Current Pharmaceutical Biotechnology - Volume 14, Issue 6, 2013

Objective: RA is one of autoimmune diseases, has drawn great attention of the world. Currently, the anti- IL-1β monoclonal antibody Canakinumab (ACZ885) for treatment of RA has entered into clinical trials. However, Full length antibody has large molecular weight, and is difficult to penetrate the tissue or the nidus. In contrast, scFv has low molecular weight and strong penetration ability, and is favorable to increase the drug concentration in the indus, hence improving the efficacy of the drug. The aim of this study is to obtain a neutralizing scFv antibody from a combinatorial scFv library against hIL-1β by the modified NLPA-based bacterial display system, for further development of the small molecule antibody drug for treatment of RA. Methods: The modified NIPA-based bacterial display system was used to construct the combinatorial scFv library derived from the spleen cDNA of immunized mice with hIL-1β. FACS was used to screen hIL- 1β-binding clones with FITC-labeled hIL-1β protein. Three clones were randomly selected from the third round of screening, and their nucleotide sequences were aligned with mouse immunoglobulin genes. The single chain antibody genes of the hIL-1β-binding clones were subcloned into the prokaryotic expression vector pET-27b for expression. The molecular mass of the purified anti-hIL-1β single chain antibody was about 28ku. The hIL-1β-binding ability of antibody were examined by ELISA and Western blot assays. Ability of the scFv antibody to neutralize hIL-1β was evaluated by the MTT test. Conclusions: In this study, it is the first time to use the NIPA-based bacteria display system to construct and screen the combinatorial scFv library. Three scFvs against hIL-1β were obtained from the scFv library of the immunized mice. Prokaryotically expressed and purified scFvs demonstrate binding ability with hIL-1β. Among the three clones. The MTT test suggests that scFv-20 is a neutralization antibody against hIL-1β. The study provides a lead candidate for further development of small molecule therapeutic antibodies for treatment of RA.

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPβ levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid β (Aβ) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aβ levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aβ levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aβ levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

Edible plant vaccines are immunogenic preparations containing antigenic proteins rather than pathogens, therefore, they sanctify situation where there is a possibility of resurgence of disease when the antigenic preparation contains the organism in any form whatsoever. Expression of antigens as vaccines and of antibodies against antigens of pathogens in transgenic plants is a convenient and inexpensive source for various bacterial, viral, helminths, protozoan and autoimmune diseases with lower capital costs. This review describes various diseases along with the production of edible transgenic plant vaccines/proteins for the same. Thus, substituting and improvising conventional immunization methods.

Plasmid vaccination is a smart gene delivery application mostly achieved through the utilisation of viral or copolymeric systems as surrogated carriers in micro or nano formulations. A common polymeric protocol for plasmid vaccine formulation, which as somewhat been successful, is via the complexation of the DNA molecules with a cationic polymer, and encapsulating in a vehicular carrier polymer. Even though plasmid vaccination research has not witnessed the much anticipated success, due a number of cellular and physicochemical reasons, application of copolymeric carriers with tight functionalities is a promising strategy to optimally deliver the DNA molecules; in view of the available chemistries and physical properties that could be tuned to enable enhanced targeted delivery, uptake and specific transfection. This also enables the targeting of specific epitopes and antigen presenting cells for the treatment of many pathogenic infections and cancer. This paper provides a brief critical review of the current state of plasmid vaccines formulation and molecular delivery with analysis of performance data obtained from clinical trials.

The ATP-binding cassette A1 (ABCA1) transporter plays a major role in the efflux of lipids by mediating the cellular transport of phospholipids and cholesterol to lipid-poor/lipid-free apolipoprotein A-I (apoA-I) particles and thereby exerting important anti-atherogenic effects. Although the mechanism whereby ABCA1 mediates cholesterol efflux is not completely understood, numerous studies have shown that, in addition to apoA-I, the expression level of the total or cell surface ABCA1 protein is a determining factor for the activity of ABCA1-mediated cholesterol efflux, and defects in ABCA1-mediated cholesterol efflux lead to various pathological conditions in different cells, including cardiovascular and metabolic disorders. It has been widely demonstrated that a growing list of natural and synthetic substances and metabolic regulators that modulate the expression of ABCA1 not only act directly on the ABCA1 gene promoter, but also occurs at the post-transcriptional level via micro-RNAs and post-translationally through the stabilization or localization of the protein. The complex regulatory network of ABCA1 results in promoting or suppressing cholesterol efflux from cells, therefore we speculate that the ABCA1 transporter is emerging as a novel therapeutic target for cardiovascular and metabolic disorders. Thus, ABCA1 is a key modulator of cellular cholesterol efflux and contributes to functional disorders in different types of cells.

The human metabolism is regulated by our internal clock; the circadian rhythm (24h-25h). There are several factors included in the regulatory pathway such as; genes (PER1-3), (CRY1-2), TIM hormones (cortisol, catecholamines, melatonin and insulin) drugs, enzymes, sleep disorders and diseases. Each one contributes in a different degree and in order to enhance the therapeutic result; we should include these factors into clusters instead of targeting each factor one by one. Malignances deregulate gene-protein expression/production, enzyme production, and in addition they induce fatigue, insomnia, stress and sleep disorders. All these factors finally contribute in minimizing the efficiency of chemotherapy treatment and quality of life. In addition, the circadian rhythm disruption induces tumor genesis, stress, and downregulates the defense and repair mechanisms of the human body. In the current mini review the underlying mechanism of the circadian rhythm is provided, along with the influence of sleep disturbances in cancer patient therapy. A proposal is presented to divide circadian rhythm and sleep disturbances into two major clusters with different management, however; with a common target to improve treatment efficiency and quality of life. Finally, a chrono-chemotherapy administration model is proposed in order to have less chemotherapy side effects.