Multivesicular liposome formulations for the sustained delivery of interferon α-2b

AIM: To develop and optimize a sustained release multivesicular liposome (MVL) formulation of interferon (IFN) alpha-2b. METHODS: IFN alpha-2b MVL were prepared using a typical double-emulsion procedure. The sustained release effects of IFN alpha-2b MVL were investigated by monitoring the blood IFN alpha-2b concentration using an enzyme-linked immunosorbent assay test after subcutaneous administration to healthy mice. RESULTS: IFN alpha-2b was successfully encapsulated in MVL with high efficiency, and the integrity of encapsulated protein was maintained. After subcutaneous injection, the MVL slowly released IFN alpha-2b into systemic circulation in a sustained manner. The estimated serum half-life of IFN alpha-2b was approximately 30 h. In addition, varying the size of the MVL preparations could further modify the in vivo release profile. CONCLUSION: IFN alpha-2b MVL may be a useful sustained release formulation in the clinical treatment of viral diseases.     

Clinical obser ration of interferon α-2b on the treatment of viral keratitis:476 cases study

Objective To observe the clinical effect of interferon on the treatment of virus keratitis. Meth-ods Review and analysis was made of 476 patients with virus keratitis who was treated with high concentration of an-ti-virus eyedrops and one million unit of α-2b interferon, the clinical safety and effect was evaluated. Result The total cure rate was 59. 1%, and the type from high to low is interstitial、endothelial、epithelial and the total cornea. The total recurrence rate is 23.5% ,and the type from high to low is epithelial,the total cornea,endothelial and inter-stitial. The incidence rate of the adverse effect is 10. 7%. Condusion Systemic administration of interferon has a direct anti-virus effect, and it can raise the cure rate of virus keratitis as well as decrease recurrencerate. One million unit of interferon has a high clinical safety and effect.     

Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.     

Antagonist peptides of human interferon-α2b isolated from phage display library inhibit interferon induced antiviral activity

AIM: To screen human interferon (IFN)-alpha2b antagonist peptides from a phage displayed heptapeptide library. METHODS: WISH cells and polyclonal anti-IFN-alpha2b antibodies were used to select IFN receptor-binding peptides from a phage displayed heptapeptide library. The specific binding of phage clones was examined by phage ELISA and immunohistochemistry. The specific binding activities of synthetic peptides to WISH cells were detected by competition assay. Effects of synthetic peptides to IFN-induced antiviral activity were analyzed by evaluating the cytopathic effect (CPE) using the MTT method. RESULTS: Twenty-three positive clones were obtained after seven rounds of selection. Ten clones were randomly picked from the positive clones and were sequenced. The corresponding amino acid sequences suggested 3 groups homologous to the 3 domains of IFN-alpha2b, defined by residues 24-41, 43-49, and 148-158 of IFN-alpha2b. As they presented as corresponding to IFN receptor-binding domains, AB loop and E helix, clone No 26 and 35 were chosen for further characterization and shown to bind to WISH cells. Two peptides corresponding to clone No 26 and 35, designated SP-7(SLSPGLP) and FY-7(FSAPVRY) were shown to compete with GFP-IFN-alpha2b for binding to its receptor and to inhibit the IFN-alpha2b-induced antiviral activity. CONCLUSION: Both IFN-alpha2b antagonist peptides, SP-7 and FY-7, were able to inhibit the IFN-induced antiviral activity, and could be helpful in laying the foundation for the molecular mechanism of the interaction between IFN and its receptor.     

Quercetin down-regulated bcl-2 gene expression in human leukemia HL-60 cells

目的：研究槲皮素对人白血病ＨＬ６０细胞ｂｃｌ２基因表达的影响．方法：采用免疫组织化学技术和ＲＮＡ点杂交观察基因表达．结果：Ｑｕｅ１５－６０μｍｏｌ·Ｌ－１处理ＨＬ６０细胞４８ｈ使ｂｃｌ２蛋白由对照组的９４％表达下调到４５％－８４％，并能明显下调ＨＬ６０细胞ｂｃｌ２ｍＲＮＡ表达．结论：Ｑｕｅ诱导ＨＬ６０细胞凋亡，其诱导凋亡的分子机制与下调ｂｃｌ２基因表达有关．     

Changes and their significances of IFN-γ, TNF-α and IL-2 level in mouse peripheral blood following intraperitoneal injection with influenza viruses

Objective To observe changes of IFN-γ, TNF-α and IL-2 levels in mouse peripheral blood following intraperitoneal injection with influenza viruses. Methods Mice were divided into 3 groups randomly and injected with human H3N2 influenza virus, avian H9N2 influenza virus and sterilized virus-free allantoic fluid, respectively. Sera in different groups were collected at several time points after virus injection, and the levels of IFN-γ, TNF-α and IL-2 in peripheral blood were tested by sandwich ELISA. Statistical analysis was made using analysis of variance and LSD-t test. Results The levels of IFN-γ and IL-2 in peripheral blood in influenza viruses injection groups were significantly higher than those in negative control group, and no significant difference in TNF-α level was found between influenza viruses injection groups and negative control group. Conclusion Intraperitoneal injection with human or avian influenza viruses can promote the production of IFN-γ and IL-2 in mouse peripheral blood, but it has little effect on the production of TNF-α.     

Changes and their significances of IFN-γ, TNF-α and IL-2 level in mouse peripheral blood following intraperitoneal injection with influenza viruses

Objective To observe changes of IFN-γ, TNF-α and IL-2 levels in mouse peripheral blood following intraperitoneal injection with influenza viruses. Methods Mice were divided into 3 groups randomly and injected with human H3N2 influenza virus, avian H9N2 influenza virus and sterilized virus-free allantoic fluid, respectively. Sera in different groups were collected at several time points after virus injection, and the levels of IFN-γ, TNF-α and IL-2 in peripheral blood were tested by sandwich ELISA. Statistical analysis was made using analysis of variance and LSD-t test. Results The levels of IFN-γ and IL-2 in peripheral blood in influenza viruses injection groups were significantly higher than those in negative control group, and no significant difference in TNF-α level was found between influenza viruses injection groups and negative control group. Conclusion Intraperitoneal injection with human or avian influenza viruses can promote the production of IFN-γ and IL-2 in mouse peripheral blood, but it has little effect on the production of TNF-α.     

Effects of intrathecal 6-hydroxydopamine, α1 and α2 adrenergic receptor antagonists on antinociception of propofol in mice

AIM: To investigate the relationship between spinal cord norepinephrine, alpha1 and alpha2 adrenergic receptors and antinociception of propofol in mice. METHODS: Kunming mice were used. Antinociceptive tests were investigated with the tail-immersion test and the acetic acid-induced writhing test. The effects of subcutaneous (sc), intrathecal (ith) and intracerebroventricular (icv) injection propofol on pain threshold were observed. The influences of pretreatment with ith 6-hydroxydopamine, alpha1R antagonist prazosin, or alpha2R antagonist yohimbine on the antinociception of propofol were studied. RESULTS: Significant antinociception was produced by propofol (25, 50 mg/kg, sc) and propofol (20, 40 microg, ith) in tail-immersion test and acetic the acid-induced writhing test (P<0.05 or P<0.01). Icv propofol (10, 20, and 40 microg) did not produce any effect on pain threshold in mice (P>0.05). The 6-hydroxydopamine (5 and 10 microg), prazosin (5 and 10 microg), or yohimbine (5 and 10 microg) ith alone did not affect basal tail-flick latency (TFL) in conscious mice, but significantly reduced the TFL as measured by tail-immersion test in propofol (50 mg/kg, sc)-treated mice, compared with basal TFL and vehicle groups (P<0.05 or P<0.01). CONCLUSION: The spinal cord is a target of propofol antinociception. In mice propofol antinociception is partly mediated by spinal norepinephrine, alpha1R and alpha2R.     

Clinical potential of electroporation for gene therapy and DNA vaccine delivery

ABSTRACT

Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.

Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.

Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.     

The antiviral effect of human interferon alpha is dependent on phosphoinositide-derived messengers

Neomycin the putative blocker of membrane polyphosphoinositide hydrolysis, inhibited the antiviral activity of human interferon alpha, when tested on human quiescent fibroblasts challenged with vesicular stomatitis virus. The anti-interferon effect of neomycin could be correlated in terms of dose dependence for both neomycin (0.05-1 mM) and interferon (100-5,000 IU/ml). The results suggest that the antiviral activity of interferon alpha depends on diacylglycerol formation. Indeed, the synthetic diacylglycerol (50 microM) was as effective as 100 IU/ml interferon in inducing the antiviral state.     

Biphasic vesicles for topical delivery of interferon alpha in human volunteers and treatment of patients with human papillomavirus infections

PURPOSE. Topical biphasic vesicle delivery system encapsulating interferon alpha (IFN α) was developed as an alternative to injections used to treat human papillomavirus (HPV) infections. METHODS. Biphasic lipid vesicles encapsulating increasing doses of IFN α (biphasic IFN α) were characterized for encapsulation efficiency, size, zeta potential and vesicle structure by centrifugation, dynamic light scattering, confocal microscopy and small-angle x-ray scattering. Biphasic IFN-α delivery into human skin in vivo and topical efficacy in patients with genital warts were evaluated. RESULTS. Average encapsulation efficiency of IFN α was 81-91%. The average particle size was 1000-1100 nm and zeta potential +70 to +78 mV. After application of 5, 15 and 40MU/g biphasic IFN α formulation in a topical patch on the upper inner arm in healthy volunteers, skin IFN α levels increased to 120±30, 380±60 and 400±80 IU/mg protein in skin homogenates (n=5, 5, and 7), respectively. Topical application of biphasic IFN α (1 MU/dose) twice daily for two weeks in a pilot study with 12 patients with external condylomata acuminata resulted in a decrease in lesion size, in 2',5'-oligoadenylate synthetase activity and in tissue viral load. CONCLUSIONS. Biphasic vesicles delivered clinically significant levels of IFN α across intact human skin and elicited marked therapeutic effect in patients.     

Inhibitory effect of Ca^2＋ on in vivo gene transfer by electroporation

AIM: To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice. METHODS: Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions. RESULTS: For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed. CONCLUSION: The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.     

Gene delivery and gene therapy of prostate cancer

Surgery, radiation or hormonal therapy are not adequate to control prostate cancer. Clearly, other novel treatment approaches, such as gene therapy, for advanced/recurrent disease are desperately needed to achieve long-term local control and particularly to develop effective systemic therapy for metastatic prostate cancer. In the last decade, significant progress in gene therapy for the treatment of localised prostate cancer has been demonstrated. A broad range of different gene therapy approaches, including cytolytic, immunological and corrective gene therapy, have been successfully applied for prostate cancer treatment in animal models, with translation into early clinical trials. In addition, a wide variety of viral and nonbiological gene delivery systems are available for basic and clinical research. Gene therapy approaches that have been developed for the treatment of prostate cancer are summarised.     

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a beta-galactosidase reporter plasmid DNA applied to ViaDerm treated skin. Skin treated with ViaDerm using 50 microm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.     

Gene expression profiling in human lung fibroblast following cadmium exposure.

Cadmium is a naturally occurring metallic element with food and smoking being the main sources of exposure in the non-occupationally exposed population. Chronic exposure to cadmium leads to tumors in a number of tissues including lung. In the present study we investigated genes whose expression is modified by Cd exposure in human lung fibroblast WI38-VA13 cells. We employed a cDNA microarray hybridization method to identify changes in the gene expression profile. Thirty five genes were identified as cadmium-responsive. Their level of expression differed significantly from controls (significance analysis of microarray; SAM, q<5%). The largest groups of gene products affected by cadmium exposure were those involved in cell cycle, immunity and defense, nucleoside metabolism and signal transduction. Repressed expression of E2f1, Tubb and Actg2 following cadmium exposure may contribute to the cell cycle arrest. Down-regulation of Eno1 indicates a potential for causing protooncogene expression and possibly for cadmium-induced carcinogenicity. These results may contribute to better understand the toxic mechanism of cadmium toxicity. Moreover, the gene expression profile of cadmium could provide potential biomarkers for cadmium exposure.     

重组人干扰素α-2b诱发急性左心衰竭

75岁老年男性脑梗死康复期患者,给予重组人干扰素α-2b300万IU肌内注射,约20min后,出现寒战、呼吸困难,不能平卧,冷汗。查体:T38.5℃,BP200/100mmHg,R40次/min,HR130次/min,精神恍惚,听诊双下肺湿性啰音,伴哮鸣音,心电图示完全左束支传导阻滞,窦性心动过速。患者经对症治疗后逐渐好转。