Nonclinical Development of Multi-targeting Biopharmaceuticals

Rodney A. Prell , ... Anu V. Connor , in Nonclinical Development of Novel Biologics, Biosimilars, Vaccines and Specialty Biologics, 2013

Fragment-based Molecules

Several potential advantages have been postulated for the small-scale fragment-based molecules. Their modular construction provides a tremendous corporeality of flexibility in size, shape, and antigen specificity. While the fragment scaffolds offer several manufacturing advantages including the power for expression in non-mammalian cell fermentation systems, manufacturing hurdles reported to date include low yields, poor product quality, and poor stability [4]. Biologically, the smaller size of these molecules may favor better tissue penetration when compared to whole IgG molecules. Nonetheless, this potential advantage may be offset past poor PK properties due to the lack of the Fc region that tin collaborate with the neonatal Fc receptor (FcRn), potentially decreasing half-life. If long-term exposure is required, one volition need to consider options such every bit stabilizing formulations (i.due east. PEGylations), protein fusions, or continuous infusion pumps to overcome rapid clearance.

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Exploiting Antigen–Antibody Interaction

Tak Due west. Mak , Mary E. Saunders , in The Immune Response, 2006

iii) Flow Cytometry

Cell populations bearing fluorochrome-tagged antibodies can be counted or otherwise evaluated according to their fluorescence using a flow cytometer. Flow cytometers are electronic instruments that can analyze diverse physical and chemic characteristics of private cells equally they are forced through a minor opening at loftier speed. Any jail cell type that tin be prepared as a "unmarried-cell intermission" tin be studied using the catamenia cytometer. The term "single-prison cell suspension" does not imply that there is a just a single cell in the sample, only that private cells accept been resuspended at relatively low density in civilization media in vitro and are non clumped together or nowadays as office of an organized tissue. Different most other prison cell types, leukocytes are able to survive quite nicely as single–cell suspensions and are non impeded or fabricated less viable by the surface binding of molecules bearing a fluorochrome tag. These characteristics have made them the mammalian prison cell type well-nigh intensely analyzed by catamenia cytometry. Menses cytometry tin can also be used to report non-mammalian cells such as leaner, yeast, and establish cells, and many marine enquiry vessels have a menstruum cytometer on board for the study of plankton.

For catamenia cytometric analysis, the single-jail cell intermission is incubated with tagged antibody specific for a prison cell surface molecule of interest and then added to a chamber in the flow cytometer known equally the "menses cell." Pressure forces the cells out the tip of a nozzle, which vibrates ultrasonically. The vibration causes each prison cell to become encased in an individual droplet of buffer. The aerosol fall ane by i past a laser beam, which excites the fluorochrome tags on the cell surface-bound antibodies. The intensity and wavelength (color) of the fluorescent light emitted by each antibiotic'due south tag is analyzed instantaneously past the computer-assisted fluorescence detectors of the flow cytometer. This assay yields information on the number of cells binding the tagged antibody and the average fluorescent intensity of each, which is a reflection of the level of expression of the molecule of interest.

It should be noted that period cytometry is a technique that can exist very broadly practical, and thus can reveal of import information about cells in a sample in improver to that provided by the bounden of fluorochrome-conjugated antibodies. Features such as size, DNA and RNA content, and calcium flux tin exist measured by analyzing how parameters such equally viscosity and refractive index are afflicted when the light amplification by stimulated emission of radiation axle hits an private prison cell. For instance, the catamenia cytometer determines the size of each jail cell by examining the "forrard low-cal scatter" associated with it, and the granularity of each cell by analyzing its "90° light besprinkle." The menstruum cytometer can be calibrated such that cells larger or smaller than those of interest are ignored, meaning that data on only a detail subpopulation of cells (for example, the lymphocytes in an unfractionated leukocyte suspension) can exist obtained. Those cells that practice not fit the specified parameters are "gated out" and ignored in the catamenia cytometer's analysis of the examination population. Thus, the output of the flow cytometer is a representation of the numbers of cells plumbing equipment a item set of low-cal besprinkle and fluorescence parameters.

Immunofluorescence in conjunction with catamenia cytometry can exist used to enumerate, characterize, and even isolate particular subsets of living cells in a sample. As a specific example of how antibodies are used in flow cytometric studies of leukocytes, let's imagine we have mouse thymus cells and we want to determine what percentage of that population is expressing the CD8 co-receptor molecule that is expressed on T cells and interacts with MHC class I molecules. A thymus cell break is prepared and incubated with fluorescein-tagged antibody recognizing CD8. The cells are then washed free of unbound antibody and passed through the flow cytometer. The menstruation cytometer analyzes the relative fluorescence intensity of each cell as it passes past the fluorescence detector, providing a relative measure of the amount of fluorescein-conjugated anti-CD8 antibody spring to each thymus cell. The information tin be reported as a graph plotting the number of cells versus fluorescence intensity (Plate 7-4A). Past also running a command sample with a fluorescein-tagged antibiotic that is not expected to bind to thymus cells, one tin can determine a groundwork level of fluorescence in a higher place which the cells tin exist said to be "CD8 positive." From this consequence, the percentage of CD8+ thymus cells in the sample can be calculated. As an example of the employ of ii-color fluorescence analysis, let'southward now suppose that in addition to analyzing the thymus cells expressing CD8, we also wish to simultaneously analyze the thymus cells expressing the CD4 co-receptor that is expressed on T cells and binds to MHC class Ii. In this case, the thymus jail cell suspension is stained by incubation with both fluorescein-tagged anti-CD8 antibody and phycoerythrin-tagged anti-CD4 antibiotic. The flow cytometer assesses each cell every bit information technology passes through the fluorescence detectors for the presence of either the fluorescein or the phycoerythrin point and keeps rails of the numbers of each. The data are shown on an attached oscilloscope organized in a graphical representation consisting of 4 quadrants (Plate vii-4B). The relative percentages of cells bearing merely CD4, cells bearing only CD8, cells bearing both CD4 and CD8, and cells bearing neither are plotted in the appropriate quadrants. More sophisticated analyses can involve staining with up to xvi different fluorescent markers and plotting for jail cell size as well as number and fluorescence. In addition, new dyes that change color upon binding to Thou+, Na+, and Ca2+ ions within a living jail cell permit a researcher to closely follow subtle intracellular reactions such as membrane pore changes and Ca2+ flux. Some examples of dyes used in these types of analyses are given in Table 7-3.

Plate 7-4. Assay of Thymocytes by Menstruum Cytometry

(A) Thymocytes have been incubated with a non-specific Ab (curve on left) and an anti-CD8–FITC tagged Ab (curve on right). The number of cells at a given level of fluorescence intensity is measured by menses cytometry. (B) Thymocytes are incubated with anti-CD4-PE and anti-CD8-FITC antibodies. Thymocytes expressing different levels of CD4 and CD8 are shown in the various quadrants.

 Reproduced courtesy of Dr. Juan-Carlos, Zúñiga-Pflücker, Department of Immunology, Academy of Toronto and Sunnybrook & Women's Enquiry Found, Toronto.

Table 7-iii. Examples of Dyes Used in Immunofluorescence Analyses

Proper noun of Dye Fluorescing Color Molecules Labeled/Detected
Alexa Fluor 430 Yellowish-green Dna, proteins
Alexa Fluor 405 Blue DNA, proteins
Alexa Fluor 488 Greenish DNA, proteins
Alexa Fluor 546 Orange Deoxyribonucleic acid, proteins
Alexa Fluor 594 Red Deoxyribonucleic acid, proteins
Fluoroscein Light-green Deoxyribonucleic acid, proteins
Rhodamine Carmine DNA, proteins
Phycoerythrin Red-orange DNA, proteins
Texas Ruby Crimson DNA, proteins
Hoechst Blueish Nucleic acids
Acridine Orange Red-orange Nucleic acids
DAPI Blue Nucleic acids
Fura-2 Cerise Calcium
Fluo-four AM Green Calcium
Indo-1 Blueish Calcium
Sodium Green Green Sodium
Corona Red Blood-red-orange Sodium
PBFI Blue Potassium
MQAE Bluish Chloride

As well as identifying and counting item cell populations, a flow cytometer tin can also sort these cells for subsequent manipulation. In fact, a keen advantage of menses cytometric assay is that fluorochrome-labeled cells can pass through the musical instrument without compromising either sterility or cell viability. The fluorescence detectors of the flow cytometer can be connected to computer-operated electromagnetic deflector plates that are programmed to straight a cell with a fluorescent indicate of a given colour and intensity to a detail collection tube, a procedure known as "jail cell-sorting." One might hear a group of immunologists talking about the results of a "FACS" analysis: FACSTM is one biotechnology company's abridgement for "fluorescence-activated cell sorter."

Allow'southward return to our instance, in which cells bearing fluorescein-tagged anti-CD8 antibody and cells bearing phycoerythrin-tagged anti-CD4 antibody were identified in a thymus prison cell population. By specifying the collection parameters of the flow cytometer, subpopulations of thymus cells begetting only CD4 or CD8, or both or neither, can be physically separated from each other. In addition, cells that stain very intensely can exist isolated from those that stain moderately, or those that stain very weakly. Each of these populations can then be recovered from its collection tube, cultured separately, and manipulated farther, providing us with a very fine resolution with which to purify and report developing T cells in the thymus that are very closely related.

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Functional genomics using high-throughput RNA interference

Dominique Vanhecke , Michal Janitz , in Drug Discovery Today, 2005

In dissimilarity to the long dsRNAs used in non-mammalian cells, siRNA molecules take to exist optimised to efficiently knockdown a particular mRNA, as the effectiveness of gene silencing depends upon the localisation of the targeted mRNA region. Despite well-adult sequence design algorithms, each target mRNA must be verified for an efficient siRNA, every bit the silencing potential of siRNAs to unlike gene regions tin can differ dramatically. Consequently, genome-broad approaches based on chemically synthesized siRNAs are very expensive. A cost-effective alternative has recently been developed whereby siRNA molecules are generated from long dsRNA by enzymatic processing using purified RNase III from Escherichia coli [41,42]. Fractional digestion of dsRNA by RNase 3 results in production of endoribonuclease-prepared siRNA (esiRNA) of twenty–25 bp in length, which efficiently mediates RNAi in cultured mammalian cells. The esiRNA prepared from a unmarried dsRNA can target multiple sites within mRNA, thus increasing effectiveness of gene silencing. Interestingly, shorter siRNAs (12–15 bp) arising as a result of more than all-encompassing RNase digestion are non able to induce an RNAi response in mammalian cells [41].

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This issue includes the special event section: Omics: Tools for Understanding and Studying Systems Toxicology

L. Verschaeve , ... Z. Xu , in Mutation Research/Reviews in Mutation Enquiry, 2010

ii.1.4 Investigations on not-mammalian prison cell systems

A limited number of studies have too been devoted to not-mammalian prison cell systems (prokaryotes and plants). In an experiment on Escherichia coli bacteria carrying the plasmid puc9, Dasdag et al. [54] found that the number of plasmid copies per cell was not inverse past exposure to 9450 and 2450   MHz microwaves for up to one hour and up to thermal levels. Belloni et al. [55] utilized a transmission line chamber to investigate the effects of 900   MHz RFR on DNA mutability and repair in E. coli. They did not discover induced Deoxyribonucleic acid damage following RFR-exposure (upwards to 66   V   one thousand−ane and 260   nT) but, on the contrary, they observed a protective result that they ascribed to an improved efficiency of the mismatch repair system. Chang et al. [56] ended from their experiments on Salmonella typhimurium and E. coli leaner that 835   MHz CDMA RFR at a SAR of 4   W   kg−1 and exposure duration of 48   h neither afflicted reverse mutation frequencies nor accelerated DNA degradation in vitro. Nevertheless, Belyaev et al. [57] did find effects of millimetre waves (51.64–51.84   GHz at a power range from 10−19 to three   ×   10−3  W   cm−two) on the chromatin conformation in E. coli cells using the method of anomalous viscosity time dependencies (AVTD).

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3D gastruloids: a novel frontier in stalk cell-based in vitro modeling of mammalian gastrulation

Susanne C. van den Brink , Alexander van Oudenaarden , in Trends in Cell Biological science, 2021

Extraembryonic tissues: essential role in development?

Comparisons between gastruloid models generated from mouse, homo, and other (non-mammalian) cells recently provided unexpected new insights into the role of the extraembryonic surroundings in development: while the morphology of gastrula-stage embryos differs vastly across species, removing embryonic cells from their extraembryonic environment and culturing them in a gastruloid protocol induces them to prefer strikingly like morphologies across species [52]. These observations indicate that differences between species might largely be attributed to differences in their extraembryonic environment and suggest that the embryonic environment plays a pivotal role in speciation events during evolution.

Taken together, the findings discussed here demonstrate that gastruloid models can provide primal new insights into the processes that directly embryogenesis. The tractable nature of the system, in combination with the accuracy with which the model captures germ layer differentiation and body axis specification, makes the system particularly well suited to study the role of the extraembryonic environment in body centrality formation and morphogenesis.

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Papillomaviruses: prophylactic vaccine prospects

Douglas R Lowy , John T Schiller , in Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1999

The antigenic characteristics of VLPs and the ability to produce preparative amounts of them in non-mammalian cells has led to several vaccine trials of VLPs in animals. Since papillomaviruses are species-specific, it has been necessary to utilise animal papillomavirus models for these vaccine studies. The models include the Shope cottontail rabbit papillomavirus (CRPV), bovine papillomavirus type iv (BPV4), and canine oral papillomavirus (COPV). BPV4 and COPV induce oral mucosal papillomas, while CRPV induces cutaneous papillomas, some of which can progress to squamous cell carcinomas. Encouraging results from systemic VLP vaccination have been obtained with all three model systems [forty–43].

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Oxidative tissue: perilipin five links storage with the furnace

Hong Wang , Carole Sztalryd , in Trends in Endocrinology & Metabolism, 2011

LD storage and the release of lipid intermediates – prison cell-specific energy homeostasis

The LD compartment is recognized as a storage organelle, merely its role in maintaining lipid homeostasis and metabolism is now also being realized. LD biogenesis is a fundamental cellular function; most if non all mammalian and non-mammalian cells convert excess FA into triglyceride for storage within LDs [xvi]. LD aggregating in 'normal' cells maintains low but sufficient intracellular FA levels for essential activities, for example β-oxidation, membrane phospholipid synthesis and steroid production, depending on cell type and developmental state. More chiefly, increasing evidence indicates that LDs play an important role in releasing endogenous ligands necessary to maintain activities of transcription factors, such as PPARs, involved in regulating lipid metabolism [17–xx].

Cell-specific FA needs crave a highly dynamic LD compartment to store and release FA tailored to accommodate cell requirements and function, ranging from highly efficient FA storage/release from or into blood (white adipocytes [21]), mitochondrial FA oxidation for thermal regulation (brown adipocytes [22]), FA oxidation for long-term mobility demands (skeletal slow-twitched fiber muscles [23]), lipidation of very low density lipoprotein product and mitochondrial β-oxidation (liver [24]), milk production (mammary epithelial cells [25]) and surfactant production (specialized lung cells [26]).

Physiological testify supports an essential role of LD in cellular lipid homeostasis. During fasting, adipose-tissue lipolysis provides a convenient source of FA fuel for energy demands in non-adipose tissues. Intriguingly, concurrent with increasing fatty acid uptake and use, oxidative tissues such as liver and eye also increase their LD content [27,28]. Progress in imaging has confirmed in humans observations made in rodent models: an inherent dynamic part of myocardial LDs, increasing threefold following a 48   h fast in healthy individuals [29].

A growing torso of evidence reveals the important role of LDs in storing excess FA in the course of TAG, in fact interim as a temporary local warehouse for subsequent cellular demands. Prove also indicates that LDs protect cells against lipotoxicity by efficiently storing bioactive lipids and toxic lipid species when FA supply exceeds need [xxx]. However, the mechanisms underlying LD biogenesis remain to be understood fully. Nonetheless, progress in understanding LD TAG hydrolysis regulation has been boosted considerably past the discovery of a family of LD surface-associated proteins, the perilipins, which provide convenient tools to explore the role of LD in cellular lipid homeostasis.

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Autophagy: shaping the tumor microenvironment and therapeutic response

Hannelore Maes , ... Patrizia Agostinis , in Trends in Molecular Medicine, 2013

Microphagy involves the direct engulfment of cytosolic components past the lysosomal membrane. During this process, the lysosomal membrane invaginates to course a specialized 'autophagic tube', which encloses portions of the cytosol including multivesicular bodies through an ATP-dependent process that is accompanied by drastic changes in the distribution of lipids and proteins within the lysosomal membrane. In mammalian cells, non-selective microphagy degrades soluble intracellular substrates, whereas selective microautophagy mechanisms have been delineated mainly in yeasts. Although these autophagy pathways are constitutively active, macroautophagy and CMA are stimulated in response to a variety of mutual metabolic and oxidative stressors and mutual compensatory mechanisms exist between these degradation pathways [118]. Microautophagy, the less characterized form of autophagy, is often stimulated in parallel with macroautophagy, especially in response to starvation or mTOR inhibition, and is idea to be a mechanism important to re-institute lysosomal membrane homeostasis and regulate lipid metabolism and endocytosis [121].

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The Golgi Complex

Brad J. Marsh , in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2005

Separate from, yet complimentary to, the concept of reconstructing a whole jail cell in toto at ≤v nm resolution is a desperate need to paradigm and analyze large cell volumes with dramatically increased frequency. 18-carat high-throughput (∼40 tomograms) studies have already been carried out for reconstructions of smaller cellular volumes in non-mammalian cells, in which the construction of involvement is mostly contained inside that book [86,87]. At present, efforts are underway to deport out similar studies for reconstructions of big jail cell volumes under a multifariousness of given physiological states or experimental conditions. Large numbers of tomograms are required (at least 30–50 different cells per condition) to more reliably narrate and quantify relatively rare or highly dynamic events, and in order for such analyses to yield statistically pregnant insights [32,34]. Still, every bit discussed above, the major impediment to these kinds of detailed comparative analyses is the capacity for expeditious data assay.

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Immunomodulatory effects and potential clinical applications of dimethyl sulfoxide

Shing-Hwa Huang , ... Gu-Jiun Lin , in Immunobiology, 2020

Abstract

Dimethyl sulfoxide (DMSO) was discovered during the 19th century past the German language chemical industry. DMSO comprises a highly polar group and two non-polar domains, which render it soluble in both aqueous solutions and organic solutions. Furthermore, DMSO tin penetrate the cell membrane of both the mammalian cells and the not-mammalian cells and prevent freeze-thaw injuries to the cells. Thus, it is often used for the cryopreservation of cells and tissues for laboratory and clinical applications. In contrast to this traditional application, DMSO has recently been shown to possess immunomodulatory furnishings, such as immune enhancement, and anti-inflammatory effects in the innate immunity. In improver, DMSO also affects the adaptive amnesty by regulating the expression of transcription factors in immune cells. This review briefly summarizes and highlights the roles and immunomodulatory effects of DMSO on the allowed system and reveals the time to come clinical therapeutic potential of DMSO treatment in cancer, in autoimmune diseases and in chronic inflammatory diseases.

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