RESUMEN
OBJECTIVE: The need for efficient drugs and early treatment of patients with SARS-CoV-2 infection developing COVID-19 symptoms is of primary importance in daily clinical practice and it is certainly among the most difficult medical challenges in the current century. Recognizing those patients who will need stronger clinical efforts could effectively help doctors anticipate the eventual need for intensification of care (IoC) and choose the best treatment in order to avoid worse outcomes. PATIENTS AND METHODS: We enrolled 501 patients, consecutively admitted to our two COVID hospitals, and collected their clinical, anamnestic and laboratory data on admission. The aim of this retrospective study was to identify those data that are strictly associated with COVID-19 outcomes (IoC and in-hospital death) and that could somehow be intended as predictors of these outcomes. This allowed us to provide a "sketch" of the patient who undergoes, more often than others, an intensification of care and/or in-hospital death. RESULTS: Males were found to have a double risk of needing an IoC (OR=2.11) and a significant role was played by both the PaO2/FiO2 ratio on admission (OR=0.99) and serum LDH (OR=1.01). The main predictors of in-hospital death were age (OR=1.08) and the PaO2/FiO2 ratio on admission (OR=0.99). CONCLUSIONS: Male patients with high serum LDH on admission are those who undergo more often an intensification of care among COVID-19 inpatients. Both age and respiratory performances on admission modify the prognosis within the hospitalization period.
Asunto(s)
COVID-19/patología , Cuidados Críticos , Mortalidad Hospitalaria , Anciano , Anciano de 80 o más Años , COVID-19/mortalidad , COVID-19/virología , Comorbilidad , Femenino , Hospitales , Humanos , Italia , L-Lactato Deshidrogenasa/sangre , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Consumo de Oxígeno , Estudios Retrospectivos , Factores de Riesgo , SARS-CoV-2/aislamiento & purificación , Factores SexualesRESUMEN
We investigated the role of the complementarity determining region 1 (CDR1) of T cell receptor (TCR) beta chain both in antigen/major histocompatibility complex I (MHC I) and in superantigen (SAg)/MHC II complex recognition. Residues 26 to 31 of the V beta 10 domain of a TCR derived from an H-2Kd-restricted cytotoxic clone were individually changed to alanine, using site-directed mutagenesis, and the mutated TCR beta chains were transfected along with the wild-type TCR alpha chain into a TCR alpha-beta-T hydridoma. These mutations affected antigen/H-2Kd complex recognition, although to a different extent, as estimated by interleukin 2 production. Certain mutations also affected differently the recognition of two Staphylococcal toxins, exfoliative toxin and Staphylococcal enterotoxin C2, presented by HLA-DR1. Whereas mutation of residues D30 or T31 affect the recognition of both toxins, residues T26, L27, and H29 are critical for the recognition of only one of the SAgs. These observations demonstrate the participation of the CDR1 region in the recognition of peptide/MHC class I as well as SAg/MHC II complexes.
Asunto(s)
Antígenos H-2/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Superantígenos/inmunología , Alanina , Alelos , Secuencia de Aminoácidos , Animales , Línea Celular , Antígenos H-2/química , Hibridomas , Células L , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , TransfecciónRESUMEN
Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4; but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here, we have evaluated the role of genetic control of the B cell response to LPS at the single cell level. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (CB6F1). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS; whereas, BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Adding CpG to the LPS stimulus pushed PEC B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the nonresponse to LPS is a specific effect. Surprisingly, PEC B cells on the F1 background behaved as high responders, while splenic B cells behaved as low responders to LPS. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an, as yet, unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control of the LPS signaling pathway in the B lymphocyte.
Asunto(s)
Linfocitos B/inmunología , Regulación de la Expresión Génica , Lipopolisacáridos/inmunología , Peritoneo/inmunología , Bazo/citología , Animales , Linfocitos B/metabolismo , Cruzamientos Genéticos , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peritoneo/citología , Bazo/inmunologíaRESUMEN
Zika virus (ZIKV) infection during pregnancy is associated with a spectrum of developmental impairments known as congenital Zika syndrome (CZS). The prevalence of this syndrome varies across ZIKV endemic regions, suggesting that its occurrence could depend on cofactors. Here, we evaluate the relevance of protein malnutrition for the emergence of CZS. Epidemiological data from the ZIKV outbreak in the Americas suggest a relationship between undernutrition and cases of microcephaly. To experimentally examine this relationship, we use immunocompetent pregnant mice, which were subjected to protein malnutrition and infected with a Brazilian ZIKV strain. We found that the combination of protein restriction and ZIKV infection leads to severe alterations of placental structure and embryonic body growth, with offspring displaying a reduction in neurogenesis and postnatal brain size. RNA-seq analysis reveals gene expression deregulation required for brain development in infected low-protein progeny. These results suggest that maternal protein malnutrition increases susceptibility to CZS.
Asunto(s)
Desnutrición/complicaciones , Infección por el Virus Zika/congénito , Infección por el Virus Zika/complicaciones , Animales , Animales Recién Nacidos , Peso Corporal , Encéfalo/enzimología , Encéfalo/patología , Brasil/epidemiología , Dieta con Restricción de Proteínas , Brotes de Enfermedades , Embrión de Mamíferos/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Desnutrición/virología , Ratones Endogámicos C57BL , Microcefalia/complicaciones , Microcefalia/virología , Neurogénesis , Tamaño de los Órganos , Embarazo , Síndrome , Carga Viral , Infección por el Virus Zika/virologíaRESUMEN
Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated in vivo the impact of congenital ZIKV infection in blood vessel development, a crucial step in organogenesis. ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly.
Asunto(s)
Neovascularización Fisiológica , Infección por el Virus Zika/congénito , Virus Zika/fisiología , Animales , Vasos Sanguíneos/patología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/virología , Modelos Animales de Enfermedad , Embrión de Mamíferos/patología , Embrión de Mamíferos/virología , Células Endoteliales/patología , Células Endoteliales/virología , Femenino , Ratones Endogámicos C57BL , Neurogénesis , Tamaño de los Órganos , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.
Asunto(s)
Linfocitos B/inmunología , Células Clonales/inmunología , Clonación Molecular/métodos , Región Variable de Inmunoglobulina/inmunología , Animales , Linfocitos B/citología , Femenino , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , ARN/química , ARN/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Addition of recombinant interleukin-2 (rIL-2) to normal adult murine thymocytes in vitro as the only exogenous stimulus leads to a dose-dependent mitogenic response characterized by two distinct dosage kinetic components. The high-affinity IL-2 thymocyte response is mounted by in vivo-activated (IL-2 receptor light chain positive) thymocytes, while the low-affinity IL-2 response, of larger amplitude, is carried out by resting thymocytes. Addition of IL-2 to thymocytes also triggers intense IL-3 secretory responses with both high and low IL-2 affinity components. Addition of high IL-2 dosages to thymocyte bulk cultures results in a dramatic increase in IL-2 responsiveness for both proliferation and IL-3 secretion on a per viable cell basis and with tightly coupled temporal kinetics. The low-affinity component of IL-2-proliferative and IL-3-secreting responses is carried out by resting mature CD4+ thymocytes, as assessed by negative selection with monoclonal antibodies (mAb) plus complement. The mechanism of resting thymocyte activation by high doses of IL-2 is partially characterized. Depletion of endogenous thymus-adherent cells abolished both proliferation and IL-3 secretion, and addition of splenic accessory cells or peritoneal macrophages to depleted thymocytes restored IL-2 responsiveness. Mature CD4+ thymocytes spontaneously form rosettes with adherent accessory cells, while CD8+ thymocytes do so with much less efficiency. Rosette formation of CD4+, but not of CD8+ thymocytes, can be blocked by anti-CD4 mAb GK1.5. At the same dosage as it prevents rosette formation, mAb GK1.5 also blocks the low-affinity thymocyte response to IL-2. The high-affinity IL-2 response is completely resistant to the action of cyclosporin A (CsA), but the low-affinity IL-2 response, although of much larger amplitude, can be almost completely suppressed by CsA. Together, these results demonstrate that resting CD4+ thymocytes can be induced to proliferation and lymphokine secretion by IL-2 alone in a process that is dependent on interaction with accessory cells, involves CD4 adhesion molecules and triggers activation through a CsA-sensitive pathway. In addition, the results demonstrate that IL-2 alone is able to enhance thymocyte IL-2 responsiveness and IL-3 secretory responses in vitro. The ability of IL-2 to induce and maintain thymocyte function is discussed in the light of these results.
Asunto(s)
Interleucina-2/farmacología , Interleucina-3/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Proteínas Recombinantes/farmacología , Linfocitos T/metabolismoRESUMEN
Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos de Superficie/fisiología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Animales , Complejo CD3 , Femenino , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Antígenos Thy-1RESUMEN
T cell receptors (TCR) recognize peptides presented by major histocompatibility complex (MHC) molecules on the surface of cells. Sequence homology between the variable regions of the T cell receptor and of antibodies suggests that similarly-folded domains participate in ligand binding in both cases. However, most current models assume that both TCR chains (alpha and beta) are required for specific binding, whereas the heavy chain alone can confer specificity on many antibodies. We have therefore constructed chimeric molecules with alpha and beta from two different TCR, one restricted by the class II MHC protein, Ek, and the other by the class I MHC, Kd. The beta chain alone was sufficient for specific recognition of a peptide, Cw3, bound to Kd, but the alpha chain contributed to the overall avidity. These results suggest that other TCR may recognize their ligands primarily through the beta chain.
Asunto(s)
Epítopos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Técnicas In Vitro , Complejo Mayor de Histocompatibilidad/inmunología , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Single alanine substitutions were introduced into the CDR1 region of the beta chain of a Kd-restricted TCR. Mutants and wild-type TCR were attached to the zeta chain of the CD3 complex and expressed at the surface of a rat basophil cell line. Transfectants were tested for the binding of purified soluble Kd-peptide complexes. With this experimental system, accessory molecules are unlikely to play a major role and the contribution of each residue to the interaction can be addressed. Results show that all positions in the CDR1 region are involved in the binding to the Kd-peptide complex but at varying degrees. These effects are discussed in relation to a molecular model of the TCR. Comparison of these results with previous data obtained in a T cell hybridoma system suggests the existence of a threshold in the TCR affinity necessary for mature T cell activation.
Asunto(s)
Antígenos H-2/inmunología , Antígenos HLA-C/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Basófilos/inmunología , Línea Celular , Antígenos H-2/metabolismo , Antígenos HLA-C/metabolismo , Modelos Moleculares , Mutación/genética , Ratas , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad , TransfecciónRESUMEN
The effects of the glycoinositolphospholipids (GIPLs) fromTrypanosoma cruzi on T lymphocyte activation were investigated in a mouse T cell hybridoma (DO-11.10). Purified GIPLs from T. cruzi strains Y and G markedly increased IL-2 mRNA transcripts and IL-2 secretion induced by mitogenic anti-CD3 and anti-Thy1 mAbs. This costimulatory function was also revealed by the induction of IL-2 secretion after the simultaneous addition of the T. cruzi GIPLs and either the calcium ionophore A23187 or phorbol ester. The capacity of the GIPL molecule to induce an increase in cytoplasmic calcium levels was also demonstrated. After exposure of T cell hybridoma to GIPL, the nuclear transcription factor NFAT1 became partially dephosphorylated, and its nuclear localization was demonstrated both in the T cell hybridoma and in Balb/c CD3(+) cells. These results demonstrate that T. cruzi GIPL molecules are capable of signaling to T cells and therefore could be valuable tools for the study of T cell activation, besides playing a potential role in subverting the T lymphocyte immune response during T. cruzi infection.