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BACKGROUND: Current US Food and Drug Administration guidance recommends that the primary endpoint for complicated urinary tract infection clinical trials be a composite of the clinical and microbiological responses, assessed at a fixed point after therapy. Although some participants meet the criteria for clinical success, they do not meet the criteria for microbiological eradication and are classified as treatment failures. These discordant outcomes have raised questions about the utility of the microbiological endpoint. METHODS: We analyzed participant data from 13 phase 3 clinical trials submitted to the US Food and Drug Administration (N = 4842). Outcomes were determined at the test of cure (TOC) visit, recommended to occur at least 5 days after therapy and at the late follow-up visit, recommended to occur 21 to 28 days after randomization. Clinical and microbiological success were defined as the resolution of complicated urinary tract infection symptoms present at study entry, with no new symptoms (clinical cure), and a reduction in density of the original pathogen to <103 CFU/mL on urine culture (microbiological eradication). RESULTS: Among included participants, 70.7% were concordant successes at the TOC visit, 18.0% were discordant failures (clinical cure/microbiological persistence), and 6.7% were concordant failures (clinical failure/microbiological persistence). Discordant participants were at an increased risk for clinical failure at the late follow-up visit, and the risk of late clinical failure increased with time. CONCLUSIONS: Discordant clinical and microbiological outcomes at the TOC visit were associated with an increased risk of late clinical failure. Microbiological outcomes appear to be an important component of the endpoint.
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Infecciones Urinarias , Humanos , Antibacterianos/uso terapéutico , Recurrencia , Infecciones Urinarias/microbiologíaRESUMEN
BACKGROUND AND AIMS: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), caused by autoimmune regulator (AIRE) mutations, manifests with chronic mucocutaneous candidiasis (CMC) and multisystem autoimmunity, most often hypoparathyroidism (HP) and adrenal insufficiency (AI). European cohorts previously reported a ~10% prevalence of APECED-associated hepatitis (APAH) with presentations ranging from asymptomatic laboratory derangements to fatal fulminant hepatic failure. Herein, we characterized APAH in a large APECED cohort from the Americas. APPROACH AND RESULTS: Forty-five consecutive patients with APECED were evaluated (2013-2015) at the National Institutes of Health (NIH; NCT01386437). Hepatology consultation assessed hepatic and autoimmune biomarkers and liver ultrasound in all patients. Liver biopsies evaluated autoimmune features and fibrosis. The 16S ribosomal RNA (rRNA) sequencing was performed in 35 patients' stools (12 with and 23 without APAH). Among 43 evaluable patients, 18 (42%) had APAH; in 33.3% of those with APAH, APAH occurred before developing classic APECED diagnostic criteria. At APAH diagnosis, the median age was 7.8 years, and patients manifested with aminotransferase elevation and/or hyperbilirubinemia. All patients with APAH were in clinical remission during their NIH evaluation while receiving immunomodulatory treatment. We found no difference in age, sex, or prevalence of CMC, AI, or HP between patients with or without APAH. Autoantibody positivity against aromatic L-amino acid decarboxylase, cytochrome P450 family 1 subfamily A member 2, histidine decarboxylase (HDC), bactericidal/permeability-increasing fold-containing B1, tryptophan hydroxlase, and 21-hydroxylase (21-OH), and the homozygous c.967_979del13 AIRE mutation were associated with APAH development. Classical serological biomarkers of autoimmune hepatitis (AIH) were only sporadically positive. AIH-like lymphoplasmacytic inflammation with mild fibrosis was the predominant histological feature. Stool microbiome analysis found Slackia and Acidaminococcus in greater abundance in patients with APAH. CONCLUSIONS: APAH is more common than previously described, may present early before classic APECED manifestations, and most often manifests with milder, treatment-responsive disease. Several APECED-associated autoantibodies, but not standard AIH-associated biomarkers, correlate with APAH.
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Hepatitis Autoinmune/etiología , Poliendocrinopatías Autoinmunes/complicaciones , Adolescente , Adulto , Américas , Autoanticuerpos/inmunología , Biomarcadores/sangre , Biopsia , Femenino , Eliminación de Gen , Hepatitis Autoinmune/patología , Hepatitis Autoinmune/terapia , Humanos , Inmunoterapia , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Masculino , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/patología , Poliendocrinopatías Autoinmunes/terapia , Adulto JovenRESUMEN
Invasive fusariosis (IF) most commonly occurs in patients with hematologic malignancies and severe neutropenia, particularly during concomitant corticosteroid use. Breakthrough infections can occur in high-risk patients despite Aspergillus-active antifungal prophylaxis. We describe a patient with rapid acute lymphoblastic leukemia (ALL) progression who presented with multifocal skin nodules thought to be choloromatous disease. These lesions were ultimately diagnosed as IF and the patient had two simultaneously active disease processes. This case highlights the importance of pathologic diagnosis of new skin lesions in ALL patients, even during leukemia progression, and demonstrates that IF can occur despite normal neutrophil counts and Aspergillus-active prophylaxis.
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Fusariosis/microbiología , Fusariosis/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adulto , Fusariosis/terapia , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMEN
BACKGROUND: Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. OBJECTIVES: To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. METHODS: MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. RESULTS: Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. CONCLUSIONS: VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.
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Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida glabrata/efectos de los fármacos , Candidiasis Bucal/tratamiento farmacológico , Candidiasis Bucal/prevención & control , Piridinas/farmacología , Tetrazoles/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Candida albicans/aislamiento & purificación , Candida glabrata/aislamiento & purificación , Candidiasis Bucal/microbiología , Farmacorresistencia Fúngica , Fluconazol/farmacología , Humanos , Ratones , Ratones Noqueados , Pruebas de Sensibilidad MicrobianaRESUMEN
Background: Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives: To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods: The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results: Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions: VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.
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Antifúngicos/farmacología , Candida/efectos de los fármacos , Candidiasis Bucal/tratamiento farmacológico , Fluconazol/farmacología , Piridinas/farmacología , Tetrazoles/farmacología , Administración Oral , Animales , Farmacorresistencia Fúngica , Humanos , Interleucina-17/genética , Ratones , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Lengua/microbiologíaRESUMEN
BACKGROUND: Clostridium difficile infection (CDI) can cause severe disease and death, especially in older adults. A better understanding of risk factors for adverse outcomes is needed. This study tests the hypotheses that infection with specific ribotypes and presence of stool toxins independently associate with severity and constructs predictive models of adverse outcomes. METHODS: Cases of non-recurrent CDI were prospectively included after positive stool tests for toxins A and/or B by enzyme immunoassay (EIA) or tcdB by polymerase chain reaction. Outcomes included severe CDI (intensive care unit admission, colectomy, or death attributable to CDI within 30 days of diagnosis) and 30-day all-cause mortality. Adjusted models were developed to test hypotheses and predict outcomes. RESULTS: In total, 1144 cases were included. The toxin EIA was positive in 37.2% and 35.6% of patients were of age >65 years. One of the 137 unique ribotypes was ribotype 027 (16.2%). Detectable stool toxin did not associate with outcomes. Adjusting for covariates, including age, Ribotype 027 was a significant predictor of severe CDI (90 cases; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.03-2.89; P = .037) and mortality (89 cases; OR, 2.02; 95% CI, 1.19-3.43; P = .009). Concurrent antibiotic use associated with both outcomes. Both multivariable predictive models had excellent performance (area under the curve >0.8). CONCLUSIONS: Detection of stool toxin A and/or B by EIA does not predict severe CDI or mortality. Infection with ribotype 027 independently predicts severe CDI and mortality. Use of concurrent antibiotics is a potentially modifiable risk factor for severe CDI.
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Toxinas Botulínicas/aislamiento & purificación , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/diagnóstico , Enterocolitis Seudomembranosa/mortalidad , Heces/microbiología , Ribotipificación , Adulto , Factores de Edad , Anciano , Clostridioides difficile/clasificación , Enterocolitis Seudomembranosa/microbiología , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Oportunidad Relativa , Reacción en Cadena de la Polimerasa , Curva ROC , Factores de Riesgo , Índice de Severidad de la EnfermedadAsunto(s)
Proteínas Adaptadoras de Señalización CARD/deficiencia , Candidiasis/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Meningoencefalitis/tratamiento farmacológico , Neutropenia/tratamiento farmacológico , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Candida albicans , Candidiasis/microbiología , Femenino , Humanos , Síndromes de Inmunodeficiencia/microbiología , Meningoencefalitis/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Neutropenia/microbiologíaAsunto(s)
Aspergilosis/genética , Esofagitis Eosinofílica/genética , Rinitis Alérgica/genética , Factor de Transcripción STAT3/genética , Adulto , Antifúngicos/uso terapéutico , Aspergilosis/diagnóstico por imagen , Aspergilosis/terapia , Desbridamiento , Esofagitis Eosinofílica/diagnóstico por imagen , Esofagitis Eosinofílica/terapia , Resultado Fatal , Haploinsuficiencia , Humanos , Inmunoterapia , Imagen por Resonancia Magnética , Masculino , Rinitis Alérgica/diagnóstico por imagen , Rinitis Alérgica/terapiaRESUMEN
Clostridium difficile is a significant nosocomial threat to human health and is the most commonly identified cause of antibiotic-associated diarrhea. The development of C. difficile colitis requires production of toxins A and/or B, but some strains do not express these proteins. These non-toxigenic C. difficile (NTCD) have garnered attention for their capacity to colonize humans and potentially reduce the risk for symptomatic colitis caused by toxigenic strains. Isolates of NTCD have been obtained from the environment as well as from animal and human sources. Studies in a hamster CDI model have demonstrated a protective effect of NTCD against toxigenic infection. The extent to which this protective effect of NTCD occurs in humans remains to be defined. Evidence for a therapeutic or preventive role for NTCD is limited but clinical prophylaxis studies are ongoing. NTCD potentially represents an exciting new tool in preventing CDI and its recurrences.
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Toxinas Bacterianas/toxicidad , Clostridioides difficile/clasificación , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/epidemiología , Diarrea/microbiología , Sustancias Protectoras/uso terapéutico , Animales , Infecciones por Clostridium/prevención & control , Cricetinae , Infección Hospitalaria/microbiología , Infección Hospitalaria/prevención & control , Modelos Animales de Enfermedad , Enterotoxinas/toxicidad , Humanos , Especificidad de la EspecieRESUMEN
Background: The prospective identification of patients at high risk for hospital-acquired/ventilator-associated bacterial pneumonia may improve clinical trial feasibility and foster antibacterial development. In a prior study conducted in the United States, clinical criteria were used to prospectively identify these patients; however, these criteria have not been applied in a European population. Methods: Adults considered high risk for pneumonia (treatment with ventilation or high levels of supplemental oxygen) in the intensive care units of 7 European hospitals were prospectively enrolled from June 12 to December 27, 2017. We estimated the proportion of high-risk patients developing pneumonia according to US Food and Drug Administration guidance and a subset potentially eligible for antibacterial trial enrollment. We compared patient characteristics, treatment exposures, and pneumonia incidence in a European cohort and a previously described US cohort. Results: Of 888 high-risk patients, 211/888 (24%) were treated for possible pneumonia, and 150/888 (17%) met the Food and Drug Administration definition for hospital-acquired/ventilator-associated bacterial pneumonia. A higher proportion of European patients treated for possible pneumonia met the pneumonia definition (150/211 [71%] vs 537/1464 [37%]; P < .001). Among patients developing pneumonia, a higher proportion of European patients met antibacterial trial eligibility criteria (124/150 [83%] vs 371/537 [69%]; P < .001). Conclusions: Clinical criteria prospectively identified high-risk patients with high rates of pneumonia in the European cohort. Despite higher rates of established risk factors and incident pneumonia, European patients were significantly less likely to receive antibiotics for possible pneumonia than US patients. Different treatment practices may contribute to lower rates of antibacterial trial enrollment in the United States.
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Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, ß-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1ß in response to ß-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1ß and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1ß-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.
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Feohifomicosis , beta-Glucanos , Animales , Humanos , Masculino , Ratones , Proteínas Adaptadoras de Señalización CARD/genética , Lectinas Tipo C/genética , Macrófagos/metabolismo , Feohifomicosis/microbiología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
We present a case of acute epiglottitis in a 16-year-old with severe aplastic anemia. He was admitted with a history suggestive of a severe upper airway infection and an absolute neutrophil count of 0 per cubic millimeter. Despite his immunocompromised state, he presented with the classical signs and symptoms of epiglottitis. We review here the presentation and comorbidities of immunocompromised patients with epiglottitis. In addition, the appropriate choice of empirical antibiotic therapy is important for the management of epiglottitis in immunocompromised patients, especially in the post-Haemophilus influenza type B vaccination era. In our patient, Enterobacter cloacae was isolated from endoscopically directed throat cultures, and treatment was successful without the need for intubation. The current literature suggests that in immunocompromised patients, particularly those who are neutropenic, there is a potentially wide range of organisms, both bacterial and fungal, that may play a role in the pathology of acute epiglottitis.
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Histoplasmosis causes a wide spectrum of clinical illness, including disseminated infection in the immunocompromised. We report a case of pulmonary histoplasmosis in an allogeneic stem cell transplant recipient and review the literature on this topic. Histoplasmosis in this patient population is uncommon, but it is associated with poor outcome.
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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by biallelic autoimmune regulator (AIRE) mutations that manifests with chronic mucocutaneous candidiasis (CMC) and autoimmunity. Patients with STAT1 gain-of-function (GOF) mutations also develop CMC and autoimmunity; they exhibit increased STAT1 protein levels at baseline and STAT1 phosphorylation (pSTAT1) upon interferon (IFN)-γ stimulation relative to healthy controls. AIRE interacts functionally with a protein that directly regulates STAT1, namely protein inhibitor of activated STAT1, which inhibits STAT1 activation. Given the common clinical features between patients with AIRE and STAT1 GOF mutations, we sought to determine whether APECED patients also exhibit increased levels of STAT1 protein and phosphorylation in CD14+ monocytes. We obtained peripheral blood mononuclear cells from 8 APECED patients and 13 healthy controls and assessed the levels of STAT1 protein and STAT1 tyrosine phosphorylation at rest and following IFN-γ stimulation, as well as the levels of STAT1 mRNA. The mean STAT1 protein levels in CD14+ monocytes exhibited a ~20% significant decrease in APECED patients both at rest and after IFN-γ stimulation relative to that of healthy donors. Similarly, the mean peak value of IFN-γ-induced pSTAT1 level was ~20% significantly lower in APECED patients compared to that in healthy controls. The decrease in STAT1 and peak pSTAT1 in APECED patients was not accompanied by decreased STAT1 mRNA or anti-IFN-γ autoantibodies; instead, it correlated with the presence of autoantibodies to type I IFN and decreased AIRE-/- monocyte surface expression of IFN-γ receptor 2. Our data show that, in contrast to patients with STAT1 GOF mutations, APECED patients show a moderate but consistent and significant decrease in total STAT1 protein levels, associated with lower peak levels of pSTAT1 molecules after IFN-γ stimulation.
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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by homozygous AIRE mutations. It classically presents with chronic mucocutaneous candidiasis and autoimmunity that primarily targets endocrine tissues; hypoparathyroidism and adrenal insufficiency are most common. Developing any two of these classic triad manifestations establishes the diagnosis. Although widely recognized in Europe, where nonendocrine autoimmune manifestations are uncommon, APECED is less defined in patients from the Western Hemisphere. We enrolled 35 consecutive American APECED patients (33 from the US) in a prospective observational natural history study and systematically examined their genetic, clinical, autoantibody, and immunological characteristics. Most patients were compound heterozygous; the most common AIRE mutation was c.967_979del13. All but one patient had anti-IFN-ω autoantibodies, including 4 of 5 patients without biallelic AIRE mutations. Urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis, and Sjögren's-like syndrome, uncommon entities in European APECED cohorts, affected 40%-80% of American cases. Development of a classic diagnostic dyad was delayed at mean 7.38 years. Eighty percent of patients developed a median of 3 non-triad manifestations before a diagnostic dyad. Only 20% of patients had their first two manifestations among the classic triad. Urticarial eruption, intestinal dysfunction, and enamel hypoplasia were prominent among early manifestations. Patients exhibited expanded peripheral CD4+ T cells and CD21loCD38lo B lymphocytes. In summary, American APECED patients develop a diverse syndrome, with dramatic enrichment in organ-specific nonendocrine manifestations starting early in life, compared with European patients. Incorporation of these new manifestations into American diagnostic criteria would accelerate diagnosis by approximately 4 years and potentially prevent life-threatening endocrine complications.
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OBJECTIVES: Previous studies suggest that colonization with non-toxigenic Clostridium difficile may protect against toxigenic C. difficile infection (CDI), yet most of the studies were conducted in men. Therefore, we conducted a study to examine this hypothesis in both genders. METHODS: Patients (n=1492) were classified by disease status at baseline and observed for 1 year. Cox proportional hazards regression was used to evaluate CDI rates within 8 weeks post-baseline (short-term) and from 8 weeks to 1 year (long-term follow-up). RESULTS: During short-term follow-up, CDI rates were 5 times greater in females with non-toxigenic Clostridium difficile compared to females without C. difficile (hazard ratio (HR) = 5.13; 95% CI: 1.47-17.83). The comparable HR in males was 0.44 (95% CI: 0.04-4.43). During long term follow-up, CDI rates were similar in those with non-toxigenic C. difficile and those without C. difficile at baseline, for both females and males. Mortality rates were significantly lower for patients colonized by non-toxigenic C. difficile than those with toxigenic C. difficile at baseline, for both genders combined (HR=0.51; 95% CI: 0.28-0.92) and were similar to those with no C. difficile at baseline (HR=0.78; 95% CI: 0.43-1.41). CONCLUSIONS: There were gender differences in the short-term risk of CDI. Mortality was similar for patients colonized with non-toxigenic C. difficile and patients without C. difficile.
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The plant cell wall is the structural basis of cellular form and thus forms a foundation on which morphogenesis builds organs and tissues. Enzymes capable of modifying major wall components are prominent candidates for regulating wall form and function. Xyloglucan endotransglucosylases/hydrolases (XTHs) are predicted to participate in xyloglucan integration and/or restructuring. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain insight into the potential physiological relevance of the distinct members of this family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data reveal that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs show XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes. These data suggest that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Glicosiltransferasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Biología Computacional/métodos , Flores/anatomía & histología , Flores/enzimología , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Glucuronidasa/análisis , Glicosiltransferasas/genética , Glicosiltransferasas/fisiología , Familia de Multigenes , Raíces de Plantas/anatomía & histología , Raíces de Plantas/enzimología , Raíces de Plantas/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/análisis , Plantones/anatomía & histología , Plantones/enzimología , Plantones/crecimiento & desarrollo , Semillas/anatomía & histología , Semillas/enzimología , Semillas/crecimiento & desarrollo , TransgenesRESUMEN
It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.
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Aciltransferasas/genética , Aciltransferasas/metabolismo , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Mutación , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Calcio/metabolismo , Catálisis , Cromatografía Liquida , Dimerización , Diploidia , Neuropatías Hereditarias Sensoriales y Autónomas/enzimología , Histidina/química , Lisina/química , Microsomas Hepáticos/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Serina C-Palmitoiltransferasa , Esfingolípidos/biosíntesis , Esfingolípidos/metabolismoRESUMEN
The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.