Search

Renal failure because of chronic kidney disease (CKD) leads to increased accumulation of uremic toxins due to decreased glomerular filtration rates. This in turn has a bearing on other metabolic functions in the body. Hemodialysis in end stage renal disease helps in elimination of toxic metabolites but long-term dialysis and the underlying chronic inflammation could affect immune cell activity, one of the main factors contributing to CKD associated complications and mortality. In this review, we tried to compile the important immune cell types and their different parameters that are altered in hemodialysis patients and the downstream impact of such alterations. Keywords: Uremia, CKD, hemodialysis, immune cells, cytokines

Diabetic foot ulcers are challenging to treat. Current strategies to treat these wounds focus on preventing infection and promoting tissue regrowth but are ineffective in many individuals. Low-grade chronic inflammation is present in individuals with diabetes, and altering the inflammatory responses at the wound site could be an alternate approach to promote healing. We hypothesized that immunomodulation of the wound microenvironment would result in accelerated healing. To test this hypothesis, we began by characterizing the changes in the myeloid cell phenotype in a mouse model [leptin receptor knockout (KO) mouse] that closely mimics the type 2 diabetes condition observed in humans. We observed increased numbers of monocytes and neutrophils in the circulation of the KO mice compared to that in wild-type control mice. We also observed several phenotypic changes in neutrophils from the KO diabetic mice, suggesting low-grade systemic inflammation. Hence, we developed a rapamycin-loaded chitosan scaffold that may be used to modulate immune responses. The use of these immunomodulatory scaffolds at a wound site resulted in accelerated healing compared to the healing using blank scaffolds. In summary, our data suggest that immunomodulation may be a viable strategy to promote the healing of wounds in individuals with diabetes.

Particle-based systems are becoming ubiquitous in the clinic. When administered in the body, phagocytic immune cells recognize these particles as foreign substances, resulting in their cellular engulfment for degradation and elimination. However, the internalization of particles may induce unintended effects on the functions of these immune cells. Understanding the consequences of particle uptake on immune cells is imperative to design biocompatible and efficient particulate formulations for diagnostic and therapeutic use. Here, we review the recent literature that describes the changes induced in immune cells following internalization of cargo-free particles. We also discuss how the inherent immunomodulatory properties of particles may be leveraged for therapeutic applications. We conclude with suggestions on newer methods to evaluate the effects of particles on immune cells.

The disease caused by SARS-CoV-2—CoVID-19—is a global pandemic that has brought severe changes worldwide. Approximately 80% of the infected patients are largely asymptomatic or have mild symptoms such as fever or cough, while rest of the patients display varying degrees of severity of symptoms, with an average mortality rate of 3–4%. Severe symptoms such as pneumonia and acute respiratory distress syndrome may be caused by tissue damage, which is mostly due to aggravated and unresolved innate and adaptive immune response, often resulting from a cytokine storm.Cytokine storm: A sudden acute increase in circulating levels of different inflammation causing cytokines including IL-6, IL-1, etc. Here, we discuss how an intricate interplay among infected cells and cells of innate and adaptive immune system can lead to such diverse clinicopathological outcomes. Particularly, we discuss how the emergent nonlinear dynamics of interaction among the components of adaptive and immune system components and virally infected cells can drive different disease severity. Such minimalistic yet rigorous mathematical modeling approaches are helpful in explaining how various co-morbidity risk factors, such as age and obesity, can aggravate the severity of CoVID-19 in patients. Furthermore, such approaches can elucidate how a fine-tuned balance of infected cell killing and resolution of inflammation can lead to infection clearance, while disruptions can drive different severe phenotypes. These results can help further in a rational selection of drug combinations that can effectively balance viral clearance and minimize tissue damage.

The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes 130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression. A retrievable, porous and anti-fibrotic macrodevice for the encapsulation of cells provides long-term protection to human cells expressing therapeutic proteins after device implantation in the intraperitoneal space of immunocompetent mice.

The antioxidant property of cerium oxide nanoparticles has increased their demand as a nanocarrier to improve the delivery and therapeutic efficacy of anticancer drugs. Here, we report the synthesis of alginate-coated ceria nanoformulations (ceria NPs) and characterization using FTIR spectroscopy, Raman microscopy, and X-ray diffraction. The synthesized ceria NPs show negligible inherent

ObjectiveThe goal of this study was to identify biochemical and immunological parameters from the blood as predictors of non-healing in early-stage diabetic foot ulcers.Research Design and MethodsWe performed a cross-sectional prospective cohort study among individuals with early-stage foot ulcers visiting the Karnataka Institute of Endocrinology Research over a 2.5-year period. Histopathological, biochemical, and immunological data (a total of 31 parameters) from 52 individuals were collected and analyzed to determine if predictors of non-healing may be identified. Data analysis was performed using traditional univariate analyses as well as univariate and multivariable logistic regression.ResultsIndividual histopathological and biochemical parameters did not show any differences between healed and non-healed individuals. However, conventional univariate analysis and univariate logistic regression analysis showed that the expression of the cell-surface proteins CD63, HLA-DR and CD11b on monocytes (CD14+) was significantly lower in non-healed individuals, but with moderate discriminative ability as assessed by area under the curve (AUC) of Receiver Operating Characteristics (ROC) curve. In comparison, a multivariable logistic regression model identified four of the 31 parameters to be salient predictors and demonstrated high discrimination ability with an AUC of ROC value of 0.87. Among the four identified parameters, LDL cholesterol (OR 18.83, CI 18.83-342) and cell-surface expression of CD63 on monocytes (OR 0.12, CI 0.12-0.45) were significant.ConclusionThrough this study we conclude that LDL cholesterol and cell-surface expression of CD63 on monocytes are strong positive and negative predictors of non-healing, respectively, in individuals with early-stage DFU. Following validation in a larger cohort, these parameters may be used by the clinician for early identification of non-healers.

Naïve helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a ‘master regulator’ – T-bet (Th1), GATA3 (Th2) and RORγT (Th17) – that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotypes – Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17 and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remains unclear. Here, through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any ‘master regulator’ can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/RORγT.

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)-block-polylactide (PEG-b-PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG-b-PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.

Graphene oxide (GO) nanoparticles have been developed for a variety of biomedical applications as a number of different therapeutic modalities may be added onto them. Here, we report the development and testing of such a multifunctional GO nanoparticle platform that contains a grafted cell-targeting functionality, active pharmaceutical ingredients, and particulates that enable the use of magnetothermal therapy. Specifically, we demonstrate the ability to covalently attach hyaluronic acid (HA) onto GO, and the resultant nanoparticulates (GO-HA) exhibited low inherent toxicity toward two different breast cancer cell lines, BT-474 and MDA-MB-231. Doxorubicin (Dox) and paclitaxel (Ptx) were successfully loaded onto GO-HA with high and moderate efficiencies, respectively. A GO-HA-Dox/Ptx system was significantly better than the GO-Dox/Ptx system at specifically killing CD44-expressing MDA-MB-231 cells but not BT-474 cells that do not express CD44. Further, modified iron oxide nanoparticles were loaded onto the GO-HA-Dox system, enabling the use of magnetic hyperthermia. Hyperthermia in combination with Dox treatment through the GO-HA system showed significantly better performance in reducing viable tumor cell numbers when compared to the individual systems. In summary, we showcase a multifunctional GO nanoparticle system that demonstrates improved efficacy in killing tumor cells.

Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing, and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils and monocytes take up sub-micrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for design of particles to evade or target these cells.

Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed byin vivotracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow and half-life in the blood reduces by about 40%, compared to non-inflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by two-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

Gliomas are heavily infiltrated with immune cells of myeloid origin. Past studies have shown that high-grade gliomas have a higher proportion of alternatively activated and suppressive myeloid cells when compared to low-grade gliomas, which correlate with poor prognosis. However, the differences in immune cell phenotypes within high-grade gliomas (between grade 3 and grade 4 or GBM) are relatively less explored, and a correlation of phenotypic characteristics between immune cells in the blood and high-grade tumors has not been performed. Additionally, myeloid cells of granulocytic origin present in gliomas remain poorly characterized. Herein, we address these questions through phenotypic characterizations of monocytes and neutrophils present in blood and tumors of individuals with glioblastoma (GBM, IDH-wild type) or grade 3 IDH-mutant gliomas. We observe that neutrophils are highly heterogeneous among individuals with glioma, and are different from healthy controls. We also show that CD163 expressing M2 monocytes are present in greater proportions in GBM tissue when compared to grade 3 IDH-mutant glioma tissue, and a larger proportion of granulocytic myeloid-derived suppressor cells are present in grade 3 IDH-mutant gliomas when compared to GBM. Finally, we demonstrate that the expression levels of CD86 and CD63 showed a high correlation between blood and tumor and suggest that these may be used as possible markers for prognosis.

Gliomas are heavily infiltrated with immune cells of myeloid origin. Past studies have shown that high-grade gliomas have a higher proportion of alternatively activated and suppressive myeloid cells when compared to low-grade gliomas, which correlate with poor prognosis. However, the differences in immune cell phenotypes within high-grade gliomas (between grade III and IV) are relatively less explored, and a correlation of phenotypic characteristics between immune cells in the blood and high grade tumors has not been performed. Additionally, myeloid cells of granulocytic origin present in gliomas remain poorly characterized. Herein, we address these questions through phenotypic characterizations of monocytes and neutrophils present in blood and tumors of individuals with glioblastoma (GBM, grade IV) or grade III gliomas. Our data show that CD163 expressing M2 monocytes are present in greater proportions in GBM tissue when compared to grade III glioma tissue. In addition, we observe that neutrophils are highly heterogeneous among individuals with glioma, and a greater proportion of granulocytic myeloid-derived suppressor cells are present in grade III gliomas when compared to GBM. Finally, we show that the expression levels of CD86 and CD63 showed a high correlation between blood and tumor, and suggest that these may be used as possible markers for prognosis.

Coumarins have been shown to possess antimicrobial, anti-quorum sensing and anti-biofilm properties against a wide range of pathogenic bacteria. This study aimed to shed light on the effects of non-substituted coumarin on biofilm formation by the foodborne pathogen

Identifying the design principles of complex regulatory networks driving cellular decision-making remains essential to decode embryonic development as well as enhance cellular reprogramming. A well-studied network motif involved in cellular decision-making is a toggle switch—a set of two opposing transcription factors A and B, each of which is a master regulator of a specific cell fate and can inhibit the activity of the other. A toggle switch can lead to two possible states—(high A, low B) and (low A, high B)—and drives the ‘either-or' choice between these two cell fates for a common progenitor cell. However, the principles of coupled toggle switches remain unclear. Here, we investigate the dynamics of three master regulators A, B and C inhibiting each other, thus forming three-coupled toggle switches to form a toggle triad. Our simulations show that this toggle triad can lead to co-existence of cells into three differentiated ‘single positive' phenotypes—(high A, low B, low C), (low A, high B, low C) and (low A, low B, high C). Moreover, the hybrid or ‘double positive' phenotypes—(high A, high B, low C), (low A, high B, high C) and (high A, low B, high C)—can coexist together with ‘single positive' phenotypes. Including self-activation loops on A, B and C can increase the frequency of ‘double positive' states. Finally, we apply our results to understand cellular decision-making in terms of differentiation of naive CD4 + T cells into Th1, Th2 and Th17 states, where hybrid Th1/Th2 and hybrid Th1/Th17 cells have been reported in addition to the Th1, Th2 and Th17 ones. Our results offer novel insights into the design principles of a multi-stable network topology and provide a framework for synthetic biology to design tristable systems.

The disease caused by SARS-CoV-2 is a global pandemic that threatens to bring long-term changes worldwide. Approximately 80% of infected patients are asymptomatic or have mild symptoms such as fever or cough, while rest of the patients have varying degrees of severity of symptoms, with 3-4% mortality rate. Severe symptoms such as pneumonia and Acute Respiratory Distress Syndrome can be caused by tissue damage mostly due to aggravated and unresolved innate and adaptive immune response, often resulting from a cytokine storm. However, the mechanistic underpinnings of such responses remain elusive, with an incomplete understanding of how an intricate interplay among infected cells and cells of innate and adaptive immune system can lead to such diverse clinicopathological outcomes. Here, we use a dynamical systems approach to dissect the emergent nonlinear intra-host dynamics among virally infected cells, the immune response to it and the consequent immunopathology. By mechanistic analysis of cell-cell interactions, we have identified key parameters affecting the diverse clinical phenotypes associated with COVID-19. This minimalistic yet rigorous model can explain the various phenotypes observed across the clinical spectrum of COVID-19, various co-morbidity risk factors such as age and obesity, and the effect of antiviral drugs on different phenotypes. It also reveals how a fine-tuned balance of infected cell killing and resolution of inflammation can lead to infection clearance, while disruptions can drive different severe phenotypes. These results will help further the case of rational selection of drug combinations that can effectively balance viral clearance and minimize tissue damage simultaneously.Significance StatementThe SARS-CoV-2 pandemic has already infected millions of people, and thousands of lives have been lost to it. The pandemic has already tested the limits of our public healthcare systems with a wide spectrum of clinicopathological symptoms and outcomes. The mechanistic underpinnings of the resultant immunopathology caused by the viral infection still remains to be elucidated. Here we propose a minimalistic but rigorous description of the interactions of the virus infected cells and the core components of the immune system that can potentially explain such diversity in the observed clinical outcomes. Our proposed framework could enable a platform to determine the efficacy of various treatment combinations and can contributes a conceptual understanding of dynamics of disease pathogenesis in SARS-CoV-2 infections.

The aim of this study was to determine the antibacterial property of extract derived from a part of the Jackfruit called ‘rag’, that is generally considered as fruit waste. Morpho-physical characterization of the Jackfruit rag extract (JFRE) was performed using gas-chromatography, where peaks indicative of furfural; pentanoic acid; and hexadecanoic acid were observed. In vitro biocompatibility of JFRE was performed using the MTT assay, which showed comparable cellular viability between extract-treated and untreated mouse fibroblast cells. Agar well disc diffusion assay exhibited JFRE induced zones of inhibition for a wide variety of laboratory and clinical strains of gram-positive and gram-negative bacteria. Analysis of electron microscope images of bacterial cells suggests that JFRE induces cell death by disintegration of the bacterial cell wall and precipitating intracytoplasmic clumping. The antibacterial activity of the JFREs was further validated in vivo using Shigella dysenteriae infected fly model, where JFRE pre-fed flies infected with S. dysenteriae had significantly reduced mortality compared to controls. JFRE demonstrates broad antibacterial property, both in vitro and in vivo, possibly by its activity on bacterial cell wall. This study highlights the importance of exploring alternative sources of antibacterial compounds, especially from plant-derived waste, that could provide economical and effective solutions to current challenges in antimicrobial therapy.

Supplementary Tables 1-15

Coumarins have been shown to possess antimicrobial, anti-quorum sensing and anti-biofilm properties against a wide range of pathogenic bacteria. This study aimed to shed light on the effects of non-substituted coumarin on biofilm formation by the foodborne pathogen Salmonella Typhimurium. Additionally, its efficacy was tested in combination with another potent anti-biofilm agent, resveratrol. Coumarin inhibited biofilm formation for prolonged periods in millimolar concentrations with marginal effects on planktonic growth. It attenuated curli and cellulose production, likely by downregulating the transcript levels of major biofilm formation genes csgD, csgA and adrA. Coumarin further restricted motility in a dose-dependent manner. In addition, coumarin with resveratrol exhibited improved anti-biofilm properties compared with the individual compounds alone. Thus, coumarin alone or with resveratrol can be employed for inhibiting biofilms in food storage and processing units.

The last few decades have seen rapid progress in the fields of drug delivery systems, implantable scaffolds, and tissue engineering. Broadly referred to as biomaterials, these inter-related areas of research have many diverse applications. One such application is in the area of immunology, where biomaterials may be used as tools to modify specific immune responses. Here, individual components of the immune system are described, followed by a discussion of the recent advances in biomaterial-based strategies for the modulation of immune responses.

The effects of senescence and aging on geriatric diseases has been well explored but how these influence infections in the elderly have been scarcely addressed. Here, we show that several innate immune responses are elevated in senescent cells and old mice, allowing them to promptly respond to bacterial infections. We have identified higher levels of iNOS as a crucial host response and show that p38 MAPK in senescent cells acts as a negative regulator of iNOS transcription. In old mice, however the ability to impede bacterial proliferation does not correlate with increased survival as elevated immune responses persist unabated eventually affecting the host. The use of anti-inflammatory drugs that could consequently be recommended also decreases iNOS disarming the host of a critical innate immune response. Overall, our study highlights that infection associated mortality in the elderly is not merely an outcome of pathogen load but is also influenced by the host’s ability to resolve inflammation induced damage. Summary statement Using cellular models and old mice we demonstrate the effect of aging on host response to bacterial infections. Aged systems mount a more effective anti-bacterial innate immune response but its persistence results in mortality of the host.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression. By studying the immune responses of animals to different types of biomaterial implants, colony stimulating factor-1 receptor is revealed as an important mediator of the foreign body reaction and a possible target for fibrosis inhibition.

Integral to the development of new biomaterials is the characterization of immune responses to biomaterial implants, and formulating methods to overcome or utilize these actions for therapeutic benefit. Neutrophils are an essential component of the immune response against biomaterials, but studies on the neutrophil-biomaterial interaction have been largely limited to characterizing their role in establishing an inflammatory microenvironment and antimicrobial activity at implant surfaces. Recent advances in neutrophil biology, especially recognition of their cellular heterogeneity, ability to suppress immune responses, the identification of a new process of cell death, and crosstalk with other immune cell types, have brought about a fundamental change in our perception regarding the activities of neutrophils. Herein, in the context of the progress in our comprehension of neutrophil function, potential avenues for effectively employing neutrophil activity to develop the next generation of regenerative biomaterials are discussed.

Surface modification of particulate systems is a commonly employed strategy to alter their interaction with proteins and cells. Past studies on nano-particles have shown that surface functionalization with polyethylene glycol (PEG) or proteins such as albumin increases circulation times by reducing their phagocytic uptake. However, studies on surface functionalized micro-particles have reported contradictory results. Here, we investigate the effects of surface functionalization using polystyrene particles with 4 different diameters ranging from 30 nm to 2.6 μm and coating them with either albumin or PEG. Our results show that with increasing particle size, surface functionalization has less to no effect on altering phagocytic uptake. The data also suggest that these differences are observed with a dense arrangement of molecules on the surface (dense brush conformation for PEG conjugation), appear to be independent of the serum proteins adsorbing on particle surfaces, and are independent of the endocytic uptake pathway. These results provide insight into the differences in the ability of surface modified nano- and micro-particles to avoid phagocytic uptake.

The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host-guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit[7]uril (CB[7])-PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB[7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy.

Neutrophils are constantly generated from hematopoietic stem and progenitor cells in the bone marrow to maintain high numbers in circulation. A considerable number of neutrophils and their progenitors have been shown to be present in the spleen too; however, their exact role in this organ remains unclear. Herein, we sought to study the function of splenic neutrophils and their progenitors using a mouse model for sterile, peritoneal inflammation. In this microcapsule device implantation model, we show chronic neutrophil presence at implant sites, with recruitment from circulation as the primary mechanism for their prevalence in the peritoneal exudate. Furthermore, we demonstrate that progenitor populations in the spleen play a key role in maintaining elevated neutrophil numbers. Our results provide new insight into the role for splenic neutrophils and their progenitors and establish a model to study neutrophil function during sterile inflammation.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton. © 2019 Mehta et al.

We report the first peptide based hDHFR inhibitors designed on the basis of structural analysis of dihydrofolate reductase (DHFR). A set of peptides were rationally designed and synthesized using solid phase peptide synthesis and characterized using nuclear magnetic resonance and enzyme immunoassays. The best candidate among them, a tetrapeptide, was chosen based on molecular mechanics calculations and evaluated in human lung adenocarcinoma cell line A549. It showed a significant reduction of cell proliferation and an IC50 of 82 µM was obtained. The interaction of the peptide with DHFR was supported by isothermal calorimetric experiments revealing a dissociation constant Kd of 0.7 µM and ΔG of −34 ± 1 kJ mol−1. Conjugation with carboxylated polystyrene nanoparticles improved further its growth inhibitory effects. Taken together, this opens up new avenues to design, develop and deliver biocompatible peptide based anti-cancer agents.

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from in vivo screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer–lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. While several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate fission or fusion remain largely unknown. In fission yeast, mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics, and determined that cells with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission. We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells. We discovered that the association of mitochondria with microtubules physically impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Tregs play important roles in maintaining immune homeostasis, and thus, therapies based on Treg are promising candidates for the treatment for a variety of immune-mediated disorders. These therapies, however, face the significant challenge of obtaining adequate numbers of Tregs from peripheral blood that maintains suppressive function following extensive expansion. Inducing Tregs from non-Tregs offers a viable alternative. Different methods to induce Tregs have been proposed and involve mainly treating cells with TGF-β-iTreg. However, use of TGF-β alone is not sufficient to induce stable Tregs. ATRA or rapa has been shown to synergize with TGF-β to induce stable Tregs. Whereas TGF-β plus RA-iTregs have been well-described in the literature, the phenotype, function, and migratory characteristics of TGF-β plus rapa-iTreg have yet to be elucidated. Herein, we describe the phenotype and function of mouse rapa-iTreg and reveal that these cells differ in their in vivo homing capacity when compared with mouse RA-iTreg and mouse TGF-β-iTreg. This difference in migratory activity significantly affects the therapeutic capacity of each subset in a mouse model of colitis. We also describe the characteristics of iTreg generated in the presence of TGF-β, RA, and rapa.

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both in vitro and in vivo and correlated with the peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. In vivo, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver. Keywords: mRNA, Base modification, Pseudouridine, Lipid nanoparticle, In vivo, Immmune response, Shire Pharmaceuticals

Deficiency of regulatory T cells (Treg), a type of lymphocyte that promotes immunological homeostasis in the healthy steady state, [1–3] is a causative factor for destructive inflammation and autoimmune disease. [2,4] Accordingly, increasing the presence of Treg at the site of autoimmunity has been suggested as a potential method to treat these types of disorders. [3,4] However, to our knowledge, techniques to increase the local presence of specific immune cell types in vivo do not yet exist. Herein, we develop and test a microscale controlled-release system for the recruitment of Treg that draws inspiration from a specific biological system: tumors. Specifically, a wide variety of tumors release the chemokine CCL22, [5,6] which is responsible for tumor-directed migration of Treg and corresponding tumor-specific immune evasion. Our hypothesis was that if a steady CCL22 release could be maintained, by tuning the material properties of a controlled-release system, Treg may be preferentially recruited to a local site in vivo. The first step towards testing this hypothesis was to design a release vehicle suitable for the steady release of CCL22. Although polymeric-microparticle-based controlled-release systems for proteins (including chemokines) have previously been developed, [7,8] the release of hydrophilic proteins from these particles typically follows a triphasic release profile. [9,10] Our goal was to achieve release of CCL22 without any periods of lag in order to produce a corresponding steady gradient of chemokine originating from a point source. To this end, new mechanistic descriptions of how controlled release from degradable materials [9,11] suggest that one of the major factors affecting the rate of release is the erosion of a dense polymer matrix into a porous macrostructure that allows for protein egress. The timing of this process and, in turn, the period of lag is determined by the type of polymer, its molecular weight (inherent viscosity) and the degree of porosity in the matrix. Accordingly, we hypothesized that addition of contiguous pores to the microparticles during fabrication would pre-establish pathways for diffusive protein egress and should bypass the need for erosion as a requisite for release. Thus, we used the polymer poly(lactic-co-glycolic acid) (PLGA) of a specific inherent viscosity (0.16–0.24 dl/g in 0.1% chloroform; in order to achieve release within a 3–8 week period) and modified the surface porosity using an osmotic gradient between the inner aqueous emulsion and the outer bulk aqueous phase (Supporting Information, Figure S1). We observed that porous microparticles prepared using a specific osmotic gradient (corresponding to 15 × 10−3 m NaCl in inner aqueous emulsion), in contrast to unmodified degradable particles ( Figure 1a), did not display the standard lag phase that would be expected with release of proteins and released CCL22 continuously over a 4-week period (Figure 1b). As an additional design parameter, the particles were made to be large enough to avoid uptake by phagocytic cells and to prohibit their movement across vascular endothelium (Supporting Information, Figure S2). As a consequence, particles would remain immobilized at the site of injection. Figure 1 CCL22MP Characteristics. a) Scanning electron micrographs of nonporous (left; top and bottom) and porous (right; top and bottom) CCL22 microparticles (CCL22MP). The top images were taken at 500× magnification, while the bottom images were taken ... In order to test the ability of rationally designed CCL22MP to attract Treg, an in vivo adoptive transfer model coupled with non-invasive live-animal imaging was used. Specifically, fluorescently labeled CCL22MP were injected into the triceps surae of normal FVB mice followed by intravenous (i.v.) infusion of ex vivo-alloactivated Treg [12] (AATreg) that constitutively expresses the luciferase gene. The hind limb muscles were chosen as the site for MP injections as these distal sites are not expected to produce large quantities of CCL22. The migration pattern of these bioluminescent AATreg was studied following the injection of non-labeled mature allogeneic dendritic cells (DC), which provide an activation stimulus (Supporting Information, Figure S3). Soon after DC stimulation, a significantly greater number of AATreg was recruited to the site of CCL22MP injection compared to an internal control of microparticles lacking CCL22 ( Figure 2a and Supporting Information Figure S4). The adoptively transferred Treg are expected to translocate to sites in the body (e.g., lungs, gut etc.) that secrete CCL22, as observed in Figure S4, but not sites such as the hind limb (as confirmed by our preliminary experiments (data not shown)). However, a significant number of Treg do migrate towards the hind limb muscles upon injection of CCL22MP but not upon addition of blank particles ( p < 0.05), suggesting that the migration must be due to the release of CCL22 from these particles. Importantly, several reports suggest that small changes in Treg numbers at local sites (leading to a change in the ratio of Treg to effector T cells) is sufficient to dramatically alter local immune responses. [13] In line with these reports, and as a test for the ability of CCL22MP to replace the function of CCL22 secreting cells in vivo (e.g., site-specific recruitment of regulatory cells), we also demonstrate that CCL22MP are effectively able to delay the rejection of transplanted allogeneic cells. Specifically, allogeneic luciferase-expressing Lewis lung carcinoma cells (which do not endogenously produce CCL22) were implanted subcutaneously into mice at the site of CCL22MP injections (or for comparison BlankMP or Bolus CCL22 as controls), and the time to rejection was recorded using non-invasive live imaging (Figure S3). We observed that the rejection rates were significantly slower in the CCL22MP group when compared to both bolus CCL22 and BlankMP controls (Figure 2b and Figure S5), supporting the hypothesis that establishing a CCL22 gradient in vivo using synthetic systems can help modulate local immune respons1es. Figure 2 CCL22MP in vivo. a) Representative fluorescence (red-gold) and luminescence images (blue-yellow) showing localization of particles and Treg, respectively; fluorescence images were used to outline (red-line) areas of particle localization (Igor Pro Living ... Potential therapeutic implications of such a bioinspired degradable controlled-release formulation capable of recruiting Treg in vivo are manifold. One obvious application is the use of CCL22MP in combination with an infusion of Treg expanded ex vivo. Current pre-clinical data suggest that freshly-isolated or ex vivo-expanded Treg infusion can prevent organ transplant rejection or suppress autoimmune diseases. [2,14,15] However, challenges such as obtaining adequate numbers and highly-purified populations of Treg have hindered progress into clinical trials. [14,15] Using formulations that release CCL22, it may be possible to lower the numbers of injected Treg, or potentially use populations with lower purity. Another use of CCL22MP would be to attract endogenous Treg populations, wherein these formulations have the potential to function as “off-the-shelf” therapeutics for the treatment of a wide variety of disorders associated with unrestrained immune reactivity. A potential drawback of using a CCL22 sustained release vehicle is that the receptor for this chemokine (CCR4) is expressed on both activated Treg and activated effector T cells, [16] suggesting that both these cell populations would be attracted towards CCL22MP. Yet in vivo studies show that CCL22 production associated with tumors [5,17] or long-surviving allografts [18] result in preferential recruitment of Treg leading to local immunosuppression. One possible explanation for these results is that Treg could express significantly more CCR4 than effector T cells [17] (Supporting Information, Figure S6). Further, it has been suggested that an optimal ratio of Treg to effector T cells, and not a complete absence of effector T cells, is necessary for effective local suppression of immunity. [14,19] Regardless, if local effector T cells abrogate a suppressive environment, CCL22MP can easily be modified to simultaneously release immunosuppressive agents (such as rapamycin) [20] to inhibit these effector T cells in situ, thereby assisting Treg to control adverse immune responses. In conclusion, we demonstrate that site-specific attraction of Treg leading to local immunomodulation can be achieved in vivo using CCL22MP. These bioinspired controlled-release formulations are particularly attractive as modular platforms for therapeutic development, as well as tools to study Treg-dependent modulation of immune responses in situ.

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be passively targeted to phagocytic cells in vivo. Dendritic cells (DC) are a class of phagocytic cells known to play important roles in transplant rejection. Previously, we have demonstrated that DC treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. Herein, we describe a strategy to deliver an intracellular depot of rapamycin to DC. To achieve this, rapamycin was encapsulated into ∼3.4 μm sized poly(lactic-co-glycolic)acid (PLGA) microparticles (rapaMPs), and release behavior was examined under intra-phagosomal (pH = 5) and extracellular (pH = 7.4) conditions. It was observed that 4 days following phagocytosis of rapaMP, DC have significantly reduced ability to activate T cells, in comparison to DC treated with soluble rapamycin. Hence, we conclude that DC-specific intracellular delivery of rapamycin results in better efficacy of the drug, with respect to its ability to modulate DC function, when compared to treating DC with extracellular rapamycin.