Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1ß, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Bioengineered Organoids Offer New Possibilities for Liver Cancer Studies: A Review of Key Milestones and Challenges.

Hepatic cancer is widely regarded as the leading cause of cancer-related mortality worldwide. Despite recent advances in treatment options, the prognosis of liver cancer remains poor. Therefore, there is an urgent need to develop more representative in vitro models of liver cancer for pathophysiology and drug screening studies. Fortunately, an exciting new development for generating liver models in recent years has been the advent of organoid technology. Organoid models hold huge potential as an in vitro research tool because they can recapitulate the spatial architecture of primary liver cancers and maintain the molecular and functional variations of the native tissue counterparts during long-term culture in vitro. This review provides a comprehensive overview and discussion of the establishment and application of liver organoid models in vitro. Bioengineering strategies used to construct organoid models are also discussed. In addition, the clinical potential and other relevant applications of liver organoid models in different functional states are explored. In the end, this review discusses current limitations and future prospects to encourage further development.

Using Liver Organoids as Models to Study the Pathobiology of Rare Liver Diseases.

Liver organoids take advantage of several important features of pluripotent stem cells that self-assemble in a three-dimensional culture matrix and reproduce many aspects of the complex organization found within their native tissue or organ counterparts. Compared to other 2D or 3D in vitro models, organoids are widely believed to be genetically stable or docile structures that can be programmed to virtually recapitulate certain biological, physiological, or pathophysiological features of original tissues or organs in vitro. Therefore, organoids can be exploited as effective substitutes or miniaturized models for the study of the developmental mechanisms of rare liver diseases, drug discovery, the accurate evaluation of personalized drug responses, and regenerative medicine applications. However, the bioengineering of organoids currently faces many groundbreaking challenges, including a need for a reasonable tissue size, structured organization, vascularization, functional maturity, and reproducibility. In this review, we outlined basic methodologies and supplements to establish organoids and summarized recent technological advances for experimental liver biology. Finally, we discussed the therapeutic applications and current limitations.

Whole Liver Derived Acellular Extracellular Matrix for Bioengineering of Liver Constructs: An Updated Review.

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

Phytochemical characterization, antioxidant and antibacterial activities of crude extracts of Anisomeles malabarica and Coldenia procumbens.

The aim of this study was to determine the phytochemical profile, antibacterial and antioxidant activities of crude aqueous leaf extracts of Anisomeles malabarica and Coldenia procumbens. The predominant components present in these crude extracts of test plants identified using gas chromatography-mass spectrometry (GC-MS) analysis in both plant extracts were phytochemicals including flavonoids, tannins, terpenoids, and phenols. The antibacterial activity of crude extracts of these plants against bacterial pathogens including Escherichia coli, Bacillus subtilis, Shigella sp., Salmonella paratyphi A and B, Proteus mirabilis, Proteus vulgaris, Pseudomonas sp. Klebsiella pneumoniae, and Staphylococcus aureus were examined. Data demonstrated that the extracts of A. malabarica and C. procumbens exhibited significant antibacterial activity against B.subtilis and P.vulgaris at the concentration of 50 mg/ml. A. malabarica aqueous extract displayed significant antioxidant activity on 2,2-diphenyl-1-picrylhydrazl (DPPH), fluorescence recovery after photobleaching (FRAP) and hydrogen peroxide (H2O2) free radicals at the concentration of 90 mg/ml. The antioxidant activity was significantly higher with A. malabarica than extract of C. procumbens. Evidence indicates that both plant extracts may possess significant pharmaceutical potential as antibacterial and antioxidant agents.

Mammalian-specific decellularized matrices derived bioink for bioengineering of liver tissue analogues: A review.

The absolute shortage of compatible liver donors and the growing number of potential recipients have led scientists to explore alternative approaches to providing tissue/ organ substitutes from bioengineered sources. Bioartificial regeneration of a fully functional tissue/organ replacement is highly dependent on the right combination of engineering tools, biological principles, and materiobiology horizons. Over the past two decades, remarkable achievements have been made in hepatic tissue engineering by converging various advanced interdisciplinary research approaches. Three-dimensional (3D) bioprinting has arisen as a promising state-of-the-art tool with strong potential to fabricate volumetric liver tissue/organ equivalents using viscosity- and degradation-controlled printable bioinks composed of hydrous microenvironments, and formulations containing living cells and associated supplements. Source of origin, biophysiochemical, or thermomechanical properties and crosslinking reaction kinetics are prerequisites for ideal bioink formulation and realizing the bioprinting process. In this review, we delve into the forecast of the potential future utility of bioprinting technology and the promise of tissue/organ- specific decellularized biomaterials as bioink substrates. Afterward, we outline various methods of decellularization, and the most relevant studies applying decellularized bioinks toward the bioengineering of in vitro liver models. Finally, the challenges and future prospects of decellularized material-based bioprinting in the direction of clinical regenerative medicine are presented to motivate further developments.

Therapeutic nexus of T cell immunometabolism in improving transplantation immunotherapy.

Immunometabolism is a therapeutic strategy to tune immune cells through metabolic reprogramming, which allows immune cells to be differentiated according to their energy requirements. Recent therapeutic strategies targeting immunometabolism suggest that intracellular metabolic reprogramming controls T cell activation, proliferation, and differentiation into effector (Teff) or regulatory (Treg) cells. Immunometabolism is being studied for the treatment of inflammatory diseases, including those associated with solid organ transplantation (SOT). Here, we review immunometabolic regulation of immune cells, with a particular focus on Treg metabolic regulation and liver kinase B1 (LKB1) signaling, which stabilize Tregs and prevent inflammation-associated tissue injuries. All in all, here we discussed how targeting T cell immunometabolism modulates Teff and Treg-mediated immune responses, which can be used to boost Treg differentiation, stability, and ultimately favor immunotolerance in clinical transplants.

Targeting Interleukin-10 Restores Graft Microvascular Supply and Airway Epithelium in Rejecting Allografts.

Interleukin-10 (IL-10) is a vital regulatory cytokine, which plays a constructive role in maintaining immune tolerance during an alloimmune inflammation. Our previous study highlighted that IL-10 mediated immunosuppression established the immune tolerance phase and thereby modulated both microvascular and epithelial integrity, which affected inflammation-associated graft malfunctioning and sub-epithelial fibrosis in rejecting allografts. Here, we further investigated the reparative effects of IL-10 on microvasculature and epithelium in a mouse model of airway transplantation. To investigate the IL-10 mediated microvascular and epithelial repair, we depleted and reconstituted IL-10, and monitored graft microvasculature, airway epithelium, and associated repair proteins. Our data demonstrated that both untreated control allografts and IL-10 (-) allografts showed a significant early (d6) increase in microvascular leakiness, drop-in tissue oxygenation, blood perfusion, and denuded airway epithelium, which is associated with loss of adhesion protein Fascin-1 and ß-catenin on vascular endothelial cells at d10 post-transplantation. However, IL-10 (+) promotes early microvascular and airway epithelial repair, and a proportional increase in endothelial Fascin-1, and ß-catenin at d10 post-transplantation. Moreover, airway epithelial cells also express a significantly higher expression of FOXJ1 and ß-catenin in syngrafts and IL-10 (+) allografts as compared to IL-10 (-) and untreated controls at d10 post-transplantation. Collectively, these findings demonstrated that IL-10 mediated microvascular and epithelial changes are associated with the expression of FOXJ1, ß-catenin, and Fascin-1 proteins on the airway epithelial and vascular endothelial cells, respectively. These findings establish a potential reparative modulation of IL-10 associated microvascular and epithelial repair, which could provide a vital therapeutic strategy to facilitate graft repair in clinical settings.

IL-10 Mediated Immunomodulation Limits Subepithelial Fibrosis and Repairs Airway Epithelium in Rejecting Airway Allografts.

Interleukin-10 plays a vital role in maintaining peripheral immunotolerance and favors a regulatory immune milieu through the suppression of T effector cells. Inflammation-induced microvascular loss has been associated with airway epithelial injury, which is a key pathological source of graft malfunctioning and subepithelial fibrosis in rejecting allografts. The regulatory immune phase maneuvers alloimmune inflammation through various regulatory modulators, and thereby promotes graft microvascular repair and suppresses the progression of fibrosis after transplantation. The present study was designed to investigate the therapeutic impact of IL-10 on immunotolerance, in particular, the reparative microenvironment, which negates airway epithelial injury, and fibrosis in a mouse model of airway graft rejection. Here, we depleted and reconstituted IL-10, and serially monitored the phase of immunotolerance, graft microvasculature, inflammatory cytokines, airway epithelium, and subepithelial collagen in rejecting airway transplants. We demonstrated that the IL-10 depletion suppresses FOXP3+ Tregs, tumor necrosis factor-inducible gene 6 protein (TSG-6), graft microvasculature, and establishes a pro-inflammatory phase, which augments airway epithelial injury and subepithelial collagen deposition while the IL-10 reconstitution facilitates FOXP3+ Tregs, TSG-6 deposition, graft microvasculature, and thereby favors airway epithelial repair and subepithelial collagen suppression. These findings establish a potential reparative modulation of IL-10-associated immunotolerance on microvascular, epithelial, and fibrotic remodeling, which could provide a vital therapeutic option to rescue rejecting transplants in clinical settings.

Corrigendum: Olax scandens Mediated Biogenic Synthesis of Ag-Cu Nanocomposites: Potential Against Inhibition of Drug-Resistant Microbes.

[This corrects the article DOI: 10.3389/fchem.2020.00103.].

Human Papillomavirus Induced Cervical and Oropharyngeal Cancers: From Mechanisms to Potential Immuno-therapeutic Strategies.

The human papillomavirus (HPV) associated infections are the hallmark of cervical and neck cancer. Almost all the cases of cervical cancer (CC) and 70% of oropharyngeal cancer (OC) are, more or less, caused by the persistent infection of HPV. CC is the fourth most common cancer globally, and is commenced by the persistent infection with human papillomaviruses (HPVs), predominantly HPV types; 16 and 18. In the light of the above facts, there is an immediate requirement to develop novel preventive and innovative therapeutic strategies that may help in lower occurrences of HPV mediated cancers. Currently, only radiation and chemical-based therapies are the treatment for HPV mediated neck cancer (NC) and CC. Recent advances in the field of immunotherapy are underway, which are expected to unravel the optimal treatment strategies for the growing HPV mediated cancers. In this review, we decipher the mechanism of pathogenesis with current immunotherapeutic advances in regressing the NC and CC, with an emphasis on immune-therapeutic strategies being tested in clinical trials and predominantly focus on defining the efficacy and limitations. Taken together, these immunological advances have enhanced the effectiveness of immunotherapy and promises better treatment results in coming future.

Hypoxia-induced complement dysregulation is associated with microvascular impairments in mouse tracheal transplants.

BACKGROUND: Complement Regulatory Proteins (CRPs), especially CD55 primarily negate complement factor 3-mediated injuries and maintain tissue homeostasis during complement cascade activation. Complement activation and regulation during alloimmune inflammation contribute to allograft injury and therefore we proposed to investigate a crucial pathological link between vascular expression of CD55, active-C3, T cell immunity and associated microvascular tissue injuries during allograft rejection. METHODS: Balb/c→C57BL/6 allografts were examined for microvascular deposition of CD55, C3d, T cells, and associated tissue microvascular impairments during rejection in mouse orthotopic tracheal transplantation. RESULTS: Our findings demonstrated that hypoxia-induced early activation of HIF-1α favors a cell-mediated inflammation (CD4+, CD8+, and associated proinflammatory cytokines, IL-2 and TNF-α), which proportionally triggers the downregulation of CRP-CD55, and thereby augments the uncontrolled release of active-C3, and Caspase-3 deposition on CD31+ graft vascular endothelial cells. These molecular changes are pathologically associated with microvascular deterioration (low tissue O2 and Blood flow) and subsequent airway epithelial injuries of rejecting allografts as compared to non-rejecting syngrafts. CONCLUSION: Together, these findings establish a pathological correlation between complement dysregulation, T cell immunity, and microvascular associated injuries during alloimmune inflammation in transplantation.

Olax scandens Mediated Biogenic Synthesis of Ag-Cu Nanocomposites: Potential Against Inhibition of Drug-Resistant Microbes.

In the present study, we have synthesized silver-copper nanocomposites (Ag-Cu NCs) using an Olax scandens leaf extract (green synthesis method) and evaluated their antimicrobial potential against less susceptible pathogens. The kinetics of Ag-Cu NCs synthesis was followed by UV-VIS and fluorescence spectroscopy. The physicochemical characterization of as-synthesized Ag-Cu NCs was executed using electron microscopy, Energy Dispersive X-Ray, Fourier Transform Infrared Spectroscopy, and a Differential Light Scattering method. As-synthesized Ag-Cu NCs induced the formation of Reactive Oxygen Species (ROS), thereby causing alteration and decrementation of cellular proteins, DNA, lipids, etc., and eventually leading to cell death, as determined by a Live/Dead assay. Next, we assessed the anti-biofilm potential of as-synthesized Ag-Cu NCs against biofilm forming bacteria. The as-synthesized Ag-Cu NCs, when compared to monometallic silver nanoparticles, exhibited significantly higher anti-microbial activity against both sensitive as well as drug resistant microbial isolates.

Cyclic undecapeptide Cyclosporin A mediated inhibition of amyloid synthesis: Implications in alleviation of amyloid induced neurotoxicity.

Amyloids are highly organized fibril aggregates arise from inappropriately folded form of the protein or polypeptide precursors under both physiological as well as simulated ambience. Amyloid synthesis is a multistep process that involves formation of several metastable intermediates. Among various intermediate species, the as-formed soluble oligomers are extremely toxic to the neuronal cells. In the present study, we evaluated cyclosporine A (CsA), an undecapeptide, for its potential to prevent aggregation of model protein ovalbumin (OVA). In an attempt to elucidate involved operative mechanism, the preliminary studies delineate that CsA affects both primary nucleation as well as other secondary pathways involved in OVA fibrillation process. By its specific interaction with amyloid intermediates, the cyclic peptide CsA seems to regulate the lag phase of the fibrillation process in concentration dependent manner. The present study further suggests that exposure to CsA during lag phase ensues in reversal of OVA fibrillation process. On the contrary, mature OVA fibril remained impervious to the CsA treatment. The cyclic undecapeptide CsA was also found to successfully alleviate amyloid induced toxicity in neuroblastoma cells.

Synergistic combination of natural bioadhesive bael fruit gum and chitosan/nano-hydroxyapatite: A ternary bioactive nanohybrid for bone tissue engineering.

In this work, we have explored the polysaccharide nature of bael fruit gum (BFG) motivated from the current findings about the substantial role of the polysaccharides in bone tissue engineering. The nanocomposite scaffold (CSH-BFG) was prepared by blending BFG, nano-hydroxyapatite (n-HA) and chitosan (CS) by co-precipitation approach and compared with n-HA and CS binary system (CSH). The analysis of different properties was carried out by SEM, TEM, FTIR, XRD and mechanical testing. The CSH-BFG scaffolds revealed a rough morphology and uniform distribution of particles along with strong chemical interactions among different components compared to the CSH scaffold. The incorporation of BFG in the scaffold resulted in significant increase of the compressive strength, compressive modulus, protein adsorption, biodegradation and swelling behaviour. The ternary system exhibited superior antibacterial activity against different bacterial pathogens compared to the binary system. The in vitro biomineralization ability was elucidated from the formation of thick apatite layer complementing the result of ARS study in the CSH-BFG nanocomposite. Our findings also revealed that BFG reinforced CSH nanocomposite exhibited enhanced cell adhesion and proliferation, osteogenic differentiation along with phenomenal cytocompatibility. Overall, our results signified that the fabricated CSH-BFG nanocomposite carries enormous potential to be applied in the bone remodelling procedures.

Nano-hydroxyapatite/ß-CD/chitosan nanocomposite for potential applications in bone tissue engineering.

Herein, we report the synthesis of a novel tri-component nanocomposite system incorporating ß-cyclodextrin (ß-CD) with nano-hydroxyapatite (n-HA) and chitosan (CS), (n-HA/ß-CD/CS) at three different temperatures via co-precipitation method. The chemical interactions and surface morphology have been evaluated by TEM, SEM and AFM techniques revealing the agglomerated nanoparticles in CS/n-HA-HA binary system whereas the ternary systems produced needle shaped nanoparticles dispersed homogeneously at low temperature with more porous and rougher surface. The addition of ß-CD in CS/n-HA at low temperature decreased the particle size and raised the thermal stability as compared to CS/n-HA. The comparative hemolytic, protein adsorption and platelet adhesion studies confirmed the better hemocompatibility of n-HA/ß-CD/CS-(RT,HT,LT) nanocomposites relative to CS/n-HA. The cell viability has been evaluated in vitro using MG-63 cell line which revealed superior non toxicity of n-HA/ß-CD/CS-LT nanocomposite in comparison to n-HA/ß-CD/CS-(RT,HT) and CS/n-HA nanocomposites. Thus it may be concluded that the orchestrated organic/inorganic n-HA/ß-CD/CS-(RT,HT,LT) nanocomposites exhibited relatively higher cell viability of human osteoblast cells, stimulated greater osteogenesis, controlled biodegradation, enhanced antibacterial activity with excellent in-vitro biomineralization and remarkable mechanical parameters as compared to CS/n-HA nanocomposite and thus may provide opportunities for potential use as an alternative biomaterial for Bone tissue engineering applications.

Amyloid form of ovalbumin evokes native antigen-specific immune response in the host: prospective immuno-prophylactic potential.

Amyloids are highly organized protein aggregates that arise from inappropriately folded versions of proteins or polypeptides under both physiological as well as simulated ambiences. Once thought to be irreversible assemblies, amyloids have begun to expose their more dynamic and reversible attributes depending upon the intrinsic properties of the precursor protein/peptide and experimental conditions such as temperature, pressure, structural modifications in proteins, or presence of chemicals in the reaction mixture. It has been repeatedly proposed that amyloids undergo transformation to the bioactive peptide/protein forms under specific conditions. In the present study, amyloids assembled from the model protein ovalbumin (OVA) were found to release the precursor protein in a slow and steady manner over an extended time period. Interestingly, the released OVA from amyloid depot was found to exhibit biophysical characteristics of native protein and reacted with native-OVA specific monoclonal as well as polyclonal antibodies. Moreover, antibodies generated upon immunization of OVA amyloidal aggregates or fibrils were found to recognize the native form of OVA. The study suggests that amyloids may act as depots for the native form of the protein and therefore can be exploited as vaccine candidates, where slow antigen release over extended time periods is a pre-requisite for the development of desired immune response.

An alternative chemical redox method for the production of bispecific antibodies: implication in rapid detection of food borne pathogens.

Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.