Identificación molecular y sensibilidad a los antifúngicos de aislamientos de sangre del complejo Candida parapsilosis en Venezuela / Molecular identification and in vitro antifungal susceptibility of blood isolates of the Candida parapsilosis species complex in Venezuela

Antecedentes. Candida parapsilosis constituye un complejo de especies formado por Candida parapsilosis sensu stricto, Candida orthopsilosis y Candida metapsilosis. Estudios a nivel mundial han descrito su epidemiología y sensibilidad a los antifúngicos. Objetivos. Los objetivos de este trabajo fueron identificar molecularmente aislamientos de sangre del complejo Candida parapsilosis y determinar su sensibilidad in vitro a los antifúngicos de uso sistémico. Métodos. Se estudiaron 86 aislamientos del complejo C. parapsilosis, provenientes de la Red de Vigilancia de Candidemia del Departamento de Micología del Instituto Nacional de Higiene Rafael Rangel, recogidos en el periodo 2008-2011. Se realizó la amplificación del gen de la deshidrogenasa alcohólica secundaria por reacción en cadena de la polimerasa y los productos fueron analizados mediante los polimorfismos de la longitud de sus fragmentos de restricción, mediante el uso de la enzima BanI. Las pruebas de sensibilidad se realizaron mediante Etest® según las instrucciones del fabricante, con modificaciones. Resultados. De los 86 aislamientos estudiados, 81 (94,2%) fueron C. parapsilosis sensu stricto, 4 (4,6%) C. orthopsilosis y uno (1,2%) C. metapsilosis. C. parapsilosis fue sensible a la anfotericina B y la caspofungina, y su porcentaje de resistencia al fluconazol y el voriconazol fue bajo. C. orthopsilosis y C. metapsilosis fueron sensibles a todos los antifúngicos probados. Conclusiones. Los resultados aportaron por primera vez en Venezuela información importante sobre la distribución del complejo C. parapsilosis en casos de candidemia, y apoyan la necesidad de continuar con los programas de vigilancia, incluyendo la discriminación molecular de las especies y las pruebas de sensibilidad a los antifúngicos, que permitirán orientar la terapia específica (AU)

Background. Candida parapsilosis is a species complex consisting of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Studies worldwide have described its epidemiology and susceptibility to antifungal agents. Aims. The aims of this study were to carry out the molecular identification of blood isolates belonging to the Candida parapsilosis species complex, and to determine their in vitro susceptibility to antifungals of systemic use. Methods. A study of 86 strains of C. parapsilosis species complex collected in 2008-2011 and obtained from the Candidaemia Surveillance Network of Mycology Department of the Rafael Rangel National Institute of Hygiene, was made. Secondary alcohol-dehydrogenase gene amplification was performed using polymerase chain reaction, and the products were analysed by restriction fragments length polymorphisms using the enzyme BanI. Susceptibility tests were performed using Etest®, following the manufacturer's instructions with modifications. Results. Of the 86 isolates studied, 81 (94.2%) were C. parapsilosis sensu stricto, 4 (4.6%) C. orthopsilosis, and one (1.2%) C. metapsilosis. C. parapsilosis isolates were susceptible to amphotericin B and caspofungin, showing low rates of resistance to fluconazole and voriconazole. C. orthopsilosis and C. metapsilosis were susceptible to all the antifungals tested. Conclusions. The results obtained in Venezuela provide for the first time important information about the distribution of C. parapsilosis species complex in cases of candidaemia, and support the need for continuing surveillance programs, including molecular discrimination of species and antifungal susceptibility tests, which may guide specific therapy (AU)

[Molecular identification and in vitro antifungal susceptibility of blood isolates of the Candida parapsilosis species complex in Venezuela]. / Identificación molecular y sensibilidad a los antifúngicos de aislamientos de sangre del complejo Candida parapsilosis en Venezuela.

BACKGROUND: Candida parapsilosis is a species complex consisting of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Studies worldwide have described its epidemiology and susceptibility to antifungal agents. AIMS: The aims of this study were to carry out the molecular identification of blood isolates belonging to the Candida parapsilosis species complex, and to determine their in vitro susceptibility to antifungals of systemic use. METHODS: A study of 86 strains of C. parapsilosis species complex collected in 2008-2011 and obtained from the Candidaemia Surveillance Network of Mycology Department of the Rafael Rangel National Institute of Hygiene, was made. Secondary alcohol-dehydrogenase gene amplification was performed using polymerase chain reaction, and the products were analysed by restriction fragments length polymorphisms using the enzyme BanI. Susceptibility tests were performed using Etest®, following the manufacturer's instructions with modifications. RESULTS: Of the 86 isolates studied, 81 (94.2%) were C. parapsilosis sensu stricto, 4 (4.6%) C. orthopsilosis, and one (1.2%) C. metapsilosis. C. parapsilosis isolates were susceptible to amphotericin B and caspofungin, showing low rates of resistance to fluconazole and voriconazole. C. orthopsilosis and C. metapsilosis were susceptible to all the antifungals tested. CONCLUSIONS: The results obtained in Venezuela provide for the first time important information about the distribution of C. parapsilosis species complex in cases of candidaemia, and support the need for continuing surveillance programs, including molecular discrimination of species and antifungal susceptibility tests, which may guide specific therapy.

Serum Adipsin Levels throughout Normal Pregnancy and Preeclampsia.

Adipsin is a protease produced at high levels by adipose tissue. It is involved in complement activation and metabolic control. The objective of this study was to determine the changes in adipsin levels during different stages of normal pregnancy, and its association with obstetric outcomes, such as preeclampsia. This nested case-control study in a longitudinal cohort included normal pregnant (n = 54) and preeclamptic (n = 18) women, both followed throughout pregnancy. Additionally, some of the normal pregnant women were followed up three months postpartum (n = 18). Healthy non-pregnant women were also studied during their menstrual cycle (n = 20). The results of this study show that in healthy non-pregnant women, adipsin levels did not change significantly during the menstrual cycle. In normal pregnant women, adipsin levels were lower (p < 0.01) when compared with non-pregnant healthy women, but these serum levels increased again during postpartum (p < 0.001). Adipsin levels were significantly elevated in preeclamptic women in late pregnancy (P < 0.01). A significant correlation was not found between leptin and adipsin during the three periods of gestation studied in healthy pregnant and preeclamptic women. Our results suggest that adipsin may be involved in pregnancy-associated metabolic changes. Moreover, the increase of adipsin levels towards late gestation in preeclamptic women could be related to the pathophysiology of this disease.

Maternal serum omentin-1 profile is similar in humans and in the rat animal model.

Omentin-1 is an adipocytokine with anti-inflammatory activity that has been associated with different metabolic disorders. The aim of this study is to investigate the serum profiles of omentin-1 throughout human and rat pregnancy. Serum omentin-1 levels were determined by ELISA in a prospective cohort study of healthy pregnant women (n=40) during the three trimesters of pregnancy and in twenty healthy non-pregnant women during the follicular and luteal phase of the menstrual cycle. In addition, serum omentin-1 levels were measured in rats during different periods of pregnancy (gestational days 8, 12, 16, 19, and 21) and in an age-matched control (virgin) group of rats (n=12rats/group). Finally, immunohistochemistry was used to demonstrate the presence of omentin-1 protein in human and rat placenta. Omentin-1 immunoreactivity was detected in cytotrophoblasts, syncytiotrophoblasts, sparse Hofbauer cells, and endothelial cells of the stem villi of human placenta. Additionally, it was detected in the labyrinthine trophoblast and yolk sac layer of the rat placenta. Human and rat serum omentin-1 levels were significantly lower in the late gestational period when compared with the non-pregnant women and virgin rats (p<0.05). Serum omentin-1 changes were not significant throughout the gestation in both species (p>0.05). Human serum omentin-1 levels have an inverse relationship with triglyceride levels during pregnancy. Our findings have not determined the exact role of omentin-1 during pregnancy, concerning the metabolic control of triglycerides and other energy sources. Whether omentin-1 decrease implies a regulatory function is still not clear. Further studies are needed to address this issue and determine the role of omentin-1 in metabolic adaptations during normal human and rat pregnancy.

Maternal Serum Meteorin Levels and the Risk of Preeclampsia.

BACKGROUND: Meteorin (METRN) is a recently described neutrophic factor with angiogenic properties. This is a nested case-control study in a longitudinal cohort study that describes the serum profile of METRN during different periods of gestation in healthy and preeclamptic pregnant women. Moreover, we explore the possible application of METRN as a biomarker. METHODS AND FINDINGS: Serum METRN was measured by ELISA in a longitudinal prospective cohort study in 37 healthy pregnant women, 16 mild preeclamptic women, and 20 healthy non-pregnant women during the menstrual cycle with the aim of assessing serum METRN levels and its correlations with other metabolic parameters. Immunostaining for METRN protein was performed in placenta. A multivariate logistic regression model was proposed and a classifier model was formulated for predicting preeclampsia in early and middle pregnancy. The performance in classification was evaluated using measures such as sensitivity, specificity, and the receiver operating characteristic (ROC) curve. In healthy pregnant women, serum METRN levels were significantly elevated in early pregnancy compared to middle and late pregnancy. METRN levels are significantly lower only in early pregnancy in preeclamptic women when compared to healthy pregnant women. Decision trees that did not include METRN levels in the first trimester had a reduced sensitivity of 56% in the detection of preeclamptic women, compared to a sensitivity of 69% when METRN was included. CONCLUSIONS: The joint measurements of circulating METRN levels in the first trimester and systolic blood pressure and weight in the second trimester significantly increase the probabilities of predicting preeclampsia.

Longitudinal analysis of maternal serum Follistatin concentration in normal pregnancy and preeclampsia.

OBJECTIVE: Follistatin (FST) is a regulator of the biological activity of activin A (Act A), binding and blocking it, which could contribute to the modulation of its pro-inflammatory activity during pregnancy. We sought to investigate, in this nested case-control study, FST serum levels during normal pregnancy and correlate it with the FST profile in preeclamptic pregnant women, normal pregnant women followed 3 months postpartum and eumenorrheic nonpregnant women throughout the menstrual cycle. SUBJECTS AND METHODS: Follistatin serum levels determined by ELISA, biochemical and anthropometric variables were measured in normal pregnant (n = 28) and preeclamptic (n = 20) women during three periods of gestation. In addition, FST serum levels were measured in a subset of normal pregnant women (n = 13) followed 3 months postpartum and in eumenorrheic nonpregnant women (n = 20) during the follicular and luteal phases of the menstrual cycle. RESULTS: Follistatin serum levels in the eumenorrheic nonpregnant and postpartum group were significantly lower when compared to levels throughout gestation (P < 0·01). Serum FST levels increased in each period of pregnancy analysed, being significantly higher towards the end of gestation (P < 0·01). FST levels were lower in late pregnancy in preeclamptic women compared to normal pregnant women (P < 0·05). Finally, FST levels were higher in the luteal phase when compared with the follicular phase of the menstrual cycle (P < 0·05). CONCLUSIONS: These analyses would permit the consideration that changes in FST levels during pregnancy contribute to the control of the Act A system.

Regulation of NucB2/Nesfatin-1 throughout rat pregnancy.

Nesfatin-1 is an anorexigenic neuropeptide derived by post-translational cleavage from the N-terminus region DNA binding/EF-hand/acidic amino acid rich region (NEFA)/nucleobindin2 (NucB2) protein through proteolytic prohormone convertases. This neuropeptide was originally localized in different appetite controlling areas such as the hypothalamic paraventricular nucleus, arcuate nucleus, supraoptic nucleus, lateral hypothalamic area, and nucleus tractus solitarius. The objective of this study was to determine the expression and the changes that occur to mRNA and protein of NucB2 and Nesfatin-1 serum levels during gestation. This study utilized molecular and immunological approaches to investigate the expression and regulation of NucB2/Nesfatin-1 protein throughout gestation in rat fed under ad libitum and food restricted conditions (30% nutrient restriction). NucB2 was immunolocalized in the amnion and decidua of the rat placenta. Nesfatin-1 serum levels were measured by radioimmunoassay on gestational days 12, 16, 19 and 21, showing a significant (p<0.01) decrease in serum levels after day 12 until the end of gestation in rats fed ad libitum. These results were correlated with the analysis of NucB2 mRNA, with a significant (p<0.01) reduction observed in both the mRNA and protein of NucB2 during the gestational days 12, 16 and 21. It was also observed that food restriction decreases Nesfatin-1 serum levels and NucB2 placental expression at day 16 of gestation when compared to pregnant rats fed ad libitum. This study illustrates for the first time through molecular and immunological approaches the NucB2 expression and regulation on rat placenta and that this peptide is regulated throughout pregnancy. Consistent with previous reports, our results provide additional evidence supporting the role of NucB2 protein as an anorexigenic peptide that may contribute to the regulation of feeding behavior and energy homeostasis. NucB2/Nesfatin-1 might play an important metabolic role during pregnancy and fetal development and its energy balance mediating role should be studied in various physiological and pathological conditions throughout gestation.

Irisin levels during pregnancy and changes associated with the development of preeclampsia.

CONTEXT: Irisin is a recently discovered adipomyokine that regulates the differentiation and phenotype of adipose tissue. OBJECTIVE: In this study, we investigated the levels of irisin over the three trimesters of gestation in healthy and preeclamptic women and during the follicular and luteal phase of the menstrual cycle in a cohort of healthy eumenoherric women. METHODS: Serum irisin was measured by an ELISA in a longitudinal prospective cohort study in 40 healthy pregnant women, 10 mild preeclamptic women, and 20 healthy eumenoherric women during the menstrual cycle to assess irisin levels and correlations with other metabolic parameters. We identified the protein expression of fibronectin type III domain-containing protein 5, the irisin precursor, in human placenta using immunohistochemical approaches in humans. RESULTS: Serum irisin levels are higher in the luteal than in the follicular phase in eumenorrheic women. Fibronectin type III domain-containing protein 5, the irisin precursor, is expressed in human placenta, and its serum levels are higher during the entire pregnancy when compared with nonpregnant women. Serum irisin correlates positively with the homeostasis model assessment of estimated insulin resistance in the first trimester of normal pregnancy. Serum irisin levels do not change throughout gestation in preeclamptic women; however, there were lower irisin levels during the third trimester when compared with the normal pregnant group. CONCLUSION: Our results suggest that irisin may be involved in reproductive function and in the pregnancy-associated metabolic changes, and this condition may be an irisin-resistant state during gestation.

Brain-derived neurotrophic factor is expressed in rat and human placenta and its serum levels are similarly regulated throughout pregnancy in both species.

OBJECTIVE: Pregnancy is characterized by several metabolic changes that promote fat gain and later onset of insulin resistance. As Brain-derived neurotrophic factor (BDNF) decreases hyperglycaemia and hyperphagia, we aimed to investigate the potential role of placental and circulating BDNF levels in these pregnancy-related metabolic changes in rats and humans. DESIGN AND METHODS: We identified the mRNA and protein expression of placental BDNF and its receptor TrkB using real-time PCR, Western blot and immunohistochemical approaches in both rat and humans. Serum BDNF was measured by ELISA. We also did a longitudinal prospective cohort study in 42 pregnant women to assess BDNF levels and correlations with other metabolic parameters. RESULTS: We found that BDNF and TrkB are expressed in both rat and human placenta. In rat, both placental mRNA and serum levels are increased throughout pregnancy, whereas their protein levels are significantly decreased at the end of gestation. Serum BDNF levels in pregnant women are significantly lower in the first trimester when compared to the second and third trimester (P < 0·0148, P < 0·0012, respectively). Serum BDNF levels were negatively correlated with gestational age at birth and fasting glucose levels. CONCLUSION: Our findings suggest that both BDNF and its receptor TrkB are expressed in rodent and human placenta being regulated during pregnancy. Taken together, these findings support a role of BDNF in the regulation of several metabolic functions during pregnancy.

Serum chemerin levels during normal human pregnancy.

During gestation there are important changes in maternal metabolism and an increase in insulin resistance, coinciding with an increase in adiposity. Chemerin is an adipocytokine which is expressed and secreted in various tissues, including placenta, and may play an important role in metabolic regulation during pregnancy. The aim of this study was to determine serum levels of chemerin during gestation and compare them to other indicators of insulin resistance. A cross-sectional study was carried out analyzing serum chemerin levels of 20 pregnant women during three gestational periods, early, middle, and late (between the 10th and 14th, the 23rd and 26th, and the 34th and 37th week) and 20 non-pregnant women were used as a control group. An analysis of chemerin levels during the menstrual cycle was performed in an eumenorrheic group (n=16) in the early follicular (cycle day 4±1) and the midluteal phase (cycle day 22±1), demonstrating that serum chemerin levels did not fluctuate significantly. Serum levels of chemerin were significantly elevated during late gestation when compared to early (P<0.001) and middle (P=0.001) gestation and a negative correlation between serum chemerin and adiponectin levels (r=-0.1643) became more significant when the non-pregnant group was included in the calculations (r=-0.2471). There was no significant association of triglycerides, total cholesterol, LDL, HDL, insulin, and HOMA levels with chemerin. Although chemerin rose significantly and is negatively associated with adiponectin levels, it is not correlated with other markers of insulin sensitivity, suggesting that more study is needed to determine whether chemerin is useful in predicting insulin resistance during gestation.

Comparative study of elderly-onset rheumatoid arthritis and young-onset rheumatoid arthritis in a Colombian population: clinical, laboratory and HLA-DRB1 findings.

OBJECTIVES: Elderly-onset rheumatoid arthritis (EORA) is considered to have different features in relation to young-onset rheumatoid arthritis (YORA). However, results from different evaluated populations worldwide have been inconsistent and in Colombia there are no known descriptions of the differences between these pathologies. The aim of this paper is to compare the clinical, laboratory and immunogenetic features in a Colombian population suffering with EORA and YORA. METHODS: EORA (≥65, n=104) and YORA (<65, n=96) patients were compared regarding clinical, laboratory and HLA-DRB1 alleles features. A control group without rheumatoid arthritis over 65 (n=179) was used to compare the HLA-DRB1 alleles. All patients met the ACR/1987 criteria for rheumatoid arthritis and the clinimetric index was calculated. RESULTS: The gender ratio (female/male) was 1.8:1 in EORA. In both groups, the main onset pattern of disease was an insidious polyarticular onset (p=0.35). EORA was characterised by more distal-proximal joint involvement in comparison to YORA (p=0.0007). In EORA, the rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) antibodies frequency was close to 50%, lower than in YORA (63%). In both groups, the DAS28 and HAQ-DI score was higher than 6 and 1, respectively. The HLA-DRB1*0403 and *1402 frequency was significantly higher in EORA than in YORA. Also, the shared epitope (p=0.0392), HLA-DRB1*01 (p=0.0068) and *0101 (p=0.0151) were associated with an anti-CCP positivity and the HLA-DRB1*0403 is protective for the anti-CCP presence in EORA (p=0.0201). CONCLUSIONS: EORA is characterised by a different clinical presentation and HLA-DRB1 alleles with respect to YORA. HLA-DRB1*0403 and *1402 are significantly more frequent in EORA compared to YORA.

Leptin and fasting regulate rat gastric glucose-regulated protein 58.

The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58) is a molecular chaperone member of the endoplasmic reticulum (ER) stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency). We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.

Vaspin and amylin are expressed in human and rat placenta and regulated by nutritional status.

Amylin (islet amyloid polypeptide) and vaspin (visceral adipose tissue specific serpin) are gut and adipocyte hormones involved in the regulation of body weight homeostasis. The aim of this study was to examine whether amylin and vaspin are expressed in human and rat placenta, as well as their regulation by nutritional status. Our results demonstrate that amylin and vaspin are localized in both human and rat placenta. In the rat term placenta vaspin was demonstrated in the trophoblast of the fetal villi, the labyrinth. Vaspin immunostaining in human placenta was localized in cytotrophoblast and syncytiotrophoblast in the first trimester placentas while in the third trimester vaspin was localized in the syncytiotrophoblast. Regarding amylin, rat placenta of 16 days of gestational age showed an intense immunostaining, mainly localized in the labyrinth. On the other hand, in the human third trimester placenta amylin immunoreactivity was intense in the syncytiotrophoblast of the chorionic villi and in decidual cells. Furthermore, placental amylin and vaspin showed an opposite pattern of expression during pregnancy, with vaspin showing the highest expression level at the end and amylin at the beginning of pregnancy. Finally, food restriction also has contrary effects on their expression, increasing vaspin but decreasing amylin placental mRNA and protein levels. Taken together, our results demonstrate that vaspin and amylin are modulated by energy status in the placenta, which suggests that these proteins may be involved in the regulation of placental metabolic functions.

Food-intake-regulating-neuropeptides are expressed and regulated through pregnancy and following food restriction in rat placenta.

BACKGROUND: Neuropeptide Y (NPY), agouti related peptide (AgRP), cocaine and amphetamine-regulated transcript (CART) and melanocortins, the products of the proopiomelanocortin (POMC), are hypothalamic peptides involved in feeding regulation and energy homeostasis. Recent evidence has demonstrated their expression in rat and human placenta. METHODS: In the current study, we have investigated the expression of those neuropeptides in the rat placenta by real-time PCR using a model of maternal food restriction. RESULTS: Our results showed that placental-derived neuropeptides were regulated through pregnancy and following food restriction. CONCLUSION: These data could indicate that placental-derived neuropeptides represent a local regulatory circuit that may fine-tune control of energy balance during pregnancy.

Expression of neuropeptide W in rat stomach mucosa: regulation by nutritional status, glucocorticoids and thyroid hormones.

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.