Metabolic and immunomodulatory control of type 1 diabetes via orally delivered bile-acid-polymer nanocarriers of insulin or rapamycin.

Oral formulations of insulin are typically designed to improve its intestinal absorption and increase its blood bioavailability. Here we show that polymerized ursodeoxycholic acid, selected from a panel of bile-acid polymers and formulated into nanoparticles for the oral delivery of insulin, restored blood-glucose levels in mice and pigs with established type 1 diabetes. The nanoparticles functioned as a protective insulin carrier and as a high-avidity bile-acid-receptor agonist, increased the intestinal absorption of insulin, polarized intestinal macrophages towards the M2 phenotype, and preferentially accumulated in the pancreas of the mice, binding to the islet-cell bile-acid membrane receptor TGR5 with high avidity and activating the secretion of glucagon-like peptide and of endogenous insulin. In the mice, the nanoparticles also reversed inflammation, restored metabolic functions and extended animal survival. When encapsulating rapamycin, they delayed the onset of diabetes in mice with chemically induced pancreatic inflammation. The metabolic and immunomodulatory functions of ingestible bile-acid-polymer nanocarriers may offer translational opportunities for the prevention and treatment of type 1 diabetes.

Successful Discontinuation of Chronic Polypsychotropic Regimen and Resolution of Withdrawal Syndrome Through Nutrition and Lifestyle Interventions: A Case Report.

BACKGROUND: A 38-year-old, female with a history of GAD, MDD, AN, and PTSD wanted to taper her multiple medications in preparation for pregnancy. Benzodiazepine medications, such as Klonopin and Restoril; antidepressants, such as Effexor; and anticonvulsant medications, such as Lamictal, can be habit-forming, and withdrawal symptoms can occur upon discontinuation of use. Polypharmacy can be implicated in poor clinical outcomes, and a strategic and supported medication taper may improve those outcomes. SUMMARY: After the primary MD unsuccessfully attempted to taper off the patient's psychotropic medications without lifestyle interventions, she was stabilized on a minimal regimen by an outside reproductive psychiatrist throughout her pregnancy. A second tapering was implemented by the primary MD after the patient had given birth and had established changes to her lifestyle. These lifestyle interventions included dietary changes, use of detoxification protocols, contemplative practices, and strategic supplement support in the setting of a powerful mindset shift. The patient experienced remarkable symptom remission after strategic discontinuation of medications through the addition of the lifestyle interventions. She also was able to heal the root-cause drivers of her psychiatric diagnoses. Currently she is symptom-free and medication-free after nearly 21 years. CONCLUSIONS: This case demonstrates the effectiveness of lifestyle interventions and psychospiritual support to enable dramatic clinical change without withdrawal syndrome after cessation of medication. More important, the initial failed tapering underpins the notion that a diligent meditation practice may be necessary to heal root-cause drivers of psychiatric symptoms and withdrawal syndrome. The results may serve to inform practitioners assisting patients who wish to discontinue benzodiazepine and other psychotropic medications or patients who would like to try a nonpharmaceutical approach as a first-line therapy.

Healing of Graves' Disease Thorough Lifestyle Changes: A Case Report.

BACKGROUND: Graves' disease is known as a chronic and incurable disease. The typical treatment is symptom-based and consists of medications, radioiodine, or surgery. These last two treatments are routinely offered to the 50% of patients that do not respond to drug therapy. Here we report the case of a patient who was able to normalize her thyroid hormones as well as her autoimmune markers in 6 months with the exclusive implementation of lifestyle interventions. SUMMARY: A 34-year-old Dutch, Caucasian female diagnosed with Graves' disease since 2014 implemented lifestyle modifications, which included dietary change to an ancestral type of diet, oral health interventions, practice of kundalini yoga, avoidance of environmental toxicants (by only eating organic food, drinking filtered water, and using natural products to clean her house or for her personal hygiene) and supportive supplements when necessary. The patient did not take any antithyroid drugs or beta-blockers during this period nor any other type of medication that could have had immunosuppressant effects. After 6 months of engaging in these lifestyle interventions, her thyroid analysis normalized and no anti-thyrotropin receptor antibodies were negative. CONCLUSION: This case report demonstrates that Graves' disease can effectively be put into lasting remission without conventional medical interventions. It also emphasizes the importance of a healthy lifestyle as a first line intervention for all patients but especially in the particular case of patients suffering from Graves' disease.

Psychotropic Drug Withdrawal and Holistic Tapering Strategies: A Case Series.

This case series aims to further the understanding of psychotropic drug withdrawal symptoms, as well as how individuals may be supported using holistic approaches for long term mood support. A secondary objective is to contribute to the evidence base for differentiating psychotropic drug withdrawal from the resurgence of psychiatric symptoms. Patients are described in two groups based on the timeline of psychotropic tapering. Group A illustrates cases of tapering safely from psychotropic medications under the supervision of the author of this case series, and Group B describes cases of individuals who sought mood support for protracted withdrawal symptoms. Both groups were treated with dietary changes, mindfulness practices, detoxification-supported gut health protocols, hormonal regulation, and treatment of comorbidities. Use of complementary medicine reduced many of the acute symptoms of psychotropic drug withdrawal, such as sleep disturbances, decreased concentration, nausea/headaches, and depression, making the process more manageable for patients. Additionally, many of the initial psychiatric complaints were kept in remission. These methods present a sustainable alternative to long-term treatment of mood symptoms and comorbid chronic illnesses. This case series indicates the benefits of integrating holistic and conventional medicine in psychotropic drug tapering, and a call for further trials to create an evidence-based database to guide future treatment and taper protocols.

The Yale Center for Biomedical Innovation and Technology (CBIT): One Model to Accelerate Impact From Academic Health Care Innovation.

The process of translating academic biomedical advances into clinical care improvements is difficult, risky, expensive, and poorly understood. Notably, many clinicians who identify health care problems do not have the time or expertise to solve the problems, and many academic researchers are unaware of important gaps in clinical care to which their expertise may apply.Recognizing an opportunity to connect people who can identify health care problems with those who can solve them, the Yale Center for Biomedical Innovation and Technology (CBIT) was established in 2014 to educate and enhance the impact of health care innovators. The authors review other health care innovation centers and describe best practices borrowed by Yale CBIT, which tailored its activities and approach to its unique ecosystem.In four years, Yale CBIT has affected over 3,000 people and established a health care innovation cycle as an efficient strategy to guide translational research. Yale CBIT has created or supported graduate and undergraduate courses, clinical immersion programs for industry partners, and large health care hackathon events. Over 200 projects have been submitted to CBIT for mentorship, and some of those projects have been commercialized and raised millions of dollars of follow-on funding.The authors present Yale CBIT as one model of accelerating the impact of academic medicine on clinical practice and outcomes. The project advising strategy is intended to be a template to maximize the efficiency of biomedical innovation and ultimately improve the outcomes and experiences of future patients.

Artificial Antigen-Presenting Cells for Immunotherapies.

Artificial antigen-presenting cells (aAPCs) overcome many of the limitations of biologically based adoptive immunotherapy protocols. While these acellular systems can be designed with a variety of parameters, including material type, diameter, and proliferative signals for T cells, we outline methods to formulate and characterize a comprehensive polymeric microparticle aAPC platform. These aAPCs, which can be reproducibly fabricated in large quantities, efficiently stimulate antigen-specific T cell activation and proliferation by both paracrine cytokine signals and engagement of T cell surface proteins.

Immunomodulatory nanoparticles ameliorate disease in the Leishmania (Viannia) panamensis mouse model.

Leishmania (Viannia) panamensis (L. (V.) panamensis) is a species of protozoan parasites that causes New World leishmaniasis, which is characterized by a hyper-inflammatory response. Current treatment strategies, mainly chemotherapeutic, are suboptimal due to adverse effects, long treatment regimens, and increasing drug resistance. Recently, immunotherapeutic approaches have shown promise in preclinical studies of leishmaniasis. As NPs may enable broad cellular immunomodulation through internalization in phagocytic and antigen-presenting cells, we tested the therapeutic efficacy of biodegradable NPs encapsulating a pathogen-associated molecular pattern (PAMP), CpG-rich oligonucleotide (CpG; NP-CpG), in mice infected with L. (V.) panamensis. NP-CpG treatment reduced lesion size and parasite burden, while neither free CpG nor empty NP showed therapeutic effects. NP-encapsulation led to CpG persistence at the site of infection along with an unexpected preferential cellular uptake by myeloid derived suppressor cells (MDSCs; CD11b(+)Ly6G(+)Ly6C(-)) as well as CD19(+) dendritic cells. This corresponded with the suppression of the ongoing immune response measured by the reduction of pathogenic cytokines IL-10 and IL-13, as well as IL-17 and IFNγ, in comparison to other treatment groups. As chronic inflammation is generally associated with the accumulation of MDSCs, this study may enable the rational design of cost-effective, safe, and scalable delivery systems for the treatment of inflammation-mediated diseases.

Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy.

BACKGROUND: Treatments to reverse peanut allergy remain elusive. Current clinical approaches using peanut oral/sublingual immunotherapy are promising, but concerns about safety and long-term benefit remain a barrier to wide use. Improved methods of delivering peanut-specific immunotherapy are needed. OBJECTIVE: We sought to investigate the efficacy and safety of peanut oral immunotherapy using CpG-coated poly(lactic-co-glycolic acid) nanoparticles containing peanut extract (CpG/PN-NPs) in a murine model of peanut allergy. METHODS: C3H/HeJ mice were rendered peanut allergic by means of oral sensitization with peanut and cholera toxin. Mice were then subjected to 4 weekly gavages with CpG/PN-NPs, vehicle (PBS), nanoparticles alone, peanut alone, CpG nanoparticles, or peanut nanoparticles. Untreated mice served as naive controls. After completing therapy, mice underwent 5 monthly oral peanut challenges. Anaphylaxis was evaluated by means of visual assessment of symptom scores and measurement of body temperature and plasma histamine levels. Peanut-specific serum IgE, IgG1, and IgG2a levels were measured by using ELISA, as were cytokine recall responses in splenocyte cultures. RESULTS: Mice with peanut allergy treated with CpG/PN-NPs but not vehicle or other treatment components were significantly protected from anaphylaxis to all 5 oral peanut challenges, as indicated by lower symptom scores, less change in body temperature, and a lower increase of plasma histamine levels. Importantly, CpG/PN-NP treatment did not cause anaphylactic reactions. Treatment was associated with a sustained and significant decrease in peanut-specific IgE/IgG1 levels and an increase in peanut-specific IgG2a levels. Compared with vehicle control animals, peanut recall responses in splenocyte cultures from nanoparticle-treated mice showed significantly decreased levels of TH2 cytokines (IL-4, IL-5, and IL-13) but increased IFN-γ levels in cell supernatants. CONCLUSIONS: Preclinical findings indicate that peanut oral immunotherapy with CpG/PN-NPs might be a valuable strategy for peanut-specific immunotherapy in human subjects.

Artificial bacterial biomimetic nanoparticles synergize pathogen-associated molecular patterns for vaccine efficacy.

Antigen-presenting cells (APCs) sense microorganisms via pathogen-associated molecular patterns (PAMPs) by both extra- and intracellular Toll-like Receptors (TLRs), initiating immune responses against invading pathogens. Bacterial PAMPs include extracellular lipopolysaccharides and intracellular unmethylated CpG-rich oligodeoxynucleotides (CpG). We hypothesized that a biomimetic approach involving antigen-loaded nanoparticles (NP) displaying Monophosphoryl Lipid A (MPLA) and encapsulating CpG may function as an effective "artificial bacterial" biomimetic vaccine platform. This hypothesis was tested in vitro and in vivo using NP assembled from biodegradable poly(lactic-co-glycolic acid) (PLGA) polymer, surface-modified with MPLA, and loaded with CpG and model antigen Ovalbumin (OVA). First, CpG potency, characterized by cytokine profiles, titers, and antigen-specific T cell responses, was enhanced when CpG was encapsulated in NP compared to equivalent concentrations of surface-presented CpG, highlighting the importance of biomimetic presentation of PAMPs. Second, NP synergized surface-bound MPLA with encapsulated CpG in vitro and in vivo, inducing greater pro-inflammatory, antigen-specific T helper 1 (Th1)-skewed cellular and antibody-mediated responses compared to single PAMPs or soluble PAMP combinations. Importantly, NP co-presentation of CpG and MPLA was critical for CD8(+) T cell responses, as vaccination with a mixture of NP presenting either CpG or MPLA failed to induce cellular immunity. This work demonstrates a rational methodology for combining TLR ligands in a context-dependent manner for synergistic nanoparticulate vaccines.

Attachment of cell-binding ligands to arginine-rich cell-penetrating peptides enables cytosolic translocation of complexed siRNA.

Cell-penetrating peptides (CPPs), such as nona-arginine (9R), poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome-to-cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation.

Cell-specific siRNA delivery by peptides and antibodies.

Cellular targeting and intracellular delivery of small interfering RNA (siRNA) remain a critical barrier to the clinical application of RNA interference. This chapter provides an overview of various delivery agents employing protein ligands mediating cell-specific delivery of siRNA. Specifically, the chapter details methodologies for the conjugation of antibody or peptide ligands to i) the cationic peptide-oligo-9-arginine (ii) the polymer poly(lactic-co-glycolic acid) (PLGA) and (iii) a lipid-vesicle (liposome).

Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines.

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.