Inhibition of Adhesion of Enteropathogenic Escherichia coli to HEp-2 Cells by Binding of a Novel Peptide to EspB Protein.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. The translocator EspB is a key virulence factor in the process of the attaching and effacing effect of EPEC and plays a critical role in the pathogenesis of the bacteria. In this study, we aimed to select the peptides binding to EspB protein by phage display library and further investigate whether these peptides can decrease the extent of invasion and virulence of EPEC on host cells by targeting to EspB protein. The expression and purification of EspB protein from E. coli was demonstrated by Western blotting. The Ph.D. 12-mer peptide phage display library was used to screen the candidate peptides binding specifically to EspB protein. Furthermore, the affinity of these candidate peptides bound to EspB was identified by enzyme-linked immunosorbent assay (ELISA). Moreover, we investigated whether these screened peptides could decrease the adherence ratio of EPEC to HEp-2 cells with increasing concentration. Successful purification of EspB protein from pET21b-EspB-transformed E. coli was identified by Western blotting. Then, the candidate peptides including phages 6, 7, 8, and 12 were screened by the Ph.D. 12-mer peptide phage display library and ELISA test demonstrated that their affinity binding to EspB protein was high compared with the control. Functional analysis indicated that synthetic peptide-6 (YFPYSHTSPRQP) significantly decreased the adherence ratio of EPEC to HEp-2 cells with increasing concentration (P < 0.01). Peptide-6 (100 µg/mL) could lead to a 40 % decrease in the adherence ratio of EPEC to HEp-2 cells compared with control (P < 0.01). However, the other three peptides at different concentrations showed only a slight ability to block the adherence of EPEC to host cells. Our data provided a potential strategy to inhibit the adhesion of EPEC to epithelial cells by a candidate peptide targeted toward EspB protein.

Electrospinning of PELA/PPY Fibrous Conduits: Promoting Peripheral Nerve Regeneration in Rats by Self-Originated Electrical Stimulation.

Peripheral nerve injuries represent a great challenge for surgeons. The conductive neural scaffold has experienced increasing interest because of its good biocompatibility and similar electrical properties as compared to those of a normal nerve. Herein, nerve conduits made from poly(d,l-lactide)-co-poly(ethylene glycol) and polypyrrole (20%, 30%, and 50%) (PELA-PPY) were prepared by electrospinning, and used in regeneration of peripheral nerve defects. The results of an in vitro experiment indicated a high biocompatibility for the as-prepared materials, supporting the attachment and proliferation of a rat pheochromocytoma PC-12 cell. Furthermore, the PELA-PPY nerve conduit implanted in the sciatic nerve defects (10 mm) of the Spraguee-Dawley rats for 12 weeks showed similar results with the autograft, while it demonstrated a better outcome than the PELA nerve conduit in electrophysiological examination, sciatic function index, total amount of regenerated myelinated nerve fibers, axon diameter, myelin thickness, and several immunohistochemistry indices (S-100, laminin, neurofilament, bromodeoxyuridine, and glial fibrillary acidic portein). We supposed that the bioactivity is mainly generated by the PPY in composite nanofibers which could transmit self-originated electrical stimulation between cells. Due to the facile preparation and excellent in vivo performance, the PPY-PELA nerve conduit is promising for use as a bioengineered biomaterial for peripheral nerve regeneration.

Directed shift of vaginal microbiota induced by vaginal application of sucrose gel in rhesus macaques.

OBJECTIVES: Sucrose gel was used to treat bacterial vaginosis in a phase III clinical trial. However, the changes of vaginal flora after treatment were only examined by Nugent score in that clinical trial, While the vaginal microbiota of rhesus macaques is characterized by anaerobic, Gram-negative bacteria, few lactobacilli, and pH levels above 4.6, similar to the microbiota of patients with bacterial vaginosis. This study is aimed to investigate the change of the vaginal microbiota of rehsus macaques after topical use of sucrose gel to reveal more precisely the bacterial population shift after the topical application of sucrose gel. METHODS: Sixteen rhesus macaques were treated with 0.5 g sucrose gel vaginally and three with 0.5 g of placebo gel. Vaginal swabs were collected daily following treatment. Vaginal pH levels and Nugent scores were recorded. The composition of the vaginal micotbiota was tested by V3∼V4 16S rDNA metagenomic sequencing. Dynamic changes in the Lactobacillus genus were analyzed by qPCR. RESULTS: The vaginal microbiota of rhesus macaques are dominated by anaerobic Gram-negative bacteria, with few lactobacilli and high pH levels above 4.6. After five days' treatment with topical sucrose gel, the component percentage of Lactobacillus in vaginal microbiota increased from 1.31% to 81.59%, while the component percentage of Porphyromonas decreased from 18.60% to 0.43%, Sneathia decreased from 15.09% to 0.89%, Mobiluncus decreased from 8.23% to 0.12%, etc.. The average vaginal pH values of 16 rhesus macaques of the sucrose gel group decreased from 5.4 to 3.89. There were no significant changes in microbiota and vaginal pH observed in the placebo group. CONCLUSIONS: Rhesus macaques can be used as animal models of bacterial vaginosis to develop drugs and test treatment efficacy. Furthermore, the topical application of sucrose gel induced the shifting of vaginal flora of rhesus macaques from a BV kind of flora to a lactobacilli-dominating flora.

Recurrent linezolid-resistant Enterococcus faecalis infection in a patient with pneumonia.

OBJECTIVE: It has been reported that LZD-resistant Enterococcus in the gastrointestinal tract of mice colonizes persistently and shows variable minimum inhibitor concentration (MIC) values. However, the colonization characteristics of Enterococcus with LZD resistance in patients remain elusive. Here, we report the case of a patient with recurrent pneumonia due to infection with LZD-resistant Enterococcus faecalis strains. The colonization characteristics of the strains isolated from this patient were analyzed. METHODS: Ten E. faecalis strains were isolated from tracheal secretions obtained from the patient during five recurrences of pneumonia over the course of 10 months. Clonal relationships were determined by pulsed-field gel electrophoresis (PFGE) with SmaI-macrorestricted genomic DNA. The susceptibility of the isolates to LZD was determined by Etest in Mueller-Hinton agar. RESULTS: The homology of these strains was demonstrated by PFGE, suggesting that occult bacterial colonization by LZD-resistant E. faecalis is possible as late as a year after exposure to LZD. These strains showed variable MICs as determined by the Etest. LZD-resistant isolates contained single or double nucleotide mutations in domain V of 23S rRNA as confirmed by PCR and sequencing. The sensitivity of the strains to vancomycin was demonstrated by broth macrodilution, and vancomycin was an effective clinical treatment on each occasion. CONCLUSIONS: Our results indicate that LZD-resistant E. faecalis strains may colonize persistently in vivo, leading to recurrent infection.

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma.

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor (HSV(GM-CSF)) in pancreatic carcinoma. METHODS: Tumor blocks were homogenized in a sterile grinder in saline. The homogenate was injected into the right armpit of each mouse. After vaccination, the mice were randomly assigned into four groups: a control group, a high dose HSV(GM-CSF) group [1 × 107 plaque forming units (pfu)/tumor], a medium dose HSV(GM-CSF) group (5 × 106 pfu/tumor) and a low dose HSV(GM-CSF) group (5 × 105 pfu/tumor). After initiation of drug administration, body weights and tumor diameters were measured every 3 d. Fifteen days later, after decapitation of the animal by cervical dislocation, each tumor was isolated, weighed and stored in 10% formaldehyde solution. The drug effectiveness was evaluated according to the weight, volume and relative volume change of each tumor. Furthermore, GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1, 2, 3 and 4 d after injection of HSV(GM-CSF). RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group, and dose-dependent effects were observed: the relative tumor proliferation rates of the low dose, medium dose and high dose groups on 15 d after injection were 45.5%, 55.2% and 65.5%, respectively. The inhibition rates of the tumor weights of the low, middle, and high dose groups were 41.4%, 46.7% and 50.5%, respectively. Furthermore, the production of GM-CSF was significantly increased in the mice infected with HSV(GM-CSF). The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups. CONCLUSION: Our study provides the first evidence that HSV(GM-CSF) could inhibit the growth of pancreatic cancer. The enhanced GM-CSF expression might be responsible for the phenomenon.

Identification of differential gene expression profiles of radioresistant lung cancer cell line established by fractionated ionizing radiation in vitro.

BACKGROUND: Radiotherapy plays a critical role in the management of non-small cell lung cancer (NSCLC). This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line established by fractionated ionizing radiation (FIR) by cDNA microarray. METHODS: The human lung adenocarcinoma cell line Anip973 was treated with high energy X-ray to receive 60 Gy in 4 Gy fractions. The radiosensitivity of Anip973R and its parental line were measured by clonogenic assay. Gene expression profiles of Anip973R and its parental line were analyzed using cDNA microarray consisting of 21 522 human genes. Identified partly different expressive genes were validated by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). RESULTS: Fifty-nine upregulated and 43 downregulated genes were identified to radio-resistant Anip973R. Up-regulated genes were associated with DNA damage repair (DDB2), extracellular matrix (LOX), cell adhesion (CDH2), and apoptosis (CRYAB). Down-regulated genes were associated with angiogenesis (GBP-1), immune response (CD83), and calcium signaling pathway (TNNC1). Subsequent validation of selected eleven genes (CD24, DDB2, IGFBP3, LOX, CDH2, CRYAB, PROCR, ANXA1 DCN, GBP-1 and CD83) by Q-RT-PCR was consistent with microarray analysis. CONCLUSIONS: Fractionated ionizing radiation can lead to the development of radiation resistance. Altered gene profiles of radioresistant cell line may provide new insights into mechanisms underlying clinical radioresistance for NSCLC.

[Preliminary study of the association between human thrombospondin and gastric cancer].

OBJECTIVE: To study the possible role of human thrombospondin (hPWTSR) in gastric cancer and explore its potential to serve as the target for gastric cancer diagnosis and intervention. METHODS: Using pLexA-hPWTSR as the bait, a premade pB42AD-based fetal brain cDNA library was constructed to identify the interacting proteins. The expression pattern of hPWTSR in gastric cancer tissues and a gastric cancer cell line was observed to investigate the correlation between hPWTSR expression and the biological behaviors of the tumor. The possibility of hPWTSR as a potential gastric cancer marker was evaluated. RESULTS: Fifty-seven independent clones were isolated from 107 clones screened. Sequence analysis indicated that the 57 positive clones represented the products of 12 genes. A RT-PCR-based expression pattern revealed that the expression of hPWTSR in gastric cancer tissues and a gastric cancer cell line was lower than that in the corresponding normal tissues, but no mutations were identified by the subsequent sequence analysis. CONCLUSIONS: hPWTSR interacts with adhesion-related proteins and tumor-related genes, and its expression is lowered in gastric cancer tissues and gastric cancer cell line. hPWTSR might play a role in gastric cancer development, especially in metastasis and might be used as a potential gastric cancer marker. The exact functions of hPWTSR and its potential clinical value still await further study.

[Multi-center phase II clinical trial of humanized anti-epidermal factor receptor monoclonal antibody h-R3 combined with radiotherapy for locoregionally advanced nasopharyngeal carcinoma].

OBJECTIVE: To evaluate the efficacy and safty of the humanized anti-epidermal factor receptor monoclonal antibody h-R3 in combination with radiotherapy for locoregionally advanced nasopharyngeal carcinoma. METHODS: Totally, 137 patients from 7 medical center around China were randomly divided into combined therapy group or control group. There was no difference in Karnofsky performance score between two groups. All patients in both groups received radical conventionally fractionated radiotherapy to the total dose of D(T) 70-76 Gy. For the combined therapy group, h-R3 was added at a dose of 100 mg i.v. weekly for 8 weeks started at the beginning of radiotherapy. RESULTS: Of the 137 eligilbe patients, 70 were in the combined therapy group treated by h-R3 plus radiotherapy and 67 in the control group by radiotherapy alone. The intent-to-treat (ITT) population consisted of 130 patients, while the per-protocol (PP) population was composed of 126 patients. The efficacy was assessed respectively at three point of time: the end of treatment, the 5th- and 17th-week after treatment. The complete response (CR) of the combined therapy group was significantly higher than that of the control group in both ITT and PP (ITT: 65.63%, 87.50%, 90.63% versus 27.27%, 42.42%, 51.52%; PP: 67.21%, 90.16%, 93.44% versus 27.69%, 43.08%, 52.31%; P < 0.05, respectively). The most common h-R3-related adverse reactions were fever (4.3%), hypotension (2.9%), nausea (1.4%), dizziness (2.9%) and rash (1.4%), which could be reversible if treated properly. Radiotherapy combined with 100 mg h-R3 i. v. weekly was tolerable and did not aggravate the side effects of radiation. The quality of life in the combined therapy group was comparable to that in the control group. CONCLUSION: This phase 1 multicenter clinical trial shows that h-R3 in combination with radiotherapy is effective and well-tolerated for the treatment of locoregionally advanced nasopharyngeal carcinoma.

[Radiosensitization of paclitaxel combined with radiation on nasopharygneal carcinoma cells (CNE-I) in vitro].

OBJECTIVE: To evaluate the radiosensitization of paclitaxel combined with radiation on nasopharygneal carcinoma cells( CNE-I) in vitro. METHODS: Human CNE-I cells were used for this study. Clonogenic assay was used to determine the drug dose of IC10, IC50 and IC90 for CNE-I Cells. The cells treated with different concentration of paclitaxel for 24 hours before or after radiation (dose ranged from 0 - 10 Gy ) were used to evaluate the radiosensitizing effect of paclitaxel combined with radiation. DNA flow cytometry was performed to define the cell cycle characteristics of cell populations treated for 0, 2, 6, 12, 18, 24 h with 0.1 nmol/L, 0.5 nmol/L, 1.0 nmol/L, 2.5 nmol/L paclitaxel, respectively. RESULTS: The dose of IC10, IC50 and IC90 for paclitaxel in CNE-I cells was 0.05 nmol/L, 1.0 nmol/L and 2.5 nmol/L, respectively. Paclitaxel treatment at concentration of 0.05 nmol/L and 1.0 nmol/L for 24 hours combined with X-ray irradiation before or after radiation showed radiosensitivity-enhansing effects in CNE-I cells. G2/M block was present when the drug concentrations were 2.5 nmol/L and 10.0 nmol/L, and it peaked at 18 hours. CONCLUSION: With an optimal paclitaxel/radiation combination, paclitaxel may exert a radiosensitizing effect on CNE-I cells. The effect might be related to the G2/M block caused by paclitaxel.

[Radiobiological effects of fast neutron/photon mixed irradiation on nasopharyngeal cancer cell line CNE-1].

OBJECTIVE: To study the radiobiological effects of fast neutron/photon mixed irradiation on human cancer cell in vitro and to discuss the mechanism in relation with cell cycle and apoptosis, thus to provide experimental support for the further application of fast neutron radiotherapy of cancer. METHODS: Exponentially growing human nasopharyngeal cancer cell line CNE-1 was irradiated in vitro with 35 MeV p-->Be fast neutron and 6 MV-X ray in grading doses (0 cGy, 40 cGy, 80 cGy, 120 cGy, 160 cGy, 240 cGy, 320 cGy and 400 cGy for neutron, and 0 cGy, 100 cGy, 200 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy and 1000 cGy for X ray). Clonogenic assay was performed, and relative biological effectiveness (RBE) of fast neutron was determined with D(10) by means of cell survival curves. Isoeffective doses of 35 MeV p-->Be fast neutron and 6 MV-X ray were obtained according to the RBE. The cells were assigned into two irradiation regimens, (1) the one-week-fractionation regimen, which adopted the radiation pattern of X x 5, N x 2 and X-N-X-X-N. After irradiation the clonogenic assay was performed to compare their survival fractions; (2) the two-dose regimen, with the radiation pattern of X + N, N + X and X + X. Flow cytometry was done at different time points after irradiation to analyze cell cycle distribution and apoptosis. Fast neutron dose was delivered on Tuesday and Friday, and all the other irradiation intervals were 24 h. RESULTS: The RBE of fast neutron to X ray in CNE-1 cells according to the D(10) ratio was 2.40. The neutron isoeffective dose for a single dose of 200 cGy of 6 MV-X ray was approximately 80 cGy. In clonogenic assay, the cell survival fractions were significantly lower in X-N-X-X-N group (0.0079) than those in X x 5 (0.018) and N x 2 (0.017) groups. The flow cytometry suggested a higher percentage of apoptotic cells after mixed irradiation, and different sequence of X ray and neutron irradiations caused varying changes in cell cycle arrest. CONCLUSION: Mixed irradiation of fast neutron and X ray showed a synergic effect in vitro on CNE-1 cell killing. Cell cycle arrest and apoptosis may play some role in the radiation damage repair mechanisms of mixed beam irradiation.

[Abrogation effect of UCN-01 on radiation-induced G2 phase arrest of tumor cells and its mechanism].

BACKGROUND & OBJECTIVE: Irradiation often causes cell cycle arrest in tumor cells. This study was to observe the abrogation of radiation-induced G(2) phase arrest of p53 mutated human cancer cell lines by 7-hydroxystaurosporine (UCN-01), and explore the mechanism. METHODS: Human nasopharyngeal carcinoma cell line CNE-1 and human lung adenocarcinoma cell line 973, both with p53 mutation, were used in this study. Human fibroblastoma cell line HT-1080 was used as control. Flow cytometry was used to observe the effects of irradiation on cell cycle of these 3 cell lines, and abrogation effect of UCN-01 on radiation-induced G(2) phase arrest. Western blot was used to detect phosphorylated CDC2-Tyr15 during the abrogation process. RESULTS: Irradiation resulted in G(2) phase arrest in CNE-1 cells and 973 cells. Proportion of cells in G(2) phase increased from 18.4% to 43.6% in CNE-1 cell line, and from 14.8% to 42.8% in 973 cell line after 2 Gy of irradiation. G(1) phase arrests, measured by "S phase cells consumption", of CNE-1 cells and 973 cells were much lower than that of HT-1080 cells (14.8% and -1.2% vs. 57.0%). Radiation-induced G(2) phase arrests were abrogated by UCN-01 from 63.5% to 16.1% in CNE-1 cell line, and from 35.4% to 16.3% in 973 cell line. UCN-01 suppressed the expression of radiation-induced phosphorylation of CDC2-Tyr15, which was in accordance with the abrogation of radiation-induced G(2) phase delay. CONCLUSIONS: Main effect of irradiation on cell cycle of p53 mutated CNE-1 cells and 973 cells is G(2) phase arrest. UCN-01 is able to abrogate radiation-induced G(2) phase, which is associated with the reduction of phosphorylated CDC2-Tyr15.

[Radiosensitivity detected by "comet" assay in two human tumor cell lines].

OBJECTIVE: To evaluate the value of the "comet" assay in detecting the radiosensitivity in human tumor cell lines. METHODS: The radiation-induced primary DNA damage and repair were detected by the comet assay in CNE-1 and 973 cell lines. The tail moment was used as the end point, to quantitate the primary DNA damage and subsequent repair ability. The cell-survival curve was plotted by the classical colony assay, to detect the D(0) value and Dq value. The results from the above two assays were compared. RESULTS: 1. With the increment of irradiation doses, under the same experimental condition, the radiation-induced primary DNA damage was more severe in CNE-1 cells than in 973 cells (P < 0.01). From the cell-survival curves, the D(0) value was 1.631 and 1.822 in CNE-1 and CNE-1 973 cells respectively, indicating that CNE-1 cells were more sensitive to irradiation than 973 cells. The radiosensitivity detected by comet assay and by colony assay in the two cell lines tended to be consistent. 2. The half-repair time of 973 and CNE-1 cell line was 33 min and 41 min detected by comet assay, which indicats that the ability of DNA damage and repair in CNE-1 cells was weaker than in 973 cells. The Dq value of the cell survival curve was 2.152 for 973 and 0.626 for CNE-1 cell line detected by the colony assay, which indicates that the sublethal damage repair in 973 cells being much faster than in CNE-1 cells. The repair ability reflected by the results in the two cell lines was consistent. CONCLUSION: The radiosensitivities reflected by the results of the primary DNA damage and repair detected by both comet assay and colony assay in CNE-1 and 973 cells are consistent. It suggests that comet assay is a good method for detecting the radiosensitivity of tumor cells.

[Abrogation of radiation-induced G2 arrest and radiosensitization by 7-hydroxystaurosporine (UCN-01) in human nasopharyngeal carcinoma cell line].

BACKGROUND & OBJECTIVE: To enhance the radiosensitivity of cancer cell is one of the most important way to improve the effect of radiotherapy. This study was designed to investigate the radiosensitization of 7-hydroxystaurosporine(UCN-01) by its abrogation of radiation-induced G2 arrest. METHODS: Flow cytometry was used to observe the effect of irradiation and UCN-01 on cell cycle of human nasopharyngeal carcinoma cells (CNE-1) with mutated p53. The effect on radiosensitivity was determined by clonogenic assay and was quantified by calculating the sensitive enhancement ratio (SER). RESULTS: Irradiation resulted in G2 arrest in a dose-dependent manner. The proportion of cells at G2 phase, comparing to the control group (18.4%), was increased to 43.6%, 77.4% and 86.4% after 12 hours with irradiation of 2, 4 and 6 Gy, respectively. Radiation-induced G2 arrest was abrogated by UCN-01 in a concentration-dependent manner. UCN-01 at the concentrations of 50, 100, 200 and 400 nmol/L decreased the proportion of cells in G2 phase from 63.5% in the control group to 28.5%, 25.0%, 16.1% and 13.7%, respectively. UCN-01 resulted in a radiosensitization in CNE-1 cells. The SERs were 2.60 and 3.09, for 100 nmol/L and 200 nmol/L of UCN-01, respectively. CONCLUSION: Radiosensitization of UCN-01 in CNE-1 cells characterized by mutated p53 is associated with its abrogation of radiation-induced G2 arrest.