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Carbohydrate-restricted diets are used by people with type 1 diabetes (T1D) to help manage their condition. However, the impact of this strategy on blood glucose responses to exercise is unknown. This study describes the nutritional strategies of an athlete with T1D, who follows a very low carbohydrate diet to manage her condition during an ultra-endurance open-water swimming event. The athlete completed the 19.7 km distance in 6 h 43 min. She experienced minimal disruptions to glycaemia, reduced need for supplemental carbohydrate, and no episodes of symptomatic hypoglycaemia. This case report will hopefully encourage further experimental studies that inform and expand current clinical practice guidelines.
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Glucemia , Diabetes Mellitus Tipo 1 , Humanos , Femenino , Diabetes Mellitus Tipo 1/terapia , Carbohidratos de la Dieta , Natación , Agua , Dieta Baja en CarbohidratosRESUMEN
Public interest in low-carbohydrate (LC) diets for type 1 diabetes (T1D) management has increased. This study compared the effects of a healthcare professional delivered LC diet compared to habitual diets higher in carbohydrates on clinical outcomes in adults with T1D. Twenty adults (18-70 yrs) with T1D (≥6 months duration) with suboptimal glycaemic control (HbA1c>7.0% or >53 mmol/mol) participated in a 16-week single arm within-participant, controlled intervention study involving a 4-week control period following their habitual diets (>150 g/day of carbohydrates) and a 12-week intervention period following a LC diet (25-75 g/day of carbohydrates) delivered remotely by a registered dietitian. Glycated haemoglobin (HbA1c -primary outcome), time in range (blood glucose: 3.5-10.0 mmol/L), frequency of hypoglycaemia (<3.5 mmol/L), total daily insulin, and quality of life were assessed before and after the control and intervention periods. Sixteen participants completed the study. During the intervention period, there were reductions in total dietary carbohydrate intake (214 to 63 g/day; P<0.001), HbA1c (7.7 to 7.1% or 61 to 54 mmol/mol; P = 0.003) and total daily insulin use (65 to 49 U/day; P<0.001), increased time spent in range (59 to 74%; P<0.001), and improved quality of life (P = 0.015), with no significant changes observed during the control period. Frequency of hypoglycaemia episodes did not differ across timepoints, and no episodes of ketoacidosis or other adverse events were reported during the intervention period. These preliminary findings suggest that a professionally supported LC diet may lead to improvements in markers of blood glucose control and quality of life with reduced exogenous insulin requirements and no evidence of increased hypoglycaemia or ketoacidosis risk in adults with T1D. Given the potential benefits of this intervention, larger, longer-term randomised controlled trials are warranted to confirm these findings. Trial Registration: https://www.anzctr.org.au/ACTRN12621000764831.aspx.
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Diabetes Mellitus Tipo 1 , Hipoglucemia , Cetosis , Humanos , Adulto , Diabetes Mellitus Tipo 1/terapia , Hemoglobina Glucada , Calidad de Vida , Dieta Baja en Carbohidratos , Insulina Regular HumanaRESUMEN
Background and methods: Circulating Loa loa antigens are often detected in individuals with heavy L. loa infections by diagnostic tests for lymphatic filariasis (LF) caused by Wuchereria bancrofti. This is a major challenge to LF mapping and elimination efforts in loiasis co-endemic areas. However, it also provides an opportunity to identify antigen biomarkers for loiasis. To determine which L. loa antigens might be promising biomarkers for distinguishing true LF from loiasis, we screened for L. loa antigens in a group of individuals with heavy L. loa infections living in the Okola Health District of Cameroon. In this longitudinal study, participants were tested for cross-reactive antigenemia by filariasis test strip (FTS), ELISA, and western blot, and were monitored for FTS status at 6, 9, 12, and 15 months post-enrollment. We then identified specific circulating L. loa antigens by liquid chromatography-tandem mass spectrometry (LC-MS/MS) from baseline and 15-month plasma samples. Principal findings and conclusions: Among 73 FTS-positive (FTS+) and 13 FTS-negative (FTS-) participants with high L. loa microfilarial loads, 83% maintained their FTS status over the course of the study, while 17% experienced at least one FTS conversion event (from FTS+ to FTS- or vice versa). Cross-reactive antigens were detected in both FTS+ and FTS- sera by western blot, and there was poor agreement in antigen detection by FTS, western blot, and ELISA methods. One protein family, a group of Nas-14 metalloproteases, was detected by LC MS/MS in >80% of tested samples, including FTS- samples. These data identify Nas-14 as a promising loiasis biomarker potentially capable of distinguishing loiasis from lymphatic filariasis.
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BACKGROUND: Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite's development and survival in its mammalian and insect hosts. RESULTS: We used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched) while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched). We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes. CONCLUSIONS: Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of infection, development, reproduction, and survival in the host. Some of these may be useful targets for vaccines or new drug treatments for filariasis.
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Brugia Malayi/genética , Perfilación de la Expresión Génica , Animales , Brugia Malayi/crecimiento & desarrollo , Análisis por Conglomerados , Desarrollo Embrionario/genética , Femenino , Estadios del Ciclo de Vida/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Oogénesis/genética , ARN de Helminto/metabolismo , Espermatogénesis/genética , Regulación hacia ArribaRESUMEN
There is well-established evidence for low-carbohydrate, high-fat (LCHF) diets in the management of chronic health conditions in adults. The natural next step is to understand the potential risks and benefits of LCHF diets for children, where they may have useful applications for general health and a variety of chronic health conditions. It is vital that any diet delivers sufficient micronutrients and energy to ensure health, wellbeing, and growth. This descriptive study assesses the nutrient and energy status of LCHF sample meal plans for children. We designed four meal plans for hypothetical weight-stable male and female children (11 years) and adolescents (16 years). Carbohydrates were limited to ≤80 g, protein was set at 15-25% of the total energy, and fat supplied the remaining calories. Using FoodWorks dietary analysis software, data were assessed against the national Australian/New Zealand nutrient reference value (NRV) thresholds for children and adolescents. All meal plans exceeded the minimum NRV thresholds for all micronutrients; protein slightly exceeded the AMDR recommendations by up to three percentage points. This study demonstrates that LCHF meal plans can be energy-, protein-, and micronutrient-replete for children and adolescents. As with any dietary approach, well-formulated meals and careful planning are key to achieving the optimal nutrient status.
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Dieta Baja en Carbohidratos , Dieta Alta en Grasa , Comidas , Adolescente , Adulto , Australia , Niño , Carbohidratos de la Dieta , Grasas de la Dieta , Femenino , Humanos , Masculino , MicronutrientesRESUMEN
Background: Rapid and accurate prevalence mapping of lymphatic filariasis (LF) is necessary to eliminate this disfiguring and disabling neglected tropical disease. Unfortunately, rapid tests such as the filariasis test strip (FTS) for Wuchereria bancrofti, the causative agent of LF in Africa, can cross-react with antigens circulating in some persons infected by the African eye worm, Loa loa, rendering the test unreliable in eleven co-endemic nations. The intended target of the FTS is a heavily glycosylated W. bancrofti circulating filarial antigen (Wb-CFA). Previously, we determined that the FTS monoclonal antibody, AD12.1, which detects a carbohydrate epitope on Wb-CFA, also detects multiple L. loa proteins in cross-reactive sera from persons with loiasis. Since the carbohydrate epitope recognized by AD12.1 is present on glycoproteins of other parasitic nematodes, including Brugia species, it is unclear why reactive glycoproteins are not detected in infections with other filarial parasites. Methods: To gain a better understanding of the proteins recognized by the FTS diagnostic antibody, we used proteomics and lectin array technology to characterize filarial glycoproteins that are bound by the AD12.1 antibody using Brugia malayi as a model. Results: Distinct but overlapping sets of AD12 glycoproteins were identified from somatic and excretory/secretory worm products. One of the identified proteins, Bm18019 was confirmed as a secreted AD12-reactive glycoprotein by in-gel proteomics and immunoassays. Based on lectin binding patterns, Brugia AD12-reactive glycoproteins express glycans including core fucose, galactose, N-acetylglucosamine and galactose (ß1-3)N-acetylgalactosamine in addition to the epitope recognized by AD12.1. None of the lectins that bound B. malayi AD12 glycoproteins had affinity for the Wb-CFA, highlighting a key difference between it and other AD12 glycoproteins. Conclusions: B. malayi somatic and excretory/secretory proteins are similar to L. loa antigens found in FTS-positive human sera, bolstering the hypothesis that circulating L. loa AD12 antigens result from worm tissue damage or death. The difference in glycan and protein composition between the Wb-CFA and other AD12 glycoproteins can be used to differentiate LF from cross-reactive loiasis.
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Galectins are among the most abundant excretory/secretory (ES) products of filarial worms, but their role in filarial biology is poorly understood. Galectin-2 (Lec-2), a major component of Brugia malayi extracellular vesicles, is released by filarial worms, and was recently identified in the serum of persons with loiasis. We therefore sought to clone and characterize Lec-2, and to develop reagents to examine its potential as a biomarker and its role in parasite biology. We cloned and expressed recombinant B. malayi Lec-2 (rBmLec-2), generated a Lec-2-specific monoclonal antibody (4B4), and used it to confirm the presence of Lec-2 in B. malayi ES products and whole worm lysate. We show that Lec-2 is absent in B. malayi oocytes, and increases in concentration as embryos mature. Recombinant BmLec-2 hemagglutinates rabbit red blood cells at concentrations less than 1 µg/mL, and this is abrogated by single amino acid substitutions in the predicted carbohydrate recognition domains. rBmLec-2 binds multiple LacNAc oligosaccharides on a mammalian carbohydrate array. Sera from 17/23 (78 %) persons with microfilaremic loiasis and 4/10 (40 %) persons with bancroftian filariasis had detectable antibody to Lec-2 by western blot. Our studies confirm the functionality of BmLec-2 and indicate anti-Lec-2 antibody responses are common in persons with filariasis. These studies set the stage for further examination of the role of Lec-2 in filarial biology and in filarial-host interactions.
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Anticuerpos Antihelmínticos/sangre , Brugia Malayi , Galectina 2 , Polisacáridos/metabolismo , Animales , Antígenos Helmínticos/inmunología , Biomarcadores , Western Blotting , Brugia Malayi/inmunología , Brugia Malayi/metabolismo , Filariasis Linfática , Filariasis , Galectina 2/biosíntesis , Galectina 2/genética , Galectina 2/inmunología , Galectina 2/metabolismo , Hemaglutinación , Interacciones Huésped-Parásitos , Loiasis , Mamíferos , Proteínas RecombinantesRESUMEN
The Global Program to Eliminate Lymphatic Filariasis (GPELF) relies heavily on a rapid diagnostic test (RDT) to a Wuchereria bancrofti circulating filarial antigen (Wb-CFA) to identify endemic areas and for determining when mass drug administration can stop. The antigen contains a carbohydrate epitope that is recognized by monoclonal antibody AD12. Og4C3, a monoclonal antibody that is used in a commercial ELISA for Wb-CFA recognizes the same moiety. Despite its diagnostic importance, little is known about the structure and function of this "AD12 epitope". It is also present on other W. bancrofti glycoproteins and on glycoproteins of other filarial worms, but such antigens are not detected in the sera of individuals with most other filarial infections. We report here functional and biochemical analyses that shed light on the interaction between filarial glycoproteins and AD12 and/or Og4C3. Binding of these monoclonal antibodies to a mammalian glycan array suggests the reactive moiety has structural similarity to terminal ß-d-glucuronic acid in a 1-3 linkage to other hexoses. However, sera collected from individuals with patent W. bancrofti infection had very low or undetectable serum antibodies to the GlcA-containing array glycans. Unlike other filarial glycoproteins, the Wb-CFA is relatively resistant to protease digestion by pronase and trypsin and completely resistant to the mucinase O-sialoglycoprotein endopeptidase (OSGE). The protease resistance of the Wb-CFA may contribute to its consistent detection in Wb-infected sera.
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Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Helmínticos/inmunología , Filariasis/diagnóstico , Filariasis/inmunología , Polisacáridos/inmunología , Wuchereria bancrofti/inmunología , Animales , Antígenos Helmínticos/sangre , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Proteínas del Helminto/inmunología , Humanos , Inmunoglobulina G/inmunología , Unión Proteica/inmunologíaRESUMEN
BACKGROUND: Filarial nematode parasites cause serious diseases such as elephantiasis and river blindness in humans, and heartworm infections in dogs. Third stage filarial larvae (L3) are a critical stage in the life cycle of filarial parasites, because this is the stage that is transmitted by arthropod vectors to initiate infections in mammals. Improved understanding of molecular mechanisms associated with this transition may provide important leads for development of new therapies and vaccines to prevent filarial infections. This study explores changes in gene expression associated with the transition of Brugia malayi third stage larvae (BmL3) from mosquitoes into mammalian hosts and how these changes are affected by radiation. Radiation effects are especially interesting because irradiated L3 induce partial immunity to filarial infections. The underlying molecular mechanisms responsible for the efficacy of such vaccines are unkown. RESULTS: Expression profiles were obtained using a new filarial microarray with 18, 104 64-mer elements. 771 genes were identified as differentially expressed in two-way comparative analyses of the three L3 types. 353 genes were up-regulated in mosquito L3 (L3i) relative to cultured L3 (L3c). These genes are important for establishment of filarial infections in mammalian hosts. Other genes were up-regulated in L3c relative to L3i (234) or irradiated L3 (L3ir) (22). These culture-induced transcripts include key molecules required for growth and development. 165 genes were up-regulated in L3ir relative to L3c; these genes encode highly immunogenic proteins and proteins involved in radiation repair. L3ir and L3i have similar transcription profiles for genes that encode highly immunogenic proteins, antioxidants and cuticle components. CONCLUSION: Changes in gene expression that normally occur during culture under conditions that support L3 development and molting are prevented or delayed by radiation. This may explain the enhanced immunogenicity of L3ir. Gene Ontology and KEGG analyses revealed altered pathways between L3 types. Energy and "immune pathways" are up-regulated and may be needed for L3i invasion and survival, while growth and development are priorities for L3c. This study has improved our understanding of molecules involved in parasite invasion and immune evasion, potential targets of protective immunity, and molecules required for parasite growth and development.
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Brugia Malayi/genética , Perfilación de la Expresión Génica , Genes de Helminto , Aedes/parasitología , Ancylostoma/genética , Animales , Brugia Malayi/inmunología , Brugia Malayi/patogenicidad , Brugia Malayi/efectos de la radiación , Caenorhabditis elegans/genética , Radioisótopos de Cesio , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Larva/genética , Larva/inmunología , Larva/patogenicidad , Larva/efectos de la radiación , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN de Helminto/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: The low-carbohydrate, high-fat (LCHF) diet is becoming increasingly employed in clinical dietetic practice as a means to manage many health-related conditions. Yet, it continues to remain contentious in nutrition circles due to a belief that the diet is devoid of nutrients and concern around its saturated fat content. This work aimed to assess the micronutrient intake of the LCHF diet under two conditions of saturated fat thresholds. DESIGN: In this descriptive study, two LCHF meal plans were designed for two hypothetical cases representing the average Australian male and female weight-stable adult. National documented heights, a body mass index of 22.5 to establish weight and a 1.6 activity factor were used to estimate total energy intake using the Schofield equation. Carbohydrate was limited to <130 g, protein was set at 15%-25% of total energy and fat supplied the remaining calories. One version of the diet aligned with the national saturated fat guideline threshold of <10% of total energy and the other included saturated fat ad libitum. PRIMARY OUTCOMES: The primary outcomes included all micronutrients, which were assessed using FoodWorks dietary analysis software against national Australian/New Zealand nutrient reference value (NRV) thresholds. RESULTS: All of the meal plans exceeded the minimum NRV thresholds, apart from iron in the female meal plans, which achieved 86%-98% of the threshold. Saturated fat intake was logistically unable to be reduced below the 10% threshold for the male plan but exceeded the threshold by 2 g (0.6%). CONCLUSION: Despite macronutrient proportions not aligning with current national dietary guidelines, a well-planned LCHF meal plan can be considered micronutrient replete. This is an important finding for health professionals, consumers and critics of LCHF nutrition, as it dispels the myth that these diets are suboptimal in their micronutrient supply. As with any diet, for optimal nutrient achievement, meals need to be well formulated.
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Dieta Baja en Carbohidratos , Dieta Alta en Grasa , Ingestión de Energía , Micronutrientes/análisis , Adulto , Australia , Índice de Masa Corporal , Femenino , Humanos , Masculino , Comidas , Persona de Mediana Edad , Política Nutricional , Adulto JovenRESUMEN
The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis.
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Antígenos Helmínticos/inmunología , Filariasis Linfática/diagnóstico , Loa/inmunología , Wuchereria bancrofti/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/sangre , Reacciones Cruzadas , Pruebas Diagnósticas de Rutina , Filariasis Linfática/sangre , Filariasis Linfática/inmunología , Filariasis Linfática/parasitología , Femenino , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Humanos , Loa/genética , Wuchereria bancrofti/genética , Wuchereria bancrofti/aislamiento & purificaciónRESUMEN
Third-stage infective larvae (L3i) of Brugia malayi are developmentally arrested in mosquitoes but must quickly adapt to a new environment when they enter mammalian hosts to initiate infections. These changes can be studied by in vitro culture of L3 (L3c) under conditions that permit molting of L3-L4. Irradiated L3 (L3ir) have stunted growth and limited lifespan in mammalian hosts, and they induce high levels of immunity to challenge infections in animal models. This study explored differences in gene expression in L3i, L3c and L3ir by expressed sequence tag EST generation and qRT-PCR. 2506 ESTs generated from cDNA libraries constructed from L3i, L3c and L3ir were grouped into 1309 gene clusters. Despite extensive prior sampling from B. malayi (>22,000 ESTs in dbEST), 73% of the L3 clusters described here are novel. Sixty-three percentage of the clusters have homology to proteins from other species including 187 specific to nematodes and 141 that have to date only been described in non-nematode species. The transcript levels of 62 candidates for up- or down-regulation in L3i, L3c and L3ir based on EST frequencies were evaluated by qRT-PCR. Twenty-eight were confirmed to have > or = 3-fold differences in expression. Genes coding for proteins believed to be involved in establishment of infection, host adaptation and targets of protective immunity were confirmed to have higher expression in L3i than in L3c. Some of the genes that were down-regulated in L3c were highly expressed in L3ir. This study provides an improved description of the adaptations that accompany the transition from L3i to L3c and the special ability of L3ir to induce protective immunity.
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Brugia Malayi/genética , Genes de Helminto , Aedes/parasitología , Animales , Brugia Malayi/crecimiento & desarrollo , Brugia Malayi/inmunología , Brugia Malayi/efectos de la radiación , Etiquetas de Secuencia Expresada , Expresión Génica/efectos de la radiación , Perfilación de la Expresión Génica , Biblioteca de Genes , Larva/genética , Larva/crecimiento & desarrollo , Larva/efectos de la radiación , Familia de Multigenes , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.
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Brugia Malayi/genética , ADN de Helmintos/genética , Proteínas del Helminto/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética , Animales , Enzimas/genética , Etiquetas de Secuencia Expresada , Femenino , Regulación de la Expresión Génica , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres SexualesRESUMEN
Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of drugs that target AChRs in filarial worms.
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Brugia Malayi/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas del Helminto/metabolismo , Receptores Colinérgicos/metabolismo , Animales , Brugia Malayi/embriología , Brugia Malayi/genética , Femenino , Genoma de los Helmintos , Proteínas del Helminto/genética , Hibridación Genética , Masculino , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Colinérgicos/química , Espermatogénesis/genética , Espermatogénesis/fisiologíaRESUMEN
Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C(t) method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.
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Brugia Malayi/genética , Animales , Secuencia de Bases , Benzotiazoles , Cartilla de ADN/genética , ADN de Helmintos/genética , Diaminas , Femenino , Colorantes Fluorescentes , Expresión Génica , Genes de Helminto , Masculino , Compuestos Orgánicos , Quinolinas , ARN de Helminto/análisis , ARN de Helminto/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Caracteres Sexuales , Transcripción GenéticaRESUMEN
BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.
RESUMEN
Glutamate-gated chloride channels (GluCl) are targets for avermectin/milbemycin (A/M) anthelmintics such as ivermectin that cause paralysis of somatic and pharyngeal muscles in gastrointestinal nematodes. Ivermectin is useful for onchocerciasis control programs because of its activity against microfilariae that often cause ocular disease and severe dermatitis. However, mechanisms responsible for reduced microfilaria production by adult worms following ivermectin treatment are poorly understood. We synthesized subunit-specific RNA probes for the Brugia malayi GluCl gene avr-14 (BmAVR-14) to localize expression of this gene in adult filarial worms. Both subunits of BmAVR-14 exhibited very similar expression patterns. In female worms, strong expression signals were detected in the ovary, developing embryos and lateral hypodermal chords, with moderate expression in the uterus wall adjacent to stretched microfilariae. These genes were also highly expressed in adult male worms (in spermatogonia, in the wall of the vas deferens, and in the lateral chords, but not in mature spermatozoa). In addition, avr-14 was highly expressed in somatic muscles adjacent to the terminal end of the vas deferens which contains mature sperm. These results show that avr-14 is highly expressed in B. malayi developing embryos and reproductive tissues, and they provide evidence for the involvement of GluCl in gamete production and embryogenesis in filarial worms. This may explain the observed suppression of microfilaria (Mf) production by female worms following treatment with avermectin/milbemycin anthelmintics.
RESUMEN
BACKGROUND: A better understanding of reproductive processes in parasitic nematodes may lead to development of new anthelmintics and control strategies for combating disabling and disfiguring neglected tropical diseases such as lymphatic filariasis and onchocerciasis. Transcriptomatic analysis has provided important new insights into mechanisms of reproduction and development in other invertebrates. We have performed the first genome-wide analysis of gender-associated (GA) gene expression in a filarial nematode to improve understanding of key reproductive processes in these parasites. METHODOLOGY/PRINCIPAL FINDINGS: The Version 2 Filarial Microarray with 18,104 elements representing â¼85% of the filarial genome was used to identify GA gene transcripts in adult Brugia malayi worms. Approximately 19% of 14,293 genes were identified as GA genes. Many GA genes have potential Caenorhabditis elegans homologues annotated as germline-, oogenesis-, spermatogenesis-, and early embryogenesis- enriched. The potential C. elegans homologues of the filarial GA genes have a higher frequency of severe RNAi phenotypes (such as lethal and sterility) than other C. elegans genes. Molecular functions and biological processes associated with GA genes were gender-segregated. Peptidase, ligase, transferase, regulator activity for kinase and transcription, and rRNA and lipid binding were associated with female GA genes. In contrast, catalytic activity from kinase, ATP, and carbohydrate binding were associated with male GA genes. Cell cycle, transcription, translation, and biological regulation were increased in females, whereas metabolic processes of phosphate and carbohydrate metabolism, energy generation, and cell communication were increased in males. Significantly enriched pathways in females were associated with cell growth and protein synthesis, whereas metabolic pathways such as pentose phosphate and energy production pathways were enriched in males. There were also striking gender differences in environmental information processing and cell communication pathways. Many proteins encoded by GA genes are secreted by Brugia malayi, and these encode immunomodulatory molecules such as antioxidants and host cytokine mimics. Expression of many GA genes has been recently reported to be suppressed by tetracycline, which blocks reproduction in female Brugia malayi. Our localization of GA transcripts in filarial reproductive organs supports the hypothesis that these genes encode proteins involved in reproduction. CONCLUSIONS/SIGNIFICANCE: Genome-wide expression profiling coupled with a robust bioinformatics analysis has greatly expanded our understanding of the molecular biology of reproduction in filarial nematodes. This study has highlighted key molecules and pathways associated with reproductive and other biological processes and identified numerous potential candidates for rational drug design to target reproductive processes.