Khaled M. Al-Qaoud

Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis

Abstract The pathways conferring immunity to human filariases are not well known, in part because human-pathogenic filariae do not complete a full life cycle in laboratory mice. We have used the only fully permissive infection of mice with filariae, i.e., infection of BALB/c mice with the rodent filarial nematode Litomosoides sigmodontis. Our previous results showed that worm development is inversely correlated with Th2 cytokine production and eosinophilia. The scope of the present study was to directly elucidate the role of interleukin-5 (IL-5) and eosinophils in controlling the development of L. sigmodontis after vaccination and in primary infection. BALB/c mice immunized with irradiated third-stage larvae (L3) were confirmed to have elevated IL-5 levels as well as high subcutaneous eosinophilia and to attack and reduce incoming larvae within the first 2 days, resulting in 70% reduction of worm load. Treatment of vaccinated mice with anti-IL-5 antibody (TRFK-5) suppressed both blood and tissue eosinophilia and completely abolished protection. This demonstrates, for the first time in a fully permissive filarial infection, that IL-5 is essential for protection induced by irradiated L3 larvae. In contrast, in primary-infected mice, anti-IL-5 treatment did not modify filarial infection within the 1st month, most likely because during primary infection IL-5-dependent mechanisms such as subcutaneous eosinophilia are induced too late to disturb worm establishment. However, there is a role for IL-5 late in primary infection where neutrophil-dependent worm encapsulation is also under the control of IL-5.

Lack of interferon-γ confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.

Human cystic echinococcosis in the West Bank of Palestine: surgical incidence and seroepidemiological study

Abstract. The surgical incidence of cystic echinococcosis (CE) due to Echinococcus granulosus was investigated in hospitals of the West Bank, Palestinian Authority between January 1990 and December 1997. Serum samples from school-children in Yata town, which showed the highest surgical incidence, were tested for anti-hydatid antibodies. A total of 390 surgically confirmed cases were recorded throughout the 8-year period, with an overall mean annual surgical incidence (MASI) of 3.1 per 100,000. A high MASI of 4.9, 5.0 and 5.1 per 100,000 was found in Hebron, Jericho and Bethlehem Governorates, respectively. Yata town, Hebron governorate, showed the highest MASI, at 16.8 per 100,000. The highest incidence was found in age groups 11–20 and 21–30 years, at 27.4% and 21.5% of the total number of cases. While there was no significant gender difference in the number of cases in the age groups of 20 years or less, the male to female case ratio was 1:3.2–4.1 in the older age groups. The liver was the most common site of hydatid cysts in 69.9% of cases. Lung cysts were predominant in younger age groups (20 years or less). The seropositivity for CE in the school-children of Yata was 2.4% and 2.1% using enzyme-linked immunosorbent assay and the indirect haemagglutination test, respectively. CE is a significant endemic disease throughout the West Bank. The disease is acquired early in life and is more prevalent among females than males. Behaviour and life-style favour the spread of the disease.

Retrospective surgical incidence and case distribution of cystic echinococcosis in Jordan between 1994 and 2000.

A retrospective follow-up study on the surgical incidence of cystic echinococcosis (CE) was carried out in major governmental, military and private hospitals throughout Jordan between 1994 and 2000. A total of 472 cases were recorded over the 7-year period and an overall mean annual surgical incidence (MASI) of 2.3 per 100,000 inhabitants was estimated. The highest number of surgical cases was recorded in hospitals of the middle region of the country. The highest MASI (3.6 per 100,000) was found in the southern region while the lowest (1.4 per 100,000) was in the northern region of the country. Taking into consideration the population size and the origin of surgically confirmed cases of each region, a relative surgical index (RSI) was calculated at which the highest (RSI=3.4) was among cases originated from the southern region and the lowest (RSI=0.7) was among those originated from the middle region of Jordan. The northern region and desert areas (badia) showed comparable RSI at 1.0 and 1.1, respectively. Males younger than 15 years of age showed significantly higher surgical incidence than females of comparable age at a ratio of 1.6:1, whereas the number of female cases of different age groups over 15 years of age was consistently higher than that of males at a ratio of 1.25-2.5:1.0. The liver was the primary site of cyst development in 69.4% of the cases and the lung involvement accounted for 13.3% of the cases. Diagnosis of CE in Jordan relies mostly on imaging methods with serological techniques being rarely used for diagnosis. The frequency of CE recurrence was 27.5% of the cases, which may be attributed to the low use of chemotherapeutic antihelminthics among surgically treated cases.

Classification, follow-up and recurrence of hepatic cystic echinococcosis using ultrasound images

Ultrasound image and morphological structure of hepatic cystic echinococcosis (CE) were analysed in 277 human cases (385 hepatic hydatid cysts). These included 65 CE cases from community surveys carried out between 1995 and 2000 in 3 countries (China, Mongolia and Jordan) and 212 cases from a clinical hospital survey from Xinjiang, China. A new simplified WHO ultrasound classification for human CE was assessed, and considered useful. It is proposed that type, size and number in particular need to be included in the ultrasound classification of hepatic CE. For comparative purposes 6 categories of type were classified in the study as Type 0 to Type 5 (T0-T5): T0, univesicular without pathognomonic signs; T1, univesicular with pathognomonic signs; T2, cysts with sagging or floating laminated membrane; T3, cysts containing daughter cysts; T4, solid mass or mixed cysts; and T5, cysts with partial or full calcifications. This differs from the WHO classification wherein Type T3 cysts (daughter cysts present) are considered a pathological stage to occur in general prior to the sagging or floating membrane (T2) stage. Recurrent hydatid cysts in the liver were also studied based on morphological structures observed directly from surgical intervention. Case follow-up over 1-5 years since endocystectomy in the community surveys indicated 10% (2/10) recurrence of cysts in the residual surgical cavity. Recurrent CE included 2 (2/4) cases after percutaneous treatment.

The seroprevalences of cystic echinococcosis, and the associated risk factors, in rural- agricultural, bedouin and semi-bedouin communities in Jordan

Abstract An ELISA was used to determine the seroprevalence of cystic echinococcosis (CE), caused by Echinococcus granulosus, in representatives of the rural–agricultural, semi-bedouin and bedouin communities of Jordan. The knowledge, attitudes and practices (KAP) pertaining to the transmission of CE in such communities were also investigated. In the ELISA, serum samples from 2388 subjects were tested for IgG antibodies reacting with antigens in crude sheep hydatid fluid (CSHF). The rural–agricultural subjects were significantly more likely to be seropositive (11.4%) than the semi-bedouin (5.0%) or bedouin (3.7%), but male and female subjects were equally likely to be seropositive. Among the rural–agricultural and semi-bedouin subjects, those aged 11–20 years were most likely to be seropositive. Among the bedouin subjects, however, seroprevalence was highest for those aged 31–40 years. When the distribution of 59 seropositive subjects detected within 36 rural–agricultural households was investigated, 12 (33.3%) of the households were each found to have at least two seropositive members. Immunoblotting indicated that 27.1% of the seropositive rural–agricultural subjects showed immunoreactivity to at least one of the CSHF antigen-B components (of 8–12, 16 and 24 kDa). The living conditions, practices and lifestyles of the rural–agricultural, semi-bedouin and bedouin communities favour the spread of CE in Jordan and warrant an effective programme for the control of the disease.

A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins

Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg Ig G ml⁻¹ resin and a dissociation constant (K(d)) of 4.78 × 10⁻⁶ M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.

Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2)

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/ 2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.

Production of Monoclonal Antibodies Against the 8 kDa Subunit of Echinococcus granulosus Antigen B (EgAgB8/2) Using DNA Immunization

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.