Systemic Immunotherapy with Micellar Resiquimod-Polymer Conjugates Triggers a Robust Antitumor Response in a Breast Cancer Model.

Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8+ T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8+ T-cells across multiple tumors suggest the potential for treating metastatic cancer.

Lactate Exposure Promotes Immunosuppressive Phenotypes in Innate Immune Cells.

INTRODUCTION: Lactate secreted by tumors is not just a byproduct, but rather an active modulator of immune cells. There are few studies aimed at investigating the true effect of lactate, which is normally confounded by pH. Such a knowledge gap needs to be addressed. Herein, we studied the immunomodulatory effects of lactate on dendritic cells (DCs) and macrophages (MΦs). METHODS: Bone marrow-derived innate immune cells were treated with 50 mM sodium lactate (sLA) and incubated for 2 days or 5 days at 37 °C. Controls included media, lipopolysaccharide (LPS), MCT inhibitors (α-cyano-4-hydroxycinnamic acid and AR-C15585). Flow cytometric analysis of immune phenotypes were performed by incubating cells with specific marker antibodies and viability dye. Differential expression analyses were conducted on R using limma-voom and adjusted p-values were generated using the Bejamini-Hochberg Procedure. RESULTS: Lactate exposure attenuated DC maturation through the downregulation of CD80 and MHCII expression under LPS stimulation. For MΦs, lactate exposure resulted in M2 polarization as evidenced by the reduction of M1 markers (CD38 and iNOS), and the increase in expression of CD163 and Arg1. We also revealed the role of monocarboxylate transporters (MCTs) in mediating lactate effect in MΦs. MCT4 inhibition significantly boosted lactate M2 polarization, while blocking of MCT1/2 failed to reverse the immunosuppressive effect of lactate, correlating with the result of gene expression that lactate increased MCT4 expression, but downregulated the expression of MCT1/2. CONCLUSIONS: This research provides valuable insight on the influence of metabolic products on tumor immunity and will help to identify novel metabolic targets for augmenting cancer immunotherapies.

Polymer-loaded hydrogels serve as depots for lactate and mimic "cold" tumor microenvironments.

The burgeoning field of biomaterials for immunotherapy has aided in the understanding of foundational mechanisms of cancer immunology. In particular, implantable biomaterials can be engineered to investigate specific aspects of the tumor microenvironment either singularly or in combination. Of note, the metabolite - lactate, a byproduct of anaerobic glycolysis, is known to reprogram immune cells, resulting in increased tumor survival. An adequate model that can recapitulate intratumoral lactate concentrations does not exist. In this study, we demonstrate that a simple biomaterial platform could be developed as an instructive tool to decipher the effects of lactate in vivo. Briefly, we demonstrate that a peptide hydrogel loaded with granulocyte-macrophage colony stimulating factor and poly-(lactic-co-glycolic acid)/(lactic acid) microparticles can generate the localized lactate concentrations (∼2-22 mM) and cellular makeup of the tumor microenvironment, following subcutaneous implantation in mice. Furthermore, infiltrating immune cells adopt phenotypes similar to those seen in other in vitro and in vivo cancer models, including immunosupressive dendritic cells. This hydrogel system is a framework to interrogate immune cell modulation in cancer-like environments using safe and degradable biomaterials. Moreover, this system can be multifaceted, as incorporation of other cancer tumor environmental factors or chemotherapeutic drugs is facile and could be insightful in developing or improving immunotherapies.

Salivary protein biomarkers associated with orthodontic tooth movement: A systematic review.

OBJECTIVE: Saliva can provide a non-invasive approach to indicate changes in the oral and systemic conditions. Salivary proteomics allows the discovery of new protein biomarkers associated with certain conditions. The effectiveness and physiological effects of orthodontic tooth movement in theory can be measured using salivary protein biomarkers. SETTING AND SAMPLE POPULATION: This study applied a systematic review to analyse current literature to define and summarize salivary biomarkers associated with orthodontic tooth movement identified by mass spectrometry proteomics and other protein detection techniques. MATERIALS AND METHODS: Peer-reviewed articles published through the 15th of November 2018 via the PubMed, EMBASE, Web of Science and Dentistry & Oral Sciences databases were reviewed. Only studies analysing protein biomarkers in saliva samples collected from human subjects associated with orthodontic treatments were included. RESULTS: Out of 482 articles, 7 studies were selected. Sample size ranged from 3 to 72 subjects. Minor variations of unstimulated whole saliva sample collection protocol were noted. Mass spectrometry proteomics and ELISA represented the majority of biomarker discovery and targeting, respectively. Twenty biomarkers were identified or chosen as target biomarkers. CONCLUSION: Salivary proteins may be used to indicate effectiveness of orthodontic treatment and orthognathic treatment as well as adverse treatment consequence, such as root resorption.

Combinatorial, Microparticle-Based Delivery of Immune Modulators Reprograms the Dendritic Cell Phenotype and Promotes Remission of Collagen-Induced Arthritis in Mice.

Safe, effective, antigen-specific therapy for rheumatoid arthritis (RA) remains an elusive clinical goal with a few lasting, viable options on the horizon. Existing therapeutic interventions are indiscriminate and inconsistently immunosuppressive, often leaving patients susceptible to infection. Herein, we investigate the use of a dual-sized, microparticle "regulatory vaccine" (REGvac) that passively targets dendritic cells for antigen-specific biomaterial-based immunotherapy of RA. This REGvac employs poly(d,l-lactic-co-glycolic-acid) (PLGA) microparticles (MPs) encapsulating (i) a dendritic cell chemoattractant, (ii) potent immunosuppressive molecules, (iii) and an RA-relevant autoantigen to provide a multifaceted approach for the treatment of collagen-induced arthritis (CIA), the primary mouse model of RA. Subcutaneous administrations of the REGvac after mice had developed moderate clinical symptoms markedly diminished overt inflammation in the paws, halted cartilage degradation, and restored gait parameters within 56 days after initial treatment. Positron emission tomography imaging corroborated reduction of inflammation in the paws of REGvac-treated mice. In-depth immunological assessments showed a decreased expression of CD80, CD86, and MHC II on CD11c+ dendritic cells in joint-associated lymph nodes. Further, we observed significant increases in conventional regulatory CD25+FOXP3+ T cells, as well as programmed cell death protein-1 (PD-1)-expressing CD4+ T cells in joint-proximal lymph nodes and the spleen. Real-time PCR analysis of joint tissues from treated mice revealed significant decreases in inflammatory cytokine expression (IL-6), while IL-10 mRNA levels were significantly increased. These observations strongly hint toward the induction of multiple tolerogenic mechanisms by administration of this MP regulatory vaccine. With regards to antigen specificity, ex vivo antigen recall assays revealed a lack of response to collagen by CD4+ T cells from the popliteal and inguinal lymph nodes of REGvac-treated mice, contrasting with the proliferative response of CD4+ T cells from CIA+ mice. Taken altogether, our results strongly support the application of this MP regulatory vaccine as a potent, biomaterial-based, antigen-specific therapy for RA.

Retrospective analysis of porous tantalum trabecular metal-enhanced titanium dental implants.

STATEMENT OF PROBLEM: The design of porous tantalum trabecular metal-enhanced titanium (TM) dental implants promises improved osseointegration, especially when grafting materials such as demineralized bone matrix are used; however, studies are lacking. PURPOSE: The purpose of this retrospective study was to compare TM implants with conventional titanium alloy (Ti) implants with and without demineralized bone matrix in terms of peri-implant bone remodeling in the first year after implant loading. MATERIAL AND METHODS: A chart review was used for all patients receiving Tapered Screw-Vent Ti and TM implants. Implants were placed and restored by a single provider between 2011 and 2015. Peri-implant bone remodeling was compared by using a paired t test (α=.05). RESULTS: A total of 82 patients received 205 implants, 44 TM and 161 Ti implants (control). No implants failed in the TM group (survival rate of 100%), and 3 implants in total, 1 immediate, failed in the Ti groups (survival rate of 98.1%). TM implants exhibited a 0.28-mm bone gain on average, whereas the control group demonstrated 0.20 mm of marginal bone loss after the first year of implant loading. Multivariate logistic regression analysis demonstrated that the odds of having bone loss was 64% less (odds ratio: 0.36; 95% confidence interval: 0.14-0.94) in the TM group than in the Ti group after controlling for bone grafting, implant location, immediate placement, bone type, and pretreatment bone level. CONCLUSIONS: TM implants exhibited less peri-implant bone loss than the control Ti implants.

Latent, Immunosuppressive Nature of Poly(lactic-co-glycolic acid) Microparticles.

Use of biomaterials to spatiotemporally control the activation of immune cells is at the forefront of biomedical engineering research. As more biomaterial strategies are employed for immunomodulation, understanding the immunogenicity of biodegradable materials and their byproducts is paramount in tailoring systems for immune activation or suppression. Poly(D,L-lactic-co-glycolic acid) (PLGA), one of the most commonly studied polymers in tissue engineering and drug delivery, has been previously described on one hand as an immune adjuvant, and on the other as a nonactivating material. In this study, the effect of PLGA microparticles (MPs) on the maturation status of murine bone marrow-derived dendritic cells (DCs), the primary initiators of adaptive immunity, was investigated to decipher the immunomodulatory properties of this biomaterial. Treatment of bone marrow-derived DCs from C57BL/6 mice with PLGA MPs led to a time dependent decrease in the maturation level of these cells, as quantified by decreased expression of the positive stimulatory molecules MHCII, CD80, and CD86 as well as the ability to resist maturation following challenge with lipopolysaccharide (LPS). Moreover, this immunosuppression was dependent on the molecular weight of the PLGA used to fabricate the MPs, as higher molecular weight polymers required longer incubation to produce comparable dampening of maturation molecules. These phenomena were correlated to an increase in lactic acid both intracellularly and extracellularly during DC/PLGA MP coculture, which is postulated to be the primary agent behind the observed immune inhibition. This hypothesis is supported by our results demonstrating that resistance to LPS stimulation may be due to the ability of PLGA MP-derived lactic acid to inhibit the phosphorylation of TAK1 and therefore prevent NF-κB activation. This work is significant as it begins to elucidate how PLGA, a prominent biomaterial with broad applications ranging from tissue engineering to pharmaceutics, could modulate the local immune environment and offers insight on engineering PLGA to exploit its evolving immunogenicity.

Unimicellar hyperstars as multi-antigen cancer nanovaccines displaying clustered epitopes of immunostimulating peptides.

Unimicellar hyperstar macromolecular chimeras displaying multiple melanoma peptide antigens were prepared primarily via a combination of click chemistry and esterification reactions starting from a biodegradable hyperbranched polymer template. Solubilization of the hyperstars in aqueous solution afforded a multi-antigen unimicellar cancer nanovaccine of about 20 nm. The nanovaccine showed good biocompatibility and uptake by dendritic cells in vitro. An in vivo evaluation of the nanovaccine therapeutic efficacy against melanoma in mice implanted with B16OVA tumors revealed significantly greater T-cell recruitment and improved survival rates for mice treated with nanovaccine and adjuvant compared to non-treated mice.

Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach.

Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

How Accurate Are Implant Surgical Guides Produced With Desktop Stereolithographic 3-Dimentional Printers?

PURPOSE: The use of tooth-supported static stereolithographic guides has greatly improved the ability to ideally place implants. This study was designed to determine the accuracy of in office-printed implant surgical guides. MATERIALS AND METHODS: Using 3shape Implant Studio, a treatment plan for implant placement for tooth 8 was developed using a digital intraoral scan from a Trios scanner and cone-beam computed tomography. Ten stereolithographic guides were printed using a Form2 3-dimensional printer. Pre- and post-implant insertion digital scans were used to determine distance and angulation differences in the mesiodistal and faciolingual positions of the implants compared with the planned position. RESULTS: The mean difference in mesiodistal direction at the alveolar crest between planned implants and placed implants was 0.28 mm (range, 0.05 to 0.62 mm) and the difference in the faciolingual direction was 0.49 mm (range, 0.08 to 0.72 mm). The mean mesiodistal angulation deviation was 0.84° (range, 0.08° to 4.48°) and the mean faciolingual angulation deviation was 3.37° (range, 1.12° to 6.43°). CONCLUSIONS: In-office fabricated stereolithographic implant surgical guides show similar accuracy to laboratory- or manufacturer-prepared guides. This technique provides a convenient and cost-effective means of assuring proper implant placement.

An introduction to biomaterial-based strategies for curbing autoimmunity.

Recently, scientists have made significant progress in the development of immunotherapeutics that correct aberrant, autoimmune responses. Yet, concerns about the safety, efficacy, and wide scale applicability continue to hinder use of contemporary, immunology-based strategies. There is a clear need for therapies that finely control molecular and cellular elements of the immune system. Biomaterial engineers have taken up this challenge to develop therapeutics with selective spatial and temporal control of immune cells. In this review, we introduce the immunology of autoimmune disorders, survey the current therapeutic strategies for autoimmune diseases, and highlight the ongoing research efforts to engineer the immune system using biomaterials, for positive therapeutic outcomes in treatment of autoimmune disorders.

Clotting activity of polyphosphate-functionalized silica nanoparticles.

We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.