Functionalized cryogel monoliths for fast and selective separation of nucleic acids directly from crude lysate.

The fast and selective separation of nucleic acids has been attractive recently because of their wide number of applications in the biomedical field such as the development of vaccines for infectious diseases, gene therapy, and diagnosis. Traditional approaches of nucleic acids separation are costlier, lengthy, and associated with possible denaturation because of the use of organic solvents in the elution step. Under this perspective, cryogels represent an attractive choice as a monolith stationary phase in column chromatography, which have proven efficient in recent chromatographic studies. Cryogels are the macroporous hydrogels with interconnecting properties between the pores. They allow the easy flow of large biomolecules with minimum mass transfer resistance. They are spongy in nature and possess good mechanical strength. Current article represents different developed functionalized cryogel monoliths for nucleic acids separation, their separation strategies, and challenges associated with further advancement in separation science.

Microneedle-Mediated Allergen-Specific Immunotherapy for the Treatment of Airway Allergy in Mice.

Subcutaneous allergen-specific immunotherapy (SCIT) qualifies as a promising approach for the permanent cure of IgE-mediated airway allergies, which can often manifest into allergic rhinitis and other allergic respiratory diseases. SCIT entails repeated administration of a high allergen dose into the subcutaneous (sc) region using a hypodermic needle for many (3-5) years, which is inconvenient and painful and reduces patient compliance. To overcome these limitations, we hypothesized that microneedles (MNs), which are minimally invasive and painless, could provide a novel approach for allergen desensitization by depositing the allergen into the superficial layers of the skin. To test this hypothesis, we compared MNs and SCIT for allergen desensitization in a mouse model of ovalbumin (Ova)-induced airway allergy. Mice were first made allergic to Ova and then treated with MNs coated with Ova (with or without CpG as an adjuvant) or via SCIT-Ova + alum (subcutaneous Ova + alum injections) for comparison. Treatment with coated MNs significantly induced Ova-specific serum IgG antibodies in a manner comparable to SCIT-Ova + alum-treated group. To test the efficacy against allergen challenge, treated mice were challenged with Ova via the nasal route. Coated MNs with Ova and CpG (MN-Ova + CpG) considerably suppressed the airway inflammation in allergic mice, evidenced by downregulation of proinflammatory cytokines (IL-5 and IL-13), upregulation of anti-inflammatory cytokine IL-10, and activation of Ova-specific immune response in bronchoalveolar (BAL) fluid. The therapeutic capacity of MN-based allergy treatment was further validated by the reduction in eosinophil and mast cell infiltration in the lung tissues of mice treated with MN-Ova + CpG, and low deposition of mucus inside their lung bronchioles. Overall, coated MNs ameliorated the symptoms of airway allergy in mice similar to SCIT and could provide a novel means of painless allergen-specific immunotherapy.

Assessment of Th1/Th2 Bias of STING Agonists Coated on Microneedles for Possible Use in Skin Allergen Immunotherapy.

Microneedle-based skin allergen-specific immunotherapy (AIT) can benefit from adjuvants that can stimulate a stronger Th1 response against the allergen. We evaluated two stimulator of interferon genes (STING) agonists, namely, cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP), as skin adjuvants using coated microneedles (MNs). For comparison, the approved subcutaneous (SC) hypodermic injection containing alum was used. Ovalbumin (Ova) was used as a model allergen. Ova-specific IgG2a antibody in serum, which is a surrogate marker for Th1 type immune response was significantly higher when STING agonists were used with coated MNs as compared to SC injection of Ova+alum in mice. In contrast, IgG1 antibody, a surrogate marker for Th2 type immune response, was at comparable levels in the MN and SC groups. Restimulation of splenocytes with Ova produced higher levels of Th1 cytokines (IFN-γ and IL-2) in the STING agonists MN groups as compared to the SC group. In conclusion, delivery of STING agonists into the skin using coated MNs activated the Th1 pathway better than SC- and MN-based delivery of alum. Thus, STING agonists could fulfill the role of adjuvants for skin AIT and even for infectious disease vaccines, where stimulation of the Th1 pathway is of interest.

Macrophage-derived reactive oxygen species protects against autoimmune priming with a defined polymeric adjuvant.

Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well-balanced polymer, poly-N-isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly-N-isopropylacrylamide-co-poly-N-tertbutylacrylamide and poly-N-tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly-N-tertbutylacrylamide induced arthritis, especially in Ncf1-deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm-CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo-responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events.

Development of a mini pig model of peanut allergy.

Introduction: The prevalence of peanut allergies is increasing, emphasizing the need for an animal model to enhance our understanding of peanut allergy pathogenesis and to advance diagnostic tools and therapeutic interventions. While mice are frequently used as model organisms, their allergic responses do not fully mirror those observed in humans, warranting the exploration of a higher animal model. The porcine gastrointestinal system closely resembles that of humans, and exhibits allergy symptoms akin to human responses, making pigs a promising model for peanut allergy research. Methods: In this study we compared two allergen sensitization protocols involving either topical allergen application after repeated tape stripping (TS) or intraperitoneal (IP) injections to induce peanut-specific allergy and anaphylaxis reactions in mini pigs. Mini pigs sensitized with a combination of peanut protein extract (PE) and cholera toxin (CT) through either the IP or the TS route. Results: Sensitized pigs via both methods developed systemic PE-specific IgG and IgE responses. Following peanut challenge via the IP route, both TS- and IP-sensitized pigs displayed allergy symptoms, including lethargy, skin rashes, vomiting, and a drop in body temperature. However, respiratory distress was observed exclusively in pigs sensitized through the TS route and not in those sensitized through the IP route. However, it is noteworthy that both groups of sensitized pigs maintained peanut hypersensitivity for up to two months post-sensitization, albeit with a reduction in the severity of allergy symptoms. Importantly, both groups exhibited sustained levels of PE-specific IgG, IgE, and elevated concentrations of mast cell protease in their blood following the IP challenges. Discussion: Overall, this study reports TS and IP as two different modes of sensitization leading to onset of peanut specific allergic reactions in mini pigs, but only the TS-sensitization led to systemic anaphylaxis (simultaneous presence of symptoms: breathing difficulty, intense skin rash, and impaired mobility). A distinctive feature of these sensitization protocols is the 100% success rate (N = 4 pigs per group) in sensitizing the subjects.

Polymer Chemistry Defines Adjuvant Properties and Determines the Immune Response against the Antigen or Vaccine.

Activation of the immune system is a needed for designing new antigen/drug delivery systems to develop new therapeutics and for developing animal disease models to study the disease pathogenesis. A weak antigen alone is insufficient to activate the immune system. Sometimes, assistance in the form of polymers is needed to control the release of antigens under in vivo conditions or in the form of an adjuvant to activate the immune system efficiently. Many kinds of polymers from different functional groups are suitable as microbial antigens for inducing therapeutic immune responses against infectious diseases at the preclinical level. The choice of the functionality of polymer varies as per the application type. Polymers from the acid and ester groups are the most common types investigated for protein-based antigens. However, electrostatic interaction-displaying polymers like cationic polymers are the most common type for nucleic acid-based antigens. Metal coordination chemistry is commonly used in polymers designed for cancer immunotherapeutic applications to suppress inflammation and induce a protective immune response. Amide chemistry is widely deployed in polymers used to develop antigen-specific disease models like the experimental autoimmune arthritis murine model.

A Perspective on Oral Immunotherapeutic Tools and Strategies for Autoimmune Disorders.

Oral immune tolerance is a physiological process to achieve tolerance against autoimmunity by oral ingestion of self-antigen(s) or other therapeutics. At the cellular level, oral tolerance suppresses autoimmune diseases by activating FoxP-positive and -negative regulatory T cells (Tregs) and/or causing clonal anergy or deletion of autoreactive T cells, affecting B cell tolerance. However, oral delivery of antigens/biologics is challenging due to their instability in the harsh environment of the gastrointestinal (GI) tract. Several antigen/drug delivery tools and approaches, including micro/nanoparticles and transgenic plant-based delivery systems, have been explored to demonstrate oral immune tolerance for different autoimmune diseases successfully. However, despite the effectiveness, variation in results, dose optimization, and undesirable immune system activation are the limitations of the oral approach to further advancement. From this perspective, the current review discusses the oral tolerance phenomenon, cellular mechanisms, antigen delivery tools and strategies, and its challenges.

Collagen type II and a thermo-responsive polymer of N-isopropylacrylamide induce arthritis independent of Toll-like receptors: a strong influence by major histocompatibility complex class II and Ncf1 genes.

We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1ß, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vß12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile.

Targeted allergen-specific immunotherapy within the skin improves allergen delivery to induce desensitization to peanut.

Aim: Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy may be limited by allergen uptake through the skin barrier. To enhance allergen uptake into the skin, the authors used peanut-coated microneedles and compared them with EPIT in a peanut allergy mouse model. Methods: Sensitized mice were treated with peanut-coated microneedles or peanut-EPIT and then challenged with peanut to determine protection. Results: Treatment with peanut-coated microneedles was safe and showed enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Protection was associated with reduced Th2 immune responses and mast cell accumulation in the intestine. Conclusion: Peanut-coated microneedles have the potential to present a safe method of improving allergen delivery for cutaneous immunotherapy.

Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy has been varied. The tight barrier provided by the skin may limit the amount of allergen taken up through the skin and thus reduce efficacy. The authors evaluated a microneedle-based approach to improve the amount of allergen deposited into the skin to improve efficacy. Mice were made allergic to peanut and then treated with peanut-coated microneedles or peanut-EPIT. Mice were challenged with peanut to determine suppression of allergic reactivity. In mice, treatment with peanut-coated microneedles was safe and enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Peanut-coated microneedles may present a novel method of improving allergen immunotherapy delivered through the skin.

Microneedles coated with peanut allergen enable desensitization of peanut sensitized mice.

The prevalence of peanut allergies has escalated over the last 20 years, yet there are no FDA approved treatments for peanut allergies. In this study we evaluated the potential of microneedles to deliver peanut protein extract (PE) into skin and assessed if the ensuing immune responses could desensitize mice that were sensitized to peanuts. Peanut sensitized mice were either treated through cutaneous immunotherapy using PE-coated microneedles or not treated, and then orally challenged with PE. After oral challenge, the clinical symptoms of peanut-induced anaphylaxis were significantly lower in the microneedle treated mice as compared to untreated mice, and this was accompanied by down-regulation of systemic anaphylaxis mediators such as histamine and mast cell protease-1 (MCPT-1) in the microneedles treated group. Overall, there was an up-regulation of Th1 cytokines (IL-2 and IFN-γ) as compared to Th2 cytokines (IL-4 and IL-5) in splenocyte culture supernatants of the microneedle treated group as compared to untreated group, suggesting that microneedles promoted immune modulation towards the Th1 pathway. Furthermore, mice treated with PE-coated microneedles were observed to retain integrity of their small intestine villi and had reduced eosinophilic infiltration as compared to the untreated but peanut sensitized mice, which further confirmed the desensitization capability of peanut cutaneous immunotherapy using coated microneedles. Thus, our current study represents a novel minimally invasive microneedle based cutaneous immunotherapy, which may provide a novel route of desensitization for the treatment of peanut allergies.

Antigen-Specific Tolerization and Targeted Delivery as Therapeutic Strategies for Autoimmune Diseases.

The prevalence of autoimmune disorders is increasing steadily and there is no permanent cure available. Immunomodulation through repeated exposure of antigens, known as antigen-specific immune tolerance or antigen-specific immunotherapy (ASI), is a promising approach to treat or prevent autoimmune disorders. Different optimization protocols (immunization routes, delivery systems, and approaches) are being developed to implement ASI against self-proteins. Including appropriate adjuvants, altered peptide ligand, and using multipeptides are approaches that can be used to specifically target autoimmunity. This review explores various ASI application methods, including different routes of antigen-specific sensitization, delivery systems, immunomodulators containing specific antigens, and other targeted approaches that have been successfully demonstrated to have therapeutic effects on autoimmune diseases.

An update on smart biocatalysts for industrial and biomedical applications.

Recently, smart biocatalysts, where enzymes are conjugated to stimuli-responsive (smart) polymers, have gained significant attention. Based on the presence or absence of external stimuli, the polymer attached to the enzyme changes its conformation to protect the enzyme from the external environment and regulate the enzyme activity, thus acting as a molecular switch. Owing to this behaviour, smart biocatalysts can be separated easily from a reaction mixture and re-used several times. Several such smart polymer-based biocatalysts have been developed for industrial and biomedical applications. In addition, they have been used in biosensors, biometrics and nano-electronic devices. This review article covers recent advances in developing different kinds of stimuli-responsive enzyme bioconjugates, including conjugation strategies, and their applications.

Three-dimensional macroporous materials for tissue engineering of craniofacial bone.

Repair of critical-size defects caused by trauma, removal of a tumour, or congenital abnormalities is a challenge in the craniomaxillofacial region because of the limitations associated with treatment. We have reviewed research papers and updated information relevant to the various types of macroporous scaffolds. We have included papers on several biomaterials and their use in various craniofacial defects such as mandibular, calvarial, and others, as well as the latest technological developments such as 3-dimensional printed scaffolds. We selected all papers about scaffolds, stem cells, and growth factors for review. Initial selection was by review of titles and abstracts, and the full texts of potentially suitable articles were then assessed. Methods of tissue engineering for repair of critical-size defects in the craniofacial bones seem to be viable options for surgical treatment in the future. Macroporous scaffolds with interconnected pores are of great value in regeneration of bone in the craniofacial region. In recent years, various natural or synthetic materials, or both, have been developed, on which macroporous scaffolds can be based. In this review we present a review on the various types of three-dimensional macroporous scaffolds that have been developed in recent years, and evaluate their potential for regeneration of craniofacial bone.

Cutaneous vaccination with coated microneedles prevents development of airway allergy.

Allergy cases are increasing worldwide. Currently allergies are treated after their appearance in patients. However, now there is effort to make a preventive vaccine against allergies. The rationale is to target patient populations that are already sensitized to allergens but have yet to develop severe forms of the allergic disease, or who are susceptible to allergy development but have not yet developed them. Subcutaneous injections and the sublingual route have been used as the primary mode of preventive vaccine delivery. However, injections are painful, especially considering that they have to be given repeatedly to infants or young children. The sublingual route is hard to use since infants can't be trained to hold the vaccine under their tongue. In the present study, we demonstrate a microneedle (MN)-based cutaneous preventive allergy treatment against ovalbumin (Ova)-induced airway allergy in mice. Insertion of MNs coated with Ova as a model allergen and CpG oligonucleotide as an adjuvant (MNs-CIT) into the skin significantly induced Ova specific systemic immune response. This response was similar to that induced by hypodermic-needle-based delivery of Ova using the clinically-approved subcutaneous immunotherapy (SCIT) route. MNs-CIT regulated Th2 cytokines (IL-4, IL-5 & IL-13) and anti-inflammatory cytokines (IL-10) in the bronchoalveolar fluid, and IL-2 and IFN-γ cytokines in restimulated splenocyte cultures. Absence of mucus deposition inside the bronchiole wall and low collagen around the lung bronchioles after Ova-allergen challenge further confirmed the protective role of MNs-CIT. Overall, MNs-CIT represents a novel minimally invasive cutaneous immunotherapy to prevent the progression of Ova induced airway allergy in mice.

Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses.

The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.

Mucosal vaccine delivery: Current state and a pediatric perspective.

Most childhood infections occur via the mucosal surfaces, however, parenterally delivered vaccines are unable to induce protective immunity at these surfaces. In contrast, delivery of vaccines via the mucosal routes can allow antigens to interact with the mucosa-associated lymphoid tissue (MALT) to induce both mucosal and systemic immunity. The induced mucosal immunity can neutralize the pathogen on the mucosal surface before it can cause infection. In addition to reinforcing the defense at mucosal surfaces, mucosal vaccination is also expected to be needle-free, which can eliminate pain and the fear of vaccination. Thus, mucosal vaccination is highly appealing, especially for the pediatric population. However, vaccine delivery across mucosal surfaces is challenging because of the different barriers that naturally exist at the various mucosal surfaces to keep the pathogens out. There have been significant developments in delivery systems for mucosal vaccination. In this review we provide an introduction to the MALT, highlight barriers to vaccine delivery at different mucosal surfaces, discuss different approaches that have been investigated for vaccine delivery across mucosal surfaces, and conclude with an assessment of perspectives for mucosal vaccination in the context of the pediatric population.

A comparative study of microneedle-based cutaneous immunization with other conventional routes to assess feasibility of microneedles for allergy immunotherapy.

Feasibility of microneedles (MNs) for cutaneous allergen specific immunotherapy (ASI) is demonstrated by comparing against currently practiced subcutaneous (SC) allergen immunotherapy, and the intramuscular (IM) and intraperitoneal (IP) routes. In Balb/c mice with ovalbumin (Ova, 25 µg) as the allergen MNs-Ova without alum induced anti-Ova IgG response comparable to IM but higher than SC and IP groups (250 µg alum was additionally used for SC, IM and IP groups). MNs-Ova induced higher anti-Ova IgG1 and IgG2a responses in comparison to other routes; however IgG2b and IgG3 responses were significantly lower than the IP group. As in SC group, anti-Ova IgE and IgA were low for MNs-Ova. Furthermore, MNs-Ova induced expression of IL-5, IL-13, IFN-γ and IL-1ß cytokines in serum, but at significantly lower levels than other routes. Overall, MNs-Ova induced allergen-specific IgG antibodies, and activated the Th1 pathway (evidenced by higher IgG2a levels), suggesting their potential use for painless ASI.

Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 µm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract.

Fabrication of macroporous cryogels as potential hepatocyte carriers for bioartificial liver support.

Two different cryogels composed of copolymer of acrylonitrile (AN) and N-vinyl-2-pyrrolidone (NVP) (poly(AN-co-NVP)) and interpenetrated polymer networks (IPN) of chitosan and poly(N-isopropylacrylamide) (poly(NiPAAm)-chitosan) were fabricated by gelation at sub-zero temperatures. The two cryogels possess an interconnected network of macropores of size 20-100 µm and efficient transport properties as determined by physiochemical analysis. Both cryogels support in vitro growth and function of fibroblasts (COS-7) and human liver hepatocarcinoma cells (HepG2). The cryogels are hemocompatible as demonstrated by low albumin adsorption and platelet adherence. Furthermore, in vivo implantation of poly(NiPAAm)-chitosan cryogel in mice shows its biocompatibility with the surrounding tissue. Primary rat hepatocytes grown on poly(NiPAAm)-chitosan cryogel for 96 h formed cellular aggregates and maintained their functions in terms of, ammonia removal, ureagenesis and drug detoxification. Cryogel-based closed continuous bioreactor systems could maintain HepG2 cells at high density for 7 days. Off-line clinical evaluation of these cryogel-based bioreactors showed the ability of immobilized cells to detoxify circulating plasma obtained from patients with acute on chronic liver failure (ACLF). Altogether, the presented data suggests cryogels as a potential bioreactor matrix for bio-artificial liver support system.