Ibrahim H. Kamal

The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachias principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Detection of Wuchereria bancrofti in mosquitoes by the polymerase chain reaction: a potentially useful tool for large-scale control programmes

Focally endemic bancroftian filariasis is targeted for elimination in the Nile delta of Egypt. Improved methods are needed for identifying endemic villages to be included in the control programme and for monitoring its success. We have evaluated the performance of a polymerase chain reaction (PCR) assay in estimating Wuchereria bancrofti infection in pools of Culex pipiens (1-25 females) from 2 adjacent villages with high (El Qolzom, 10.8%) and low (Kafr Shorafa, 2.1%) prevalence rates of human filariasis. This assay detects a repeated sequence in W. bancrofti deoxyribonucleic acid (DNA). Mosquitoes resting within houses were captured by aspiration and pooled by house. Houses were classified as positive or negative for human filarial infection based on night blood examinations of residents. The assay detected parasite DNA in mosquitoes from 60% of 25 infected houses and 24% of 25 uninfected houses. PCR processing of mosquitoes caught within houses of unknown filariasis infection status (44 in El Qolzom, 37 in Kafr Shorafa) identified 31.8% and 8.1% of houses, respectively, as containing infected mosquitoes. These results support the validity of the PCR assay for evaluating filarial prevalence in different villages. C. pipiens collected outdoors in dry ice-baited traps and tested by PCR (266 in Qolzom, 82 in Kafr Shorafa) did not contain parasite DNA. Pools of female mosquitoes (296 in Qolzom, 240 in Kafr Shorafa) captured in oviposition traps were also negative. We concluded that the PCR based assay is a powerful epidemiological tool that can be used for evaluating W. bancrofti infection in villages in the Nile delta and for monitoring the application of control programmes in filariasis endemic areas.

Helminth genome analysis: The current status of the filarial and schistosome genome projects

Genome projects for the parasitic helminths Brugia malayi (a representative filarial nematode) and Schistosoma were initiated in 1995 by the World Health Organization with the ultimate objectives of identifying new vaccine candidates and drug targets and of developing low resolution genome maps. Because no genetic maps are available, and very few genes have been characterized from either parasite group, the first goal of both Initiatives has been to catalogue new genes for future placement on chromosome and physical maps. These genes have been identified by the expressed sequence tag (EST) approach, utilising cDNA libraries constructed from diverse life cycle stages. To date, the Initiatives have deposited over 16,000 Brugia ESTs and nearly 8000 Schistosoma ESTs in Genbanks dbEST database, corresponding to 6000 and over 3600 genes respectively (33% of Brugias estimated gene compliment, 18-24% of that of Schistosoma). Large fragment, genomic libraries have been constructed in BAC and YAC vectors for studies of genomic organization and for physical and chromosome mapping, and public, hypertext genomic databases have been established to facilitate data access. We present a summary of progress within the helminth genome initiatives and give several examples of important gene discoveries and future applications of these data.

Development of a quantitative, competitive polymerase chain reaction-enzyme-linked immunosorbent assay for the detection of Wuchereria bancrofti DNA

Abstract A quantitative, competitive polymerase chain reaction (QC-PCR) assay for the sensitive detection of Wuchereria bancrofti DNA was developed. A competitor sequence was constructed by an exchange of nucleotides in the Wuchereria-specific Ssp I repeat. The PCR products were hybridized to specific DNA probes and their amounts, determined by an enzyme-linked immunosorbent assay (ELISA). In laboratory-prepared samples the QC-PCR-ELISA assay was capable of detecting the amount of DNA equivalent to 0.1 microfilaria (mf) added to 200 μl of blood lysate. The assay was also tested on 78 blood samples collected in endemic areas in Egypt. All 28 samples that were positive both for mf and for circulating antigen were also QC-PCR-ELISA-positive. In addition, one mf-negative but antigen-positive sample was also positive as determined by QC-PCR-ELISA. A positive correlation of mf density with the QC-PCR-ELISA was observed. Samples containing 10 or fewer mf/ml had a mean relative amount of Ssp I PCR product of 19.7 units, whereas samples with 11–100 mf/ml had a mean of 36.3 units and those with more than 100 mf/ml had a mean of 84.6 units. Because of the high standard deviation within each group, estimates of worm burdens in infected individuals using the QC-PCR-ELISA are not recommended. However, we present data indicating that the W. bancrofti QC-PCR-ELISA is a powerful new tool for evaluation of parasitic loads for community-based diagnosis of bancroftian filariasis.

Two-stage PCR assay for detection of human brucellosis in endemic areas.

BackgroundBrucellosis is a common zoonosis that can cause a severe febrile illness in humans. It constitutes a persistent health problem in many developing countries around the world. It is one of the most frequently reported diseases in Saudi Arabia and incidence is particularly high in the Central region, and around the city of Riyadh. The aim of this study was to evaluate a two-stage PCR assay for detection of human brucellosis particularly in endemic areas.MethodsA total of 101 serum samples were collected from patients with acute febrile illness (AFI) of unknown cause from two different locations in the Western region of Saudi Arabia. The first location (Northern) is characterized by a nomadic rural population while the second (Central) is a modern urban city. All samples were subjected to DNA extraction and Brucella genus-specific PCR amplification using B4/B5 primers of the bcsp31 gene. Positive B4/B5 samples were subjected to multiplex species-specific Brucella PCR amplification.ResultsIn the Northern location, 81.9% of the AFI samples were confirmed Brucella positive, while all the samples collected from the Central region proved to be Brucella negative. Samples positive for Brucella were subjected to multiplex species-specific Brucella amplification. B. abortus was detected in 10% and B. melitensis in 8% of the samples, while the majority (82%) of samples showed both B. abortus and B. melitensis. As expected, B. suis was not detected in any of the samples.ConclusionsThis study concluded that a two-stage PCR assay could be useful as a rapid diagnostic tool to allow the consideration of brucellosis as a possible cause of AFI, particularly in non-urban locations. It also recommends the collection of epidemiological data for such patients to obtain further information that may help in rapid diagnosis.

Evaluation of a PCR-ELISA to detect Wuchereria bancrofti in Culex pipiens from an Egyptian village with a low prevalence of filariasis

The programmes for the elimination of bancroftian filariasis that have been implemented in the Nile delta of Egypt are expected to lead to substantial reductions in filarial loads in the treated populations. Better methods than those currently available are needed for monitoring the efficacy of these and similar efforts at intervention. A PCR-ELISA was therefore evaluated as an epidemiological tool for the detection of the Wuchereria-bancrofti-specific SspI repeat in pools of Culex pipiens collected in a village with a low prevalence of filarial infection in its human residents (2.1%). Indoor-resting mosquitoes were collected by aspiration from 114 randomly selected houses (during one to nine visits/house) and separated into 673 pools, each of which held the mosquitoes collected during one night from one house. Although 18 (2.7%) of the pools showed PCR inhibition and had to be excluded, filarial DNA was detected, using the PCR-ELISA, in 91 (13.9%) of the 655 remaining mosquito pools. The minimum prevalence of W. bancrofti infection in the mosquitoes caught (assuming one infected mosquito/positive pool) was 2.8%. The mean (S.D.) number of mosquitoes/pool did not vary significantly between positive [5.5 (3.4)] and negative [4.9 (3.5)] pools. The assay detected parasite DNA in mosquitoes from 19.3% of 114 houses when only the first visit was considered and from 73.9% of the 88 houses visited more than once. The PCR-ELISA yielded results comparable with those of the regular PCR-SspI assay. The latter assay is recommended for the routine examination, in laboratories in endemic areas, of mosquito pools from randomly selected houses, as the ELISA component of the PCR-ELISA is exceedingly time-consuming, expensive and requires special equipment.

Hypermethylation of P15, P16, and E-cadherin genes in ovarian cancer

Both p16 and p15 proteins are inhibitors of cyclin-dependent kinases that prevent the cell going through the G1/S phase transaction. E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent interactions between adjacent epithelial cells. Two groups of patients were selected: the first group suffered from epithelial serous ovarian tumors and the second group suffered from benign ovarian lesions; ovarian tissue samples from all the subjects (benign and malignant) were subjected to methylation-specific polymerase chain reaction for methylated and unmethylated alleles of the genes (E-cadherin, p15, and p16). Results obtained showed that aberrant methylation of p15 and p16 genes were detected in 64.29 and 50% of ovarian cancer patients, while E-cadherin hypermethylation was detected in 78.57% of ovarian cancer patients. Methylation of E-cadherin was significantly correlated with different stage of disease (p < 0.05). It was found that the risk of E-cadherin hypermethylation was 1.347-fold, while risk of p15 hypermethylation was 1.543-fold and p16 was 1.2-fold among patients with ovarian cancer than that among patients with benign ovarian lesions. In conclusion, Dysfunction of the cell cycle and/or the cell–cell adhesion molecule plays a role in the pathogenesis of ovarian cancer and that the analysis of the methylation of p15 and E-cadherin genes can provide clinically important evidence on which to base the treatment.

Early predictors of microvascular complications in type 1 diabetic patients.

OBJECTIVES To investigate the possibility of depending on adiponectin and leptin as early predictors of microvascular complications in type 1 diabetic subjects. DESIGN AND METHODS We studied 63 type 1 diabetic subjects from the National Institute of Diabetes (30 normoalbuminuric and 33 microalbuminuric). Clinical, demographic characteristics and kidney function tests were monitored. Plasma levels of adiponectin, leptin, interlukein-6 (IL-6), and the high sensitive C-reactive protein (CRP) were measured in these subjects. RESULTS Microalbuminuric subjects showed a significant elevation in adiponectin levels and a significant decrease in leptin levels as compared to normoalbuminuric subjects. Adiponectin showed a significant positive correlation with microalbuminuria concentrations while leptin showed a significant negative correlation with both fasting blood glucose and glycated hemoglobin A(1c). CONCLUSION The results of this study introduced the possibility of depending on adiponectin and leptin as early, reliable, and sensitive predictors for the microvascular complications monitored by microalbuminuria concentration and glycemic control indices.

Adiponectin, leptin, and lipid profile in type 1 diabetic children and adolescents

BACKGROUND Adipose tissue is known to produce and secrete a variety of bioactive substances known as adipocytokines. Adiponectin and leptin are considered to be among the most important adipocytokines: OBJECTIVES We sought to explore the relationships between adipocytokines (adiponectin and leptin), plasma lipoprotein lipid, and diabetic control indices in type 1 diabetic subjects. SUBJECTS AND METHODS In this study 63 clinically diagnosed type 1 diabetic subjects and 30 age- and sex-matched healthy control subjects were analyzed. Age, sex, diabetic duration, family history of diabetes, daily insulin dose, weight, height, body mass index, and systolic and diastolic blood pressure were recorded. Fasting blood glucose, glycated hemoglobin A(1c), total hemoglobin, plasma lipoprotein, lipid and plasma concentrations of adiponectin and leptin were measured in type 1 diabetic subjects and control subjects. RESULTS In this study a significant increase in triglycerides and high-density lipoprotein cholesterol in plasma of type 1 diabetics was found as compared with normal control subjects. In type 1diabetic subjects, plasma adiponectin was significantly elevated, whereas leptin showed a significant decrease as compared to a normal control group. Leptin concentrations showed a positive correlation with body mass index and systolic blood pressure but a negative correlation with both fasting blood glucose and glycated hemoglobinA(1c). CONCLUSION The results of this study suggest that blood leptin but not adiponectin concentrations have a significant correlation with indices of glycemic control.

Possible inhibition of hydroxy methyl glutaryl CoA reductase activity by nicotinic acid and ergosterol: as targeting for hypocholesterolemic action

OBJECTIVE Coronary artery diseases including atherosclerosis is considered as commonest problem worldwide. Ergosterols are the main components of vegetable oils and nuts. The objective of this study was to evaluate the potential hypoplipidemic and hypocholesterolemic effects of ergosterol in combination with niacin in rats fed high fat diet (HFD). METHODS Eighty male albino rats were included in this study divided into two main groups: Group I: Normal rats fed standard diet treated with either niacin (8.5 mg /kg b.w) or ergosterol (100 mg/Kg b.w) or both. Group II; rats fed HFD treated with either niacin (8.5 mg /kg b.w) or ergosterol (100 mg/Kg b.w) or both The feeding and treatment lasted for 8 weeks. RESULTS A significant elevation in the levels of total cholesterol, triacylglycerol, VLDL-c, LDL-c and atherogenic factor (p<0.001) in rats fed on HFD compared with normal control while HDL-c was significantly reduced in HFD rats compared with control group. Supplementation of diet with niacin or ergosterol or combined exerts improvement in the studied parameters by lowering triacylglycerol, total cholesterol, LDL-c and atherogenic factor and elevate HDL-c near to the value of control. Niacin combined with ergosterol were effective in the reduction of hydroxy methyl glutaryl-CoA reducatase (HMGCoA) compared with control (p<0.001). The combined effect was more potent than individual alone. CONCLUSION Utilization of niacin and ergosterol may prevent the hypercholesterolemia and incidence of coronary heart diseases. These functional foods act as nutriceutical as dyslipidemics.