Effector Role Research Articles

Mediators of human ureteral smooth muscle contraction-a role for erythropoietin, tamsulosin and Gli effectors.

BackgroundUreteral contractility is a poorly understood process. Contractions have been demonstrated to occur in the smooth muscle layers of the ureter. Previous work suggests the involvement of Gli family proteins and erythropoietin (EPO) in regulating mammalian ureteral smooth muscle contraction. We sought to devise a method by which the effects of these proteins and tamsulosin on distal human ureteral tissue contractility could be investigated to better understand mechanisms regulating human ureteral function.MethodsIRB approval was obtained to procure portions of extraneous distal ureteral tissue from living donor renal transplants. Contractility was measured by placing the tissue in Krebs buffer and stimulating via a uniform electric current. Contractile force was recorded with each stimulation with and without the presence of a Gli inhibitor (GANT61) or EPO. Each ureteral specimen was subsequently fixed and tested by immunohistochemistry to determine Gli, EPO and alpha-adrenergic receptor activity.ResultsElectrical field stimulation successfully elicited contractions in the ureteral tissue. Administering tamsulosin decreased force and duration of ureteral contractions. Inhibiting Gli signaling decreased contractility and EPO decreased ureteral contractile forces within 5 minutes of administration versus untreated controls. Staining confirmed Gli1 protein and α-adrenergic receptor expression in ureteral smooth muscle and epithelial tissue with EPO receptor expression confined to the epithelial layer.ConclusionsDistal ureteral contractile forces are decreased by inhibition of Gli family proteins and the α-adrenergic receptor. EPO acts within five minutes, suggesting ion channel involvement instead of changes in gene expression. Continuing work will elucidate the role of these proteins in coordinating ureteral contractions. This has implications for the use of pharmacologic methods to address ureteral contractility and dysfunctional peristalsis during stone passage, ureteroscopy, in transplant patients and potentially to reduce symptoms from ureteral stents.

Abstract 1100: Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer

Abstract 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. In many cases the molecular resistance mechanisms remain unknown. A systematic identification of the involved genes and pathways is critical to overcome osimertinib resistance. To define the resistance landscape of EGFR kinase inhibition we performed genome-wide gain and loss of function CRISPR screens in EGFR mutant lung cancer cell lines treated with osimertinib. Resistance hits were enriched for genes in previously identified resistance pathways including PI3K (PTEN, TSC2), MAPK (NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, LZTR1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockouts demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, indicating transcriptional activation of the Hippo pathway. According to our screening data, many resistance hits mapped onto the Hippo signaling axis - upstream regulators (NF2, AMOTL2), core signaling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signaling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and elevated activity of a TEAD reporter system, indicating activation of Hippo transcriptional programs. To maintain osimertinib resistance, expression of both YAP1 and WWTR1 was necessary, suggesting non-redundant roles for both Hippo main effectors in mediating osimertinib resistance. The combination of osimertinib and a TEAD inhibitor (MYF-01-37) reversed the resistance phenotype in NF2 KO cell line models in long-term proliferation assays and also substantially repressed the emergence of drug-tolerant persister cells in PC-9, HCC827 and HCC4006 cell lines following osimertinib treatment. These cells exhibited an enhanced apoptotic response when treated with combination of osimertinib and TEAD inhibitor. Consequently, we propose Hippo signaling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan S. Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Erica Banks, Nin Guan, Shanade Dunn, Maria Luisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer [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 1100.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (ΔspPbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. ΔspPbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of ΔspPbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 – known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

Targeting MicroRNA-192-5p, a Downstream Effector of NOXs (NADPH Oxidases), Reverses Endothelial DHFR (Dihydrofolate Reductase) Deficiency to Attenuate Abdominal Aortic Aneurysm Formation.

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Means, Motive, and Opportunity: Do Non-Islet-Reactive Infiltrating T Cells Contribute to Autoimmunity in Type 1 Diabetes?

In human type 1 diabetes and animal models of the disease, a diverse assortment of immune cells infiltrates the pancreatic islets. CD8+ T cells are well represented within infiltrates and HLA multimer staining of pancreas sections provides clear evidence that islet epitope reactive T cells are present within autoimmune lesions. These bona fide effectors have been a key research focus because these cells represent an intellectually attractive culprit for β cell destruction. However, T cell receptors are highly diverse in human insulitis. This suggests correspondingly broad antigen specificity, which includes a majority of T cells for which there is no evidence of islet-specific reactivity. The presence of “non-cognate” T cells in insulitis raises suspicion that their role could be beyond that of an innocent bystander. In this perspective, we consider the potential pathogenic contribution of non-islet-reactive T cells. Our intellectual framework will be that of a criminal investigation. Having arraigned islet-specific CD8+ T cells for the murder of pancreatic β cells, we then turn our attention to the non-target immune cells present in human insulitis and consider the possible regulatory, benign, or effector roles that they may play in disease. Considering available evidence, we overview the case that can be made that non-islet-reactive infiltrating T cells should be suspected as co-conspirators or accessories to the crime and suggest some possible routes forward for reaching a better understanding of their role in disease.

Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort.

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study’s objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

Necroptosis is an inflammatory form of lytic programmed cell death that is thought to have evolved to defend against pathogens. Genetic deletion of the terminal effector protein—MLKL—shows no overt phenotype in the C57BL/6 mouse strain under conventional laboratory housing conditions. Small molecules that inhibit necroptosis by targeting the kinase activity of RIPK1, one of the main upstream conduits to MLKL activation, have shown promise in several murine models of non-infectious disease and in phase II human clinical trials. This has triggered in excess of one billion dollars (USD) in investment into the emerging class of necroptosis blocking drugs, and the potential utility of targeting the terminal effector is being closely scrutinised. Here we review murine models of disease, both genetic deletion and mutation, that investigate the role of MLKL. We summarize a series of examples from several broad disease categories including ischemia reperfusion injury, sterile inflammation, pathogen infection and hematological stress. Elucidating MLKL’s contribution to mouse models of disease is an important first step to identify human indications that stand to benefit most from MLKL-targeted drug therapies.

GPCR-mediated YAP/TAZ inactivation in fibroblasts via EPAC1/2, RAP2C, and MAP4K7.

Yes-associated protein (YAP) and PDZ-binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs-coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2-mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.

RUNX inhibitor suppresses graft-versus-host disease through targeting RUNX-NFATC2 axis.

Patients with refractory graft‐versus‐host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt‐related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb‐M’, which is the inhibitor that switches off the entire RUNX family by alkylating agent–conjugated pyrrole‐imidazole (PI) polyamides, inhibited T‐cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX‐binding sequences (TGTGGT). Chb‐M’ also suppressed the expression of NFATC2 and pro‐inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb‐M’ showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD‐associated cytokines including tissue necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor were reduced in the peripheral blood of Chb‐M’ injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX‐NFATC2 axis can be a novel therapeutic target against GVHD.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin.

Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.

Biochemical and structural insights into Rab12 interactions with RILP and its family members

Alongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

SLI1 confers broad-spectrum resistance to phloem-feeding insects.

Resistance (R) genes usually compete in a coevolutionary arms race with reciprocal effectors to confer strain‐specific resistance to pathogens or herbivorous insects. Here, we investigate the specificity of SLI1, a recently identified R gene in Arabidopsis that encodes a small heat shock‐like protein involved in resistance to Myzus persicae aphids. In a panel with several aphid and whitefly species, SLI1 compromised reproductive rates of three species: the tobacco aphid M. persicae nicotianae, the cabbage aphid Brevicoryne brassicae and the cabbage whitefly Aleyrodes proletella. Electrical penetration graph recording of aphid behaviour, revealed shorter salivations and a 3‐to‐5‐fold increase in phloem feeding on sli1 loss‐of‐function plants. The mustard aphid Lipaphis erysimi and Bemisia tabaci whitefly were not affected by SLI1. Unlike the other two aphid species, L. erysimi exhibited repetitive salivations preceding successful phloem feeding, indicating a role of salivary effectors in overcoming SLI1‐mediated resistance. Microscopic characterization showed that SLI1 proteins localize in the sieve tubes of virtually all above‐ and below‐ground tissues and co‐localize with the aphid stylet tip after penetration of the sieve element plasma membrane. These observations reveal an unconventional R gene that escapes the paradigm of strain specificity and confers broad‐spectrum quantitative resistance to phloem‐feeding insects.

CD8αα expression increases during demyelinating disease progression

Abstract Experimental autoimmune encephalomyelitis (EAE) is a well-described mouse model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). While the role of CD4 T cells is well established, other immune cell types, particularly CD8 T cells, are key contributors to disease pathogenesis. Our study aims to categorize the functional contributions of CD8 T cell subsets to EAE kinetics and disease outcomes. The use of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide alongside an adjuvant to inoculate mice, provides a reproducible disease model. Demyelinating disease severity was analyzed at defined timepoints including, onset (d10–14), peak (d17–21), early chronic (d28–35) and late chronic (d80–120) stages. The total number of CD3+ T cells in the CNS remained consistent during EAE. However, it goes from a CD4 T cell dominated response at peak EAE to an equalized 1:1 ratio of CD4 and CD8 T cells at late chronic stage. Furthermore, differentiation of CD8 T cell subsets based on CD8α and CD8β protein expression revealed that CD8αα T cells numbers increase to comprise ~40% of the CD8+ population at late chronic EAE. In addition, CD8βLO cells become a prominent subset during late chronic stage, with a concomitant decrease in CD8αβHI T cells. Moreover, CD8αα T cells are activated and exhibit a central memory phenotype along with downregulation of exhaustion markers by FACS analysis, and yet have significantly reduced cytokine expression. These results are consistent with single cell RNA sequencing data obtained at the peak and late chronic time points. Thus, CD8αα T cells constitute a phenotypically distinct class of CD8 T cell subsets, with a blunted effector role in demyelinating disease outcome.

Innate (and Innate-like) Lymphoid Cells: Emerging Immune Subsets With Multiple Roles Along Transplant Life.

Transplant immunology is currently largely focused on conventional adaptive immunity, particularly T and B lymphocytes, which have long been considered as the only cells capable of allorecognition. In this vision, except for the initial phase of ischemia/reperfusion, during which the role of innate immune effectors is well established, the latter are largely considered as "passive" players, recruited secondarily to amplify graft destruction processes during rejection. Challenging this prevalent dogma, the recent progresses in basic immunology have unraveled the complexity of the innate immune system and identified different subsets of innate (and innate-like) lymphoid cells. As most of these cells are tissue-resident, they are overrepresented among passenger leukocytes. Beyond their role in ischemia/reperfusion, some of these subsets have been shown to be capable of allorecognition and/or of regulating alloreactive adaptive responses, suggesting that these emerging immune players are actively involved in most of the life phases of the grafts and their recipients. Drawing upon the inventory of the literature, this review synthesizes the current state of knowledge of the role of the different innate (and innate-like) lymphoid cell subsets during ischemia/reperfusion, allorecognition, and graft rejection. How these subsets also contribute to graft tolerance and the protection of chronically immunosuppressed patients against infectious and cancerous complications is also examined.

Sos1 Modulates Extracellular Matrix Synthesis, Proliferation, and Migration in Fibroblasts.

Non-reversible fibrosis is common in various diseases such as chronic renal failure, liver cirrhosis, chronic pancreatitis, pulmonary fibrosis, rheumatoid arthritis and atherosclerosis. Transforming growth factor beta 1 (TGF-β1) is involved in virtually all types of fibrosis. We previously described the involvement of Ras GTPase isoforms in the regulation of TGF-β1-induced fibrosis. The guanine nucleotide exchange factor Son of Sevenless (Sos) is the main Ras activator, but the role of the ubiquitously expressed Sos1 in the development of fibrosis has not been studied. For this purpose, we isolated and cultured Sos1 knock-out (KO) mouse embryonic fibroblasts, the main extracellular matrix proteins (ECM)-producing cells, and we analyzed ECM synthesis, cell proliferation and migration in the absence of Sos1, as well as the role of the main Sos1-Ras effectors, Erk1/2 and Akt, in these processes. The absence of Sos1 increases collagen I expression (through the PI3K-Akt signaling pathway), total collagen proteins, and slightly increases fibronectin expression; Sos1 regulates fibroblast proliferation through both PI3K-Akt and Raf-Erk pathways, and Sos1-PI3K-Akt signaling regulates fibroblast migration. These study shows that Sos1 regulates ECM synthesis and migration (through Ras-PI3K-Akt) and proliferation (through Ras-PI3K-Akt and Ras-Raf-Erk) in fibroblasts, and describe for the first time the role of the Sos1-Ras signaling axis in the regulation of cellular processes involved in the development of fibrosis.

Silencing of inflammatory pathways through CRISPR CAS9 knockout of tak1 in human chondrocytes

Purpose: Osteoarthritis (OA) is a debilitating disease with few effective therapeutic options. Chronic inflammation is a risk factor for poorer outcome, and it remains unclear how the presence of proinflammatory cytokines can affect cartilage repair. To address this question, we investigated the role of the main effector of the inflammatory pathways, Tak1, a MAP kinase associated with OA progression. Tak1 was knocked out in chondrocytes using CRISPR/Cas9 and the ability of edited chondrocytes to undergo chondrogenesis in the presence of IL-1β, lipopolysaccharide (LPS) and TNFα was studied.

MiR-486-5p: A Prognostic Biomarker for Chronic Myeloid Leukemia.

MicroRNA miR-486-5p has been reported as a potential biomarker for diagnosis, prognosis, and as a therapeutic target in various cancers. In this study, we analyzed alterations in the expression of miR-486-5p in chronic Myeloid Leukemia (CML) patients. Initially, the expression of miR-486-5p was studied in the BCR-ABL1+ve CML K562 cell line by quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the miR-486-5p expression was significantly upregulated in K562 cells after imatinib exposure, as compared to untreated K562 cells (p-value = 0.047). These observations were corroborated by a hospital-based study of the miR-486-5p expression in peripheral blood leucocytes of 36 CML patients in the chronic phase (CP) and compared with age and sex-matched healthy volunteers as control subjects. qRT-PCR-based quantification revealed significant downregulation of the miR-486-5p expression in newly diagnosed untreated CP-CML patients’ samples (2–ΔCt = 13.19 ± 14.41) as compared to control samples (2–ΔCt = 254.5 ± 274.8) (p-value < 0.0001). Levels of miR-486-5p were found to be distinctly elevated in the post-imatinib treatment samples of CML patients (2–ΔCt = 469.7 ± 312.9) as compared to pre-treatment samples (p-value < 0.0001). CML patients’ clinical and hematological responses to imatinib therapy (oral dose of 400 mg OD) were monitored for 12 months. The correlation of pre-treatment miR-486-5p levels with Sokal score indicated that patients with a higher expression of miR-486-5p had better prognoses. Patients with higher pre-imatinib miR-486-5p levels also showed a major hematologic response to imatinib in a shorter time and vice versa. To the best of our knowledge, this is the first report of alterations in the miR-486-5p expression in peripheral blood leucocytes of CML patients. Our observations support a tumor suppressor role of miR-486-5p in CML. The downregulation of the miR-486-5p expression may be critically important in the disease progression of CML patients. The upregulation of the miR-486-5p expression in post-imatinib exposure K562 cells and CML patients after 12 months of imatinib treatment suggests an onco-suppressor effector role of miR-486-5p in the BCR-ABL downstream signaling pathway. miR-486-5p can be explored as a novel biomarker for the early detection of CML.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Caloric Restriction Impairs Regulatory T cells Within the Tumor Microenvironment After Radiation and Primes Effector T cells.

Outcomes for triple negative breast cancer (TNBC) are poor and may be improved by increasing CD8+ tumor infiltrating lymphocytes (TIL) to augment antitumor immunity. Radiation (RT) can promote immunogenic cell death with increased antitumor T cell activity but also stimulates suppressive regulatory T cells (Tregs). Because metabolic alterations affect immune homeostasis and prior studies show caloric restriction (CR) combined with RT improves preclinical TNBC outcomes, we hypothesized that CR augments RT, in part, by altering intratumoral immunity. Using an invivo model of TNBC, we treated mice with ad libitum (AL) diet, radiation, a CR diet, or CR + RT, and demonstrated an immune suppressive environment with a significant increase in CD4+ CD25+Foxp3+ Tregs after RT but not in CR-fed mice. CD8:Treg ratio in CR + RT TIL increased 4-fold compared with AL + RT mice. Invivo CD8 depletion was performed to assess the role of effector T cells in mitigating the effects of CR, and it was found that in mice undergoing CR, depletion of CD8 T cells resulted in increased tumor progression and decreased median survival compared with isotype control-treated mice. In addition, PD-1 expression on CD3+CD8+ Tcells within the tumor microenvironment was significantly increased in CR + RT versus AL + RT treated mice as per immunofluorescence. Serum from breast cancer patients undergoing RT alone or CR and RT was collected pre- and postintervention, and a cytokine array demonstrated that patients treated with CR + RT had notable decreases in immunosuppressive cytokines such as IL-2Rγ, IL-10Rβ, and TGF-β2 and 3 compared with patients receiving RT alone. In conclusion, combining CR with RT decreases intratumoral Tregs, increases CD8:Treg, and increases PD-1 expression via a process dependent on CD8 T cells in a TNBC model. Breast cancer patients undergoing CR concurrently with RT also had significant reduction in immunosuppressive cytokine levels compared with those receiving RT alone.

The Root Lesion Nematode Effector Ppen10370 Is Essential for Parasitism of Pratylenchus penetrans.

The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of crops. Like other plant pathogens, P. penetrans deploys a battery of secreted protein effectors to manipulate plant hosts and induce disease. Although several candidate effectors of P. penetrans have been identified, detailed mechanisms of their functions and particularly their host targets remain largely unexplored. In this study, a repertoire of candidate genes encoding pioneer effectors of P. penetrans was amplified from mixed life stages of the nematode, and candidate effectors were cloned and subjected to transient expression in a heterologous host, Nicotiana benthamiana, using potato virus X-based gene vector. Among seven analyzed genes, the candidate effector designated as Ppen10370 triggered pleiotropic phenotypes substantially different from those produced by wild type infection. Transcriptome analysis of plants expressing Ppen10370 demonstrated that observed phenotypic changes were likely related to disruption of core biological processes in the plant due to effector-originated activities. Cross-species comparative analysis of Ppen10370 identified homolog gene sequences in five other Pratylenchus species, and their transcripts were found to be localized specifically in the nematode esophageal glands by in situ hybridization. RNA silencing of the Ppen10370 resulted in a significant reduction of nematode reproduction and development, demonstrating an important role of the esophageal gland effector for parasitism.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.