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In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [3H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells. [Copyright &y& Elsevier]

Discusses the constitutive activation of the m5 muscarinic receptor by a series of mutations at the extracellular end of transmembrane 6. Factors affecting the increase of the constitutive activity of the receptor; Description of the maximum response of the wild-type receptor to the agonist carbachol; Results of binding and functional assays.

Simple Summary: The use of checkpoint antibodies has revolutionized the treatment of cancer. Tumor-infiltrating T cells, key mediators of anti-tumor immune responses, are often actively silenced by the tumor microenvironment. Checkpoint antibodies block inhibitory signals or enhance positive signaling pathways in these T cells to overcome silencing, resulting in an improved anti-tumor T-cell response. To date, many clinical studies have focused on blocking inhibitory pathways (e.g., CTLA-4 and PD-1), with varying success. Increasingly, alternative checkpoint molecules are being identified and used as monotherapies, or in combination with existing PD-1/CTLA-4 treatments. This review dissects the potential role of checkpoint antibodies against PD-1, VISTA and 4-1BB in the future treatment of cutaneous skin cancers. Non-melanoma skin cancers (NMSC) have a higher incidence than all other cancers combined with cutaneous squamous cell carcinoma (cSCC), capable of metastasis, representing approximately 20% of NMSCs. Given the accessibility of the skin, surgery is frequently employed to treat localized disease, although certain localities, the delineation of clear margins, frequency and recurrence of tumors can make these cancers inoperable in a subset of patients. Other treatment modalities, including cryotherapy, are commonly used for individual lesions, with varying success. Immunotherapy, particularly with checkpoint antibodies, is increasingly a promising therapeutic approach in many cancers, offering the potential advantage of immune memory for protection against lesion recurrence. This review addresses a role for PD-1, 4-1BB and VISTA checkpoint antibodies as monotherapies, or in combination as a therapeutic treatment for both early and late-stage cSCC. [ABSTRACT FROM AUTHOR]

Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier to use and more cost-effective method for administration of vaccines when compared to the needle and syringe. Since MAPs deliver vaccine to the dermis and epidermis, a degree of local immune response at the site of application is expected. In a phase 1 clinical trial (ACTRN 12618000112268), the Vaxxas high-density MAP (HD-MAP) was used to deliver a monovalent, split inactivated influenza virus vaccine into the skin. HD-MAP immunisation led to significantly enhanced humoral responses on day 8, 22 and 61 compared with IM injection of a quadrivalent commercial seasonal influenza vaccine (Afluria Quadrivalent®). Here, the aim was to analyse cellular responses to HD-MAPs in the skin of trial subjects, using flow cytometry and immunohistochemistry. HD-MAPs were coated with a split inactivated influenza virus vaccine (A/Singapore/GP1908/2015 [H1N1]), to deliver 5 μg haemagglutinin (HA) per HD-MAP. Three HD-MAPs were applied to the volar forearm (FA) of five healthy volunteers (to achieve the required 15 μg HA dose), whilst five control subjects received three uncoated HD-MAPs (placebo). Local skin response was recorded for over 61 days and haemagglutination inhibition antibody titres (HAI) were assessed on days 1, 4, 8, 22, and 61. Skin biopsies were taken before (day 1), and three days after HD-MAP application (day 4) and analysed by flow-cytometry and immunohistochemistry to compare local immune subset infiltration. HD-MAP vaccination with 15 μg HA resulted in significant HAI antibody titres compared to the placebo group. Application of uncoated placebo HD-MAPs resulted in mild erythema and oedema in most subjects, that resolved by day 4 in 80% of subjects. Active, HA-coated HD-MAP application resulted in stronger erythema responses on day 4, which resolved between days 22–61. Overall, these erythema responses were accompanied by an influx of immune cells in all subjects. Increased cell infiltration of CD3+, CD4+, CD8+ T cells as well as myeloid CD11b+ CD11c+ and non-myeloid CD11b- dendritic cells were observed in all subjects, but more pronounced in active HD-MAP groups. In contrast, CD19+/CD20+ B cell counts remained unchanged. Key limitations include the use of an influenza vaccine, to which the subjects may have had previous exposure. Different results might have been obtained with HD-MAPs inducing a primary immune response. In conclusion, influenza vaccine administered to the forearm (FA) using the HD-MAP was well-tolerated and induced a mild to moderate skin response with lymphocytic infiltrate at the site of application. [ABSTRACT FROM AUTHOR]

Many G protein-coupled receptors (GPCRs) are therapeutic targets, with most drugs acting at the orthosteric site. Some GPCRs also possess allosteric sites, which have become a focus of drug discovery. In the M2 muscarinic receptor, allosteric modulators regulate the binding and functional effects of orthosteric ligands through a mix of conformational changes, steric hindrance and electrostatic repulsion transmitted within and between the constituent protomers of an oligomer. Tacrine has been called an atypical modulator because it exhibits positive cooperativity, as revealed by Hill coefficients greater than 1 in its negative allosteric effect on binding and response. Radioligand binding and molecular dynamics simulations were used to probe the mechanism of that modulation in monomers and oligomers of wild-type and mutant M2 receptors. Tacrine is not atypical at monomers, which indicates that its atypical effects are a property of the receptor in its oligomeric state. These results illustrate that oligomerization of the M2 receptor has functional consequences. [ABSTRACT FROM AUTHOR]

Prophylactic human papillomavirus (HPV) vaccines are commercially available for prevention of infection with cancerogenic HPV genotypes but are not able to combat pre-existing HPV-associated disease. In this study, we designed a nanomaterial-based therapeutic HPV vaccine, comprising manganese (Mn4+)-doped silica nanoparticles (Mn4+-SNPs) and the viral neoantigen peptide GF001 derived from the HPV16 E7 oncoprotein. We show in mice that Mn4+-SNPs act as self-adjuvants by activating the inflammatory signaling pathway via generation of reactive oxygen species, resulting in immune cell recruitment to the immunization site and dendritic cell maturation. Mn4+-SNPs further serve as Ag carriers by facilitating endo/lysosomal escape via depletion of protons in acidic endocytic compartments and subsequent Ag delivery to the cytosol for cross-presentation. The Mn4+-SNPs+GF001 nanovaccine induced strong E7-specific CD8+ T cell responses, leading to remission of established murine HPV16 E7-expressing solid TC-1 tumors and E7-expressing transgenic skin grafts. This vaccine construct offers a simple and general strategy for therapeutic HPV and potentially other cancer vaccines. [ABSTRACT FROM AUTHOR]

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity. [ABSTRACT FROM AUTHOR]

Pathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T–B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer. OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool. [ABSTRACT FROM AUTHOR]

Background: The Vaxxas high-density microarray patch (HD-MAP) consists of a high density of microprojections coated with vaccine for delivery into the skin. Microarray patches (MAPs) offer the possibility of improved vaccine thermostability as well as the potential to be safer, more acceptable, easier to use, and more cost-effective for the administration of vaccines than injection by needle and syringe (N&S). Here, we report a phase I trial using the Vaxxas HD-MAP to deliver a monovalent influenza vaccine that was to the best of our knowledge the first clinical trial to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs.Methods and Findings: HD-MAPs were coated with a monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/2015 H1N1 haemagglutinin (HA). Between February 2018 and March 2018, 60 healthy adults (age 18-35 years) in Melbourne, Australia were enrolled into part A of the study and vaccinated with either: HD-MAPs delivering 15 μg of A/Singapore/GP1908/2015 H1N1 HA antigen (A-Sing) to the volar forearm (FA); uncoated HD-MAPs; intramuscular (IM) injection of commercially available quadrivalent influenza vaccine (QIV) containing A/Singapore/GP1908/2015 H1N1 HA (15 μg/dose); or IM injection of H1N1 HA antigen (15 μg/dose). After 22 days' follow-up and assessment of the safety data, a further 150 healthy adults were enrolled and randomly assigned to 1 of 9 treatment groups. Participants (20 per group) were vaccinated with HD-MAPs delivering doses of 15, 10, 5, 2.5, or 0 μg of HA to the FA or 15 μg HA to the upper arm (UA), or IM injection of QIV. The primary objectives of the study were safety and tolerability. Secondary objectives were to assess the immunogenicity of the influenza vaccine delivered by HD-MAP. Primary and secondary objectives were assessed for up to 60 days post-vaccination. Clinical staff and participants were blind as to which HD-MAP treatment was administered and to administration of IM-QIV-15 or IM-A/Sing-15. All laboratory investigators were blind to treatment and participant allocation. Two further groups in part B (5 participants per group), not included in the main safety and immunological analysis, received HD-MAPs delivering 15 μg HA or uncoated HD-MAPs applied to the forearm. Biopsies were taken on days 1 and 4 for analysis of the cellular composition from the HD-MAP application sites. The vaccine coated onto HD-MAPs was antigenically stable when stored at 40°C for at least 12 months. HD-MAP vaccination was safe and well tolerated; any systemic or local adverse events (AEs) were mild or moderate. Observed systemic AEs were mostly headache or myalgia, and local AEs were application-site reactions, usually erythema. HD-MAP administration of 2.5 μg HA induced haemagglutination inhibition (HAI) and microneutralisation (MN) titres that were not significantly different to those induced by 15 μg HA injected IM (IM-QIV-15). HD-MAP delivery resulted in enhanced humoral responses compared with IM injection with higher HAI geometric mean titres (GMTs) at day 8 in the MAP-UA-15 (GMT 242.5, 95% CI 133.2-441.5), MAP-FA-15 (GMT 218.6, 95% CI 111.9-427.0), and MAP-FA-10 (GMT 437.1, 95% CI 254.3-751.3) groups compared with IM-QIV-15 (GMT 82.8, 95% CI 42.4-161.8), p = 0.02, p = 0.04, p < 0.001 for MAP-UA-15, MAP-FA-15, and MAP-FA-10, respectively. Higher titres were also observed at day 22 in the MAP-FA-10 (GMT 485.0, 95% CI 301.5-780.2, p = 0.001) and MAP-UA-15 (367.6, 95% CI 197.9-682.7, p = 0.02) groups compared with the IM-QIV-15 group (GMT 139.3, 95% CI 79.3-244.5). Results from a panel of exploratory immunoassays (antibody-dependent cellular cytotoxicity, CD4+ T-cell cytokine production, memory B cell (MBC) activation, and recognition of non-vaccine strains) indicated that, overall, Vaxxas HD-MAP delivery induced immune responses that were similar to, or higher than, those induced by IM injection of QIV. The small group sizes and use of a monovalent influenza vaccine were limitations of the study.Conclusions: Influenza vaccine coated onto the HD-MAP was stable stored at temperatures up to 40°C. Vaccination using the HD-MAP was safe and well tolerated and resulted in immune responses that were similar to or significantly enhanced compared with IM injection. Using the HD-MAP, a 2.5 μg dose (1/6 of the standard dose) induced HAI and MN titres similar to those induced by 15 μg HA injected IM.Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR.org.au), trial ID 108 ACTRN12618000112268/U1111-1207-3550. [ABSTRACT FROM AUTHOR]

The article informs about Polyamino acids as a potent self-adjuvanting delivery system for peptide-based nanovaccines. Topics discussed include potent vaccine delivery system fulfilling the above requirements; particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant; and developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Actinic Keratosis (AK), Intraepidermal Carcinoma (IEC), and Squamous Cell Carcinoma (SCC) are generally considered to be advancing stages of the same disease spectrum. However, while AK often regress spontaneously, and IEC often regress in response to immune-activating treatments, SCC typically do not regress. Therefore, it is vital to define whether fundamental immunological changes occur during progression to SCC. Here we show that proinflammatory cytokine expression, chemokine expression, and immune cell infiltration density change during progression to SCC. Our findings suggest a switch from predominantly proinflammatory cytokine production to chemokine production is a key feature of progression from precancer to cancer. Together, these observations propose a model that can underpin current research and open new avenues of exploration into the clinical significance of these profiles with respect to immunotherapeutic or other treatment outcomes. [ABSTRACT FROM AUTHOR]

Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in individuals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach; potentially ensuring that large numbers of immunized individuals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44–57) administered with Montanide ISA51 adjuvant. [ABSTRACT FROM AUTHOR]

Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk human papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the keratin 14 promoter. We show that HPV16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV16 E7-expressing skin secreted high levels of thymic stromal lymphopoietin (TSLP) and contained increased numbers of innate lymphoid cells (ILCs). High levels of circulating immunoglobulin E were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ- imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]- methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMPPNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. [ABSTRACT FROM AUTHOR]

Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1β, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1β; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chon-drocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1β phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) or hematopoietic stem cell (HSC) transplantation is used as curative therapy for hematologic malignancies. Incorporation of gene therapy to drive tolerogenic expression of antigens is a promising strategy to overcome the limited long-term efficacy of autologous HSC transplantation for autoimmune diseases. HSC engraftment and tolerance induction is readily achieved after myeloablative or immune-depleting conditioning regardless of the cellular compartment in which antigen is expressed. It is unclear whether the efficiency of engraftment and tolerance induction is influenced by targeting antigen to specific cellular compartments. This is particularly important when using clinically feasible low-intensity conditioning aimed at preserving infectious immunity in individuals where immunologic memory exists to the autoantigen to be expressed. Here we demonstrate that, under immune-preserving conditions, confining expression of a transgenically expressed antigen to dendritic cells permits stable, long-term engraftment of genetically modified BM even when recipients are immune to the expressed antigen. In contrast, broader expression within the hematopoietic compartment leads to graft rejection and therapeutic failure because of antigen expression in HSCs. These findings are relevant to the clinical application of genetically engineered HSCs and provide evidence that careful selection of promoters for HSC-mediated gene therapy is important, particularly where tolerance is sought under immune-preserving conditions. [ABSTRACT FROM AUTHOR]

Abstract: Background: G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion. Methods: Fusion proteins of the M2 muscarinic receptor and the α-subunit of Gi1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots. Results: Receptor fused to native αi1 underwent proteolysis near the point of fusion to release a fragment with the mobility of αi1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[3H]methylscopolamine was 60% of that for [3H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. Conclusions: A stable fusion protein of the M2 receptor and truncated αi1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. General significance: Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein. [Copyright &y& Elsevier]

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence life-time imaging, has been used to estimate the size of oligomers formed by the M2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M2 receptor to enhanced green fluorescent protein-M2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values ore as follows: n = 2, 0.495 ± 0.019; n = 4, 0.202 ± 0.010; n = 6, 0.128 ± 0.006; n = 8, 0.093 ± 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 - 0.24, identifying the M2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process. [ABSTRACT FROM AUTHOR]

Abstract: M2 muscarinic receptor extracted from Sf9 cells in cholate–NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588–604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]NMS at either 30 or 4°C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30°C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4°C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M2 muscarinic receptor. [Copyright &y& Elsevier]

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAIPyP4) and meso-tetrakis(4N-metal- lylpyridinium-4-yl) porphyrin (TMetAIPyP4) with the poly(rA)poly(rU) and poly(rl)poly(rC) ANA duplexes between 18 and 45 °C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAIPyP4 intercalate into the poly- (rA)poly(rU) and poly(rl)poly(rC) host duplexes, while TMetAIPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, ΔGb, enthalpies, ΔHb, and entropies, ΔSb. For each RNA duplex, the binding enthalpy, ΔHb, is the most favorable for TEtOHPyP4 (an intercalator) followed by TAIPyP4 (an intercalator) and TMetAIPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAIPyP4 produces the most favorable change in entropy, ΔSb, followed by the intercalators TAIPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex. [ABSTRACT FROM AUTHOR]

Abstract: The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials. [Copyright &y& Elsevier]

Examines the cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Role of oligomers in the coexpression of isoforms; Effects on adenylate cyclase by desensitized mutant Β[sub 2]-adrenergic receptor; Solubilization in sodium cholate.

Simple Summary: Therapies selectively targeting cancer-specific defects have the advantage of minimising damage to normal tissue including the immune system. The work described here investigates a therapy that targets replication stress, a common feature of many cancer types including melanoma. We demonstrate that this therapy not only selectively kills tumours but also triggers pro-immunogenic signals from the tumour to attract immune cells. In animal models, it has no adverse effects on immune response and triggers a strong anti-tumour immune response. The major component of this response are specialised immune cells, but the tumour itself trigger a conversion of this anti-tumour response to an immune suppressive response that cannot be overcome with current immunotherapies. The work demonstrates that understanding the immune response triggered is essential to guide the selection of the optimal immunotherapy to promote long term tumour control. Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response. [ABSTRACT FROM AUTHOR]

T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8+ Cytotoxic T lymphocytes (CTL), as well as therapeutic strategies trialled to overcome these tumour-evasion mechanisms. Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models. [ABSTRACT FROM AUTHOR]

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lu, Kong, Zhang, Firdaus, Wells, Stephenson, Toth, Skwarczynski and Cruz.)

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG., (© 2024. The Author(s).)

Background: Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity., Methods: The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections., Results: The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4 + FoxP3 + cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor., Conclusions: Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0-21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sartain, Viecelli, Veitch, Franklin, Dymock, Wells and Campbell.)

Barrier-to-autointegration factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1's DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1's N-terminal domain.

Background: Immunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus also suppresses T-cell function in the skin, contributing to a high incidence of skin cancer and associated mortality and morbidity in solid organ transplant recipients. Here, we aimed to identify a compound capable of re-establishing antitumor T-cell control in the skin despite the presence of tacrolimus., Methods: In this study, we performed time-resolved fluorescence resonance energy transfer to identify molecules capable of antagonizing the interaction between tacrolimus and FKBP12. The capacity of these molecules to rescue mouse and human T-cell function in the presence of tacrolimus was determined in vitro, and the antitumor effect of the lead compound, Q-2361, was assessed in "regressor" models of skin cancer in immunosuppressed mice. Systemic CD8 T-cell depletion and analyses of intratumoral T-cell activation markers and effector molecule production were performed to determine the mechanism of tumor rejection. Pharmacokinetic studies of topically applied Q-2361 were performed to assess skin and systemic drug exposure., Results: Q-2361 potently blocked the interaction between tacrolimus and FKBP12 and reversed the inhibition of the nuclear factor of activated T cells activation by tacrolimus following T-cell receptor engagement in human Jurkat cells. Q-2361 rescued T-cell function in the presence of tacrolimus, rapamycin, and everolimus. Intratumoral injection of Q-2361-induced tumor regression in mice systemically immune suppressed with tacrolimus. Mechanistically, Q-2361 treatment permitted T-cell activation, proliferation, and effector function within tumors. When CD8 T cells were depleted, Q-2361 could not induce tumor regression. A simple solution-based Q-2361 topical formulation achieved high and sustained residence in the skin with negligible drug in the blood., Conclusions: Our findings demonstrate that the local application of Q-2361 permits T-cells to become activated driving tumor rejection in the presence of tacrolimus. The data presented here suggests that topically applied Q-2361 has great potential for the reactivation of T-cells in the skin but not systemically, and therefore represents a promising strategy to prevent or treat skin malignancies in immunosuppressed organ transplant recipients., Competing Interests: Competing interests: The use of Q-2361 as disclosed in this study has been patented (WO2021248189), with JWW, BWD, AH, T-AC, RP, and KB as coinventors. All other authors declare that they have no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Osteoarthritis (OA) is a leading cause of disability with no cure and only supportive therapy. Adeno-associated virus (AAV) serotype 2.5 is being used in a Phase I clinical trial to deliver the interleukin-1 receptor antagonist into knee joints with OA. Neutralizing antibodies (Nab) directed against AAV2.5, if present, could inhibit gene transfer. Here, we report the prevalence of AAV2.5 Nab in the sera and synovial fluids of patients with OA. Nab titers were measured by their ability to inhibit in vitro transduction by AAV2.5 encoding GFP. Of 44 synovial fluids from patients with mid-stage and advanced OA, 43% had undetectable Nab; 25% had low titers (<1:100), 16% had medium titers (1:100-1:1000) and 16% had high titers (>1:1000) of Nab. Titers of AAV2.5 Nabs correlated with those of AAV2, but not with those of AAV5. Serum titers of AAV2.5 Nab correlated positively with titers in synovial fluid, and were never less than the matched synovial fluid titers. These findings suggest that high titers of Nab against AAV2.5 are uncommon in the synovial fluids of patients with OA, and individuals with high synovial fluid Nab titers can be identified by measuring titers in the serum., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Inflammatory skin conditions are the 4 th leading cause of non-fatal health burden in the general population worldwide. The diagnosis of skin lesions due to systemic drug reactions, viral or bacterial exanthems, or in patients with psoriasis, atopic dermatitis or contact dermatitis is often difficult and relies heavily upon conventional histopathologic examination. Conversely, it is widely accepted that the cutaneous profile of inflammatory markers, or 'inflammatory signature', is differentially expressed in various skin conditions. In this pilot study, we investigated the possibility of inflammatory skin disease diagnosis from an immunological perspective in small punch biopsies. We collected lesional and perilesional punch biopsies from 139 patients suffering from a variety of inflammatory skin conditions and attending the Dermatology Department at the Princess Alexandra Hospital in Brisbane, Australia. Using bead-based immunoassays we were able to measure 13 out of 17 inflammatory markers from a pre-selected multi-analyte panel and to detect significant differences between lesional and perilesional biopsies from each individual patient. Hierarchical and unbiased clustering methods based on inflammatory signatures grouped psoriasis and atopic dermatitis lesions into individual clusters in contrast to other skin conditions, highlighting the potential of inflammatory signatures to be used as diagnostic differentiators and to inform alternative targets in anti-inflammatory treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Lin, McMeniman, Wu, Kao, Kumari, Boyle, Wells, Soyer and Gonzalez-Cruz.)

We employed a previously described procedure, based on circular dichroism (CD) spectroscopy, to quantify the distribution of conformational states adopted by equimolar mixtures of complementary G-rich and C-rich DNA strands from the promoter regions of the VEGF and Bcl-2 oncogenes. Spectra were recorded at different pHs, concentrations of KCl, and temperatures. The temperature dependences of the fractional populations of the duplex, G-quadruplex, i -motif, and coiled conformations of each promoter were then analyzed within the framework of a thermodynamic model to obtain the enthalpy and melting temperature of each folded-to-unfolded transition involved in the equilibrium. A comparison of the conformational data on the VEGF and Bcl-2 DNA with similar results on the c-MYC DNA, which we reported previously, reveals that the distribution of conformational states depends on the specific DNA sequence and is modulated by environmental factors. Under the physiological conditions of room temperature, neutral pH, and elevated concentrations of potassium ions, the duplex conformation coexists with the G-quadruplex conformation in proportions that depend on the sequence. This observed conformational diversity has biological implications, and it further supports our previously proposed thermodynamic hypothesis of gene regulation. In that hypothesis, a specific distribution of duplex and tetraplex conformations in a promoter region is fine-tuned to maintain the healthy level of gene expression. Any deviation from a healthy distribution of conformational states may result in pathology stemming from up- or downregulation of the gene.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient's autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies., Competing Interests: Declaration of interests The authors declare no competing interests other than the following: The avelumab work was conducted under investigator-initiated contract with Merck KGaA. The costs of the experiments were paid for by Merck KGaA and avelumab supplied by Merck. This work utilizes technology subject to the following patents: 2015 WO2014063206-A1 “classifying epidermal growth factor receptor positive tumor into subtype eg epidermal growth factor receptor antagonist sensitive subtype, involves analyzing ligand-induced epidermal growth factor receptor internalization status of tumor.” Simpson, F., Saunders, N.A.; 2015 WO2014063205-A1 “composition useful in kit for treating tumor, preferably cell surface antigen positive tumor eg cancerous tumors, comprises antibody that binds to cell surface antigen of tumor and inhibitor of receptor mediated endocytosis” Simpson, F., Saunders, N.A., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We characterized the conformational preferences of DNA in an equimolar mixture of complementary G-rich and C-rich strands from the promoter region of the c-MYC oncogene. Our CD-based approach presupposes that the CD spectrum of such a mixture is the spectral sum of the constituent duplex, G-quadruplex, i -motif, and coiled conformations. Spectra were acquired over a range of temperatures at different pHs and concentrations of KCl. Each spectrum was unmixed in terms of the predetermined spectra of the constituent conformational states to obtain the corresponding weighting factors for their fractional contributions to the total population of DNA. The temperature dependences of those contributions then were analyzed in concert according to a model based on a thermodynamic representation of the underlying equilibria. Fitted estimates of the melting enthalpy and temperature obtained for the duplex, G-quadruplex, and i -motif imply that the driving force behind dissociation of the duplex and the concomitant formation of tetrahelical structures is the folding of the G-strand into the G-quadruplex. The liberated C-strand adopts the i -motif conformation at acidic pH and exists in the coiled state at neutral pH. The i -motif alone cannot induce dissociation of the duplex even at pH 5.0, at which it is most stable. Under the physiological conditions of neutral pH, elevated potassium, and room temperature, the duplex and G-quadruplex conformations coexist with the C-strand in the coiled state. Taken together, our results suggest a novel, thermodynamically controlled mechanism for the regulation of gene expression.

Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro . Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA i n vivo . The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development.

EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in three-dimensional culture. These studies have been completed via genetic sequencing, cell line, or three-dimensional in vitro and in vivo murine models. Here, we describe an imaging method that allows ex vivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumors. We analyzed sets of tumor samples from advanced cutaneous squamous cell carcinoma and head and neck squamous cell carcinoma, actinic keratosis, intraepidermal carcinoma, and cutaneous squamous cell carcinoma. We show that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intraepidermal carcinoma, and well-differentiated cutaneous squamous cell carcinoma, different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live ex vivo human tumors confirm that endocytosis dysregulation is a physiological event in human tumors and has therapeutic implications., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunotherapy for skin malignancies has ushered in a new era for cancer treatments by demonstrating unprecedented durable responses in the setting of metastatic Melanoma. Consequently, checkpoint inhibitors are now the first-line treatment of metastatic melanoma and widely used as adjuvant therapy for stage III disease. With the observation that higher tumor mutational burden correlates with a better response, checkpoint inhibitors are tested in other skin cancer types of known high tumor mutational burden with promising results and recently became the first-ever FDA-approved treatment for metastatic Merkel cell carcinoma. The emerging new standards-of-care will necessitate more precise biomarkers and predictors for treatment response and immune-related adverse events. Measurable immune-related mediators are currently under investigation as factors that promote or block the response to cancer immunotherapy and may provide insights into the underlying immune response to the tumor. Cytokines and chemokines are such mediators and are crucial for facilitating the recruitment and activation of specific subsets of leukocytes within the microenvironment of skin cancers. The exact mechanisms of how these meditators, both immunological and non-immunological, operate in the tumor microenvironment is an area of active research, so to reliable biomarkers of responses to cancer immunotherapy. Here, we will review and summarize the expanding body of literature for immune-related biomarkers pertaining to Melanoma, Basal cell carcinoma, Squamous cell carcinoma, and Merkel cell carcinoma, highlighting clinically relevant checkpoint inhibitor therapy biomarker advancements.

Chemokines and their receptors play an important role in the recruitment, activation and differentiation of immune cells. The chemokine receptor, CXCR3, and its ligands, CXCL9, CXCL10, and CXCL11 are key immune chemoattractants during interferon-induced inflammatory responses. Inflammation of the skin resulting from infections or autoimmune disease drives expression of CXCL9/10/11 and the subsequent recruitment of effector, CXCR3 + T cells from the circulation. The relative contributions of the different CXCR3 chemokines and the three variant isoforms of CXCR3 (CXCR3A, CXCR3B, CXCR3alt) to the inflammatory process in human skin requires further investigation. In skin cancers, the CXCR3 receptor can play a dual role whereby expression on tumor cells can lead to cancer metastasis to systemic sites while receptor expression on immune cells can frequently promote anti-tumor immune responses. This review will discuss the biology of CXCR3 and its associated ligands with particular emphasis on the skin during inflammation and carcinogenesis.

Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M 2 muscarinic receptor and of G i1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M 2 and mCherry-M 2 ) and G proteins (i.e., eGFP-Gα i1 β 1 γ 2 and mCherry-Gα i1 β 1 γ 2 ) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and G i1 can exist as homo-oligomers. Oligomerization of differently tagged G i1 decreased upon the activation of co-expressed wild-type M 2 receptor by an agonist. Measurements on a tagged M 2 receptor (M 2 -mCherry) and eGFP-Gα i1 β 1 γ 2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M 2 (D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gα i1 β 1 γ 2 , indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M 2 receptor on the one hand and of G i1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Patients receiving immunosuppressive drugs to prevent organ transplant rejection exhibit a greatly increased risk of developing cutaneous squamous cell carcinoma (SCC). However, not all immunosuppressive drugs confer the same risk. Randomised, controlled trials demonstrate that switching renal transplant recipients receiving calcineurin inhibitor-based therapies to mammalian target of rapamycin (mTOR) inhibitors results in a reduced incidence of de novo SSC formation, and can even result in the regression of pre-existing premalignant lesions. However, the contribution played by residual immune function in this setting is unclear. We examined the hypotheses that mTOR inhibitors promote the enhanced differentiation and function of CD8 + memory T cells in the skin. Here, we demonstrate that the long-term oral administration of rapamycin to achieve clinically-relevant whole blood drug target thresholds, creates a "low rapamycin dose" environment in the skin. While both rapamycin and the calcineurin inhibitor tacrolimus elongated the survival of OVA-expressing skin grafts, and inhibited short-term antigen-specific CD8 + T cell responses, rapamycin but not tacrolimus permitted the statistically significant infiltration of CD8 + effector memory T cells into UV-induced SCC lesions. Furthermore, rapamycin uniquely enhanced the number and function of CD8 + effector and central memory T cells in a model of long-term contact hypersensitivity provided that rapamycin was present during the antigen sensitization phase. Thus, our findings suggest that patients switched to mTOR inhibitor regimens likely experience enhanced CD8 + memory T cell function to new antigen-challenges in their skin, which could contribute to their lower risk of de novo SSC formation and regression of pre-existing premalignant lesions.

Detecting naïve antigen-specific B cells can be challenging. Use of multiple, complementary tetramers with different fluorochromes enhances sensitivity and specificity allowing naïve antigen-specific B cells to be readily distinguished within a polyclonal repertoire. Activated, affinity-matured B cells, however, can be detected effectively using a single tetramer., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD4 + CD8 + double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice. DP T cells were also present in Jα18 -/- and CD1d -/- mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number. We explored the possibility that DP T cells could represent aggregates between CD4 + and CD8 + single-positive T cells and found strong evidence that a large proportion of apparent DP T cells were indeed aggregates. However, the existence of true CD4 + CD8 + DP T cells was confirmed by Amnis ImageStream (Millipore Sigma, Billerica, MA, USA) imaging. Multiple rounds of FACS sorting separated true DP cells from aggregates and indicated that conventional analyses may lead to ∼10-fold overestimation of DP T cell numbers. The high degree of aggregate contamination and overestimation of DP abundance using conventional analysis techniques may explain discrepancies reported in the literature for DP T cell origin, phenotype, and function., (© Society for Leukocyte Biology.)

We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.