RESUMEN
Treatments of colitis, inflammation of the intestine, rely on induction of immune suppression associated with systemic adverse events, including recurrent infections. This treatment strategy is specifically problematic in the increasing population of patients with cancer with immune checkpoint inhibitor (ICI)-induced colitis, as immune suppression also interferes with the ICI-treatment response. Thus, there is a need for local-acting treatments that reduce inflammation and enhance intestinal healing. Here, we investigated the effect and safety of bacterial delivery of short-lived immunomodulating chemokines to the inflamed intestine in mice with colitis. Colitis was induced by dextran sulfate sodium (DSS) alone or in combination with ICI (anti-PD1 and anti-CTLA-4), and Limosilactobacillus reuteri R2LC (L. reuteri R2LC) genetically modified to express the chemokine CXCL12-1α (R2LC_CXCL12, emilimogene sigulactibac) was given perorally. In addition, the pharmacology and safety of the formulated drug candidate, ILP100-Oral, were evaluated in rabbits. Peroral CXCL12-producing L. reuteri R2LC significantly improved colitis symptoms already after 2 days in mice with overt DSS and ICI-induced colitis, which in benchmarking experiments was demonstrated to be superior to treatments with anti-TNF-α, anti-α4ß7, and corticosteroids. The mechanism of action involved chemokine delivery to Peyer's patches (PPs), confirmed by local CXCR4 signaling, and increased numbers of colonic, regulatory immune cells expressing IL-10 and TGF-ß1. No systemic exposure or engraftment could be detected in mice, and product feasibility, pharmacology, and safety were confirmed in rabbits. In conclusion, peroral CXCL12-producing L. reuteri R2LC efficiently ameliorates colitis, enhances mucosal healing, and has a favorable safety profile.NEW & NOTEWORTHY Colitis symptoms are efficiently reduced by peroral administration of probiotic bacteria genetically modified to deliver CXCL12 locally to the inflamed intestine in several mouse models.
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Quimiocina CXCL12 , Colitis , Sulfato de Dextran , Modelos Animales de Enfermedad , Limosilactobacillus reuteri , Animales , Colitis/inmunología , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/terapia , Colitis/metabolismo , Ratones , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Administración Oral , Conejos , Probióticos/administración & dosificación , Ratones Endogámicos C57BL , Femenino , Colon/metabolismo , Colon/microbiología , Colon/inmunología , MasculinoRESUMEN
[Figure: see text].
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Quimiocina CXCL12/metabolismo , Isquemia/terapia , Macrófagos/fisiología , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo II/metabolismo , Flujo Sanguíneo Regional/fisiología , Animales , Movimiento Celular , Proliferación Celular , Quimiocina CCL2/metabolismo , Humanos , Isquemia/fisiopatología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo III/metabolismo , Plásmidos/metabolismo , Receptores CCR2/metabolismo , Receptores CXCR4/metabolismoRESUMEN
BACKGROUND: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts. METHODS: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points. RESULTS: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%). CONCLUSIONS: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.
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Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , COVID-19/inmunología , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/inmunología , Reinfección , Adulto , Anticuerpos Antivirales/inmunología , Infecciones Asintomáticas , Prueba de Ácido Nucleico para COVID-19 , Prueba Serológica para COVID-19 , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Células T de Memoria , Persona de Mediana Edad , Pandemias , SARS-CoV-2 , Factores de TiempoRESUMEN
Neutrophils promptly accumulate in large numbers at sites of tissue injury. Injuries to the skin or mucosae disrupt barriers against the external environment, and the bactericidal actions of neutrophils are important in preventing microbial invasion. Neutrophils have also been associated with exacerbated inflammation, for example in non-healing wounds or in conditions such as inflammatory bowel disease (IBD). However, additional neutrophil functions important for angiogenesis and tissue restoration have been uncovered in models of sterile and ischemic injury, as well as in tumors. These functions are also relevant in healing skin and mucosal wounds, and can be impaired in conditions associated with non-healing wounds, such as diabetes. Here, we discuss our current understanding of neutrophil contributions to healing, and how the latter can be compromised in disease.
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Neutrófilos/inmunología , Neutrófilos/metabolismo , Cicatrización de Heridas , Animales , Susceptibilidad a Enfermedades , Homeostasis , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Neutrófilos/patología , Especificidad de Órganos , Piel/inmunología , Piel/metabolismo , Piel/patología , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunologíaRESUMEN
OBJECTIVE: The full spectrum of coronavirus disease 2019 (COVID-19) infection ranges from asymptomatic to acute respiratory distress syndrome, characterized by hyperinflammation and thrombotic microangiopathy. The pathogenic mechanisms are poorly understood, but emerging evidence suggest that excessive neutrophil extracellular trap (NET) formation plays a key role in COVID-19 disease progression. Here, we evaluate if circulating markers of NETs are associated with COVID-19 disease severity and clinical outcome, as well as to markers of inflammation and in vivo coagulation and fibrinolysis. Approach and Results: One hundred six patients with COVID-19 with moderate to severe disease were enrolled shortly after hospital admission and followed for 4 months. Acute and convalescent plasma samples as well as plasma samples from 30 healthy individuals were assessed for markers of NET formation: citrullinated histone H3, cell-free DNA, NE (neutrophil elastase). We found that all plasma levels of NET markers were elevated in patients with COVID-19 relative to healthy controls, that they were associated with respiratory support requirement and short-term mortality, and declined to those found in healthy individuals 4 months post-infection. The levels of the NET markers also correlated with white blood cells, neutrophils, inflammatory cytokines, and C-reactive protein, as well as to markers of in vivo coagulation, fibrinolysis, and endothelial damage. CONCLUSIONS: Our findings suggest a role of NETs in COVID-19 disease progression, implicating their contribution to an immunothrombotic state. Further, we observed an association between circulating markers of NET formation and clinical outcome, demonstrating a potential role of NET markers in clinical decision-making, as well as for NETs as targets for novel therapeutic interventions in COVID-19.
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COVID-19/sangre , COVID-19/complicaciones , Trampas Extracelulares/metabolismo , Anciano , Biomarcadores/sangre , Síndrome de Liberación de Citoquinas/sangre , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Femenino , Fibrinólisis , Humanos , Inflamación/sangre , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Trombosis/virologíaRESUMEN
Impaired wound closure is a growing medical problem associated with metabolic diseases and aging. Immune cells play important roles in wound healing by following instructions from the microenvironment. Here, we developed a technology to bioengineer the wound microenvironment and enhance healing abilities of the immune cells. This resulted in strongly accelerated wound healing and was achieved by transforming Lactobacilli with a plasmid encoding CXCL12. CXCL12-delivering bacteria administrated topically to wounds in mice efficiently enhanced wound closure by increasing proliferation of dermal cells and macrophages, and led to increased TGF-ß expression in macrophages. Bacteria-produced lactic acid reduced the local pH, which inhibited the peptidase CD26 and consequently enhanced the availability of bioactive CXCL12. Importantly, treatment with CXCL12-delivering Lactobacilli also improved wound closure in mice with hyperglycemia or peripheral ischemia, conditions associated with chronic wounds, and in a human skin wound model. Further, initial safety studies demonstrated that the topically applied transformed bacteria exerted effects restricted to the wound, as neither bacteria nor the chemokine produced could be detected in systemic circulation. Development of drugs accelerating wound healing is limited by the proteolytic nature of wounds. Our technology overcomes this by on-site chemokine production and reduced degradation, which together ensure prolonged chemokine bioavailability that instructed local immune cells and enhanced wound healing.
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Quimiocina CXCL12/administración & dosificación , Quimiocina CXCL12/farmacología , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/metabolismo , Cicatrización de Heridas , Animales , Proliferación Celular , Regulación de la Expresión Génica , Terapia Genética , Humanos , Macrófagos/metabolismo , Ratones , Plásmidos , Piel , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta/metabolismo , Heridas y Lesiones/terapiaRESUMEN
The unique role of neutrophils in host defense is not only based on their abilities to kill bacteria but is also due to their abundance in circulation and their ability to quickly migrate and accumulate in great numbers at afflicted sites. The high number of circulating neutrophils is the result of regulated release of new neutrophils from bone marrow as well as from marginated pools to balance their recruitment to tissue. Marginated pools, such as the spleen and lung, have previously been attributed to passively delay neutrophil transit time due to their large capillary network, but recent reports demonstrate that they are comprised of neutrophils with specific functions. The spleen, for instance, holds neutrophil subpopulations at different anatomical locations with distinct functions important for, e.g., bacterial eradication, and the lung was recently shown to re-educate neutrophils that had trafficked from a site of sterile injury to home back to bone marrow for elimination. Further, recent reports demonstrate subpopulations of neutrophils with different actions during homeostasis, infection, tissue restitution and cancer. It is becoming increasingly clear that this cannot be due to different stages of neutrophil activation during their life span but instead points towards distinct subpopulations of neutrophils with different effector functions. Whether these cellular distinctions are due to different education or origin is, however, not yet known. Together, the accumulating information about the heterogeneous neutrophils presents important insights into their role in development of pathologies, as well as revealing novel targets in the form of certain subpopulations to treat disease.
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Neutrófilos/citología , Animales , Humanos , Modelos Biológicos , Neoplasias/patología , Cicatrización de HeridasRESUMEN
PURPOSE OF REVIEW: This review describes the mechanisms by which neutrophils contribute to angiogenesis in hypoxic tissues during different conditions and diseases (e.g., menstrual cycle, wound healing, ischemic diseases, cancers), with particular focus on the recently described proangiogenic neutrophil subpopulation. RECENT FINDINGS: The importance of neutrophils in initiation of angiogenesis has been described during the past decade, and is believed to occur through release of the well-known proangiogenic factors Bv8, vascular endothelial growth factor A, and matrix metalloproteinase 9. However, additional proangiogenic actions of neutrophils have been outlined this year, mediated through for example pyruvate kinase M2, 14,15-epoxyeicosatrienoic acid, and formation of neutrophil extracellular traps, although their distinct mechanisms of action remain partly unknown. Neutrophils can also limit angiogenesis by secreting for example neutrophil elastase and α-defensins, which generate angiostatic molecules and proteolytically inactivate proangiogenic factors, respectively. These opposing neutrophil actions can be the consequence of on-site education or recruitment of distinct subpopulations from circulation. Indeed, a circulating proangiogenic neutrophil subpopulation was recently described in mice and men, which was rapidly recruited to hypoxic tissues by vascular endothelial growth factor A. SUMMARY: These recent findings have highlighted the diversity of actions performed by neutrophils in the angiogenic process and identified new opportunities to regulate angiogenesis.
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Neovascularización Patológica/inmunología , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Neutrófilos/fisiología , Animales , Humanos , Leucocitos/fisiología , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.
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Integrina alfa4/metabolismo , Islotes Pancreáticos/metabolismo , Músculo Esquelético/metabolismo , Neutrófilos/metabolismo , Receptores CXCR4/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Western Blotting , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Integrina alfa4/genética , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía por Video , Músculo Esquelético/citología , Neovascularización Fisiológica , Infiltración Neutrófila , Neutrófilos/citología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The availability of paracrine factors in the islets of Langerhans, and the constitution of the beta cell basement membrane can both be affected by proteolytic enzymes. This study aimed to investigate the effects of the extracellular matrix-degrading enzyme gelatinase B/matrix metalloproteinase-9 (Mmp-9) on islet function in mice. Islet function of Mmp9-deficient (Mmp9(-/-)) mice and their wild-type littermates was evaluated both in vivo and in vitro. The pancreata of Mmp9(-/-) mice did not differ from wild type in islet mass or distribution. However, Mmp9(-/-) mice had an impaired response to a glucose load in vivo, with lower serum insulin levels. The glucose-stimulated insulin secretion was reduced also in vitro in isolated Mmp9(-/-) islets. The vascular density of Mmp9(-/-) islets was lower, and the capillaries had fewer fenestrations, whereas the islet blood flow was threefold higher. These alterations could partly be explained by compensatory changes in the expression of matrix-related proteins. This in-depth investigation of the effects of the loss of MMP-9 function on pancreatic islets uncovers a deteriorated beta cell function that is primarily due to a shift in the beta cell phenotype, but also due to islet vascular aberrations. This likely reflects the importance of a normal islet matrix turnover exerted by MMP-9, and concomitant release of paracrine factors sequestered on the matrix.
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Envejecimiento/fisiología , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Neovascularización Fisiológica , Animales , Capilares/efectos de los fármacos , Capilares/patología , Capilares/ultraestructura , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/patología , Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Masculino , Metaloproteinasa 9 de la Matriz/deficiencia , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Flujo Sanguíneo Regional , Transcripción Genética/efectos de los fármacosRESUMEN
Recruitment and retention of leukocytes at a site of blood vessel growth are crucial for proper angiogenesis and subsequent tissue perfusion. Although critical for many aspects of regenerative medicine, the mechanisms of leukocyte recruitment to and actions at sites of angiogenesis are not fully understood. In this study, we investigated the signals attracting leukocytes to avascular transplanted pancreatic islets and leukocyte actions at the engraftment site. Expression of the angiogenic stimulus VEGF-A by mouse pancreatic islets was elevated shortly after syngeneic transplantation to muscle. High levels of leukocytes, predominantly CD11b(+)/Gr-1(+)/CXCR4(hi) neutrophils, were observed at the site of engraftment, whereas VEGF-A-deficient islets recruited only half of the amount of leukocytes when transplanted. Acute VEGF-A exposure of muscle increased leukocyte extravasation but not the levels of SDF-1α. VEGF-A-recruited neutrophils expressed 10 times higher amounts of MMP-9 than neutrophils recruited to an inflammatory stimulus. Revascularization of islets transplanted to MMP-9-deficient mice was impaired because blood vessels initially failed to penetrate grafts, and after 2 weeks vascularity was still disturbed. This study demonstrates that VEGF-A recruits a proangiogenic circulating subset of CD11b(+)/Gr-1(+) neutrophils that are CXCR4(hi) and deliver large amounts of the effector protein MMP-9, required for islet revascularization and functional integration after transplantation.
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Trasplante de Islotes Pancreáticos/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Neovascularización Fisiológica/fisiología , Neutrófilos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Antígeno CD11b/metabolismo , Quimiocina CXCL12/metabolismo , Femenino , Hipoxia , Inmunohistoquímica , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía por Video , Neovascularización Fisiológica/genética , Infiltración Neutrófila , Receptores CXCR4 , Receptores de Quimiocina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Lack of sleep greatly affects our immune system. The present study investigates the acute effects of total sleep deprivation on blood neutrophils, the most abundant immune cell in our circulation and the first cell type recruited to sites of infection. Thus, the population diversity and function of circulating neutrophils were compared in healthy young men following one night of total sleep deprivation (TSD) or after 8h regular sleep. We found that neutrophil counts were elevated after nocturnal wakefulness (2.0 ± 0.2 × 10(9)/l vs. 2.6 ± 0.2 × 10(9)/l, sleep vs. TSD, respectively) and the population contained more immature CD16(dim)/CD62L(bright) cells (0.11 ± 0.040 × 10(9)/l [5.5 ± 1.1%] vs. 0.26 ± 0.020 × 10(9)/l [9.9 ± 1.4%]). As the rise in numbers of circulating mature CD16(bright)/CD62L(bright) neutrophils was less pronounced, the fraction of this subpopulation showed a significant decrease (1.8 ± 0.15 × 10(9)/l [88 ± 1.8%] vs. 2.1 ± 0.12 × 10(9)/l [82 ± 2.8%]). The surface expression of receptors regulating mobilization of neutrophils from bone marrow was decreased (CXCR4 and CD49d on immature neutrophils; CXCR2 on mature neutrophils). The receptor CXCR2 is also involved in the production of reactive oxygen species (ROS), and in line with this, total neutrophils produced less ROS. In addition, following sleep loss, circulating neutrophils exhibited enhanced surface levels of CD11b, which indicates enhanced granular fusion and concomitant protein translocation to the membrane. Our findings demonstrate that sleep loss exerts significant effects on population diversity and function of circulating neutrophils in healthy men. To which extent these changes could explain as to why people with poor sleep patterns are more susceptible to infections warrants further investigation.
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Neutrófilos/inmunología , Privación de Sueño/inmunología , Enfermedad Aguda , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Núcleo Celular/ultraestructura , Quimiotaxis de Leucocito , Proteínas Ligadas a GPI/análisis , Voluntarios Sanos , Humanos , Selectina L/análisis , Recuento de Leucocitos , Masculino , Neutrófilos/química , Neutrófilos/clasificación , Neutrófilos/metabolismo , Polisomnografía , Especies Reactivas de Oxígeno/metabolismo , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Receptores de IgG/análisis , Receptores de Interleucina-8B/biosíntesis , Receptores de Interleucina-8B/genética , Estallido Respiratorio , Adulto JovenRESUMEN
Cancer detection is challenging, especially in patients with unspecific cancer symptoms. Biomarkers could identify patients at high risk of cancer. Prior studies indicate that neutrophil extracellular traps (NETs) are associated with cancer, but also with autoimmune and infectious diseases. The objective of this prospective study was to investigate markers associated with NET formation (nucleosomal citrullinated histone 3 [H3Cit-DNA], cell free DNA [cfDNA] and neutrophil elastase [NE]), and c-reactive protein (CRP) in patients with unspecific cancer symptoms, such as fatigue, weight loss or radiological sign of malignancy without an apparent primary tumor, referred to the Diagnostic Center at Danderyd Hospital in Sweden. Blood samples were drawn on admission, before cancer diagnosis. Out of 475 patients, 160 (34%) were diagnosed with cancer, 56 (12%) with autoimmune disease, 32 (7%) with infectious disease, 71 (15%) with other diseases and 156 (33%) received no diagnosis. H3Cit-DNA, cfDNA, NE and CRP were significantly higher in patients with cancer compared to patients without cancer (p < 0.0001, p < 0.0001, p = 0.004, and p = 0.0002 respectively). H3Cit-DNA, but not cfDNA, NE or CRP, was significantly elevated in patients with cancer compared to patients with autoimmune disease (p = 0.0001). H3Cit-DNA, cfDNA, NE or CRP did not differ between cancer and infectious disease. In conclusion, H3Cit-DNA is elevated in patients diagnosed with cancer compared to non-cancer patients with the same symptomatology. Further studies should evaluate if H3Cit-DNA could aid in selecting patients that would benefit the most from a rapid cancer diagnostic work-up.
RESUMEN
Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRß. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRß deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing.
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Modelos Animales de Enfermedad , Isquemia , Macrófagos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Isquemia/metabolismo , Isquemia/patología , Isquemia/genética , Fenotipo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/lesiones , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Ratones Endogámicos C57BL , Ratones , Masculino , Miembro Posterior/irrigación sanguínea , Neovascularización Fisiológica/genética , Regulación hacia Arriba , Transcriptoma , Análisis de la Célula Individual , Biomarcadores/metabolismo , Recuperación de la Función , Ratones NoqueadosRESUMEN
Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.
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Transportador 1 de Aminoácidos Excitadores , Fibroblastos , Erección Peniana , Pene , Receptores Notch , Animales , Masculino , Ratones , Circulación Sanguínea , Transportador 1 de Aminoácidos Excitadores/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Erección Peniana/fisiología , Pene/irrigación sanguínea , Pene/fisiología , Receptores Notch/metabolismo , Transducción de Señal , Vasoconstricción , VasodilataciónRESUMEN
αB-crystallin is a small heat shock protein, which has pro-angiogenic properties by increasing survival of endothelial cells and secretion of vascular endothelial growth factor A. Here we demonstrate an additional role of αB-crystallin in regulating vascular function, through enhancing tumor necrosis factor α (TNF-α) induced expression of endothelial adhesion molecules involved in leukocyte recruitment. Ectopic expression of αB-crystallin in endothelial cells increases the level of E-selectin expression in response to TNF-α, and enhances leukocyte-endothelial interaction in vitro. Conversely, TNF-α-induced expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin is markedly inhibited in endothelial cells isolated from αB-crystallin-deficient mice. This is associated with elevated levels of IκB in αB-crystallin deficient cells and incomplete degradation upon TNF-α stimulation. Consistent with this, endothelial adhesion molecule expression is reduced in inflamed vessels of αB-crystallin deficient mice, and leukocyte rolling velocity is increased. Our data identify αB-crystallin as a new regulator of leukocyte recruitment, by enhancing pro-inflammatory nuclear factor κ B-signaling and endothelial adhesion molecule expression during endothelial activation.
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Moléculas de Adhesión Celular/biosíntesis , Células Endoteliales/citología , Rodamiento de Leucocito/fisiología , Leucocitos/citología , FN-kappa B/metabolismo , Cadena B de alfa-Cristalina/fisiología , alfa-Cristalinas/deficiencia , Transporte Activo de Núcleo Celular , Animales , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Línea Celular , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas I-kappa B/biosíntesis , Proteínas I-kappa B/genética , Inflamación , Células Jurkat , Masculino , Ratones , Microvasos/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción Genética , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba , Cadena B de alfa-Cristalina/genética , alfa-Cristalinas/genéticaRESUMEN
The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1ß [IL-1ß]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.
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Proteínas de Unión al Calcio/fisiología , Permeabilidad Capilar/fisiología , Endotelio/metabolismo , Neutrófilos/fisiología , Migración Transendotelial y Transepitelial , Animales , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio/citología , Endotelio/efectos de los fármacos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Confocal/instrumentación , Microscopía Electrónica de Transmisión , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/ultraestructura , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.
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Infección Irruptiva , COVID-19 , Humanos , Carga Viral , COVID-19/prevención & control , SARS-CoV-2 , Personal de Salud , Inmunoglobulina A , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
Background: Impaired wound healing is a growing medical problem and very few approved drugs with documented clinical efficacy are available. CXCL12-expressing lactic acid bacteria, Limosilactobacillus reuteri (ILP100-Topical), has been demonstrated to accelerate wound healing in controlled preclinical models. In this first-in-human study, the primary objective was to determine safety and tolerability of the drug candidate ILP100-Topical, while secondary objectives included assessments of clinical and biologic effects on wound healing by traditionally accepted methods and explorative and traceable assessments. Methods: SITU-SAFE is an adaptive, randomised, double-blind, placebo-controlled, first-in-human phase 1 trial (EudraCT 2019-000680-24) consisting of a single (SAD) and a multiple ascending dose (MAD) part of three dose cohorts each. The study was performed at the Phase 1 Unit, Uppsala University Hospital, Uppsala, Sweden. Data in this article were collected between Sep 20th, 2019 and Oct 20th 2021. In total 240 wounds were induced on the upper arms in 36 healthy volunteers. SAD: 12 participants, 4 wounds (2/arm), MAD: 24 participants, 8 wounds (4/arm). Wounds in each participant were randomised to treatment with placebo/saline or ILP100-Topical. Findings: In all individuals and doses, ILP100-Topical was safe and well-tolerated with no systemic exposure. A combined cohort analysis showed a significantly larger proportion of healed wounds (p = 0.020) on Day 32 by multi-dosing of ILP100-Topical when compared to saline/placebo (76% (73/96) and 59% (57/96) healed wounds, respectively). In addition, time to first registered healing was shortened by 6 days on average, and by 10 days at highest dose. ILP100-Topical increased the density of CXCL12+ cells in the wounds and local wound blood perfusion. Interpretation: The favourable safety profile and observed effects on wound healing support continued clinical development of ILP100-Topical for the treatment of complicated wounds in patients. Funding: Ilya Pharma AB (Sponsor), H2020 SME Instrument Phase II (#804438), Knut and Alice Wallenberg foundation.