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International Journal of Molecular... Dec 2022Hypertrophic scars continue to be a major burden, especially after burns. Persistent inflammation during wound healing appears to be the precipitating aspect in...
Hypertrophic scars continue to be a major burden, especially after burns. Persistent inflammation during wound healing appears to be the precipitating aspect in pathologic scarring. The lack of a standardized model hinders research from fully elucidating pathophysiology and therapy, as most therapeutic approaches have sparse evidence. The goal of this project was to investigate the mechanisms of scar formation after prolonged wound inflammation and to introduce a method for generating standardized hypertrophic scars by inducing prolonged inflammation. Four wound types were created in Duroc pigs: full-thickness wounds, burn wounds, and both of them with induced hyperinflammation by resiquimod. Clinical assessment (Vancouver Scar Scale), tissue oxygenation by hyperspectral imaging, histologic assessment, and gene expression analysis were performed at various time points during the following five months. Native burn wounds as well as resiquimod-induced full-thickness and burn wounds resulted in more hypertrophic scars than full-thickness wounds. The scar scale showed significantly higher scores in burn- and resiquimod-induced wounds compared with full-thickness wounds as of day 77. These three wound types also showed relative hypoxia compared with uninduced full-thickness wounds in hyperspectral imaging and increased expression of levels. The highest number of inflammatory cells was detected in resiquimod-induced full-thickness wounds with histologic features of hypertrophic scars in burn and resiquimod-induced wounds. Gene expression analysis revealed increased inflammation with only moderately altered fibrosis markers. We successfully created hypertrophic scars in the Duroc pig by using different wound etiologies. Inflammation caused by burns or resiquimod induction led to scars similar to human hypertrophic scars. This model may allow for the further investigation of the exact mechanisms of pathological scars, the role of hypoxia and inflammation, and the testing of therapeutic approaches.
Topics: Animals; Burns; Cicatrix, Hypertrophic; Inflammation; Swine; Wound Healing
PubMed: 36613761
DOI: 10.3390/ijms24010316 -
Frontiers in Immunology 2017Toll-like receptors (TLR) triggering of B cells are known to promote B cell expansion, differentiation of B cells into antibody-producing and memory cells, but the TLR...
Toll-like receptors (TLR) triggering of B cells are known to promote B cell expansion, differentiation of B cells into antibody-producing and memory cells, but the TLR responses of porcine B cells is poorly characterized. Therefore, this study investigated the response pattern of porcine B cell subsets to a large collection of TLR ligands and demonstrates that the TLR2 ligand Pam3Cys-SK4 and the TLR7/8 ligands gardiquimod and resiquimod are particularly efficient at inducing proliferation, CD25 and CCR7. This activation was also determined in B-cell subpopulations including a CD21IgM subset, an IgG subset and two putative B1-like subsets, defined as CD21IgMCD11R1CD11cCD14 and CD21IgM CD11R1CD11cCD14 B cells. The latter two were larger and expressed higher levels of CD80/86 and spontaneous phospholipase C-γ2 phosphorylation. All porcine B-cell subsets were activated by TLR2, TLR7, and TLR9 ligands. Naïve and memory conventional B cells responded similar to TLR ligands. The CD11R1 B1-like subset had the highest proliferative responses. While both B1-like subsets did not spontaneously secrete IgM, they were the only subsets to produce high level of TLR-induced IgM. Similar to polyclonal IgM responses, memory B cells were efficiently induced to produce specific antibodies by CpG oligodinucleotide, resiquimod, and to a weaker extend by Pam3Cys-SK4. Depletion of plasmacytoid dendritic cells (pDCs) enhanced TLR-induced antibodies. The same set of TLR ligands also induced CD40 on cDCs, pDCs, and monocytes with the exception of TLR4 ligand being unable to activate pDCs. Gardiquimod and resiquimod were particularly efficient at inducing CCR7 on pDCs. Porcine B cells expressed high levels of TLR7, but relatively little other TLR mRNA. Nevertheless, TLR2 on B cells was rapidly upregulated following stimulation, explaining the strong responses following stimulation. Subset-specific analysis of TLR expression demonstrated a comparable expression of TLR2, TLR7, and TLR9 in all B cell subsets, but TLR3 was restricted to B1-like cells, whereas TLR4 was only expressed on conventional B cells, although both at low levels. Altogether, our data describe porcine innate B1-like cells, and how different B cell subsets are involved in innate sensing.
PubMed: 28890720
DOI: 10.3389/fimmu.2017.01044 -
Journal of Controlled Release :... Dec 2018Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to...
Lipid conjugation of TLR7 agonist Resiquimod ensures co-delivery with the liposomal Cationic Adjuvant Formulation 01 (CAF01) but does not enhance immunopotentiation compared to non-conjugated Resiquimod+CAF01.
Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to pathogens and microorganisms: the rapid innate immune response (as characterised by the production of Th1 promoting cytokines and type 1 interferons) and the adaptive immune response. Through this activation, ligands and agonists of TLRs can enhance immunotherapeutic efficacy. Resiquimod is a small (water-soluble) agonist of the endosome-located Toll-like receptors 7 and 8 (TLR7/8). However due to its molecular attributes it rapidly distributes throughout the body after injection. To circumvent this, these TLR agonists can be incorporated within delivery systems, such as liposomes, to promote the co-delivery of both antigen and agonists to antigen presenting cells. In this present study, resiquimod has been chemically conjugated to a lipid to form a lipid-TLR7/8 agonist conjugate which can be incorporated within immunogenic cationic liposomes composed of dimethyldioctadecylammonium bromide (DDA) and the immunostimulatory glycolipid trehalose 6,6' - dibehenate (TDB). This DDA:TDB-TLR7/8 formulation offers similar vesicle characteristics to DDA:TDB (size and charge) and offers high retention of both resiquimod and the electrostatically adsorbed TB subunit antigen Ag85B-ESAT6-Rv2660c (H56). Following immunisation through the intramuscular (i.m.) route, these cationic DDA:TDB-TLR7/8 liposomes form a vaccine depot at the injection site. However, immunisation studies have shown that this biodistribution does not translate into notably increased antibody nor Th1 responses at the spleen and draining popliteal lymph node compared to DDA:TDB liposomes. This work demonstrates that the conjugation of TLR7/8 agonists to cationic liposomes can promote co-delivery but the immune responses stimulated do not merit the added complexity considerations of the formulation.
Topics: Adjuvants, Immunologic; Animals; Female; Glycolipids; Imidazoles; Lipids; Liposomes; Mice, Inbred BALB C; Quaternary Ammonium Compounds; Toll-Like Receptor 7; Vaccines
PubMed: 30291987
DOI: 10.1016/j.jconrel.2018.10.002 -
Journal of Controlled Release :... Nov 2022Effective drug delivery requires ample dosing at the target tissue while minimizing negative side effects. Drug delivery vehicles such as polymeric nanoparticles (NPs)...
Effective drug delivery requires ample dosing at the target tissue while minimizing negative side effects. Drug delivery vehicles such as polymeric nanoparticles (NPs) are often employed to accomplish this challenge. In this work, drug release of numerous drugs from surface eroding polymeric NPs was evaluated in vitro in physiologically relevant pH 5 and neutral buffers. NPs were loaded with paclitaxel, rapamycin, resiquimod, or doxorubicin and made from an FDA approved polyanhydride or from acetalated dextran (Ace-DEX), which has tunable degradation rates based on cyclic acetal coverage (CAC). By varying encapsulate, pH condition, and polymer, a range of distinct drug release profiles were achieved. To model the obtained drug release curves, a mechanistic mathematical model was constructed based on drug diffusion and polymer degradation. The resulting diffusion-erosion model accurately described drug release from the variety of surface eroding NPs. For drug release from varied CAC Ace-DEX NPs, the goodness of fit of the developed diffusion-erosion model was compared to several conventional drug release models. The diffusion-erosion model maintained optimal fit compared to conventional models across a range of conditions. Machine learning was then employed to estimate effective diffusion coefficients for the diffusion-erosion model, resulting in accurate prediction of in vitro release of dexamethasone and 3'3'-cyclic guanosine monophosphate-adenosine monophosphate from Ace-DEX NPs. This predictive modeling has potential to aid in the design of future Ace-DEX formulations where optimized drug release kinetics can lead to a desired therapeutic effect.
Topics: Drug Liberation; Dextrans; Polymers; Nanoparticles; Drug Delivery Systems; Pharmaceutical Preparations
PubMed: 36208792
DOI: 10.1016/j.jconrel.2022.09.067 -
Nature Communications May 2024In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed... (Randomized Controlled Trial)
Randomized Controlled Trial
In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.
Topics: Humans; Dendritic Cells; Glioma; Female; Male; Interferons; Middle Aged; Cancer Vaccines; CD8-Positive T-Lymphocytes; Poly I-C; Adult; Toll-Like Receptors; Imidazoles; Aged; Vaccination; Monocytes; Brain Neoplasms; CD4-Positive T-Lymphocytes; Immunotherapy; Toll-Like Receptor Agonists; Carboxymethylcellulose Sodium; Polylysine
PubMed: 38719809
DOI: 10.1038/s41467-024-48073-y -
Oncoimmunology Aug 2013Toll-like receptors (TLRs) have long been known for their ability to initiate innate immune responses upon exposure to conserved microbial components such as... (Review)
Review
Toll-like receptors (TLRs) have long been known for their ability to initiate innate immune responses upon exposure to conserved microbial components such as lipopolysaccharide (LPS) and double-stranded RNA. More recently, this family of pattern recognition receptors has been attributed a critical role in the elicitation of anticancer immune responses, raising interest in the development of immunochemotherapeutic regimens based on natural or synthetic TLR agonists. In spite of such an intense wave of preclinical and clinical investigation, only three TLR agonists are currently licensed by FDA for use in cancer patients: bacillus Calmette-Guérin (BCG), an attenuated strain of that operates as a mixed TLR2/TLR4 agonist; monophosphoryl lipid A (MPL), a derivative of that functions as a potent agonist of TLR4; and imiquimod, a synthetic imidazoquinoline that activates TLR7. One year ago, in the August and September issues of , we described the main biological features of TLRs and discussed the progress of clinical studies evaluating the safety and therapeutic potential of TLR agonists in cancer patients. Here, we summarize the latest developments in this exciting area of research, focusing on preclinical studies that have been published during the last 13 mo and clinical trials launched in the same period to investigate the antineoplastic activity of TLR agonists.
PubMed: 24083080
DOI: 10.4161/onci.25238 -
Pharmaceutics Mar 2021Recent advances in immunotherapy have revolutionized cancer therapy. Immunotherapies can engage the adaptive and innate arms of the immune system. Therapeutics targeting...
Recent advances in immunotherapy have revolutionized cancer therapy. Immunotherapies can engage the adaptive and innate arms of the immune system. Therapeutics targeting immune checkpoint inhibitors (i.e., CTLA-4; PD-1, and PD-L1) have shown efficacy for subsets of cancer patients by unleashing an adaptive antitumor immune response. Alternatively, small molecule immune modulators of the innate immune system such as toll-like receptor (TLR) agonists are being developed for cancer therapy. TLRs function as pattern recognition receptors to microbial products and are also involved in carcinogenesis. Reisquimod is a TLR 7/8 agonist that has antitumor efficacy. However, systemic delivery free resiquimod has proven to be challenging due to toxicity of nonspecific TLR 7/8 activation. Therefore, we developed a targeted peptide-drug conjugate strategy for systemic delivery of resiquimod. We designed an activatable cell penetrating peptide to deliver resiquimod specifically to the tumor tissue while avoiding normal tissues. The activatable cell penetrating peptide (ACPP) scaffold undergoes enzymatic cleavage by matrix metalloproteinases 2/9 in the extracellular matrix followed by intracellular lysosomal cathepsin B mediated release of the free resiquimod. Importantly, when conjugated to ACPP; the tumor tissue concentration of resiquimod was more than 1000-fold greater than that of surrounding non-cancerous tissue. Moreover, systemic ACPP-resiquimod delivery produced comparable therapeutic efficacy to localized free resiquimod in syngeneic murine tumors. These results highlight a precision peptide-drug conjugate delivery.
PubMed: 33801967
DOI: 10.3390/pharmaceutics13030365 -
International Journal of Molecular... Jun 2019Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The cause of multiple sclerosis is unknown but there are several...
BACKGROUND
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The cause of multiple sclerosis is unknown but there are several evidences that associate the genetic basis of the disease with environmental causes. An important association between viral infection and development of MS is clearly demonstrated. Viruses have a strong impact on innate immune cells. In particular, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), are able to respond to viruses and to activate the adaptive immune response.
METHODS
In this study we mimic viral infection using synthetic single-strand RNA, Resiquimod, and we compared the response of both DC subsets derived from healthy donors and MS patients by characterizing the expression of costimulatory molecules on the DC surface.
RESULTS
We found that pDCs from MS patients express higher levels of OX40-L, HLA-DR, and CD86 than healthy donors. Moreover, we found that blood cells from MS patients and healthy donors upon Resiquimod-stimulation are enriched in a subpopulation of pDCs, characterized by a high amount of costimulatory molecules.
CONCLUSION
Overall, these results indicate that activation of pDCs is enhanced in MS, likely due to a latent viral infection, and that costimulatory molecules expressed on pDCs could mediate a protective response against the viral trigger of autoimmunity.
Topics: B7-2 Antigen; Dendritic Cells; HLA-DR Antigens; Humans; Imidazoles; Multiple Sclerosis, Relapsing-Remitting; OX40 Ligand
PubMed: 31181776
DOI: 10.3390/ijms20112811 -
Indian Journal of Hematology & Blood... Oct 2023Targeting toll-like receptors (TLRs), via TLR agonists, has been implicated in the regulation of immunometabolism. B-chronic lymphocytic leukemia (B-CLL) represents a...
UNLABELLED
Targeting toll-like receptors (TLRs), via TLR agonists, has been implicated in the regulation of immunometabolism. B-chronic lymphocytic leukemia (B-CLL) represents a suitable model for B-cell derived malignancies with shifted metabolic adaptations. Several signaling pathways have been found to be critical in metabolic reprogramming of CLL, including mechanistic target of rapamycin- hypoxia inducible factor-1α (mTOR- HIF-1α) pathway, the main metabolic regulator of glycolysis. Here, we investigated the effect of TLR7/8 agonist (Resiquimod) on the expression of mTOR and HIF-1α in patients with CLL. B cells were purified using Rosettesep Human B cell Enrichment Cocktail (Stem cell Technologies, Vancouver, BC, Canada#15,024) from peripheral venous blood of CLL patients (n = 20) and healthy individuals (n = 15). Isolated B cells were then cultured in both presence and absence of Resiquimod. Gene expression of mTOR and HIF-1α were assessed using qRT-PCR. Resiquimod significantly decreased mTOR and HIF-1α gene expression in both CLL ( < 0.001and < 0.001, respectively) and Normal B cells ( = 0.004 and = 0.001, respectively). Resiquimod may reprogram immunometabolism of malignant B-CLL cells via down-regulation of key glycolytic metabolic actors, mTOR and HIF-1α genes. Accordingly, Resiquimod may be an adjuvant as a therapeutic tool for CLL, which needs to be studied further.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s12288-023-01649-y.
PubMed: 37786827
DOI: 10.1007/s12288-023-01649-y -
Advanced Science (Weinheim,... Dec 2023Patients with colorectal cancer (CRC) and diffuse peritoneal metastasis (PM) are not eligible for surgical intervention. Thus, palliative treatment remains the standard...
Patients with colorectal cancer (CRC) and diffuse peritoneal metastasis (PM) are not eligible for surgical intervention. Thus, palliative treatment remains the standard of care in clinical practice. Systemic chemotherapy fails to cause drug accumulation at the lesion sites, while intraperitoneal chemotherapy (IPC) is limited by high clearance rates and associated complications. Given the poor prognosis, a customized OxP/R848@PLEL hydrogel delivery system has been devised to improve the clinical benefit of advanced CRC with diffuse PM. This system is distinguished by its simplicity, security, and efficiency. Specifically, the PLEL hydrogel exhibits excellent injectability and thermosensitivity, enabling the formation of drug depots within the abdominal cavity, rendering it an optimal carrier for IPC. Oxaliplatin (OxP), a first-line drug for advanced CRC, is cytotoxic and enhances the immunogenicity of tumors by inducing immunogenic cell death. Furthermore, OxP and resiquimod (R848) synergistically enhance the maturation of dendritic cells, promote the expansion of cytotoxic T lymphocytes, and induce the formation of central memory T cells. Moreover, R848 domesticates macrophages to an anti-tumor phenotype. OxP/R848@PLEL effectively eradicates peritoneal metastases, completely inhibits ascites production, and significantly prolongs mice lifespan. As such, it provides a promising approach to managing diffuse PM in patients with CRC without surgical indications.
Topics: Humans; Animals; Mice; Hydrogels; Peritoneal Neoplasms; Colorectal Neoplasms; Antineoplastic Agents; Oxaliplatin; Immunotherapy
PubMed: 37875399
DOI: 10.1002/advs.202303819