RESUMEN
TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.
Asunto(s)
Antígeno B7-H1 , Neoplasias de la Mama , Linfocitos T CD8-positivos , Inhibidores de Puntos de Control Inmunológico , Factor de Crecimiento Transformador beta , Femenino , Animales , Ratones , Diferenciación Celular , Linfocitos T CD8-positivos/inmunología , Células Madre , Antígeno B7-H1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Interferón gamma/inmunología , Agotamiento de Células T , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones Endogámicos BALB C , Línea Celular Tumoral , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , RNA-SeqRESUMEN
The actions of the immune system are finely tuned, involving complex communication and coordination between diverse immune and non-immune cells across the tissues of the body. A healthy immune system requires a precise balance between immunity and tolerance. Regulatory T cells (Tregs) have long been appreciated as one of the master regulators of this balance; their importance is underscored by the autoimmunity that develops in mice and humans when Tregs are missing or dysfunctional. In addition to the immunoregulatory roles of Tregs in suppressing autoimmunity and inflammation via control of adaptive and innate immune responses, several non-immune modulatory functions of Tregs have been identified in recent years. In this review, we have highlighted the growing literature on the action of Tregs in metabolism, stem cell maintenance, tissue repair, and angiogenesis. Alongside Tregs' immune suppressive role, these non-suppressive activities comprise a key function of Tregs in regulating health and disease. As Tregs receive increasing attention as therapeutic targets, understanding their non-canonical functions may become an important feature of Treg-directed interventions.
Asunto(s)
Tolerancia Inmunológica , Linfocitos T Reguladores , Humanos , Animales , Ratones , AutoinmunidadRESUMEN
The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Fibroblastos Asociados al Cáncer/patología , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Isoantígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Microambiente Tumoral , Apoptosis , Biomarcadores de Tumor/genética , Fibroblastos Asociados al Cáncer/inmunología , Fibroblastos Asociados al Cáncer/metabolismo , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Isoantígenos/genética , Glicoproteínas de Membrana/genética , Neutrófilos/inmunología , Neutrófilos/metabolismo , Pronóstico , Receptores de Superficie Celular/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Asunto(s)
Células Dendríticas Foliculares/inmunología , Fibroblastos/inmunología , Ganglios Linfáticos/inmunología , Células del Estroma/inmunología , Anciano , Animales , Apoptosis/genética , Apoptosis/inmunología , Proliferación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Dendríticas Foliculares/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/inmunología , Técnicas de Sustitución del Gen , Humanos , Inmunidad Celular/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ganglios Linfáticos/citología , Masculino , Ratones , Ratones Transgénicos , RNA-Seq , Análisis de la Célula Individual , Células del Estroma/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN- population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFß signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide-dependent manner, whereas FAP+PDPN- pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.
Asunto(s)
Neoplasias de la Mama/patología , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Células del Estroma/metabolismo , Microambiente Tumoral/inmunología , Animales , Neoplasias de la Mama/inmunología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Proliferación Celular , Endopeptidasas , Femenino , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Pericitos/metabolismo , Pericitos/patología , Células del Estroma/patología , Linfocitos T/patologíaRESUMEN
Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/inmunología , Urotelio/patología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Estudios de Cohortes , Colágeno/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Inmunoterapia , Ratones , Mutación , Metástasis de la Neoplasia , Fenotipo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Resultado del Tratamiento , Microambiente Tumoral/inmunología , Neoplasias Urológicas/genética , Neoplasias Urológicas/patología , Urotelio/efectos de los fármacos , Urotelio/inmunologíaRESUMEN
Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αß (LTαß) on Th17 cells and LTßR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTßR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Linfotoxina-alfa/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Células del Estroma/inmunología , Células Th17/inmunología , Adulto , Animales , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Inflamación/inmunología , Masculino , Meninges/citología , Meninges/inmunología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunologíaRESUMEN
A dynamic and mutualistic interaction between tumour cells and the surrounding stroma promotes the initiation, progression, metastasis and chemoresistance of solid tumours. Far less understood is the relationship between the stroma and tumour-infiltrating leukocytes; however, emerging evidence suggests that the stromal compartment can shape antitumour immunity and responsiveness to immunotherapy. Thus, there is growing interest in elucidating the immunomodulatory roles of the stroma that evolve within the tumour microenvironment. In this Review, we discuss the evidence that stromal determinants interact with leukocytes and influence antitumour immunity, with emphasis on the immunological attributes of stromal cells that may foster their protumorigenic function.
Asunto(s)
Células del Estroma/inmunología , Microambiente Tumoral , Animales , Comunicación Celular , Movimiento Celular , Citotoxicidad Inmunológica , Matriz Extracelular/fisiología , Fibroblastos/fisiología , Humanos , Leucocitos/fisiología , Células Madre Mesenquimatosas/fisiologíaRESUMEN
In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/citología , Lectinas Tipo C/metabolismo , Ganglios Linfáticos/citología , Glicoproteínas de Membrana/metabolismo , Amidas/farmacología , Animales , Supervivencia Celular/inmunología , Colágeno/inmunología , Citoesqueleto/inmunología , Citoesqueleto/ultraestructura , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/inmunología , Fibroblastos/ultraestructura , Lectinas Tipo C/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/ultraestructura , Masculino , Glicoproteínas de Membrana/inmunología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Fosforilación , Piridinas/farmacología , Organismos Libres de Patógenos EspecíficosRESUMEN
After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.
Asunto(s)
Células Dendríticas/fisiología , Fibroblastos/citología , Ganglios Linfáticos/citología , Células del Estroma/citología , Actomiosina/metabolismo , Animales , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/inmunología , Femenino , Fibroblastos/fisiología , Inflamación/inmunología , Lectinas Tipo C/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Células del Estroma/fisiología , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Proteína rhoC de Unión a GTPRESUMEN
The AcrAB-TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli. Here we show that AcrZ (formerly named YbhT), a 49-amino-acid inner membrane protein, associates with the AcrAB-TolC complex. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. The highly conserved acrZ gene is coregulated with acrAB through induction by the MarA, Rob, and SoxS transcription regulators. In addition, mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ may enhance the ability of the AcrAB-TolC pump to export certain classes of substrates.
Asunto(s)
Farmacorresistencia Microbiana , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Immunoblotting , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación , Periplasma/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Podoplanin (PDPN) is a well-conserved, mucin-type transmembrane protein expressed in multiple tissues during ontogeny and in adult animals, including the brain, heart, kidney, lungs, osteoblasts, and lymphoid organs. Studies of PDPN-deficient mice have demonstrated that this molecule plays a critical role in development of the heart, lungs, and lymphatic system. PDPN is widely used as a marker for lymphatic endothelial cells and fibroblastic reticular cells of lymphoid organs and for lymphatics in the skin and tumor microenvironment. Much of the mechanistic insight into PDPN biology has been gleaned from studies of tumor cells; tumor cells often upregulate PDPN as they undergo epithelial-mesenchymal transition and this upregulation is correlated with increased motility and metastasis. The physiological role of PDPN that has been most studied is its ability to aggregate and activate CLEC-2-expressing platelets, as PDPN is the only known endogenous ligand for CLEC-2. However, more recent studies have revealed that PDPN also plays crucial roles in the biology of immune cells, including T cells and dendritic cells. This review will provide a comprehensive overview of the diverse roles of PDPN in development, immunology, and cancer.
RESUMEN
To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.
Asunto(s)
Movimiento Celular/fisiología , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Actinas/metabolismo , Inmunidad Adaptativa/fisiología , Animales , Células Presentadoras de Antígenos/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Embrión de Mamíferos , Células Endoteliales/metabolismo , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Cadenas Ligeras de Miosina/metabolismo , Activación Plaquetaria , Embarazo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal/fisiología , Piel/citología , Piel/metabolismo , Técnicas de Cultivo de Tejidos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino acids (small proteins) are encoded in the Escherichia coli genome. The vast majority of the corresponding genes have no known function. We screened 125 DNA bar-coded mutants to identify novel cell envelope stress and acute acid shock phenotypes associated with deletions of genes coding for sRNAs and small proteins. Nine deletion mutants (ssrA, micA, ybaM, ryeF, yqcG, sroH, ybhT, yobF, and glmY) were sensitive to cell envelope stress and two were resistant (rybB and blr). Deletion mutants of genes coding for four small proteins (yqgB, mgrB, yobF, and yceO) were sensitive to acute acid stress. We confirmed each of these phenotypes in one-on-one competition assays against otherwise-wild-type lacZ mutant cells. A more detailed investigation of the SsrA phenotype suggests that ribosome release is critical for resistance to cell envelope stress. The bar-coded deletion collection we generated can be screened for sensitivity or resistance to virtually any stress condition.