-GMP) signaling system comprising a single diguanylate cyclase, Rrp1, and two phosphodiesterases. This system is essential for spirochete survival in ticks, in part because it controls the expression of the glp operon involved in glycerol utilization. In this study, we showed that a B. burgdorferi c-di-GMP receptor, PlzA, functions as both a positive and a negative regulator for glp expression. Deletion of plzA or mutation in plzA that impaired c-di-GMP binding abolished glp expression. On the other hand, overexpression of plzA resulted in glp repression, which could be rescued by simultaneous overexpression of rrp1. plzA overexpression in the rrp1 mutant, which is devoid of c-di-GMP, or overexpression of a plzA mutant incapable of c-di-GMP binding further enhanced glp repression. Combined results suggest that c-di-GMP-bound PlzA functions as a positive regulator, whereas ligand-free PlzA acts as a negative regulator for glp expression. Thus, PlzA of B. burgdorferi with a streamlined c-di-GMP signaling system not only controls multiple targets, as previously envisioned, but has also evolved different modes of action.IMPORTANCE The Lyme disease pathogen, Borrelia burgdorferi, has a simple cyclic dimeric GMP (c-di-GMP) signaling system essential for adaptation of the pathogen to the complicated tick environment. The c-di-GMP effector of B. burgdorferi, PlzA, has been shown to regulate multiple cellular processes, including motility, osmolality sensing, and nutrient utilization. The findings of this study demonstrate that PlzA not only controls multiple targets but also has different functional modalities, allowing it to act as both positive and negative regulator of the glp operon expression. This work highlights how bacteria with a small genome can compensate for the limited regulatory repertoire by increasing the complexity of targets and modes of action in their regulatory proteins.

Sodium butyrate exerts protective effect against Parkinson's disease in mice via stimulation of glucagon like peptide-1.

Sodium butyrate (NaB) has exhibited protective activity in neurological disorders. Here, we investigated the neuroprotective effect and potential mechanisms of NaB in a mouse model of Parkinson's disease (PD). A mouse was intraperitoneally treated with MPTP (30mg/kg) for 7 consecutive days to induce PD model and NaB (200mg/kg) was intragastrically treated for 3weeks. The behavioral tests were then conducted. Dopaminergic degeneration was evaluated by western blot and immunohistochemistry of tyrosine hydroxylase (TH) in the SN. Brain damage was assessed by histologic (Nissl staining for cell death), apoptosis-associated protein and tight junction (TJ) proteins studies. Meanwhile, the levels of colonic glucagon-like peptide-1 (GLP-1) and cerebral GLP-1 receptor (GLP-1R) expression were assessed. Our results showed that NaB improved neurobehavioral impairment including cognitive behavior and coordination performance. Moreover, NaB treatment prevented the MPTP-induced dopaminergic degeneration and decreased expression level of TH in the striatum. NaB treatment attenuated the PD-associated disruption of BBB by upregulation of Occludin and zonula occludens (ZO)-1. In addition, NaB resulted in increased level of Bcl-2 and decreased level of Bax. Particularly, NaB-treated mice with PD exhibited increased colonic GLP-1 level as well as upregulation of brain GLP-1R expression compared with PD group. Our findings suggest that NaB has potential as a novel therapeutic for treatment of PD, and its mechanism was associated with stimulating colonic GLP-1secretion.

Investigation of ospC Expression Variation among Borrelia burgdorferi Strains.

Outer surface protein C (OspC) is the most studied major virulence factor of Borrelia burgdorferi, the causative agent of Lyme disease. The level of OspC varies dramatically among B. burgdorferi strains when cultured in vitro, but little is known about what causes such variation. It has been proposed that the difference in endogenous plasmid contents among strains contribute to variation in OspC phenotype, as B. burgdorferi contains more than 21 endogenous linear (lp) and circular plasmids (cp), and some of which are prone to be lost. In this study, we analyzed several clones isolated from B. burgdorferi strain 297, one of the most commonly used strains for studying ospC expression. By taking advantage of recently published plasmid sequence of strain 297, we developed a multiplex PCR method specifically for rapid plasmid profiling of B. burgdorferi strain 297. We found that some commonly used 297 clones that were thought having a complete plasmid profile, actually lacked some endogenous plasmids. Importantly, the result showed that the difference in plasmid profiles did not contribute to the ospC expression variation among the clones. Furthermore, we found that B. burgdorferi clones expressed different levels of BosR, which in turn led to different levels of RpoS and subsequently, resulted in OspC level variation among B. burgdorferi strains.

Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension.

Mutations in mitochondrial (mt)transfer (t)RNA (mt­tRNA) have been reported to serve important roles in hypertension. To determine the underlying molecular mechanisms of mt­tRNA mutations in hypertension, the present study screened for mt­tRNA mutations in a Chinese family with a high incidence of essential hypertension. Sequence analysis of the mt­tRNA genes in this family revealed the presence of an A4401G mutation in the glycine­and methionine­tRNA genes, and a G5821A mutation in the cysteine­tRNA (tRNACys) gene. The G5821A mutation was located at a position conserved in various species, and disrupted G6­C67 base­pairing. It was hypothesized that the G5821A mutation may decrease the baseline expression levels of tRNACys, and consequently result in failure of tRNA metabolism. The A4401G mutation was reported to cause the mitochondrial dysfunction responsible for hypertension. Thus, the combination of G5821A and A4401G mutations may contribute to the high incidence of hypertension in this family. Mt­tRNA mutations may serve as potential biomarkers for hypertension.

Association Between IL-17, IL-8 and IL-18 Expression in Peripheral Blood and Helicobacter Pylori Infection in Mongolian Gerbils.

BACKGROUND: Persistent Helicobacter pylori infection confers an increased risk for serious illnesses such as peptic ulcers and gastric cancer. Various cytokines are involved in the regulation of inflammatory immune response in H. pylori-infected gastric mucosa. OBJECTIVES: The current study aimed to obtain evidence regarding the association between IL-17, IL-8 and IL-18 expression in peripheral blood and H. pylori infection in Mongolian gerbils. MATERIALS AND METHODS: Mongolian gerbils were inoculated with H. pylori by a metal stomach catheter. After sacrifice, their gastric mucosae were examined in macroscopic, histological and electron microscopy levels. In addition, enzyme linked immunosorbent assay (ELISA) assay was performed on the IL-17, IL-8 and IL-18 cytokines in the blood samples. RESULTS: Serum levels of IL-17, IL-8 and IL-18 were remarkably up-regulated compared to those of the control group. There was an obvious correlation between the increase of IL-17 and the serious extent of gastritis in the current study. However, the serum levels of IL-8 and IL-18 without getting increasingly more for repetitive intragastric administration. There were plenty of neutrophils infiltrating in the infected group mucosal. Intestinal metaplasia and gastric ulcers were also founded in H. pylori infected animals after enhanced inoculation. The edema, degeneration and necrosis changes could be found in organelles by transmission electron microscopy. More serious pathological changes were detected in the enhanced inoculation groups compared to the cycle group. CONCLUSIONS: The serum levels of IL-17, but not IL-8 and IL-18 may serve as a potential biomarker for diagnosis and predicting the prognosis of gastritis caused by H. pylori.

Relationship between CIP2A expression, and prognosis and MDR-related proteins in patients with advanced gastric cancer.

CIP2A is highly expressed in a variety of malignancies. We determined the expression and clinical significance of CIP2A in patients with advanced gastric cancer. CIP2A protein was expressed in 25 of 37 cancer tissue specimens. There was no correlation between CIP2A and PGP, GST-π, Topo-II, and LRP expression. Survival analysis showed significant differences between the survival rate of the CIP2A protein-positive and -negative groups (χ(2)=4.509, P=0.034), but the degree of positive expression was unrelated to survival time (χ(2)=4.639, P=0.098). CIP2A expression may have no prospective value for optimizing chemotherapy regimens, but it can be an indicator for patient prognosis.

[Analysis of cervical cancer incidence and mortality in cancer registries of Zhejiang province, 2000 to 2009].

OBJECTIVE: To investigate the cervical cancer incidence and mortality in cancer registries of Zhejiang province during 2000 to 2009. METHODS: The data of cervical cancer incidence and mortality were collected from six cancer registries in Zhejiang province. Staff of Zhejiang Provincial Cancer Prevention and Control Office checked, sorted and analyzed the data to calculate crude, standardized rate and trend. Chinese census in 1982 and Segi's population were used for age-standardized incidence and mortality rates. RESULTS: The incidence rate of cervical cancer in Zhejiang cancer registration areas was 11.78/100 000 during 2000 to 2009, and age-standardized incidence rates by Chinese standard population and by world standard population were 7.05/100 000 and 8.62/100 000, respectively. The mortality rate was 1.89/100 000, and age-standardized mortality rates by Chinese standard population and by world standard population were 0.95/100 000 and 1.23/100 000, respectively. The age-specific incidence rates showed different trends, increased significantly after the age of 25, peaked at 45-year-old group, which was 23.03/100 000 (578/2 510 099) , and decreased at the age of 50, while the age-specific mortality rates gentlely increased, peaked at 85 years of age group, which was 11.94/100 000 (33/276 414) . The cervical Cancer Incidence from 5.96/100 000 (86/1 443 589) in 2000, increased to 18.90/100 000 (898/4 751 426) in 2009, the annual percent change (APC) was 16.64% (95%CI:11.87%-21.61%). The mortality showed a gentle upward trend from 1.45/100 000 (21/1 443 589) , increased to 2.53/100 000 (120/4 751 426) in 2009, the APC was 6.63% (95%CI:1.73%-11.77%). CONCLUSION: Cervical cancer showed younger trend, the incidence and mortality trends showed an increasing trend, should strengthen the prevention and control of cervical cancer.

[Investigation of etiology of massive infection with porcine pseudorabies virus in Henan and neighboring Provinces].

In early 2011, the serious outbreak of porcine pseudorabies virus (PRV) infection suddenly recurred in Henan and neighboring Provinces. To investigate the etiology of massive infection with PRV, 16 800 serum samples, 905 porcine epidemic diarrhea virus (PEDV) back-feeding tissues, and 56 PR gene deleted live vaccines were colleted from January 2011 to May 2013 to detect PRV field infection using a PRV gE antibody test kit. The gE and TK genes of 11 new epidemic PRV strains were sequenced by PCR, and their molecular characteristics were analyzed. Moreover, virus titer determination, protective test against PRV, and vaccine potency testing were performed. The results showed that the detection rate of PRV field infection-positive pig farms was 68.06%, and the overall positive rate of PRV field infection in serum was 38.47%; the positive rates in breeding sows, breeding boars, reserve pigs, and commercial pigs were 40.12%, 30.88%, 54.67%, and 26.52%, respectively. The new epidemic strains were in the same evolutionary branch and belonged to the virulent strain group. Compared with the classical PRV strain, the virulence of new epidemic strains changed a little. The length of gE gene was 1 787 bp, and the length of TK gene was 963 bp. The nucleotide homologies of gE and TK genes to Chinese reference strains were 98.2%-99.8% and 98.90%-99.6%, respectively, and the amino acid homologies were 97.1%-99.8% and 97.5%-99.4%, respectively. Commercial vaccine had a 100% protective effect against the new epidemic strains. The positive rate of PRV field infection was 0% in vaccine and 40.44% in back-feeding tissues. The results confirmed that PRV field infection rates were rising sharply among pigs in Henan and neighboring Provinces after 2011. The main virulence genes of new epidemic PRV strains did not change significantly over the years. PR gene deleted live vaccines had no PRV field infection and could completely resist the attack of new strains. The virus carriage of breeding boars and reserve pigs and the serious PRV field infection in PEDV back-feeding tissues were the main causative factors for massive infection with PRV and epidemic outbreak in Henan and neighboring Provinces from 2011 to 2013.

Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus.

Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935) S. aureus isolates, including 28 (3.0%, 28/935) MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%), ampicillin/sulbactam (83.3%) and trimethoprim/sulfamethoxazole (93.1%). 82%, (23/28) of the MRSA isolates and 66% (77/116) of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin--an agent commonly used for nasal decolonization. 16 different sequence types (STs), as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779) and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.

Inhibition of p38 MAPK activity in B-NHL Raji cells by treatment with engineered CD20-specific T cells.

Adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors is a promising approach to lymphoma therapy. However, modification of the cellular signaling pathways in target tumor cells by treatment with engineered CD20-specific T cells has yet to be fully elucidated. In this study, the non-Hodgkin's lymphoma Raji cell line was co-cultured with T cells that were genetically modified with anti-CD20scFvFc/CD28/CD3ζ or anti-CD20scFvFc gene. The cytolytic activity of engineered CD20-specific T cells and IL-10 secretion was quantitated by Cytotoxicity and ELISA assays, respectively. The engineered CD20-specific T cells and Raji cells were sorted using flow cytomety for the Western blot analysis. Treatment of Raji cells with T cells genetically modified with anti-CD20scFvFc/CD28/CD3ζ chimera (compared to anti-CD20scFvFc) yielded a higher cytotoxicity against Raji cells in vitro. Additionally, we found that engineered CD20-specific T cells caused a decrease in IL-10 secretion and inhibition of phosphor-STAT3 and Bcl-2 expression in Raji cells, possibly through the down-regulation of p38 MAPK and NF-κB activity. These results indicate that the treatment of Raji cells with engineered CD20-specific T cells inhibited the cellular p38 MAPK signaling pathways, which enhanced its antitumor activities against CD20-positive tumor cells.

Potential therapeutic strategy for non-Hodgkin lymphoma by anti-CD20scFvFc/CD28/CD3zeta gene tranfected T cells.

BACKGROUND: Anti-CD20 monoclonal antibody treatment has not only increased survival and cure rates in many non-Hodgkin lymphomas, but also has prompted an explosion in the development of novel antibodies and biologically active substances with specific cellular targets in the field of malignancies treatment. Since the robust immune responses are elicited by the gene-modified T cells, gene based T cell therapy may also provide a powerful tool for cancer immunotherapy. METHODS: In this study, we developed a vector construction encoding a chimeric T cell receptor that recognizes the CD20 antigen and delivers co-stimulatory signals to achieve T cell activation. One non-Hodgkin lymphoma cell line Raji cells co-cultured with peripheral blood-derived T cells were stably transfected with anti-CD20scFvFc/CD28/CD3zeta gene or anti-CD20scFvFc gene. T cells expressing anti-CD20scFvFc/CD28/CD3zeta or anti-CD20scFvFc gene co-cultured with CD20 positive Raji cells for different times. Cell lysis assay was carried by [3H]TdR release assay. The expressions of Fas, Bcl-2 and Caspase-3 of Raji cells were detected by flow cytometric. The secretion of IFN-gamma and IL-2 in co-culture medium was tested by ELISA assay. Activity of AP-1 was analyzed by EMSA. RESULTS: Following efficient transduction of peripheral blood-derived T cells with anti-CD20scFvFc/CD28/CD3zeta gene, an obvious cell lysis of Raji cells was observed in co-culture. T cells transduced anti-CD20scFvFc/CD28/CD3zeta gene had superior secretion of IFN-gamma and IL-2 compared to T cells transduced anti-CD20scFvFc gene. Also it led to a much stronger Fas-induced apoptosis signaling transduction in target cancer cells. CONCLUSION: So adoptively T cells transduced anti-CD20scFvFc/CD28/CD3zeta gene mediates enhanced anti-tumor activities against CD20 positive tumor cells, suggesting a potential of gene-based immunotherapy for non-Hodgkin lymphoma.

[Identification of differentially expressed genes in recurrent nasopharyngeal carcinoma and analysis of their chromosomal location].

OBJECTIVE: To identify differentially expressed genes in recurrent nasopharyngeal carcinoma (rNPC) by DNA microarrays, and analyze chromosomal localizations and molecular function by bioinformatics. METHODS: The primary nasopharyngeal carcinoma (pNPC) tissue samples and rNPC tissue samples were selected, and Affymetrix Gene1.0 ST gene chips were used to identify differential expressed genes in rNPC, and the bioinformatics was used to analyze their chromosomal localizations as well as molecular functions. RESULTS: A total of 44 genes were identified to be differential expressed in rNPC. Thirty-six genes were down regulated, 8 genes were up regulated. Functional classification of down-regulation genes showed that most genes (10 genes, 27.8%) belonged to the enzyme activity genes, followed by calcium ion binding genes (7 genes, 19.4%), protein binding genes (5 genes, 13.9%), receptor activity genes (4 genes, 11.1%), ATP binding genes (2 genes, 5.6%), transcription factor genes (2 genes, 5.6%), extracellular matrix binding and growth factor binding have 1 gene respectively (each accounted for 2.8%). In addition, the functions of 4 genes (11.1%) were unknown. Functional classification of up-regulation genes showed most genes (3 genes, 37.5%) were unknown, followed enzyme activity genes (2 genes, 25.0%), receptor activity, calcium ion binding and voltage-gated ion channel activity genes have 1 genes respectively (each accounted for 12.5%). These genes were localized randomly on the most the chromosomes, with a majority of them localized on chromosomes 1, 17. Chromosome 1 contained the most differentially expressed genes (10, 22.7%), followed by chromosomes 17 (5, 11.3%). CONCLUSIONS: The differential expressed genes in rNPC were supposed to be randomly distributed on most chromosomes, but the majorities were found on chromosomes 1, 17. Abnormality in three groups of genes, including in enzyme activity, calcium ion binding and protein binding associate genes, might play important roles in rNPC. Those genes need to be further studied.