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A study was conducted to analyze the molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors. The cyclic synthetic octapeptides based on the sequence of a loop from Agouti protein or Agouti-related protein were functional antagonists of the melanocortin-4 receptor. Results indicated that the antagonist activity of Agouti-related protein correlated with the loop determined by Cys-10 and Cys-117.

To elucidate the molecular basis of the interaction of the native dodecapeptide γ-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced L-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of γ-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when L-Ala was substituted for His[sup 6], Phe[sup 7], Arg[sup 8] and Trp[sup 9]. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in γ-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met³ was replaced with L-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of γ-MSH. [ABSTRACT FROM AUTHOR]

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non-small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14 + , CD68 + , and CD163 + monocytes and CK + tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies., Significance: The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1 + /CD68 + cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background: CD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers., Methods: Tissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated., Results: CD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma., Conclusions: The high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40., (© 2023. The Author(s).)

Pancreatic cancer is marked by a desmoplastic tumor microenvironment and low tumor immunogenicity, making it difficult for immunotherapy drugs to improve outcomes for patients. Tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts (CAFs) are seen in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma (PDAC). In this work, we sought to characterize the expression levels and potential prognostic value of TILs (CD4, CD8, and CD20) and CAFs (Thy-1, FAP, and SMA) in a large retrospective cohort of PDAC patients. Additionally, we investigated the expression levels and prognostic significance of CD200, an immunoinhibitory protein that has shown interest as a potential target for immune checkpoint blockade. We measured the expression levels of these seven proteins with multiplexed immunofluorescence staining and quantitative immunofluorescence (QIF). We found CD8 and FAP to be independent predictors of progression-free survival and overall survival. CD200 was found to be heterogeneously expressed in both the tumor and stromal compartments of PDAC, with the majority of patients having positive stromal expression and negative tumor expression. This work demonstrates the potential clinical utility of CD8 and FAP in PDAC patients, and it sheds light on the expression patterns of CD200 in pancreatic cancer as the protein is being tested as a target for immune checkpoint blockade.

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world's population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector., Competing Interests: The authors have declared that no competing interests exist.

Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.

CD200/CD200R is an immune checkpoint with broad expression patterns and a potential target for immune therapy. In this study, we assess both CD200 and CD200R expression in solid tumors, with a focus on lung cancer, and evaluate their association with clinicopathologic characteristics, mutation status, outcome, and programmed death-ligand 1 (PD-L1) expression. We used multiplexed quantitative immunofluorescence (QIF) to measure the expression of CD200 and CD200R in a total of 455 patients from three lung cancer cohorts. Using carefully validated antibodies, we performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. CD200 tumor positivity was found in 29.7% of non-small cell lung cancer (NSCLC) patients and 33.3% of lung large cell neuroendocrine carcinoma (LCNEC) patients. CD200 demonstrated notable intratumoral heterogeneity. CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation ( p < 0.0001). Neither CD200 nor CD200R were associated with other clinicopathologic characteristics or mutation status. Both biomarkers were not prognostic for disease-free or overall survival in NSCLC. CD200 showed moderate correlation with PD-L1. CD200/CD200R pathway is frequently expressed in lung cancer patients. Differential expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone in patients with NSCLC.

Antibodies play a crucial role in basic research and clinical decision-making. However, there are no standardized algorithms or guidelines to ensure their accuracy and validity. There have been efforts to generate consensus, but, with the exception of clinical labs, antibody validation remains variable in the literature and sometimes in clinical practice. Here we focus on immunohistochemistry, an example of a scientific and clinical tool where validation of antibodies is critical. We describe a protocol that we use to validate antibodies specifically for immunohistochemistry, including some of the pillars of antibody validation from Uhlen et al. 2016, as an example of a rigorous approach to build antibody-based tests for both basic and translational science labs and for the clinic.

Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase in flavivirus-susceptible cells at the infected bite site. In addition, an antibody response to specific salivary gland (SG) components changes the pathogenesis of flaviviruses in human populations. To investigate whether antigenic SG proteins are capable of enhancing infection with Zika virus (ZIKV), a reemerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library and demonstrate that a previously undescribed SG protein we term neutrophil stimulating factor 1 (NeSt1) activates primary mouse neutrophils ex vivo Passive immunization against NeSt1 decreases pro-interleukin-1β and CXCL2 expression, prevents macrophages from infiltrating the bite site, protects susceptible IFNAR -/- IFNGR -/- (AG129) mice from early ZIKV replication, and ameliorates virus-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenvironment, allowing a higher level of early viral replication and enhancing ZIKV pathogenesis. IMPORTANCE When a Zika virus-infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identified a molecule that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We named this molecule neutrophil-stimulating factor 1 (NeSt1). When we used antibodies to block NeSt1 in mice and then allowed Zika virus-infected mosquitoes to feed on these mice, they survived much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection., (Copyright © 2019 American Society for Microbiology.)

Background: Cryosurgery is the most commonly used method to treat actinic keratosis (AK). Cryosurgical methods are not standardized., Objective: To examine differences in the spray techniques used for liquid nitrogen cryosurgery when treating AKs of the head, and the effect of these variations in technique on rates of complete clearance of AKs., Materials and Methods: Patients were those from the FIELD-1 study, who received cryosurgery as per the investigators' usual practice to all AKs. This was followed by topical treatment with either vehicle gel or ingenol mebutate gel, 0.015%, after 3 weeks. The investigator recorded the average duration of cryosurgery spray used, the number of freeze-thaw cycles, and the distance from the tip of the spray device to the AK. Clearance rates were determined at Week 11., Results: Less-aggressive freezing techniques were used for AKs on the face than for those on the scalp. However, higher rates of complete clearance on the face and scalp were associated with more-aggressive freezing techniques., Conclusion: Patients with AKs on the face receive less-aggressive cryosurgery than do patients with AKs on the scalp.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Selective estrogen receptor modulators (SERMs) interact with estrogen receptors as agonists or antagonists depending on the target tissue. Currently available SERMs are used to treat and prevent breast cancer and osteoporosis, to treat ovulatory dysfunction in women, and for contraception. Because current therapies do not adequately treat menopausal symptoms, the search continues for the optimal SERM for postmenopausal women, which would relieve hot flushes, treat vaginal atrophy, and prevent fractures, while protecting the endometrium, breast, and cardiovascular system. Future use of SERMs may also include their use in a tissue selective estrogen complex (TSEC), a therapy that combines a SERM with estrogen(s), designed to deliver the efficacy of each component with improved overall tolerability for the treatment of postmenopausal women. The future of SERMs may also include their use in men for the treatment of osteoporosis and various syndromes associated with secondary hypogonadism and possibly prostate cancer. Continued research should allow the full potential of SERMs to be uncovered., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed., (2010 Elsevier Ltd. All rights reserved.)

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models., (2010 Elsevier Ltd. All rights reserved.)

Objective: The objective of this study was to investigate whether epicardial cryoablation could achieve sufficient endocardial hypothermia to create transmural lesions leading to acute and sustained pulmonary vein isolation in a normothermic beating-heart model., Methods: Thirty-five- to 45-kg pigs underwent median sternotomy. Epicardial cryoablation was performed on the right ventricle after insertion of thermocouples. Endocardial temperatures from thermocouples were recorded continuously and correlated with the thickness of the myocardium. Thirteen animals underwent pulmonary vein isolation as a box lesion by using 5-minute epicardial cryoablation. Endocardial temperatures were measured in 5 of these animals. Ten animals survived for 7 or 30 days. Electrical isolation was tested at the time of surgical intervention and again at death. Hearts were removed en bloc and submitted for gross and microscopic examination., Results: Endocardial temperature varied inversely with tissue thickness, ranging from -60 degrees C in 5-mm-thick tissue to staying unchanged in tissue more than 10 mm thick. During pulmonary vein isolation, median endocardial temperatures were between -1 degrees C and -22 degrees C. Acute electrical isolation was achieved in all 13 animals. All except one of the animals maintained electrical isolation long-term. Histologic analysis revealed transmurality in 89% of sections, although none of the box lesions were completely transmural., Conclusion: Epicardial cryoablation can produce long-term pulmonary vein isolation in a beating heart. Dose-response studies demonstrate consistent endocardial hypothermia in tissues up to 7 mm thick. To our knowledge, this is the first report documenting endocardial hypothermia during epicardial cryoablation. This technology holds promise for performing the complete maze procedure on a beating heart.

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.