Optimizing in vitro spherulation cues in the fungal pathogen Coccidioides.

Coccidioides spp . are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly under characterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae, and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly-harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal media to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.

Functional gastrointestinal disorders frequency by Rome IV criteria.

INTRODUCTION: Functional gastrointestinal disorders (FGIDs) are a very common pediatric disease, with strong implications for children and their families. We aimed to determine their frequency in our environment (per Rome IV criteria) and to establish if there is seasonal variability in diagnosis. METHODS: Descriptive, prospective study. For 12 months, children under 16 years of age with suspected FGIDs who had a first pediatric gastroenterology consultation were included and classified according to Rome IV criteria. Statistical analysis was done with SPSS v22. RESULTS: 574 children received consultations, 67% were >4 years of age. FGIDs were suspected in 44.6% of the patients, 32.4% were diagnosed according to Rome IV criteria (16.4% <4 years, 40.3% >4 years). 51.1% were female, average age of 8.4 ±â€¯4.2 years and mean of 7 months of symptoms until diagnosis (range 3-150). In patients <4 years, the most common disorders were functional constipation (48.4%), regurgitation (22.5%) and functional diarrhea (16.1%); in patients >4 years of age, functional abdominal pain (29%), functional dyspepsia (28.4%) and functional constipation (16.8%) were most frequent. We didn't discern seasonal variations in diagnosis in the global study population (p = 0.96) or by age group (<4 p = 0.51; >4 p = 0.57). CONCLUSIONS: FGIDs account for one third of our patients' consultations. While the Rome IV criteria are more inclusive than before, almost 30% of patients with suspected FGIDs don't meet said criteria. Although a seasonal difference regarding diagnosis was observed, it wasn't statistically significant either in the sample group as a whole or by age group.

Frecuencia de trastornos gastrointestinales funcionales según criterios Roma IV / Functional gastrointestinal disorders frequency by Rome IV criteria

Introducción: Los trastornos funcionales gastrointestinales (TFGI) son una enfermedad común en pediatría, con fuertes implicaciones para el niño y su familia. Nuestro objetivo es determinar su frecuencia en nuestro medio según criterios Roma IV y la posible variación estacional al diagnóstico. Material y métodos: Estudio descriptivo con recogida de información prospectiva. Durante un año se incluyó a menores de 16 años que acudieron a primera consulta de Gastroenterología Pediátrica con sospecha de TFGI, a los que se clasificó según Roma IV. Análisis estadístico mediante SPSS v22. Resultados: Acudieron en total 574 niños, el 67% mayores de 4 años. Se sospechó TFGI en el 44,6%, siendo diagnosticados según criterios Roma IV el 32,4% (16,4% <4 años, 40,3%> 4 años). El 51,1% eran mujeres, edad media de 8,4± 4,2 años y mediana de 7 meses de síntomas al diagnóstico (rango: 3-150). Por frecuencia, en <4 años destacan el estreñimiento (48,4%), la regurgitación (22,5%) y la diarrea funcional (16,1%), y en> 4 años el dolor abdominal funcional no especificado (29%), la dispepsia funcional (28,4%) y el estreñimiento (16,8%). En el diagnóstico por trimestres no se objetivaron diferencias en el total (p=0,96) ni por grupos de edad (< 4 años, p=0,51;> 4 años, p=0,57) Conclusión: Según Roma IV, los TFGI suponen un tercio de los pacientes de nuestra consulta. A pesar de ser más inclusivos que previamente, casi un 30% de los pacientes con sospecha no cumple criterios. Aunque existe cierta variación estacional en la frecuencia diagnóstica, no fue significativa ni en el total ni por grupos de edad. (AU)

Introduction: Functional gastrointestinal disorders (FGIDs) are a very common pediatric disease, with strong implications for children and their families. We aimed to determine their frequency in our environment (per Rome IV criteria) and to establish if there is seasonal variability in diagnosis. Material and methods: Descriptive, prospective study. For 12 months, children under 16 years of age with suspected FGIDs who had a first pediatric gastroenterology consultation were included and classified according to Rome IV criteria. Statistical analysis was done with SPSS v22. Results: 574 children received consultations, 67% were >4 years of age. FGIDs were suspected in 44.6% of the patients, 32.4% were diagnosed according to Rome IV criteria (16.4%, <4 years; 40.3%, >4 years). 51.1% were female, average age of 8.4±4.2 years and mean of 7 months of symptoms until diagnosis (range 3–150). In patients <4 years, the most common disorders were functional constipation (48.4%), regurgitation (22.5%) and functional diarrhea (16.1%); in patients >4 years of age, functional abdominal pain (29%), functional dyspepsia (28.4%) and functional constipation (16.8%) were most frequent. We did not discern seasonal variations in diagnosis in the global study population (p=.96) or by age group (< 4, P=.51; > 4, P=.57). Conclusions: FGIDs account for one third of our patients’ consultations. While the Rome IV criteria are more inclusive than before, almost 30% of patients with suspected FGIDs do not meet said criteria. Although a seasonal difference regarding diagnosis was observed, it was not statistically significant either in the sample group as a whole or by age group. (AU)

[Functional gastrointestinal disorders frequency by Rome IV criteria]. / Frecuencia de trastornos gastrointestinales funcionales según criterios Roma IV.

INTRODUCTION: Functional gastrointestinal disorders (FGIDs) are a very common pediatric disease, with strong implications for children and their families. We aimed to determine their frequency in our environment (per Rome IV criteria) and to establish if there is seasonal variability in diagnosis. MATERIAL AND METHODS: Descriptive, prospective study. For 12 months, children under 16 years of age with suspected FGIDs who had a first pediatric gastroenterology consultation were included and classified according to Rome IV criteria. Statistical analysis was done with SPSS v22. RESULTS: 574 children received consultations, 67% were >4 years of age. FGIDs were suspected in 44.6% of the patients, 32.4% were diagnosed according to Rome IV criteria (16.4%, <4 years; 40.3%, >4 years). 51.1% were female, average age of 8.4±4.2 years and mean of 7 months of symptoms until diagnosis (range 3-150). In patients <4 years, the most common disorders were functional constipation (48.4%), regurgitation (22.5%) and functional diarrhea (16.1%); in patients >4 years of age, functional abdominal pain (29%), functional dyspepsia (28.4%) and functional constipation (16.8%) were most frequent. We did not discern seasonal variations in diagnosis in the global study population (p=.96) or by age group (< 4, P=.51; > 4, P=.57). CONCLUSIONS: FGIDs account for one third of our patients' consultations. While the Rome IV criteria are more inclusive than before, almost 30% of patients with suspected FGIDs do not meet said criteria. Although a seasonal difference regarding diagnosis was observed, it was not statistically significant either in the sample group as a whole or by age group.

Prevention of estradiol 17beta-D-glucuronide-induced canalicular transporter internalization by hormonal modulation of cAMP in rat hepatocytes.

In estradiol 17ß-d-glucuronide (E17G)-induced cholestasis, the canalicular hepatocellular transporters bile salt export pump (Abcb11) and multidrug-resistance associated protein 2 (Abcc2) undergo endocytic internalization. cAMP stimulates the trafficking of transporter-containing vesicles to the apical membrane and is able to prevent internalization of these transporters in estrogen-induced cholestasis. Hepatocyte levels of cAMP are regulated by hormones such as glucagon and adrenaline (via the ß2 receptor). We analyzed the effects of glucagon and salbutamol (a ß2 adrenergic agonist) on function and localization of Abcb11 and Abcc2 in isolated rat hepatocyte couplets exposed to E17G and compared the mechanistic bases of their effects. Glucagon and salbutamol partially prevented the impairment in Abcb11 and Abcc2 transport capacity. E17G also induced endocytic internalization of Abcb11 and Abcc2, which partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained internalized and mainly colocalizing with Rab11a in the perinuclear region after incubation with glucagon. Glucagon prevention was dependent on cAMP-dependent protein kinase (PKA) and independent of exchange proteins activated directly by cAMP (Epac) and microtubules. In contrast, salbutamol prevention was PKA independent and Epac/MEK and microtubule dependent. Anticholestatic effects of glucagon and salbutamol were additive in nature. Our results show that increases in cAMP could activate different anticholestatic signaling pathways, depending on the hormonal mediator involved.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver.

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Oxidative stress in primary culture hepatocytes isolated from partially hepatectomized rats.

The aim of this study was to evaluate the influence of partial hepatectomy prior to cell isolation on hepatocytes in vitro. We characterized the possible changes of various stress oxidative parameters within the first 24 h after seeding. Male Wistar rats served as donors. Hepatocytes were isolated by collagenase digestion from either liver of simulated surgery (SH) or from liver 1 h after 70% hepatectomy (PH), and the changes in stress parameters were analyzed after 1, 3, 18, and 24 h in culture. At 24 h, only hepatocytes from PH maintained significantly increased reactive oxygen species production, oxidized glutathione percentage, and Cu/Zn superoxide dismutase and catalase activities. Our results show that hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells from SH metabolically recover from this stress after 18 h. After this time, primary culture hepatocytes primed by PH maintain their in vivo-like metabolic activities (increase in both oxidative stress and antioxidant status).

Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein.

We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters. The studies were performed 24h after administration of the drug. APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy. In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter. Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2. APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate. Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug. In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.

Fibronectin expression in proximal tubules from ischemic rat kidneys without reperfusion.

The expression of fibronectin (FN), one of the extracellular matrix proteins, was studied in isolated renal proximal tubules in a in vivo rat model of unilateral renal ischemia without reperfusion. FN is involved in cell-extracellular matrix interactions and defective cell-extracellular matrix interactions have been hypothesized to contribute to ischemic renal failure. The expression of FN was investigated by reverse transcription-polymerase chain reaction (RT-PCR), Elisa and Western blot. Isolated proximal tubules from control and post-ischemic rat kidneys were used. ATP, intracellular calcium content, and alkaline phosphatase were also measured to describe the effects associated to 40 min of ischemia. Control tubules expressed FN. Forty minutes of ischemia promoted diminished ATP levels and phosphatase alkaline activity, and increased intracellular calcium in isolated proximal tubules. An increased abundance of FN was observed by ischemic tubules as compared with control tubules. To determine quantitatively the value of FN content, ELISA method was performed. The ischemic tubules also expressed higher amount of FN mRNA. Three amplification products were obtained from both ischemic and control proximal tubular cDNA. The relative amounts of each of the obtained products were the same, strongly suggesting that the augmentation of the FN gene transcription during ischemia is not associated to a modification in the splicing pattern. Moreover, this expression is increased after 40 min of ischemia, not followed by reperfusion.

Protein kinase C-dependent inhibition of the lysosomal degradation of endocytosed proteins in rat hepatocytes.

We studied the role of protein kinase C (PKC) in the lysosomal processing of endocytosed proteins in isolated rat hepatocytes. We used [14C]sucrose-labeled horseradish peroxidase ([14C]S-HRP) to simultaneously evaluate endocytosis and lysosomal proteolysis. The PKC activator phorbol 12-myristate 13-acetate (PMA) inhibited the lysosomal degradation of [14C]S-HRP (1 microM PMA: 40% inhibition, P<.05), without affecting either the endocytic uptake or the delivery to lysosomes. However, PMA was not able to affect the lysosomal processing of the beta-galactosidase substrate dextran galactosyl umbelliferone. The PKC inhibitors, chelerytrine (Che), staurosporine (St) and Gö 6976, prevented PMA inhibitory effect on lysosomal proteolysis. Nevertheless, purified PKC failed to alter proteolysis in [14C]S-HRP-loaded isolated lysosomes, suggesting that intracellular intermediates are required. PMA induced phosphorylation and hepatocyte membrane-to-lysosome redistribution of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, raising the possibility that MARCKS mediates the PKC-induced inhibition of lysosomal proteolysis.

Preservacion hipotermica del higado: evaluacion de la solucion Eurocollins utilizando cortes de higado de rata / Liver hypothermic preservation: evaluation of Eurocollins solution using rats liver slices

Se analizan los efectos producidos en el hígado por la isquemia fría en solución eurocollins (EC). Para ello se evalúa la función de cortes provenientes de hígados preservados en EC. Se utilizaron hígados de ratas Wistar, perfundidos con Krebs-Henseleit (KH) luego con EC y conservados a 4º en la misma durante 7, 24 y 48 hs. Finalizados los períodos de preservación, se hicieron cortes, los que fueron incubados 1 hora a 37ºC en KH y se determinaron los parámetros: distribución del agua y electrolitos tisulares, liberación de LDH al medio de incubación, producción de sustancias Ac. Tiobarbitúrico reactivas (SATBR) y Ureagénesis. Los cortes provenientes de hígados preservados desde 7hs, mostraron un incremento del agua tisular, expresado en la expansión del espacio extracelular. También se observaron, una disminución progresiva de la conc. tisular de K+, aumentos de SATBR de la liberación de LDH con el incremento del tiempo de presevación. No se observaron cambios en la ureogénesis. Estos resultados indican que la isquemia fría en EC, provoca cambios en la permeabilidad de membranas, fenómeno que afecta severamente el mecanismos de regulación del volumen tisular y compromete la viabilidad funcional del órgano a transplantar

Preservacion hipotermica del higado: evaluacion de la solucion Eurocollins utilizando cortes de higado de rata / Liver hypothermic preservation: evaluation of Eurocollins solution using rats liver slices

Se analizan los efectos producidos en el hígado por la isquemia fría en solución eurocollins (EC). Para ello se evalúa la función de cortes provenientes de hígados preservados en EC. Se utilizaron hígados de ratas Wistar, perfundidos con Krebs-Henseleit (KH) luego con EC y conservados a 4º en la misma durante 7, 24 y 48 hs. Finalizados los períodos de preservación, se hicieron cortes, los que fueron incubados 1 hora a 37ºC en KH y se determinaron los parámetros: distribución del agua y electrolitos tisulares, liberación de LDH al medio de incubación, producción de sustancias Ac. Tiobarbitúrico reactivas (SATBR) y Ureagénesis. Los cortes provenientes de hígados preservados desde 7hs, mostraron un incremento del agua tisular, expresado en la expansión del espacio extracelular. También se observaron, una disminución progresiva de la conc. tisular de K+, aumentos de SATBR de la liberación de LDH con el incremento del tiempo de presevación. No se observaron cambios en la ureogénesis. Estos resultados indican que la isquemia fría en EC, provoca cambios en la permeabilidad de membranas, fenómeno que afecta severamente el mecanismos de regulación del volumen tisular y compromete la viabilidad funcional del órgano a transplantar (AU)