The purpose of this study was to investigate the damage mechanism of pathogenic E.coli on mouse brain microvascular endothelial cells(BMEC cells)and mouse alveolar macrophages(MH-S cells),as well as the lung and brain of healthy mice.In this study,BMEC cells and MH-S cells were infected with pathogenic E.coli strains,and cell morphological changes were observed.Plate counting method was used to detect the adhesion and invasion ability of the strains to cells and the number of bacteria in the lungs and brains of mice.RT-qPCR was used to detect the ex-pression of TNF-α,IL-1β and IL-6 genes in cells and mouse organs at different time periods.West-ern blot was used to detect the expression of p-NF-κB,p-JAK2 and p-STAT3 proteins related to inflammation in cells and mouse organs after infection.The results showed that the cell culture medium of the infection group was turbid,the cell vision became dark and blurred,some cells shrank and died,and more fragments were produced.The adhesion rate and invasion rate of BMEC cells at 3 h were significantly lower than those at 6 h(P＜0.050),and the adhesion rate and inva-sion rate of MH-S cells at 3 h were significantly higher than those at 6 h(P＜0.010).Infected mice had a large area of swelling and bleeding in the brain,and the lungs had different degrees of swell-ing and bleeding.The bacterial load in the brain and lung was the highest at 12 h.Compared with the control group,the mRNA expression levels of IL-1β,IL-6 and TNF-α in the infection group were significantly increased at 3 h and 6 h(P＜0.050),and the mRNA expression levels of inflam-matory factors in BMEC cells and MH-S cells were the highest at 6 and 3 h,respectively.The mR-NA expression of inflammatory factors in the brain and lung of infected mice showed a trend of in-creasing first and then decreasing with time,with the highest expression at 12 h after infection.The expression levels of p-NF-κB protein in BMEC cells,MH-S cells,lung and brain tissues of mice in the infection group were significantly higher than those in the control group(P＜0.001),and the expression levels of p-JAK2 protein and p-STAT3 protein were significantly lower than those in the control group(P＜0.050).The above results showed that pathogenic E.coli could adhere and invade BMEC cells and MH-S cells,colonize in lung and brain tissues of mice,promote the expres-sion of NF-κB protein in cells and tissues,inhibit the expression of JAK2 protein and STAT3 pro-tein,and then stimulate cells and tissues to produce inflammatory response.

Objective: 18F-N-(2-(Diethylamino)ethyl)-5-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy) picolinamide ( 18F-PFPN) is a novel positron emission tomography (PET) probe designed to specifically targets melanin. This study aimed to evaluate the diagnostic feasibility of 18F-PFPN in patients with ocular or orbital melanoma. Materials and Methods: Three patients with pathologically confirmed ocular or orbital melanoma (one male, two females; age 41–59 years) were retrospectively reviewed. Each patient underwent comprehensive 18F-PFPN and 18F-fluorodeoxyglucose ( 18F-FDG) PET scans. The maximum standardized uptake value (SUV max) of the lesion and the interference caused by background tissue were compared between 18F-PFPN and 18F-FDG PET imaging. In addition, the effect of intrinsic pigments in the uvea and retina on the interpretation of the results was examined. The contralateral non-tumorous eye of each patient served as a control. Results: All primary tumors (3/3) were detected using 18F-PFPN PET, while only two primary tumors were detected using 18F-FDG PET. Within each lesion, the SUV max of 18F-PFPN was 2.6 to 8.3 times higher than that of 18F-FDG. Regarding the quality of PET imaging, the physiological uptake of 18F-FDG PET in the brain and periocular tissues limited the imaging of tumors. However, 18F-PFPN PET minimized this interference. Notably, intrinsic pigments in the uvea and retina did not cause abnormal concentrations of 18F-PFPN, as no anomalous uptake of 18F-PFPN was detected in the healthy contralateral eyes. Conclusion Compared to 18F-FDG, 18F-PFPN demonstrated higher detection rates for ocular and orbital melanomas with minimal interference from surrounding tissues. This suggests that 18F-PFPN could be a promising clinical diagnostic tool for distinguishing malignant melanoma from benign pigmentation in ocular and orbital melanomas.

This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro.The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers:inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers (inner layer,outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15％ FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells (RGCs％) was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7),were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ).Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15％ FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed:(1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs％ was (19.9±1.2)％,(0.5±0.2)％,and (6.2±1.7)％ respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant (P＜0.05); (2)fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs％,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that:(1) RGCs％ in the inner layer retina was higher than that in the outer layer retina;(2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.

This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro. The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers: inner layer and outer layer, under a surgical microscope. Retinal cells isolated from different layers (inner layer, outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS. After 3-day culture, the RGCs in the retinal cells obtained from mixed culture of inner, outer, and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1, and the rate of RGCs to retinal cells (RGCs%) was calculated. Two monoclonal antibodies, anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7), were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ). Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS. Immunocytochemical staining for Thy-1.1, MTT, and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs. The results showed: (1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was (19.9±1.2)%, (0.5±0.2)%, and (6.2±1.7)% respectively in the mixed culture of inner, outer, and whole retinal tissue, with differences being significant (P<0.05); (2) fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%, RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks. The viability of RGCs was declined with the decreasing seeding density, and most cells presented round or oval in shape with thin neurites. It was concluded that: (1) RGCs% in the inner layer retina was higher than that in the outer layer retina; (2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.

Clopidogrel was believed to be superior to aspirin by the well-known CAPRIE trial. However, no other large clinical trials demonstrated the same results, but all focused on the combina-tion use of clopidogrel with aspirin, and combination therapy in CREDO was called the "Emperor's New Clothes". However, no one overturned the results of these clinical trials by quantitatively ana-lyzing them. We reviewed ten large-scale clinical trials about clopidogrel. On the basis of results of CAPRIE, CREDO and CHARISMA trials, we re-estimated their minimal sample sizes and their powers by three well-established statistical methodologies. From the results of CAPRIE, we inferred that the minimal sample size should be 85 086 or 84 968 but its power was only 30.70%. A huge gap existed. The same was also true of CREDO and CHARISMA trials. Moreover, in CAPRIE trial, 0 was included in the 95% confidence interval and 1 was included in the 95% confidence interval for the relative risk. There were some paradoxical data in CAPRIE trial. We are led to conclude that the results in CAPRIE, CREDO, and from the subgroup analysis in CHARISMA trials were questionable. These results failed to demonstrate that clopidogrel was superior to aspirin or that clopidogrel used in combination with aspirin was better than aspirin alone. The cost-effectiveness analyses by some pre-vious studies were not reliable.

Clopidogrel was believed to be superior to aspirin by the well-known CAPRIE trial. However, no other large clinical trials demonstrated the same results, but all focused on the combination use of clopidogrel with aspirin, and combination therapy in CREDO was called the "Emperor's New Clothes". However, no one overturned the results of these clinical trials by quantitatively analyzing them. We reviewed ten large-scale clinical trials about clopidogrel. On the basis of results of CAPRIE, CREDO and CHARISMA trials, we re-estimated their minimal sample sizes and their powers by three well-established statistical methodologies. From the results of CAPRIE, we inferred that the minimal sample size should be 85 086 or 84 968 but its power was only 30.70%. A huge gap existed. The same was also true of CREDO and CHARISMA trials. Moreover, in CAPRIE trial, 0 was included in the 95% confidence interval and 1 was included in the 95% confidence interval for the relative risk. There were some paradoxical data in CAPRIE trial. We are led to conclude that the results in CAPRIE, CREDO, and from the subgroup analysis in CHARISMA trials were questionable. These results failed to demonstrate that clopidogrel was superior to aspirin or that clopidogrel used in combination with aspirin was better than aspirin alone. The cost-effectiveness analyses by some previous studies were not reliable.

To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.
Animals ; Cattle ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Light ; Phagocytosis ; radiation effects ; Trabecular Meshwork ; cytology ; radiation effects

To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.
Cell Proliferation/radiation effects ; Cells, Cultured ; *Light ; Phagocytosis/radiation effects ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*radiation effects

To investigate whether the TGF-beta 1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-beta 1 in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMT-GF-beta 1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-beta 1 protein expression specific for pMAMTGF-beta 1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23.37%. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-beta 1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.
Animals ; Cell Division ; Cells, Cultured ; Epithelium, Corneal ; cytology ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; Lipids ; pharmacology ; Plasmids ; genetics ; Rabbits ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.
Animals ; Anterior Chamber ; Corneal Transplantation ; Endothelium, Corneal ; drug effects ; pathology ; Gene Transfer Techniques ; Immune Tolerance ; Rabbits ; Transforming Growth Factor beta ; administration & dosage ; biosynthesis ; genetics