RESUMEN
The assembly of Ig genes in developing B lymphocytes by V(D)J recombination is initiated by the RAG1-RAG2 endonuclease complex. We previously identified an interaction between RAG1 and viral protein R binding protein (VprBP) (also known as DNA damage binding protein 1 cullin 4-associated factor 1 [DCAF1]), a substrate receptor for the cullin 4-really interesting new gene (RING) E3 ubiquitin ligase (CRL4). We report in this article that in mice, B cell-intrinsic loss of VprBP increases RAG1 protein levels and disrupts expression of the endoribonuclease Dicer, which is essential for microRNA maturation. Rag1/2 transcription is known to be derepressed by loss of microRNA-mediated suppression of phosphatase and tensin homolog, raising the possibility that the elevated level of RAG1 observed in VprBP-deficient B cells is caused indirectly by the loss of Dicer. However, we show that VprBP restrains RAG1 expression posttranscriptionally and independently of Dicer. Specifically, loss of VprBP stabilizes RAG1 protein, which we show is normally degraded via a mechanism requiring both 20S proteasome and cullin-RING E3 ubiquitin ligase activity. Furthermore, we show that RAG1 stabilization through small molecule inhibition of cullin-RING E3 ubiquitin ligase activation promotes V(D)J recombination in a murine pre-B cell line. Thus, in addition to identifying a role for VprBP in maintaining Dicer levels in B cells, our findings reveal the basis for RAG1 turnover and provide evidence that the CRL4VprBP(DCAF1) complex functions to maintain physiological levels of V(D)J recombination.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Homeodominio/genética , Animales , Linfocitos B/fisiología , Células Cultivadas , Proteínas Cullin/genética , Ratones , MicroARNs/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Serina-Treonina Quinasas , Proteolisis , Interferencia de ARN/fisiología , Transcripción Genética/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
IL10 plays a dual role in supporting humoral immunity and inhibiting inflammatory conditions. B cells producing IL10 are thought to play a key regulatory role in maintaining self-tolerance and suppressing excessive inflammation during autoimmune and infectious diseases, primarily by inhibiting associated T cell responses. The extent to which B cells, through the provision of IL10, might function to sustain or inhibit autoantibody production is less clear. We previously described transgenic mice expressing catalytically inactive RAG1 (dnRAG1 mice), which show expansion of an IL10-compentent CD5+ B cell subset that phenotypically resembles B10 B cells, hypogammaglobulinemia, and a restricted B cell receptor repertoire with features indicative of impaired B cell receptor editing. We show here that B10-like B cells in dnRAG1 mice bind the membrane-associated autoantigen phosphatidylcholine (PtC), and that in vitro lipopolysaccharide (LPS) stimulation of dnRAG1 splenocytes induces a robust IgM response enriched in reactivity toward lupus-associated autoantigens. This outcome was correlated with detection of sIgMhi B cell populations that were distinct from, but in addition to, sIgMint populations observed after similar treatment of wild-type splenocytes. Loss of IL10 expression in dnRAG1 mice had no significant effect on B10-like B cell expansion or the frequency of PtC+ B cells. Compared to IL10+/+ dnRAG1 mice, levels of serum IgM, but not serum IgG, were highly elevated in some naïve IL10-/- dnRAG1 mice, and was correlated with a significant increase in serum BAFF levels. Differentiation of sIgMint B cells from LPS-stimulated dnRAG1 splenocytes was enhanced by loss of IL10 expression and IL10 blockade, but was suppressed by treatment with recombinant IL10. In vitro LPS-induced differentiation and antibody production was inhibited by treatment with JAK/STAT inhibitors or a synthetic corticosteroid, independent of IL10 expression and genotype. Taken together, these data suggest that IL10 expression in dnRAG1 mice maintains suppression of IgM levels in part by inhibiting BAFF production, and that regulatory B10-like B cells, through the provision of IL10, constrains B cell differentiation in response to mitogenic stimuli. Furthermore, autoantibody profiling raises a possible link between CD5+ B cell expansion, mitogenic stimulation, and autoantibodies associated with autoimmune complications observed in lupus and lupus-related disorders.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Proteínas de Homeodominio/inmunología , Interleucina-10/inmunología , Animales , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Antígenos CD5/inmunología , Antígenos CD5/metabolismo , Diferenciación Celular/inmunología , Proliferación Celular , Células Cultivadas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Ratones Noqueados , Ratones Transgénicos , Autotolerancia/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
Resistant hypertension (RH) is defined as blood pressure (BP) that remains above target levels despite adherence to at least three different antihypertensive medications, typically including a diuretic. Epidemiological studies estimate that RH is increasing in prevalence, and is associated with detrimental health outcomes. The pathophysiology underlying RH is complex, involving multiple, overlapping contributors including activation of the renin-angiotensin aldosterone system and the sympathetic nervous system, volume overload, endothelial dysfunction, behavioural and lifestyle factors. Hypertension guidelines currently recommend specific pharmacotherapy for 1st, 2nd and 3rd-line treatment, however no specific fourth-line pharmacotherapy is provided for those with RH. Rather, five different antihypertensive drug classes are generally suggested as possible alternatives, including: mineralocorticoid receptor antagonists, α1-adrenergic antagonists, α2-adrenergic agonists, ß-blockers, and peripheral vasodilators. Each of these drug classes vary in their efficacy, tolerability and safety profile. This review summarises the available data on each of these drug classes as a potential fourth-line drug and reveals a lack of robust clinical evidence for preferred use of most of these classes in the setting of RH. Moreover, there is a lack of direct comparative trials that could assist in identifying a preferred fourth-line pharmacologic approach and in providing evidence for hypertensive guidelines for adequate treatment of RH.
Asunto(s)
Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Presión Sanguínea/efectos de los fármacos , Diuréticos/uso terapéutico , Humanos , Sistema Renina-AngiotensinaRESUMEN
B cell development past the pro-B cell stage in mice requires the Cul4-Roc1-DDB1 E3 ubiquitin ligase substrate recognition subunit VprBP. Enforced Bcl2 expression overcomes defects in distal VH-DJH and secondary Vκ-Jκ rearrangement associated with VprBP insufficiency in B cells and substantially rescues maturation of marginal zone and Igλ(+) B cells, but not Igκ(+) B cells. In this background, expression of a site-directed Igκ L chain transgene increases Igκ(+) B cell frequency, suggesting VprBP does not regulate L chain expression from a productively rearranged Igk allele. In site-directed anti-dsDNA H chain transgenic mice, loss of VprBP function in B cells impairs selection of Igκ editor L chains typically arising through secondary Igk rearrangement, but not selection of Igλ editor L chains. Both H and L chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together, these data argue that VprBP is required for the efficient receptor editing and selection of Igκ(+) B cells, but is largely dispensable for Igλ(+) B cell development and selection, and that VprBP is necessary to rescue autoreactive B cells from anergy induction.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/genética , Selección Clonal Mediada por Antígenos/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Alelos , Animales , Linfocitos B/inmunología , Membrana Celular/metabolismo , Anergia Clonal/genética , Expresión Génica , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas lambda de Inmunoglobulina/metabolismo , Factores Reguladores del Interferón/genética , Ratones , Ratones Transgénicos , Factor de Transcripción PAX5/genética , Fenotipo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Recombinación V(D)JRESUMEN
AIM: Acute postinfectious glomerulonephritis is common in indigenous communities in the Northern Territory, Australia. It is a major risk factor for the high prevalence of chronic kidney disease. We aimed to analyse the clinical presentation, pathological spectra, treatment and outcomes of biopsy-proven acute postinfectious glomerulonephritis in the Northern Territory. METHODS: We performed a retrospective cohort analysis of all adult patients (≥18 years) who were diagnosed with acute postinfectious glomerulonephritis on native renal biopsies from 01/01/2004 to 31/05/2014. The outcome measure was end-stage renal disease requiring long-term dialysis. RESULTS: Forty-three of 340 patients who had renal biopsies had acute postinfectious glomerulonephritis. Most were Aboriginals (88.4%). They had co-morbidities; diabetes mellitus (60.5%), hypertension (60.5%) and smoking (56.4%). Forty-nine per cent had multiple pathologies on biopsy. Predominant histological pattern was diffuse proliferative glomerulonephritis (72%). Main sites of infections were skin (47.6%) and upper respiratory tract infection (26.2%) with streptococcus and staphylococcus as predominant organisms. Fifty per cent of patients developed end-stage renal disease. On multivariable logistic regression analysis, those on dialysis had higher baseline creatinine (P = 0.003), higher albumin/creatinine ratio at presentation (P = 0.023), higher serum creatinine at presentation (P = 0.02) and lower estimated glomerular filtration rate at presentation (P = 0.012). CONCLUSION: Overall, most patients had pre-existing pathology with superimposed acute postinfectious glomerulonephritis that led to poor outcomes in our cohort.
Asunto(s)
Enfermedades Transmisibles/etnología , Glomerulonefritis/etnología , Glomerulonefritis/patología , Riñón/patología , Nativos de Hawái y Otras Islas del Pacífico , Enfermedad Aguda , Adulto , Biopsia , Enfermedades Transmisibles/diagnóstico , Comorbilidad , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Glomerulonefritis/fisiopatología , Glomerulonefritis/terapia , Humanos , Riñón/fisiopatología , Fallo Renal Crónico/etnología , Fallo Renal Crónico/patología , Fallo Renal Crónico/terapia , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Northern Territory/epidemiología , Diálisis Renal , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.
Asunto(s)
Linfocitos B/citología , Proteínas Portadoras/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Serina-Treonina Quinasas , TransgenesRESUMEN
BACKGROUND: CD1d is a widely expressed lipid antigen presenting molecule required for CD1d-restricted invariant natural killer T (iNKT) cell development. Elevated CD1d expression is detected in CD5(+) IL10-producing B cells, called B10 B cells, and is correlated with poorer prognosis in chronic lymphocytic leukemia (CLL), a CD5(+) B cell malignancy with B10-like functional properties. Whether CD1d expression regulates CD5(+) B cell accumulation, IL10 competence, and antibody production in naïve mice with pathologic CD5(+) B cell expansion remains untested. RESULTS: Using three different transgenic mouse models of benign or leukemic CD5(+) B cell expansion, we found that CD1d was differentially expressed on CD5(+) B cells between the three models, but loss of CD1d expression had no effect on CD5(+) B cell abundance or inducible IL10 expression in any of the models. Interestingly, in the CLL-prone Eµ-TCL1 model, loss of CD1d expression suppressed spontaneous IgG (but not IgM) production, whereas in the dnRAG1xEµ-TCL1 (DTG) model of accelerated CLL, loss of CD1d expression was associated with elevated numbers of splenic CD4(+) and CD8(+) T cells and an inverted CD4(+):CD8(+) T cell ratio. Unexpectedly, before leukemia onset, all three transgenic CD1d-deficient mouse strains had fewer splenic transitional B cells than their CD1d-proficient counterparts. CONCLUSIONS: The results show that CD1d expression and iNKT cells are dispensable for the development, accumulation, or IL10 competence of CD5(+) B cells in mice prone to benign or leukemic CLL-like B cell expansion, but reveal a novel role for iNKT cells in supporting B cell progression through the transitional stage of development in these animals. These results suggest CD1d-directed therapies to target CLL could be evaded by downregulating CD1d expression with little effect on continued leukemic CD5(+) B cell survival. The data also imply that iNKT cells help restrain pro-leukemic CD8(+) T cell expansion in CLL, potentially explaining a reported correlation in human CLL between disease progression, the loss of NKT cells, and a paradoxical increase in CD8(+) T cells.
Asunto(s)
Antígenos CD1d/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD5/metabolismo , Interleucina-10/biosíntesis , Animales , Formación de Anticuerpos/inmunología , Antígenos CD1d/genética , Linfocitos B/citología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismoRESUMEN
Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eµ-TCL1 mice to determine whether dnRAG1 expression in Eµ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eµ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas/genética , Aceleración , Animales , Catálisis , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Activación Enzimática/genética , Expresión Génica , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Recombinación V(D)J/genéticaRESUMEN
B1 B cells reactive to phosphatidyl choline (PtC) exhibit restricted immunoglobulin heavy chain (HC) and light chain (LC) combinations, exemplified by VH12/Vκ4/5H. Two checkpoints are thought to focus PtC+ B cell maturation in VH12-transgenic mice (VH12 mice): V-J rearrangements encoding a "permissive" LC capable of VH12 HC pairing are selected first, followed by positive selection based on PtC binding, often requiring LC receptor editing to salvage PtC- B cells and acquire PtC reactivity. However, evidence obtained from breeding VH12 mice to editing-defective dnRAG1 mice and analyzing LC sequences from PtC+ and PtC- B cell subsets instead suggests that receptor editing functions after initial positive selection to remove PtC+ B cells in VH12 mice. This offers a mechanism to constrain natural, polyreactive B cells to limit their frequency. Sequencing also reveals occasional in-frame hybrid LC genes, reminiscent of type 2 gene replacement, that, testing suggests, arise via a recombination-activating gene (RAG)-independent mechanism.
Asunto(s)
Región Variable de Inmunoglobulina , Fosfatidilcolinas , Animales , Linfocitos B , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Transgénicos , BazoRESUMEN
During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild-type mice. The dnRAG1 mice accumulate splenic B cells with a B1-like phenotype that exhibit defects in B-cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B-cell development at the immature-to-mature transition, immunoglobulin deficiency, and poorer immune responses to thymus-independent antigens. Interestingly, dnRAG1 mice expressing the anti-dsDNA 3H9H56R heavy chain fail to accumulate splenic B1-like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1-like B cells in the spleen.
Asunto(s)
Linfocitos B/inmunología , Genes RAG-1 , Recombinación V(D)J , Animales , Linfocitos B/citología , Dominio Catalítico , Proliferación Celular , Activación Enzimática , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Bazo/citología , Bazo/inmunología , VDJ Recombinasas/genética , VDJ Recombinasas/inmunologíaRESUMEN
Alport syndrome is a common hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes that results in progressive glomerular and interstitial renal disease. Interstitial monocytes that accumulate in the renal cortex from Alport mice are immunopositive for integrin α1ß1, while only a small fraction of circulating monocytes are immunopositive for this integrin. We surmised that such a disparity might be due to the selective recruitment of α1ß1-positive monocytes. In this study, we report the identification of collagen XIII as a ligand that facilitates this selective recruitment of α1ß1 integrin-positive monocytes. Collagen XIII is absent in the vascular endothelium from normal renal cortex and abundant in Alport renal cortex. Neutralizing antibodies against the binding site in collagen XIII for α1ß1 integrin selectively block VLA1-positive monocyte migration in transwell assays. Injection of these antibodies into Alport mice slows monocyte recruitment and protects against renal fibrosis. Thus, the induction of collagen XIII in endothelial cells of Alport kidneys mediates the selective recruitment of α1ß1 integrin-positive monocytes and may potentially serve as a therapeutic target for inflammatory diseases in which lymphocyte/monocyte recruitment involves the interaction with α1ß1 integrin.
Asunto(s)
Colágeno Tipo XIII/metabolismo , Endotelio Vascular/metabolismo , Integrina alfa1beta1/metabolismo , Monocitos/fisiología , Nefritis Hereditaria/patología , Nefritis Hereditaria/fisiopatología , Migración Transendotelial y Transepitelial/fisiología , Animales , Anticuerpos/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Células CHO , Células Cultivadas , Colágeno Tipo XIII/genética , Cricetinae , Cricetulus , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Fibroblastos/citología , Fibroblastos/fisiología , Fibrosis , Integrina alfa1beta1/genética , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Ratones , Ratones Noqueados , Monocitos/citologíaAsunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/economía , Compuestos Heterocíclicos con 3 Anillos/economía , Costos de los Medicamentos , Infecciones por VIH/economía , Inhibidores de Integrasa VIH/uso terapéutico , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Humanos , Oxazinas , Piperazinas , PiridonasRESUMEN
Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.
Asunto(s)
Subgrupos de Linfocitos B/efectos de los fármacos , Densidad Ósea/genética , Células de la Médula Ósea , Relación CD4-CD8 , Cese del Hábito de Fumar , Fumar/efectos adversos , Contaminación por Humo de Tabaco , Animales , Subgrupos de Linfocitos B/inmunología , Densidad Ósea/efectos de los fármacos , Densidad Ósea/inmunología , Recuento de Células , Células Cultivadas , Femenino , Proteínas Relacionadas con Receptor de LDL/deficiencia , Proteínas Relacionadas con Receptor de LDL/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fumar/inmunología , Especificidad de la Especie , Bazo/citología , Bazo/efectos de los fármacos , Timo/citología , Timo/efectos de los fármacosRESUMEN
Rapidly assaying cell viability for diverse bacteria species is not always straightforward. In eukaryotes, cell viability is often determined using colorimetric dyes; however, such dyes have not been identified for bacteria. We screened different dyes and found that erythrosin B (EB), a visibly red dye with fluorescent properties, functions as a vital dye for many Gram-positive and -negative bacteria. EB worked at a similar concentration for all bacteria studied and incubations were as short as 5 min. Given EB's spectral properties, diverse experimental approaches are possible to rapidly visualize and/or quantitate dead bacterial cells in a population. As the first broadly applicable colorimetric viability dye for bacteria, EB provides a cost-effective alternative for researchers in academia and industry.
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Bacterias/química , Técnicas Bacteriológicas/métodos , Eritrosina/química , Colorantes Fluorescentes/química , Membrana Celular/química , Colorimetría , Citometría de FlujoRESUMEN
PURPOSE: The purpose of this study was to evaluate the impact of sugammadex on operating room (OR) times versus neostigmine in patients recovering from rocuronium or vecuronium induced neuromuscular blockade. METHODS: This retrospective cohort study evaluated patients 18 years or older with an American Society of Anesthesiologists (ASA) physical status of I-III who received sugammadex or neostigmine (January- October 2017) for reversal of rocuronium or vecuronium at a 500 bed, community hospital. Patients who were pregnant or breastfeeding were excluded. The primary outcome measure was the time from sugammadex or neostigmine administration to OR exit. The primary outcome was evaluated using a linear regression model adjusting for inpatient procedures, age, sex, body mass index, and ASA score. Secondary outcomes included the incidence of bradycardia as well as nausea and vomiting. RESULTS: The baseline characteristics of the patients in the cohort (sugammadex=134, neostigmine=143) were similar. The median time from drug administration to OR exit was similar for neostigmine and sugammadex (16 vs. 15.5 minutes, p=0.11). Sugammadex had a statistically significant reduction in time from drug administration to OR exit (coefficient -2.7 minutes, 95% confidence interval -5.2 to -0.2 minutes) in the multivariable linear regression model. Sugammadex had lower rates of bradycardia (5.6 vs. 2.2%) or nausea and vomiting (18 vs. 11%) that did not reach statistical significance. CONCLUSIONS: Sugammadex had statistically shorter OR exit times after drug administration in the cohort. The mean 2.7 minute benefit is unlikely to be clinically meaningful and limits its application in practice unless larger cohorts detect a benefit due to a significant reduction.in.adverse.events.
RESUMEN
PURPOSE: The purpose of this study was to evaluate the impact of sugammadex on operating room (OR) times versus neostigmine in patients recovering from rocuronium or vecuronium induced neuromuscular blockade. METHODS: This retrospective cohort study evaluated patients 18 years or older with an American Society of Anesthesiologists (ASA) physical status of I-III who received sugammadex or neostigmine (January- October 2017) for reversal of rocuronium or vecuronium at a 500 bed, community hospital. Patients who were pregnant or breastfeeding were excluded. The primary outcome measure was the time from sugammadex or neostigmine administration to OR exit. The primary outcome was evaluated using a linear regression model adjusting for inpatient procedures, age, sex, body mass index, and ASA score. Secondary outcomes included the incidence of bradycardia as well as nausea and vomiting. RESULTS: The baseline characteristics of the patients in the cohort (sugammadex=134, neostigmine=143) were similar. The median time from drug administration to OR exit was similar for neostigmine and sugammadex (16 vs. 15.5 minutes, p=0.11). Sugammadex had a statistically significant reduction in time from drug administration to OR exit (coefficient -2.7 minutes, 95% confidence interval -5.2 to -0.2 minutes) in the multivariable linear regression model. Sugammadex had lower rates of bradycardia (5.6 vs. 2.2%) or nausea and vomiting (18 vs. 11%) that did not reach statistical significance. CONCLUSIONS: Sugammadex had statistically shorter OR exit times after drug administration in the cohort. The mean 2.7 minute benefit is unlikely to be clinically meaningful and limits its application in practice unless larger cohorts detect a benefit due to a significant reduction.in.adverse.events.
Asunto(s)
Enfermedades Endémicas , Fallo Renal Crónico/epidemiología , Melioidosis/epidemiología , Adulto , Anciano , Antibacterianos/uso terapéutico , Burkholderia pseudomallei/aislamiento & purificación , Femenino , Humanos , Incidencia , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/microbiología , Fallo Renal Crónico/mortalidad , Fallo Renal Crónico/terapia , Masculino , Melioidosis/diagnóstico , Melioidosis/tratamiento farmacológico , Melioidosis/microbiología , Melioidosis/mortalidad , Persona de Mediana Edad , Northern Territory/epidemiología , Diálisis Peritoneal , Estudios Prospectivos , Diálisis Renal , Medición de Riesgo , Factores de Riesgo , Estaciones del Año , Factores de TiempoRESUMEN
BACKGROUND: We showed that the pluripotent platelet growth factor and mediator lysophosphatidic acid (LPA) controls key regenerative responses of human gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) and positively modulates their responses to platelet-derived growth factor (PDGF). This study determined which LPA receptor (LPAR) subtype(s) LPA signals through to stimulate mitogenic extracellular signal-regulated kinase (ERK) 1/2 signaling and chemotaxis and to elicit intracellular Ca(2+) increases in GFs and PDLFs because many healing responses are calcium-dependent. METHODS: Activation of mitogen-activated protein kinase was determined using Western blotting with an antibody to phosphorylated ERK1/2. Migration responses were measured using a microchemotaxis chamber. GF and PDLF intracellular Ca(2+) mobilization responses to multiple LPA species and LPAR subtype-specific agonists were measured by using a cell-permeable fluorescent Ca(2+) indicator dye. RESULTS: LPA stimulated ERK1/2 phosphorylation via LPA(1)(-3). For GFs, LPA(1) preferentially elicited chemotaxis, and LPA(1-3) for PDLFs, as confirmed using subtype-specific agonists. Elevation of intracellular calcium seems to be mediated through LPA(1) and LPA(3), with little, if any, contribution from LPA(2). CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that LPA signals through specific LPAR subtypes to stimulate human oral fibroblast regenerative responses. These data, in conjunction with our previous findings showing that LPA modulates GF and PDLF responses to PDGF, suggest that LPA is a factor of emerging importance to oral wound healing.