Current Pharmaceutical Biotechnology - Volume 16, Issue 8, 2015

Targeted cellular immunotherapy with bifunctional antibodies (bsAbs) has emerged as a promising therapeutic approach for cancer over the last two decades. Progress in antibody engineering has led to the generation of many different types of antibody-derived entities that display at least two binding specificities. Most bsAbs consist of large IgG-like proteins with multiple antigen-binding regions containing Fc parts or smaller entities without Fc. BsAbs have the potential to engage effector cells of the immune system, thereby overcoming some of the immune response escape mechanisms of tumor cells. Preclinical and clinical trials of various bsAb constructs have demonstrated impressive results in terms of immune effector cell retargeting and induction of efficient anti-tumor responses. This review provides an overview of the established bsAbs focusing on improvements in format and design as well as the mechanisms of action of the most promising candidates and describes the results of the most recent clinical studies.

Recombinant adeno-associated virus (rAAV) vector is one of the promising delivery tools for gene therapy. Currently, hundreds of clinical trials are performed but the major barrier for clinical application is the absence of any ideal large scale production technique to obtain sufficient and highly pure rAAV vector. The large scale production technique includes upstream and downstream processing. The upstream processing is a vector package step and the downstream processing is a vector purification step. For large scale downstream processing, the scientists need to recover rAAV from dozens of liters of cell lysate or medium, and a variety of purification strategies have been developed but not comprehensively compared till now. Consequently, this review will evaluate the scalable downstream purification strategies systematically, especially those based on the physicochemical properties of AAV virus, and attempt to find better scalable downstream strategies for rAAV vectors.

The “amyloid cascade hypothesis” posits that an extracellular build-up of amyloid-β oligomers (Aβ-os) and polymers (fibrils) subsequently inducing toxic hyperphosphorylated (p)-Tau oligomers (p-Tau-os) and neurofibrillary tangles starts the sporadic late-onset Alzheimer’s disease (LOAD) in the aged lateral entorhinal cortex. Conversely, mutated genes cause a diffuse cerebral Aβs/Aβ-os overproduction promoting early-onset familiar AD (EOFAD). Surplus exogenous Aβ-os exert toxic actions at several levels. They reach the nuclei of human astrocyte-neurons teams (ANTs) to enhance the transcription of Aβ precursor protein (APP) and β-secretase/BACE1 genes. The overexpressed APP and BACE1 proteins act in concert with γ-secretase to overproduce endogenous Aβs/Aβ-os, of which a few enter the nuclei to upkeep Aβs overproduction, while the rest gather in the cytoplasm, damage mitochondria, and are oversecreted. Simultaneously, extracellular Aβ-os bind the ANTs’ calcium-sensing receptors (CaSRs) activating signalings that hinder the proteolysis and hence favor the surplus hoarding/secretion of Aβs/Aβ-os. Overreleased Aβ-os spread, reach growing numbers of adjacent ANTs to recruit them to overproduce/oversecrete further Aβ-os amounts via the just mentioned mechanisms. Alongside, Aβ•CaSR signalings elicit a noxious overproduction/overrelease of nitric oxide (NO) and vascular endothelial growth factor (VEGF)-A from ANTs’ astrocytes. While astrocytes survive the toxic onslaught, neurons die. Thus, AD progression is driven by ceaselessly self-sustaining neurotoxic cycles, which engender first Aβ-os and later p-Tau-os that cooperatively destroy increasingly wider cognition-related cortical areas. Notably, a highly selective allosteric CaSR antagonist (calcilytic), like NPS 2143, does preserve human cortical postnatal HCN-1A neurons viability notwithstanding the presence of exogenous Aβ-os by suppressing the otherwise elicited oversecretion and spread of newly synthesized Aβ-os. Therefore, if given at minimal cognitive impairment or earlier stages, calcilytics could halt AD progression and preserve the patients’ cortical neurons, cognitive abilities, and eventually life.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Although there have been various attempts to express and purify human TNF-α, the current work introduces a simple, rapid, and efficient method for its production without loss of biological activity. The protein was expressed based on GST-tagged fusion system in Escherichia coli under optimized condition. The expressed GST fusion protein was applied to glutathione affinity column and then, TNF-α was cleaved off the GST using thrombin protease. The purity of the product was more than 95% and further size exclusion chromatography slightly improved the purity. The purified human TNF-α was tested for its biological activity and structural analysis, using MTT assay (EC50 of 4.1 ×10E–12 M in L929 cell death assay) and circular dichroism spectropolarimetry, respectively. The results showed that the method used in this study enables successful production of highly purified and fully functional TNF-α.

Aimed to explore an agglutination test which can simultaneously detect two pathogenic bacteria, an agglutination test based on colored silica nanoparticles (colored-SiNps) was established in this work. Monodisperse colored-SiNps were used as agglutination test carriers; red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red 136 and C.I. Reactive Blue 14. Then the red-SiNps were sensitized with antibodies against E. sakazaki and denoted as IgG-red-SiNps; The blue-SiNps were coated with antibodies against S. pullorum and S. Gallinarum and denoted as IgGblue- SiNps. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could simultaneously agglutinate with E. sakazakii and S. pullorum and S. gallinarum on glass slide. The E. sakazakii and S. pullorum and S. gallinarum could be simultaneously detected by agglutination test with obvious agglutination phenomena. The E. sakazakii and S. pullorum and S. gallinarum could both be detected in a range from 4×103 to 4×109 CFU/mL. The pullorum and S. gallinarum and E. sakazakii in the infected food sample were detected by mixture solution of IgG-red-SiNps and IgG-blue-SiNps too. This agglutination test was easy and rapid, it might be useful for in situ rapid detection method for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.

Herpesvirus and poliovirus are responsible for important diseases in human and animal. Trichilia catigua a Brazilian native plant known as catiguá has several medicinal properties among them antimicrobial for bacteria and protozoa, however, no antiviral activity has been reported yet. This study evaluated the antiviral activity of the crude extract (CE) and aqueous and ethyl acetate fractions (AF, EAF) obtained from T. catigua in the replication of the Herpes simplex virus (HSV-1), bovine herpesvirus (BoHV-1) and poliovirus (PV-1). The cytotoxicity was analyzed by MTT assay and the antiviral effect was determined by the addition of extracts (0.25 to 100.0μg/ml), before (-2h and -1h), during (0h) and after (1h and 2h) the viral infection, by plaque reduction assay, in HEp-2 cell culture. The virucidal activity and inhibition of viral adsorption were also evaluated. In addition, the combination index (CI) with Acyclovir (ACV - reference drug) was determined for HSV-1. CE, AF and EAF showed a low toxicity (CC50 >400µg/ml) and low inhibitory concentration (IC50), ranging from 2.44-34.25 µg/ml for herpesvirus and 0.67 to 1.8 µg/ml for PV-1, associated with high selectivity index. The tested compounds showed high virucidal effect and high ability to inhibit viral adsorption, for all virus. The CI demonstrated a synergic effect (CI<1) for AF and EAF comparatively to acyclovir (ACV). Our study demonstrated that the extract and fractions of T. catigua is promising for future antiviral drug design with economically feasible production.

This work was focused on in vitro evaluation of anti-biofilm and anti-quorum sensing effects of four selected flavonoid compounds /(+)-catechin, caffeic acid, quercetin and morin/ using the strain Pseudomonas aeruginosa PAO1. At a concentration of 0.5 MIC quercetin was the only compound found to potently reduce both P. aeruginosa biofilm formation (95%) and its twitching motility. The chemical scaffold of quercetin, a common dietary polyphenol, may actually inspire development of novel and more effective medicinal agents targeting P. aeruginosa, the bacterium well known for its resistance.

Several epidemiological studies show that fruits, vegetables and cereals can play a nutraceutical role for their content of many antioxidant phytochemicals such as carotenoids, ascorbic acid and phenolics. A commercial inoculant (MICOSAT F®) containing arbuscular mycorrhizal fungi (AMF) could improve the nutritional value in crops. The goal of this work was to evaluate the effect of AMF on the production level of carotenoids, AsA, phenols including antocyanins and saponins, proteins, total antioxidant activity and nitrates in fruits, vegetables, legumes and durum wheat var. grecale, whose consumption is largely recommended according to Mediterranean diet. The treatment increased the antioxidant activity in strawberries (37.50%), in giant lentils (29.17%) and in durum wheat (63.63%) but decreased it in kiwi (31.81%) and in grape (19.81%). Nitrate levels decreased significantly in strawberries (39.78%) and in tomato intended for transformation (37.79%). The application of MICOSAT F® enhanced the levels of several secondary metabolites. However, the amount of phytochemicals and respective by-products were reduced in some cases. Environmental conditions and modality of AMF inoculation could module both primary and secondary metabolites.

Gold nanoparticles have found prominence in pharmaceutical applications due to their unique physical properties as well as their inert nature. Mycosynthesis of noble metal nanoparticles is less stringent and eco-friendly. In this paper, we have reported the economically-viable synthesis of gold nanoparticles, mediated by five different fungal strains Aspergillus flavus NCIM650, Phoma exigua NCIM1237, Aspergillus niger NCIM 616, Aspergillus niger NCIM 1025 and Trichoderma reesei NCIM 1186. An efficient approach for fungal growth was discussed wherein the biomass was cultivated in non-limiting conditions, followed by addition of gold salt solution. Cyclic Voltammetry studies were conducted to show the varying reducing capacities of these strains. The surface plasmon peaks for gold nanoparticles produced by Aspergillus flavus NCIM650, Phoma exigua NCIM1237, Aspergillus niger NCIM 616, Aspergillus niger NCIM 1025 and Trichoderma reesei NCIM 1186 were recorded as 536nm, 543nm, 542nm, 560nm, 537nm respectively. Based on the cyclic voltammetry studies and UV-Visible spectroscopy, transmission electron microscopy (TEM) analysis was done. Among the five strains, gold nanoparticles fabricated by Aspergillus niger NCIM 616 gave quite promising results. The antioxidant activities were evaluated using DPPH quenching assay and hydrogen peroxide assay.