Soluble Chemokines Research Articles

Sensing Soluble Immune Checkpoint Molecules and Disease-Relevant Cytokines in Cancer: A Novel Paradigm in Disease Diagnosis and Monitoring

Conventional detection of cancer involves highly invasive and expensive diagnostic procedures, often leading to non-compliance from patients. Therefore, there is a strong requirement for the development of non-invasive techniques that can facilitate rapid and timely diagnosis of the disease. The tumor-immune interaction often leads to anomalous expression of different soluble immune signaling molecules like cytokines and chemokines, thus making them promising candidates for sensing disease development and progression. Furthermore, differential expression of soluble isoforms of several immune-checkpoint molecules like PD-L1, CTLA-4 etc., has been found to have strong correlation with tissue-specific tumor development, disease progression and in many cases, disease prognosis. Therefore, development of biosensors, to rapidly detect and analyze the levels of these soluble immune molecules in different body fluids, requiring minimal sample volume, has the potential to be a game-changer in the field of cancer diagnosis. In addition, real time monitoring of these soluble immune checkpoint molecules in patient-derived biofluids may serve as decision support tools for patient selection for immunotherapeutic interventions. Majority of the biosensors designed to detect the soluble immune biomarkers, have used a two-antibody based sandwich system to capture the target analyte. However, new technologies using bioreceptors like the aptamers or nano-yeast scFv antibody fragments have made possible multiplexed detection of several analytes simultaneously. The use of gold nanoparticles or carbon nanotubes on the electrode surface serves to increase the sensitivity of detection, due to their high electrical conductivity. Further, fabrication of the biosensors on microfluidic platforms enable the detection of these analytes at ultra-low levels. This review discusses the recent advances made in the development of biosensors for specific and selective detection of these immune-markers that can be successfully translated to the clinics as a new paradigm in disease diagnosis and monitoring.

Molecular and immune interactions between β- and γ-herpesviruses in the immunocompromised host.

β- and γ-herpesviruses persistently infect most of the world population, largely without clinical manifestations. However, in immunosuppressive settings like transplantation, these viruses are often jointly reactivated, associating with graft dysfunction/rejection, HCMV disease, and lymphoproliferation. In HIV/AIDS, direct interaction mechanisms have been described for EBV and KSHV in primary effusion lymphoma, demonstrating that the cooperation between both viruses enhances lymphomagenesis. Here, we discuss the clinical evidence supporting that the simultaneous reactivation of these viruses increases the probability of mutual interactions, also providing a conceptual framework explaining how one virus can influence another. Specifically, we propose mechanisms of indirect communication through immune soluble mediators, mainly cytokines, chemokines, and IFN regulatory molecules, based on common features of their infectious cycles and the convergent need on immunomodulatory mechanisms. This latter point should be experimentally addressed in feature research.

Integrated analysis of circulating immune cellular and soluble mediators reveals specific COVID19 signatures at hospital admission with utility for prediction of clinical outcomes.

Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis.Methods: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity.Results: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity.Conclusions: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Tumor-Associated Macrophages: New Horizons for Pituitary Adenoma Researches

Macrophages are one of the most common infiltrating immune cells and an essential component of tumor microenvironment. Macrophages and the soluble cytokines and chemokines produced play an important role in tumorigenesis, progression, invasion and metastasis in solid tumors. Despite the multiple studies in other solid tumors, there is little known about macrophages in pituitary adenomas. Recently, studies about pituitary adenoma-infiltrated macrophages have been emerging, including the immunohistochemical and immunophenotypic analysis of the pituitary adenomas and further studies into the mechanism of the crosstalk between macrophages and tumor cells in vivo and in vitro. These studies have offered us new insights into the polarization of macrophages and its role in tumorigenesis, progression and invasion of pituitary adenomas. This review describes the advances in the field of pituitary adenoma-infiltrated macrophages and the prospect of targeting macrophages as cancer therapy in pituitary adenoma.

An Immunocompetent Microphysiological System to Simultaneously Investigate Effects of Anti-Tumor Natural Killer Cells on Tumor and Cardiac Microtissues.

Existing first-line cancer therapies often fail to cope with the heterogeneity and complexity of cancers, so that new therapeutic approaches are urgently needed. Among novel alternative therapies, adoptive cell therapy (ACT) has emerged as a promising cancer treatment in recent years. The limited clinical applications of ACT, despite its advantages over standard-of-care therapies, can be attributed to (i) time-consuming and cost-intensive procedures to screen for potent anti-tumor immune cells and the corresponding targets, (ii) difficulties to translate in-vitro and animal-derived in-vivo efficacies to clinical efficacy in humans, and (iii) the lack of systemic methods for the safety assessment of ACT. Suitable experimental models and testing platforms have the potential to accelerate the development of ACT. Immunocompetent microphysiological systems (iMPS) are microfluidic platforms that enable complex interactions of advanced tissue models with different immune cell types, bridging the gap between in-vitro and in-vivo studies. Here, we present a proof-of-concept iMPS that supports a triple culture of three-dimensional (3D) colorectal tumor microtissues, 3D cardiac microtissues, and human-derived natural killer (NK) cells in the same microfluidic network. Different aspects of tumor-NK cell interactions were characterized using this iMPS including: (i) direct interaction and NK cell-mediated tumor killing, (ii) the development of an inflammatory milieu through enrichment of soluble pro-inflammatory chemokines and cytokines, and (iii) secondary effects on healthy cardiac microtissues. We found a specific NK cell-mediated tumor-killing activity and elevated levels of tumor- and NK cell-derived chemokines and cytokines, indicating crosstalk and development of an inflammatory milieu. While viability and morphological integrity of cardiac microtissues remained mostly unaffected, we were able to detect alterations in their beating behavior, which shows the potential of iMPS for both, efficacy and early safety testing of new candidate ACTs.

Differentiation of adipose-derived stem cells to chondrocytes using electrospraying

An important challenge in the fabrication of tissue engineered constructs for regenerative medical applications is the development of processes capable of delivering cells and biomaterials to specific locations in a consistent manner. Electrospraying live cells has been introduced in recent years as a cell seeding method, but its effect on phenotype nor genotype has not been explored. A promising candidate for the cellular component of these constructs are human adipose-derived stem cells (hASCs), which are multipotent stem cells that can be differentiated into fat, bone, and cartilage cells. They can be easily and safely obtained from adipose tissue, regardless of the age and sex of the donor. Moreover, these cells can be maintained and expanded in culture for long periods of time without losing their differentiation capacity. In this study, hASCs directly incorporated into a polymer solution were electrosprayed, inducing differentiation into chondrocytes, without the addition of any exogenous factors. Multiple studies have demonstrated the effects of exposing hASCs to biomolecules—such as soluble growth factors, chemokines, and morphogens—to induce chondrogenesis. Transforming growth factors (e.g., TGF-β) and bone morphogenetic proteins are particularly known to play essential roles in the induction of chondrogenesis. Although growth factors have great therapeutic potential for cell-based cartilage regeneration, these growth factor-based therapies have presented several clinical complications, including high dose requirements, low half-life, protein instability, higher costs, and adverse effects in vivo. The present data suggests that electrospraying has great potential as hASCs-based therapy for cartilage regeneration.

Novel Cytokine-Mediated Mechanism of Action Identified By Quantitative Seroproteomics in Multiple Myeloma Patients Treated with Tagraxofusp, a Novel CD123-Directed Targeted Therapy

Introduction Plasmacytoid dendritic cells (pDCs) express CD123/IL-3Rα and promote tumor growth and immunosuppression in multiple myeloma (MM) (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia, 2018, 32:843-846). Tagraxofusp is a novel targeted therapy directed against CD123, and is FDA-approved for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm [BPDCN]). Tagraxofusp can also trigger anti-MM activity by reducing the viability of immunologically defective and tumor-promoting pDCs in MM. Furthermore, tagraxofusp synergistically enhances the anti-MM activity of anti-MM agents bortezomib and pomalidomide. Our preclinical findings led to a recently completed phase 1/2 clinical trial of tagraxofusp with pomalidomide/dexamethasone in relapsed/refractory MM patients (NCT02661022). Results demonstrated preliminary safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR) (ASH 2019). Here, we report the early results of our translational correlative studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort.Methods Tagraxofusp is a bioengineered targeted therapy directed to CD123 comprised of human IL-3 fused to a truncated diphtheria toxin (DT) payload (Stemline Therapeutics, NY). pDCs and patient MM cells were purified from BM/PB samples after informed consent, and quantified using FACS, as described (Ray et al, Leukemia, 2018). A novel high throughput seroproteomics platform SOMAscan was used to analyze 1,310 protein analytes in serum samples from MM patients (n = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp.Results Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a distinct reduction in the frequency of viable pDCs [average 2% at screening vs 0.75% post-tagraxofusp; n = 6; p = 0.036]. Of note, pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. SOMAscan analysis of patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p < 0.05]. Importantly, tagraxofusp treatment reduced pDC-related soluble proteins including IFN-α (fold change: 0.8, treated vs untreated; p < 0.05). Pathway analysis further show that treatment affected immune signaling. For example, tagraxofusp decreased the levels of immunosuppressive proteins, soluble CD40L and IL1R2 (0.071-fold and 0.088 fold vs untreated; p = 0.02 and p = 0.013, respectively), promoting immune response. Moreover, analysis of end of treatment samples showed decreased soluble C-reactive protein, affecting the complement cascade after treatment (0.53-fold, p = 0.0173) via the downregulation of several C-C motif soluble chemokines (p < 0.05). Our earlier study showed that pDC-MM interactions triggered secretion of IL-3, which in turn promotes both pDC survival and MM cell growth. Importantly, tagraxofusp in this trial decreased serum IL-3 levels (fold change 0.75, treated vs untreated; p < 0.05).Conclusions In the present study, we validate the target specificity of tagraxofusp against MM pDCs in relapsed and refractory MM patients enrolled in a phase 1/2 clinical trial. A future clinical trial of tagraxofusp in combination with bortezomib and pomalidomide will examine the utility of tagraxofusp to improve outcome in patients with relapsed refractory MM. DisclosuresMo: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eli Lilly: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy. Olguin: Stemline Therapeutics, New York, NY: Current Employment. Chen: Stemline Therapeutics, New York, NY: Current Employment. Brooks: Stemline Therapeutics: Current Employment. Mughal: Stemline: Current Employment, Current holder of stock options in a privately-held company; Oxford University Press, Informa: Other: financial benefit and/or patents . Richardson: Janssen: Consultancy; Celgene/BMS: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy, Research Funding; AstraZeneca: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Protocol Intelligence: Consultancy; Regeneron: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: Oncopeptides: Consultancy; C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

A highly efficient method for the production and purification of recombinant human CXCL8.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.

Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues.

Macrophages are versatile cells of the innate immune system that perform diverse functions by responding to dynamic changes in their microenvironment. While the effects of soluble cues, including cytokines and chemokines, have been widely studied, the effects of physical cues, including mechanical stimuli, in regulating macrophage form and function are less well understood. In this study, we examined the effects of static and cyclic uniaxial stretch on macrophage inflammatory and healing activation. We found that cyclic stretch altered macrophage morphology and responses to IFNγ/LPS and IL4/IL13. Interestingly, we found that both static and cyclic stretch suppressed IFNγ/LPS induced inflammation. In contrast, IL4/IL13 mediated healing responses were suppressed with cyclic but enhanced with static stretch conditions. Mechanistically, both static and cyclic stretch increased expression of the integrin CD11b (αM integrin), decreased expression of the mechanosensitive ion channel Piezo1, and knock down of either CD11b or Piezo1 through siRNA abrogated stretch-mediated changes in inflammatory responses. Moreover, we found that knock down of CD11b enhanced the expression of Piezo1, and conversely knock down of Piezo1 enhanced CD11b expression, suggesting the potential for crosstalk between integrins and ion channels. Finally, stretch-mediated differences in macrophage activation were also dependent on actin, since pharmacological inhibition of actin polymerization abrogated the changes in activation with stretch. Together, this study demonstrates that the physical environment synergizes with biochemical cues to regulate macrophage morphology and function, and suggests a role for CD11b and Piezo1 crosstalk in mechanotransduction in macrophages.

A Comprehensive Analysis of Immune Constituents in Blood and Bronchoalveolar Lavage Allows Identification of an Immune Signature of Severe Asthma in Children

BackgroundTargeted approaches may not account for the complexity of inflammation involved in children with severe asthma (SA), highlighting the need to consider more global analyses. We aimed to identify sets of immune constituents that distinguish children with SA from disease-control subjects through a comprehensive analysis of cells and immune constituents measured in bronchoalveolar lavage (BAL) and blood.MethodsTwenty children with SA and 10 age-matched control subjects with chronic respiratory disorders other than asthma were included. Paired blood and BAL samples were collected and analyzed for a large set of cellular (eosinophils, neutrophils, and subsets of lymphocytes and innate lymphoid cells) and soluble (chemokines, cytokines, and total antibodies) immune constituents. First, correlations of all immune constituents between BAL and blood and with demographic and clinical data were assessed (Spearman correlations). Then, all data were modelled using supervised multivariate analyses (partial least squares discriminant analysis, PLS-DA) to identify immune constituents that significantly discriminate between SA and control subjects. Univariate analyses were performed (Mann-Whitney tests) and then PLS-DA and univariate analyses were combined to identify the most discriminative and significant constituents.ResultsConcentrations of soluble immune constituents poorly correlated between BAL and blood. Certain constituents correlated with age or body mass index and, in asthmatics, with clinical symptoms, such as the number of exacerbations in the previous year, asthma control test score, or forced expiratory volume. Multivariate supervised analysis allowed construction of a model capable of distinguishing children with SA from control subjects with 80% specificity and 100% sensitivity. All immune constituents contributed to the model but some, identified by variable-important-in-projection values > 1 and p < 0.1, contributed more strongly, including BAL Th1 and Th2 cells and eosinophilia, CCL26 (Eotaxin 3), IgA and IL-19 concentrations in blood. Blood concentrations of IL-26, CCL13, APRIL, and Pentraxin-3 may also help in the characterization of SA.ConclusionsThe analysis of a large set of immune constituents may allow the identification of a biological immune signature of SA. Such an approach may provide new leads for delineating the pathogenesis of SA in children and identifying new targets for its diagnosis, prediction, and personalized treatment.

Getting on in Old Age: How the Gut Microbiota Interferes With Brain Innate Immunity.

The immune system is crucial for defending against various invaders, such as pathogens, cancer cells or misfolded proteins. With increasing age, the diminishing immune response, known as immunosenescence, becomes evident. Concomitantly, some diseases like infections, autoimmune diseases, chronic inflammatory diseases and cancer, accumulate with age. Different cell types are part of the innate immunity response and produce soluble factors, cytokines, chemokines, and type I interferons. Improper maturation of innate immune cells or their dysfunction have been linked to numerous age-related diseases. In parallel to the occurrence of the many functional facets of the immune response, a symbiotic microbiota had been acquired. For the relevant and situation-dependent function of the immune system the microbiome plays an essential role because it fine-tunes the immune system and its responses during life. Nevertheless, how the age-related alterations in the microbiota are reflected in the innate immune system, is still poorly understood. With this review, we provide an up-to-date overview on our present understanding of the gut microbiota effects on innate immunity, with a particular emphasis on aging-associated changes in the gut microbiota and the implications for the brain innate immune response.

Abstract 316: Effect of schweinfurthins on malignant plasma cells

Abstract Multiple myeloma (MM), a bone marrow neoplasm is currently incurable and effective alternative therapies are needed. Schweinfurthin-3114 (S-3114) purified from a plant extract, inhibits growth of established MM cell lines. The mechanism(s) for this effect are not fully characterized. To advance understanding, MM cell lines RPMI-8226 and MM.1s were incubated with increasing concentrations of S-3114 for up to 48 hours. S-3114 decreased cell viability (trypan blue assay) and mitochondrial activity (MTT assay) in a concentration and time-dependent manner (p≤0.05). RPMI-8226 cells were more sensitive (EC50 2.36 nM) than MM.1s (EC50 11.1 nM) at 48 hours. Flow cytometry analyses revealed increased apoptosis in a concentration and time-dependent manner in both cell lines (64.0% in RPMI-8226 with 20 nM S-3114; 86.3% in MM.1s with 270 nM S-3114, both at 48 hours). S-3114 also induced cell cycle arrest at G2/M. In RPMI-8226, the percent of cells arrested in G2/M increased from 45.9 to 66.1 with 3.5 nM S-3114 at 24 and 48 hours, respectively. In RPMI-8226, 20 nM S-3114 for 24 and 48 hours caused a 100% G2/M arrest. In both cell lines, S-3114 reduced protein kinase B (Akt) activation (Western blot) in a concentration and time-dependent manner. In MM.1s the apoptotic extrinsic pathway (caspase 8), and not the intrinsic pathway (caspase 9), is highly involved. In fact, S-3114 (20 nM for 48 hours, and 270 nM for 24 and 48 hours) caused a major cleavage for both 43 and 18 KDa caspase 8 fragments. Also cleaved Poly ADP-Ribose Polymerase 1 (PARP-1) increased at S-3114 concentrations of 20 and 270 nM at 24 and 48 hours. S-3114 influence on Golgi-mediated secretion was evaluated by measuring the soluble chemokine Macrophage Inflammatory Protein-1 alpha (MIP-1α), which is secreted by the Golgi and it is involved in the progression of MM disease. S-3114 treatment for 48 hours stimulated the MIP-1α negative cell line (RPMI-8226) to significantly increase MIP-1α RNA levels and to secrete MIP-1α at low concentrations. On the contrary, despite that prolonged S-3114 treatment significantly increased MIP-1α RNA levels, its secretion is decreased in the MIP-1α positive cell line (MM.1s). These in vitro studies demonstrate that S-3114 impairs the Phosphoinositide 3-Kinases (PI3K)/Akt signaling pathway resulting in cell death and/or cell cycle arrest. Data also shows an increased gene expression of the pro-survival MIP-1α by S-3114. Our ongoing studies are focused on schweinfurthin effects on MIP-1 α and Golgi secretion. We postulate that the cytotoxic impact of S-3114 requires disruption of MIP-1α signaling to regulate cell cycle, survival, and impairment of Golgi architecture and secretion in malignant plasma cells. In aggregate, these results reveal mechanism(s) for S-3114 effects and support further work advancing S-3114 as a novel therapy for MM. Citation Format: Barbara Manfredi, Raymond J. Hohl. Effect of schweinfurthins on malignant plasma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 316.

Interactions between Cancer-Associated Fibroblasts and T Cells in the Pancreatic Tumor Microenvironment and the Role of Chemokines.

Simple SummaryDespite advances in therapeutic modalities, the five-year overall survival for pancreatic cancer is still less than 10%. Pancreatic tumors are characterized by a highly fibrotic stroma comprised of activated cancer-associated fibroblasts (CAFs) which surrounds the cancer cells. Pancreatic CAFs are involved in creating an immunosuppressive tumor microenvironment by secretion of immunoregulatory and chemoattractive factors, which prevent tumor-reactive T-cell responses. This review article discusses recent discoveries about the role of different subsets of CAFs as regulators of anti-tumor immunity in pancreatic cancer, with emphasis on chemokines and suppressive factors. Understanding the interactions between T cells and CAFs as well as their spatial distribution within the tumor is of great importance for the development of novel targeted therapies to overcome immunosuppression and to enhance immunotherapy.Less than 10% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) survive 5 years or more, making it one of the most fatal cancers. Accumulation of T cells in pancreatic tumors is associated with better prognosis, but immunotherapies to enhance the anti-tumor activity of infiltrating T cells are failing in this devastating disease. Pancreatic tumors are characterized by a desmoplastic stroma, which mainly consists of activated cancer-associated fibroblasts (CAFs). Pancreatic CAFs have emerged as important regulators of the tumor microenvironment by contributing to immune evasion through the release of chemokines, cytokines, and growth factors, which alters T-cell migration, differentiation and cytotoxic activity. However, recent discoveries have also revealed that subsets of CAFs with diverse functions can either restrain or promote tumor progression. Here, we discuss our current knowledge about the interactions between CAFs and T cells in PDAC and summarize different therapy strategies targeting the CAF–T cell axis with focus on CAF-derived soluble immunosuppressive factors and chemokines. Identifying the functions of different CAF subsets and understanding their roles in T-cell trafficking within the tumor may be fundamental for the development of an effective combinational treatment for PDAC.

Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway.

The endoplasmic reticulum (ER) regulates protein folding, post-translational modifications, lipid synthesis, and calcium signaling to attenuate the accumulation of misfolded proteins causing ER stress and maintains cellular homeostasis. The tumor microenvironment is rich in soluble cytokines, chemokines, growth, and angiogenic factors and can drive the ER’s abnormal functioning in healthy cells. Cancer cells adapt well to the tumor microenvironment induced ER stress. We identified that the inflammatory breast cancer (IBC) cells abundantly express osteoprotegerin (OPG) and their tumor microenvironment is rich in OPG protein. OPG also called osteoclast differentiation factor/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for receptor activator of nuclear factor-kappa B ligand (RANKL). Employing mass spectrometry analysis, we identified a set of ER chaperones associated with OPG in IBC cell lysates (SUM149PT, SUM1315MO2) compared to healthy human mammary epithelial cells (HMEC). Proximity ligation assay (PLA) and immunoprecipitation assay validated the interaction between OPG and ER chaperone and master regulator of unfolded protein response (UPR) GRP78/BiP (glucose-regulated protein/Binding immunoglobulin protein). We detected remarkably high gene expression of CCAAT enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), protein disulfide-isomerase (PDI), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) in SUM149PT and SUM190PT cells when compared to HMEC. Similarly, tissue sections of human IBC expressed high levels of ER stress proteins. We evaluated cell death and apoptosis upon Salubrinal and phenylbutyrate treatment in healthy and IBC cells by caspase-3 activity and cleaved poly (ADP-ribose) polymerase (PARP) protein assay. IBC (SUM149PT and SUM190PT) cells were chemosensitive to Salubrinal treatment, possibly via inhibition in OPG secretion, upregulating ATF4, and CHOP, thus ultimately driving caspase-3 mediated IBC cell death. Salubrinal treatment upregulated PDI, which connects ER stress to oxidative stress. We observed increased ROS production and reduced cell proliferation of Salubrinal treated IBC cells. Treatment with antioxidants could rescue IBC cells from ROS and aborted cell proliferation. Our findings implicate that manipulating ER stress with Salubrinal may provide a safer and tailored strategy to target the growth of inflammatory and aggressive forms of breast cancer.

Behavior of Chemokine Receptor 6 (CXCR6) in Complex with CXCL16 Soluble form Chemokine by Molecular Dynamic Simulations: General Protein‒Ligand Interaction Model and 3D-QSAR Studies of Synthetic Antagonists.

The CXCR6‒CXCL16 axis is involved in several pathological processes, and its overexpression has been detected in different types of cancer, such as prostate, breast, ovary, and lung cancer, along with schwannomas, in which it promotes invasion and metastasis. Moreover, this axis is involved in atherosclerosis, type 1 diabetes, primary immune thrombocytopenia, vitiligo, and other autoimmune diseases, in which it is responsible for the infiltration of different immune system cells. The 3D structure of CXCR6 and CXCL16 has not been experimentally resolved; therefore, homology modeling and molecular dynamics simulations could be useful for the study of this signaling axis. In this work, a homology model of CXCR6 and a soluble form of CXCL16 (CXCR6‒CXCL16s) are reported to study the interactions between CXCR6 and CXCL16s through coarse-grained molecular dynamics (CG-MD) simulations. CG-MD simulations showed the two activation steps of CXCR6 through a decrease in the distance between the chemokine and the transmembrane region (TM) of CXCR6 and transmembrane rotational changes and polar interactions between transmembrane segments. The polar interactions between TM3, TM5, and TM6 are fundamental to functional conformation and the meta-active state of CXCR6. The interactions between D77-R280 and T243-TM7 could be related to the functional conformation of CXCR6; alternatively, the interaction between Q195-Q244 and N248 could be related to an inactive state due to the loss of this interaction, and an arginine cage broken in the presence of CXCL16s allows the meta-active state of CXCR6. A general protein‒ligand interaction supports the relevance of TM3‒TM5‒TM6 interactions, presenting three relevant pharmacophoric features: HAc (H-bond acceptor), HDn (H-bond donator), and Hph (hydrophobic), distributed around the space between extracellular loops (ECLs) and TMs. The HDn feature is close to TM3 and TM6; likewise, the HAc and Hph features are close to ECL1 and ECL2 and could block the rotation and interactions between TM3‒TM6 and the interactions of CXCL16s with the ECLs. Tridimensional quantitative structure-activity relationships (3D-QSAR) models show that the positive steric (VdW) and electrostatic fields coincide with the steric and positive electrostatic region of the exo-azabicyclo[3.3.1]nonane scaffold in the best pIC50 ligands. This substructure is close to the E274 residue and therefore relevant to the activity of CXCR6. These data could help with the design of new molecules that inhibit chemokine binding or antagonize the receptor based on the activation mechanism of CXCR6 and provoke a decrease in chemotaxis caused by the CXCR6‒CXCL16 axis.

Biologics and their delivery systems: Trends in myocardial infarction.

Cardiovascular disease is the leading cause of death around the world, in which myocardial infarction (MI) is a precipitating event. However, current therapies do not adequately address the multiple dysregulated systems following MI. Consequently, recent studies have developed novel biologic delivery systems to more effectively address these maladies. This review utilizes a scientometric summary of the recent literature to identify trends among biologic delivery systems designed to treat MI. Emphasis is placed on sustained or targeted release of biologics (e.g. growth factors, nucleic acids, stem cells, chemokines) from common delivery systems (e.g. microparticles, nanocarriers, injectable hydrogels, implantable patches). We also evaluate biologic delivery system trends in the entire regenerative medicine field to identify emerging approaches that may translate to the treatment of MI. Future developments include immune system targeting through soluble factor or chemokine delivery, and the development of advanced delivery systems that facilitate the synergistic delivery of biologics.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1β, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P<0.0001), MCP-1 (P<0.0001), HGF (P= 0.0001), and VEGF (P<0.0001) in the acute phase. Fourteen molecules (IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.

Loss of Speckle-Type POZ Protein Promotes Prostate Cancer Cell Migration and Invasion Through Upregulation of MCP-1.

BackgroundThe goal of this study is to verify that the loss of speckle-type POZ protein (SPOP) promotes the migration and invasion of prostate cancer cells, and that this process is brought about by an increase in MCP-1.Material/MethodsSPOP knockout C4-2 cells (C4-2 SPOP−/−) were verified by western blotting. Transwell and wound-healing assays were applied to verify different migration and invasion abilities between the C4-2 SPOP−/− and control cells. We used an antibody array to find different soluble chemokine factors in the C4-2 SPOP−/− cells. ELISA and qRT-PCR were applied for confirmation. To test MCP-1 function in conditioned medium, a transwell assay was applied with or without anti-MCP-1 antibody.ResultsThe western blot showed that SPOP was knocked out in sgSPOP-1 and sgSPOP-2 (different clones of C4-2 SPOP−/−). The transwell and wound-healing assays indicated that, compared with control cells, sgSPOP-1 and sgSPOP-2 had stronger migration and invasion abilities. The antibody array found that the expression of MCP-1 was upregulated in sgSPOP-1 and sgSPOP-2 conditioned medium. This result was verified by ELISA and qRT-PCR. In the prostate cancer cells, migration and invasion activity was greatly increased in C4-2 SPOP−/− conditioned medium, while this activity was decreased after anti-MCP-1 antibody neutralization.ConclusionsOur findings suggest that the loss of SPOP in C4-2 cells promotes increased cell migration and invasion abilities. This may be realized by upregulating the expression of MCP-1. The inhibition of MCP-1 expression may be an effective treatment for SPOP-mutant prostate cancer.

Epithelial Cells and Inflammation in Pulmonary Wound Repair.

Respiratory diseases are frequently characterised by epithelial injury, airway inflammation, defective tissue repair, and airway remodelling. This may occur in a subacute or chronic context, such as asthma and chronic obstructive pulmonary disease, or occur acutely as in pathogen challenge and acute respiratory distress syndrome (ARDS). Despite the frequent challenge of lung homeostasis, not all pulmonary insults lead to disease. Traditionally thought of as a quiescent organ, emerging evidence highlights that the lung has significant capacity to respond to injury by repairing and replacing damaged cells. This occurs with the appropriate and timely resolution of inflammation and concurrent initiation of tissue repair programmes. Airway epithelial cells are key effectors in lung homeostasis and host defence; continual exposure to pathogens, toxins, and particulate matter challenge homeostasis, requiring robust defence and repair mechanisms. As such, the epithelium is critically involved in the return to homeostasis, orchestrating the resolution of inflammation and initiating tissue repair. This review examines the pivotal role of pulmonary airway epithelial cells in initiating and moderating tissue repair and restitution. We discuss emerging evidence of the interactions between airway epithelial cells and candidate stem or progenitor cells to initiate tissue repair as well as with cells of the innate and adaptive immune systems in driving successful tissue regeneration. Understanding the mechanisms of intercellular communication is rapidly increasing, and a major focus of this review includes the various mediators involved, including growth factors, extracellular vesicles, soluble lipid mediators, cytokines, and chemokines. Understanding these areas will ultimately identify potential cells, mediators, and interactions for therapeutic targeting.