Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.     

Nigella sativa L.: Uses in traditional and contemporary medicines – An overview

There are several wild and cultivated plants that offer excellent opportunities for being used as herbal and therapeutic agents. The identification of medicinal properties of plants and their effectiveness in treating diseases are important components in medicinal plant research and this can pave ways for further improvements in traditional drug use. Climatic conditions and phytogeography in Pakistan are ideal for naturally occurring diverse flora and managed cultivation of hundreds of plants of medicinal significance. Nigella sativa commonly known as the black seed is an important medicinal plant that has been widely used as a multipurpose medicinal agent in different countries since old times. The plant is abundantly cultivated in Pakistan for uses as condiment and medicines. It possesses important classes of bioactive compounds among which thymoquinone has attracted significant attention from the scientific community because of its active role in treating a diverse spectrum of diseases. The black seeds are used for reducing adverse effects of arthritis, asthma, inflammation, liver and gastro disorders besides their potential role in diabetes and cancers. The focus of this review is to highlight the medicinal significance of N. sativa in traditional medicine and opportunities for exploitation in contemporary medicine.     

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.     

Lipid and Carbohydrate Modifications of α-Galactosylceramide Differently Influence Mouse and Human Type I Natural Killer T Cell Activation

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators.     

Intrinsic Neuronal Properties Switch the Mode of Information Transmission in Networks

Diverse ion channels and their dynamics endow single neurons with complex biophysical properties. These properties determine the heterogeneity of cell types that make up the brain, as constituents of neural circuits tuned to perform highly specific computations. How do biophysical properties of single neurons impact network function? We study a set of biophysical properties that emerge in cortical neurons during the first week of development, eventually allowing these neurons to adaptively scale the gain of their response to the amplitude of the fluctuations they encounter. During the same time period, these same neurons participate in large-scale waves of spontaneously generated electrical activity. We investigate the potential role of experimentally observed changes in intrinsic neuronal properties in determining the ability of cortical networks to propagate waves of activity. We show that such changes can strongly affect the ability of multi-layered feedforward networks to represent and transmit information on multiple timescales. With properties modeled on those observed at early stages of development, neurons are relatively insensitive to rapid fluctuations and tend to fire synchronously in response to wave-like events of large amplitude. Following developmental changes in voltage-dependent conductances, these same neurons become efficient encoders of fast input fluctuations over few layers, but lose the ability to transmit slower, population-wide input variations across many layers. Depending on the neurons'' intrinsic properties, noise plays different roles in modulating neuronal input-output curves, which can dramatically impact network transmission. The developmental change in intrinsic properties supports a transformation of a networks function from the propagation of network-wide information to one in which computations are scaled to local activity. This work underscores the significance of simple changes in conductance parameters in governing how neurons represent and propagate information, and suggests a role for background synaptic noise in switching the mode of information transmission.     

Biotechnological applications and potential of wood-degrading mushrooms of the genus <Emphasis Type="Italic">Pleurotus</Emphasis>

The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and important biotechnological and environmental applications. The cultivation of Pleurotus spp is an economically important food industry worldwide which has expanded in the past few years. P. ostreatus is the third most important cultivated mushroom for food purposes. Nutritionally, it has unique flavor and aromatic properties; and it is considered to be rich in protein, fiber, carbohydrates, vitamins and minerals. Pleurotus spp are promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic and immunomodulation activities. The bioactive molecules isolated from the different fungi are polysaccharides. One of the most important aspects of Pleurotus spp is related to the use of their ligninolytic system for a variety of applications, such as the bioconversion of agricultural wastes into valuable products for animal feed and other food products and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants. In this Mini-Review, we describe the properties of Pleurotus spp in relation to their biotechnological applications and potential.     

Targeting the Biophysical Properties of the Myeloma Initiating Cell Niches: A Pharmaceutical Synergism Analysis Using Multi-Scale Agent-Based Modeling

Multiple myeloma, the second most common hematological cancer, is currently incurable due to refractory disease relapse and development of multiple drug resistance. We and others recently established the biophysical model that myeloma initiating (stem) cells (MICs) trigger the stiffening of their niches via SDF-1/CXCR4 paracrine; The stiffened niches then promote the colonogenesis of MICs and protect them from drug treatment. In this work we examined in silico the pharmaceutical potential of targeting MIC niche stiffness to facilitate cytotoxic chemotherapies. We first established a multi-scale agent-based model using the Markov Chain Monte Carlo approach to recapitulate the niche stiffness centric, pro-oncogenetic positive feedback loop between MICs and myeloma-associated bone marrow stromal cells (MBMSCs), and investigated the effects of such intercellular chemo-physical communications on myeloma development. Then we used AMD3100 (to interrupt the interactions between MICs and their stroma) and Bortezomib (a recently developed novel therapeutic agent) as representative drugs to examine if the biophysical properties of myeloma niches are drugable. Results showed that our model recaptured the key experimental observation that the MBMSCs were more sensitive to SDF-1 secreted by MICs, and provided stiffer niches for these initiating cells and promoted their proliferation and drug resistance. Drug synergism analysis suggested that AMD3100 treatment undermined the capability of MICs to modulate the bone marrow microenvironment, and thus re-sensitized myeloma to Bortezomib treatments. This work is also the first attempt to virtually visualize in 3D the dynamics of the bone marrow stiffness during myeloma development. In summary, we established a multi-scale model to facilitate the translation of the niche-stiffness centric myeloma model as well as experimental observations to possible clinical applications. We concluded that targeting the biophysical properties of stem cell niches is of high clinical potential since it may re-sensitize tumor initiating cells to chemotherapies and reduce risks of cancer relapse.     

Biophysical Model of Bacterial Cell Interactions with Nanopatterned Cicada Wing Surfaces

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on their physical surface structure. The wings provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. We propose a biophysical model of the interactions between bacterial cells and cicada wing surface structures, and show that mechanical properties, in particular cell rigidity, are key factors in determining bacterial resistance/sensitivity to the bactericidal nature of the wing surface. We confirmed this experimentally by decreasing the rigidity of surface-resistant strains through microwave irradiation of the cells, which renders them susceptible to the wing effects. Our findings demonstrate the potential benefits of incorporating cicada wing nanopatterns into the design of antibacterial nanomaterials.     

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.     

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.     

Regulatory perspective on in vitro potency assays for human dendritic cells used in anti-tumor immunotherapy

Dendritic cells (DCs) are key connectors between the innate and adaptive immune system and have an important role in modulating other immune cells. Therefore, their therapeutic application to steer immune responses is considered in various disorders, including cancer. Due to differences in the cell source and manufacturing process, each DC medicinal product is unique. Consequently, release tests to ensure consistent quality need to be product-specific.Although general guidance concerning quality control testing of cell-based therapies is available, cell type-specific regulation is still limited. Especially guidance related to potency testing is needed, because developing an in vitro assay measuring cell properties relevant for in vivo functionality is challenging. In this review, we provide DC-specific guidance for development of in vitro potency assays for characterisation and release. We present a broad overview of in vitro potency assays suggested for DC products to determine their anti-tumor functionality. Several advantages and limitations of these assays are discussed. Also, we provide some points to consider for selection and design of a potency test. The ideal functionality assay for anti-tumor products evaluates the capacity of DCs to stimulate antigen-specific T cells. Because this approach may not be feasible for release, use of surrogate potency markers could be considered, provided that these markers are sufficiently linked to the in vivo DC biological activity and clinical response. Further elucidation of the involvement of specific DC subsets in anti-tumor responses will result in improved manufacturing processes for DC-based products and should be considered during potency assay development.     

Maturation promoting factor activation in early amphibian embryos: Temporal and spatial control

The cytoplasmic localisation of factors capable of influencing the behaviour of nuclei has long been considered a potential mechanism for generating cell differences during development. Yoshio Masui was instrumental in identifying two cytoplasmic factors, maturation promoting factor (MPF) and cytostatic factor (CSF), defining the first biological assay for their isolation and characterisation. These biological assays involved the transfer of cytoplasm between amphibian oocytes, MPF being able to promote meiotic maturation (progression to MII) and CSF to stabilise the MII state. Masui was subsequently involved in developing a ‘cell-free’ system with the potential for analysis not just of MPF and CSF, but many aspects of nucleo-cytoplasmic interaction. Masui and Markert initially showed that MPF activity could be generated in enucleate oocytes following progesterone stimulation, indicating a cytoplasmic origin. Masui subsequently showed that MPF activity was distributed unevenly through the egg of Rana pipiens during maturation. In this review we will consider the historical context in which the MPF assays were established, then briefly consider some of the molecular components that are now known to influence MPF activation. We will then consider evidence for the asymmetric activation of MPF and the possibility that the nucleus contributes to MPF activation in early embryos.     

Structures and mechanisms of formation of liprotides

Many proteins form complexes called liprotides with oleic acid and other cis-fatty acids under conditions where the protein is partially unfolded. The complexes vary in structure depending on the ratio of protein and lipid, but the most common structural organization is the core-shell structure, in which a layer of dynamic, partially unfolded and extended proteins surrounds a micelle-like fatty acid core. This structure, first reported for α-lactalbumin together with OA, resembles complexes formed between proteins and anionic surfactants like SDS. Liprotides first rose to fame through their anti-carcinogenic properties which still remains promising for topical applications though not yet implemented in the clinic. In addition, liprotides show potential in drug delivery thanks to the ability of the micelle core to solubilize and stabilize hydrophobic compounds, though applications are challenged by their sensitivity to acidic pH and dynamic exchange of lipids which makes them easy prey for serum “hoovers” such as albumin. However, liprotides are also of fundamental interest as a generic “protein complex structure”, demonstrating the many and varied structural consequences of protein-lipid interactions. Here we provide an overview of the different types of liprotide complexes, ranging from quasi-native complexes via core-shell structures to multi-layer structures, and discuss the many conditions under which they form. Given the many variable types of complexes that can form, rigorous biophysical analysis (stoichiometry, shape and structure of the complexes) remains crucial for a complete understanding of the mechanisms of action of this fascinating group of protein-lipid complexes both in vitro and in vivo.     

Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

Bipolaris papendorfii has been reported as a fungal plant pathogen that rarely causes opportunistic infection in humans. Secondary metabolites isolated from this fungus possess medicinal and anticancer properties. However, its genetic fundamental and basic biology are largely unknown. In this study, we report the first draft genome sequence of B. papendorfii UM 226 isolated from the skin scraping of a patient. The assembled 33.4 Mb genome encodes 11,015 putative coding DNA sequences, of which, 2.49% are predicted transposable elements. Multilocus phylogenetic and phylogenomic analyses showed B. papendorfii UM 226 clustering with Curvularia species, apart from other plant pathogenic Bipolaris species. Its genomic features suggest that it is a heterothallic fungus with a putative unique gene encoding the LysM-containing protein which might be involved in fungal virulence on host plants, as well as a wide array of enzymes involved in carbohydrate metabolism, degradation of polysaccharides and lignin in the plant cell wall, secondary metabolite biosynthesis (including dimethylallyl tryptophan synthase, non-ribosomal peptide synthetase, polyketide synthase), the terpenoid pathway and the caffeine metabolism. This first genomic characterization of B. papendorfii provides the basis for further studies on its biology, pathogenicity and medicinal potential.     

Biophysical properties of tear film lipid layer II. Polymorphism of FAHFA

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of endogenous lipids that consist of two acyl chains connected through a single ester bond. Being a unique species of FAHFAs, (O-acyl)-ω-hydroxy fatty acids (OAHFAs) differ from other FAHFAs in that their hydroxy fatty acid backbones are ultralong and their hydroxy esterification is believed to be solely at the terminal (ω-) position. Only in recent years with technological advances in lipidomics have OAHFAs been identified as an important component of the tear film lipid layer (TFLL). It was found that OAHFAs account for approximately 4 mol% of the total lipids and 20 mol% of the polar lipids in the TFLL. However, their biophysical function and contribution to the TFLL is still poorly understood. Here we studied the molecular biophysical mechanisms of OAHFAs using palmitic-acid-9-hydroxy-stearic-acid (PAHSA) as a model. PAHSA and OAHFAs share key structural similarities that could result in comparable biophysical properties and molecular mechanisms. With combined biophysical experiments, atomic force microscopy observations, and all-atom molecular dynamics simulations, we found that the biophysical properties of a dynamic PAHSA monolayer under physiologically relevant conditions depend on a balance between kinetics and thermal relaxation. PAHSA molecules at the air-water surface demonstrate unique polymorphic behaviors, which can be explained by configurational transitions of the molecules under various lateral pressures. These findings could have novel implications in understanding biophysical functions that FAHFAs, in general, or OAHFAs, specifically, play in the TFLL.     

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.     

New strategies for fluorescently labeling proteins in the study of amyloids

Amyloid proteins are widely studied, both for their unusual biophysical properties and their association with disorders such as Alzheimer’s and Parkinson’s disease. Fluorescence-based methods using site-specifically labeled proteins can provide information on the details of their structural dynamics and their roles in specific biological processes. Here, we describe the application of different labeling methods and novel fluorescent probe strategies to the study of amyloid proteins, both for in vitro biophysical experiments and for in vivo imaging. These labeling tools can be elegantly used to answer important questions on the function and pathology of amyloid proteins.