PolySciTech mPEG-PLGA/PLGA-rhodamine used in the development of nanoparticle-based intracellular MRSA treatment

MRSA is a bacterial infection that is highly resistant to conventional antibiotic treatments or other therapies. It is still affected by vancomycin, but the bacterial spores have the capability to ‘hide’ inside of cells making it very difficult to treat. One means around this is to use nanoparticles for delivery of the antibiotic to the cells to ensure suitable vancomycin in a local concentration to kill off the bacteria. Recently, researchers at Purdue University used mPEG-PLGA (Polyvivo AK030) and rhodamine-B labelled PLGA (PolyVivo AV011) from PolySciTech (www.polyscitech.com) to create pH sensitive nanoparticles designed for intracellular delivery of vancomycin. This research holds promise to improve treatments of this deadly bacterial infection. Read more: Pei, Yihua, Mohamed F. Mohamed, Mohamed N. Seleem, and Yoon Yeo. “Particle engineering for intracellular delivery of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA)-infected macrophages.” Journal of Controlled Release (2017). http://www.sciencedirect.com/science/article/pii/S0168365917307745

“Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious threat to the public health. MRSA is particularly difficult to treat when it invades host cells and survive inside the cells. Although vancomycin is active against MRSA, it does not effectively kill intracellular MRSA due to the molecular size and polarity that limit its cellular uptake. To overcome poor intracellular delivery of vancomycin, we developed a particle formulation (PpZEV) based on a blend of polymers with distinct functions: (i) poly(lactic-co-glycolic acid) (PLGA, P) serving as the main delivery platform, (ii) polyethylene glycol-PLGA conjugate (PEG-PLGA, p) to help maintain an appropriate level of polarity for timely release of vancomycin, (iii) Eudragit E100 (a copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate, E) to enhance vancomycin encapsulation, and (iv) a chitosan derivative called ZWC (Z) to trigger pH-sensitive drug release. PpZEV NPs were preferentially taken up by the macrophages due to its size (500–1000 nm) and facilitated vancomycin delivery to the intracellular pathogens. Accordingly, PpZEV NPs showed better antimicrobial activity than free vancomycin against intracellular MRSA and other intracellular pathogens. When administered intravenously, PpZEV NPs rapidly accumulated in the liver and spleen, the target organs of intracellular infection. Therefore, PpZEV NPs is a promising carrier of vancomycin for the treatment of intracellular MRSA infection. Keywords: Nanoparticles, Intracellular drug delivery, pH-sensitive, Macrophages, Intracellular MRSA, Vancomycin”

Improved targeted-delivery system using oriented antibody fragments developed using PLGA-PEG-Azide from PolySciTech.

A powerful tool for medicinal delivery is the use of a nanoparticle with a surface covered in a specific antibody or targeting ligand. Because these antibodies and ligands bind specifically to certain protein factors these can be tailored to target to specific cells, notably cancer cells. Since antibody bonding is a stereochemical process, shape and orientation of the antibody matters in terms of its capability to bind. If the active site of the ligand is facing inwards, towards the nanoparticle, it may not work well at all. Recently, researchers working at Queen’s University Belfast, University College London, (UK) and Universidade de Lisboa (Portugal) used PLGA-PEG-Azide from PolySciTech (www.polyscitech.com, PolyVivo AI085) to generate nanoparticles which had very precisely controlled antibody orientation on their surface allowing for improved functionality and targeting. They tested this system for its ability to bind to HER2 (a factor that is overexpressed in cancer cells) and found it had substantially higher binding than a randomly oriented nanoparticle system. This research holds promise for developing a wide-array of targeted delivery systems for treating a variety of diseases, most notably cancer. Read more: M. Greene, D. A. Richards, J. Nogueira, K. Campbell, P. Smyth, M. Fernandez, C. J. Scott and V. Chudasama “Generating Next-Generation Antibody-Nanoparticle Conjugates through the Oriented Installation of Non-Engineered Antibody Fragments” Chem. Sci., 2017, DOI: 10.1039/C7SC02747H. (http://pubs.rsc.org/en/Content/ArticleLanding/2017/SC/C7SC02747H#!divAbstract)

“Abstract: The successful development of targeted nanotherapeutics is contingent upon the conjugation of therapeutic nanoparticles to target-specific ligands, with particular emphasis being placed on antibody-based ligands. Thus, new methods that enable the covalent and precise installation of targeting antibodies to nanoparticle surfaces are greatly desired, especially those which do not rely on costly and time-consuming antibody engineering techniques. Herein we present a novel method for the highly controlled and oriented covalent conjugation of non-engineered antibody F(ab) fragments to PLGA-PEG nanoparticles using disulfide-selective pyridazinedione linkers and strain-promoted alkyneazide click chemistry. Exemplification of this method with trastuzumab and cetuximab showed significant improvements in both conjugation efficiency and antigen binding capability, when compared to commonly employed strategies for antibody-nanoparticle construction. This new approach paves the way for the development of antibody-targeted nanomedicines with improved paratope availability, reproducibility and uniformity to enhance both biological activity and ease of manufacture.”

PolySciTech PLGA-NH2 used in research thesis on nanoparticle-surface interactions with living cells

Nanoparticles have been around for many years but we are still, as a species, just scratching at the surface of understanding them. Of course, the surface is the most important part of a nanoparticle since, due to their incredibly small size, they have an incredible surface area to volume ratio. For example, 1g of PLGA nanoparticles (100 nm) would have a surface area of 7.8 square meters (a little larger than a typical parking space for a car). For this reason, the surface and how it interacts with living organisms is the most important aspect of nanoparticle technology. Recently, Angie (Morris) Thorn at University of Iowa published a PhD thesis which details efforts to broaden our understanding of nanoparticle surface interactions with cells. This includes work with PLGA-NH2 (PolyVivo AI063) from PolySciTech (www.polyscitech.com) to generate nanoparticles covered with either chitosan (for mucoadhesion) or TPP (mitochondria-targeting) to create targeted nanoparticles as drug-delivery vectors. Read more: Thorn, Angie Sue Morris. “The impact of nanoparticle surface chemistry on biological systems.” (2017). http://ir.uiowa.edu/etd/5659/

“Abstract: The unique properties of nanomaterials, such as their small size and large surface area-to-volume ratios, have attracted tremendous interest in the scientific community over the last few decades. Thus, the synthesis and characterization of many different types of nanoparticles has been well defined and reported on in the literature. Current research efforts have redirected from the basic study of nanomaterial synthesis and their properties to more application-based studies where the development of functionally active materials is necessary. Today such nanoparticle-based systems exist for a range of biomedical applications including imaging, drug delivery and sensors. The inherent properties of the nanomaterial, although important, aren’t always ideal for specific applications. In order to optimize nanoparticles for biomedical applications it is often desirable to tune their surface properties. Researchers have shown that these surface properties (such as charge, hydrophobicity, or reactivity) play a direct role in the interactions between nanoparticles and biological systems can be altered by attaching molecules to the surface of nanoparticles. In this work, the effects of physicochemical properties of a wide variety of nanoparticles was investigated using in vitro and in vivo models. For example, copper oxide (CuO) nanoparticles were of interest due to their instability in biological media. These nanoparticles undergo dissolution when in an aqueous environment and tend to aggregate. Therefore, the cytotoxicity of two sizes of CuO NPs was evaluated in cultured cells to develop a better understanding of how these propertied effect toxicity outcomes in biological systems. From these studies, it was determined that CuO NPs are cytotoxic to lung cells in a size-dependent manner and that dissolved copper ions contribute to the cytotoxicity however it is not solely responsible for cell death. Moreover, silica nanoparticles are one of the most commonly used nanomaterials because they are easy to synthesize and their properties (such as size, porosity and surface chemistry) can be fine-tuned. Silica nanoparticles can be found in thousands of commercially available products such as toothpastes, cosmetics and detergents and are currently being developed for biomedical applications such as drug delivery and biomedical imaging. Our findings herein indicate that the surface chemistry of silica nanoparticles can have an effect on lung inflammation after exposure. Specifically, amine-modified silica NPs are considered to be less toxic compared to bare silica nanoparticles. Together, these studies provide insight into the role that material properties have on toxicity and allow for a better understanding of their impact on human and environmental health. The final aim of this thesis was to develop surface-modified nanoparticles for drug delivery applications. For this, biodegradable, polymeric NPs were used due to their inert nature and biocompatibility. Furthermore, polymeric NPs are excellent for loading drugs and using them as drug delivery vehicles. In this work, poly (lactic-co-glycolic acid) (PLGA) NPs were loaded with a therapeutic peptide. These NPs were then coated with chitosan (a mucoadhesive polymer) for the treatment of allergic asthma or coated with a small cationic mitochondrial targeting agent for the treatment of ischemia/reperfusion injury. Taken as a whole, this thesis sheds light on the impact of NPs on human health. First by providing useful toxological data for CuO and silica NPs as well as highlighting the potential of surface-modified polymeric NPs to be used in drug delivery-based applications. Keywords: Cell Culture, Nanoparticle, Toxicity”

PEG-PLGA/PLGA from PolySciTech used by Precision NanoSystems, Inc. as part of microfluidics NanoAssemblr™ method optimization and testing

Microfluidics references the use of systems which have extremely small fluid-channels on the order of magnitude of microns in scale. Precision NanoSystems, Inc. is a company which has developed an array of microfluidic instruments and machines designed for a variety of applications including the synthesis of micro and nanoparticles. For generating micro/nano-particles of polymers, the typical process is to dissolve the polymer in an organic solvent and then mix it with water so that the polymer (which is not water soluble) precipitates out into sub-micron sized particles. Unlike simple emulsion, where the solvent and water are randomly mixed together, the mixing of the organic phase with the water phase in a microfluidics system is highly controlled which allows for the generation of very precise micro/nanoparticles. Recently, Precision Nanosystems, Inc. used a series of mPEG-PLGA’s from PolySciTech (www.polyscitech.com, PolyVivo AK010, AK037, AK106) to make a series of test pegylated nanoparticles with highly controlled properties. They reported these results in a scientific poster presented at controlled release society (CRS) 2017 meeting. This research holds promise for a wide array of applications involving the use of nanoparticles/microparticles as drug delivery systems. Read more here: S.M. Garg, M. Parmar, A. Thomas, E. Ouellet, M. Deleonardis, P. Johnson, A. Armstead, S. Ip, T.J. Leaver, A.W. Wild, R.J. Taylor, E.C. Ramsay “Microfluidics-based Manufacture of PEG-b-PLGA Block Copolymer Nanoparticles for the Delivery of Small Molecule Therapeutics” Controlled Release Society 2017 Poster Session. (https://www.precisionnanosystems.com/resources/?_sf_s=PEG-b-PLGA&_sft_resource-type=poster)

“(Poster introduction): Purpose: In recent years, numerous methods have been developed for the production of block copolymer nanoparticles as drug delivery vehicles. However, these methods pose numerous challenges in maintaining consistent nanoparticle quality, tuning size depending on the application, optimization for scale-up, and reproducibility. The NanoAssemblr™ platform is an automated microfluidics-based system that eliminates user variability and is capable of reproducible, and scalable manufacture of nanoparticles. Here, we describe the use of microfluidic mixing to manufacture PEG-b-PLGA nanoparticles using the NanoAssemblr™ Benchtop instrument. We further describe optimization strategies and investigate the physical encapsulation of a hydrophobic model drug coumarin-6.Results: Microfluidic mixing enabled the rapid and consistent manufacturing of PEG-b-PLGA nanoparticles having diameters below 100 nm. Instrument parameters such as aqueous:organic Flow Rate Ratio and Total Flow Rate had a significant impact on the size of the resulting nanoparticles. Increasing the molecular weight of the PLGA block from 10000 – 95000 Da resulted in an increase in the size of the nanoparticles from 40 – 80 nm. However, changes in the total flow rate of the instrument enabled all the nanoparticles to be tuned to a similar size of 60 nm which is difficult to control using conventional techniques. Coumarin-6 was successfully loaded into PEG-b-PLGA nanoparticles with an encapsulation efficiency of 52% w/w which was significantly higher than that obtained by co-solvent evaporation technique (34% w/w). The size of the nanoparticles prepared using the NanoAssemblr platform were tunable over a broad range while co-solvent evaporation does not provide a reliable means to tune size.”

In addition to this poster, Precision Nanosystems, Inc. has utilized PLGA from PolySciTech (PolyVivo AP121) in generating a wide array of technical data relevant to their microfluidic system. You can see these technical whitepapers here (https://www.precisionnanosystems.com/resources/?_sf_s=plga&_sft_resource-type=tech-bulletins-and-white-papers)

PLA-amine from PolySciTech used in development of atherosclerosis-targeted nanoparticles for treatment of heart disease

Heart disease, typically due to atherosclerotic lesions, is one of the leading causes of death in USA. Most treatments for this disease focus on surgical interventions (e.g. stent placement), which is often utilized in acute situations, or on systemic medicines such as statins, which are typically applied as a preventative. There is a need for therapies to be applied in non-emergency situations but where atherosclerotic lesions are known to be present. Conventionally, nanoparticles have been applied for use against cancer, however they can be targeted to lesions by using appropriate targeting moieties. Recently, researchers working jointly at Harvard Medical School, New York University, Technical University of Denmark, Korea Institute of Ceramic Engineering and Technology, Korea Advanced Institute of Science and Technology, and King Abdulaziz University used PLA-NH2 (PolyVivo AI041, www.polyscitech.com) from PolySciTech as a reactive precursor for generating a fluorescently-conjugated tracer as part of a novel nanoparticle-based system for treatment of artherosclerosis. Read more: Yu, Mikyung, Jaume Amengual, Arjun Menon, Nazila Kamaly, Felix Zhou, Xiaoding Xu, Phei Er Saw et al. “Targeted Nanotherapeutics Encapsulating Liver X Receptor Agonist GW3965 Enhance Antiatherogenic Effects without Adverse Effects on Hepatic Lipid Metabolism in Ldlr−/− Mice.” Advanced Healthcare Materials (2017). http://onlinelibrary.wiley.com/doi/10.1002/adhm.201700313/full

“Abstract: The pharmacological manipulation of liver X receptors (LXRs) has been an attractive therapeutic strategy for atherosclerosis treatment as they control reverse cholesterol transport and inflammatory response. This study presents the development and efficacy of nanoparticles (NPs) incorporating the synthetic LXR agonist GW3965 (GW) in targeting atherosclerotic lesions. Collagen IV (Col IV) targeting ligands are employed to functionalize the NPs to improve targeting to the atherosclerotic plaque, and formulation parameters such as the length of the polyethylene glycol (PEG) coating molecules are systematically optimized. In vitro studies indicate that the GW-encapsulated NPs upregulate the LXR target genes and downregulate proinflammatory mediator in macrophages. The Col IV-targeted NPs encapsulating GW (Col IV–GW–NPs) successfully reaches atherosclerotic lesions when administered for 5 weeks to mice with preexisting lesions, substantially reducing macrophage content (≈30%) compared to the PBS group, which is with greater efficacy versus nontargeting NPs encapsulating GW (GW–NPs) (≈18%). In addition, mice administered the Col IV–GW–NPs do not demonstrate increased hepatic lipid biosynthesis or hyperlipidemia during the treatment period, unlike mice injected with the free GW. These findings suggest a new form of LXR-based therapeutics capable of enhanced delivery of the LXR agonist to atherosclerotic lesions without altering hepatic lipid metabolism.”

Protein phosphorylation assay kits by Tymora Analytical now available through PolySciTech

The selective phosphorylation of proteins is a key step in many pathways regulating their functions. Abnormal phosphorylation is involved in a wide variety of diseases including cancer. To perform antibody labeling, an effective antibody has to be made for each phosphorylated protein, which is an expensive and time-consuming process. Recently, Tymora Analytical has developed a titanium-based reagent assay kit to allow for detection of protein phosphorylation in a rapid, efficient, and sensitive assay. Due to a recent distribution agreement, these products are now available through PolySciTech division of Akina, Inc. (https://akinainc.com/polyscitech/products/tymora/). Learn more about this powerful assay method in a recent publication here: Iliuk, Anton, Li Li, Michael Melesse, Mark C. Hall, and W. Andy Tao. “Multiplexed Imaging of Protein Phosphorylation on Membranes Based on TiIV Functionalized Nanopolymers.” ChemBioChem 17, no. 10 (2016): 900-903. http://europepmc.org/articles/4870103

“Abstract: Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. Keywords: antibodies, dendrimers, membranes, multiplexed analysis, phosphoproteins”

PLGA from PolySciTech used in the development of nanoparticle-based obesity treatment

Obesity in humans is a contributing factor to many other health concerns, such as arthritis and cardiovascular problems. Recently, researchers at Purdue University utilized PLGA from PolySciTech (www.polyscitech.com) (PolyVivo AP101) to develop nanoparticles which deliver dibenzazepine to reduce the overgrowth of adipocytes (fat-cells). This research holds promise to provide for improved treatments of obesity. Read more: Jiang, Chunhui, Mario Alberto Cano-Vega, Feng Yue, Liangju Kuang, Naagarajan Narayanan, Gozde Uzunalli, Madeline P. Merkel, Shihuan Kuang, and Meng Deng. “Dibenzazepine-loaded nanoparticles induce local browning of white adipose tissue to counteract obesity.” Molecular Therapy (2017). http://www.cell.com/molecular-therapy-family/molecular-therapy/abstract/S1525-0016(17)30256-3

“Inhibition of Notch signaling via systemic drug administration triggers conversion of white adipocytes into beige adipocytes (browning) and reduces adiposity. However, translation of this discovery into clinical practice is challenged by potential off-target side effects and lack of control over the location and temporal extent of beige adipocyte biogenesis. Here, we demonstrate an alternative approach to stimulate browning using nanoparticles (NPs) composed of FDA-approved poly(lactide-co-glycolide) that enable sustained local release of a Notch inhibitor (dibenzazepine, DBZ). These DBZ-loaded NPs support rapid cellular internalization and inhibit Notch signaling in adipocytes. Importantly, focal injection of these NPs into the inguinal white adipose tissue depots of diet-induced obese mice results in localized NP retention and browning of adipocytes, consequently improving the glucose homeostasis and attenuating body-weight gain of the treated mice. These findings offer new avenues to develop a potential therapeutic strategy for clinical treatment of obesity and its associated metabolic syndrome. Keywords: drug delivery; nanoparticle; browning; adipocyte; Notch signaling; obesity; PLGA; dibenzazepine; adipose tissue; Notch inhibitor”

Tissue scaffolds with improved delivery of growth factor developed using PLGA-PEG-Mal from PolySciTech

A powerful tool in medicine would be the ability to produce a tissue-scaffold which allows for tissue which has been lost due to disease or trauma to be replaced with fresh stem-cells. There are many barriers to the developemtn of this tool one of which is ensuring that the stem-cells have the appropriate anchoring sites as well as the correct growth factors to ensure their appropriate growth and development. Recently, researchers working jointly at Fudan University (China), Tianjin Medical University (China), Ewha Women’s University (Korea), and University of Michigan utilized Maleimide-PEG-PLGA (PolyVivo AI136) and fluorescently conjugated PLGA-FPR648 (Polyvivo AV008) from PolySciTech (www.polyscitech.com) to generate a scaffold which allowed for controlled release of differentiation factors. They used the developed scaffold to repair ischemic tissue in a mouse model. This research holds promise to enable tissue repair and regeneration by successfully growing differentiated stem-cells into damaged areas. Read more: Li, Ruixiang, Zhiqing Pang, Huining He, Seungjin Lee, Jing Qin, Jian Wu, Liang Pang, Jianxin Wang, and Victor C. Yang. “Drug depot-anchoring hydrogel: A self-assembling scaffold for localized drug release and enhanced stem cell differentiation.” Journal of Controlled Release (2017). http://www.sciencedirect.com/science/article/pii/S016836591730706X

“Abstract: Localized and long-term delivery of growth factors has been a long-standing challenge for stem cell-based tissue engineering. In the current study, a polymeric drug depot-anchoring hydrogel scaffold was developed for the sustained release of macromolecules to enhance the differentiation of stem cells. Self-assembling peptide (RADA16)-modified drug depots (RDDs) were prepared and anchored to a RADA16 hydrogel. The anchoring effect of RADA16 modification on the RDDs was tested both in vitro and in vivo. It was shown that the in vitro leakage of RDDs from the RADA16 hydrogel was significantly less than that of the unmodified drug depots (DDs). In addition, the in vivo retention of injected hydrogel-incorporated RDDs was significantly longer than that of hydrogel-incorporated unmodified DDs. A model drug, vascular endothelial growth factor (VEGF), was encapsulated in RDDs (V-RDDs) as drug depot that was then anchored to the hydrogel. The release of VEGF could be sustained for 4 weeks. Endothelial progenitor cells (EPCs) were cultured on the V-RDDs-anchoring scaffold and enhanced cell proliferation and differentiation were observed, compared with a VEGF-loaded scaffold. Furthermore, this scaffold laden with EPCs promoted neovascularization in an animal model of hind limb ischemia. These results demonstrate that self-assembling hydrogel-anchored drug-loaded RDDs are promising for localized and sustained drug release, and can effectively enhance the proliferation and differentiation of resident stem cells, thus lead to successful tissue regeneration. Graphical abstract: Schematic illustration of a vascular endothelial growth factor (VEGF)-loaded RDDs-anchoring hydrogel. The RADA16 peptide is the basic self-assembling unit forming fiber and constructing hydrogel; poly (lactic-co-glycolic acid) (PLGA) based, VEGF-loaded drug depots (DDs) were modified using the RADA16 peptide (V-RDDs) to anchor them to the skeleton of the hydrogel; PEG was applied as a spacer to ensure the full stretch of the RADA16 peptide. VEGF demonstrated sustained release into the hydrogel to enhance the proliferation and differentiation of resident EPCs. Keywords: PLGA; RADA16 hydrogel; Sustained release; Endothelial progenitor cells; Vascular endothelial growth factor; Tissue regeneration”

mPEG-PLGA from PolySciTech used in development of immune-control treatment for allergic reactions

Allergic contact dermatitis is a common inflammatory skin condition caused by a pathological immune response to a given trigger such as poison ivy oils or nickel metal. This aggravating skin condition can be prevented and treated by reducing the local formation of allergen specific t-cells. Doing so, however, requires careful localized delivery of specific set of molecules including proteins and small-molecule signals to discourage an overly responsive immune attack. This same strategy has great application towards other uses such as autoimmune disease disorders and transplant rejection. Recently, Researchers at University of Pittsburgh used mPEG-PLGA from PolySciTech (www.polyscitech.com) (PolyVivo AK037) to generate microparticles which can locally deliver TGF-β1, Rapamycin, and IL-2 to the skin. They discovered these particles were successful in prevent or reversing allergic responses in sensitized mice. This research holds promise to treat a wide-array of immune-mediated disease state. Read more: Balmert, Stephen C., Cara Donahue, John R. Vu, Geza Erdos, Louis D. Falo, and Steven R. Little. “In vivo induction of regulatory T cells promotes allergen tolerance and suppresses allergic contact dermatitis.” Journal of Controlled Release (2017). http://www.sciencedirect.com/science/article/pii/S0168365917307046

“Abstract: Allergic contact dermatitis (ACD) is a common T-cell mediated inflammatory skin condition, characterized by an intensely pruritic rash at the site of contact with allergens like poison ivy or nickel. Current clinical treatments use topical corticosteroids, which broadly and transiently suppress inflammation and symptoms of ACD, but fail to address the underlying immune dysfunction. Here, we present an alternative therapeutic approach that teaches the immune system to tolerate contact allergens by expanding populations of naturally suppressive allergen-specific regulatory T cells (Tregs). Specifically, biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) microparticles were engineered to release TGF-β1, Rapamycin, and IL-2, to locally sustain a microenvironment that promotes Treg differentiation. By expanding allergen-specific Tregs and reducing pro-inflammatory effector T cells, these microparticles inhibited destructive hypersensitivity responses to subsequent allergen exposure in an allergen-specific manner, effectively preventing or reversing ACD in previously sensitized mice. Ultimately, this approach to in vivo Treg induction could also enable novel therapies for transplant rejection and autoimmune diseases.”