Protective efficacy of Toxoplasma gondii infected cells-derived exosomes against chronic murine toxoplasmosis.

Exosomes were isolated from T. gondii infected human hepatoblastoma cells using the exosome isolation kit and characterized by electron microscopy and Western blotting. Exosomes adsorbed to alum adjuvant were evaluated as a potential immunizing agent against murine chronic toxoplasmosis compared to excretory secretory antigens (ESA)-alum. Mice were immunized at days 1, 15 and 29. The levels of IgG, IFN-γ, IL-4 and IL-10, CD4+ and CD8+ T cells were determined using sandwich enzyme-linked immunosorbent assay (sandwich ELISA) at days 14, 28 and 56 of the experiment. Then mice were infected orally with 10 cysts of T. gondii. The protective efficacy of the antigens were evaluated by counting the brain cysts and measuring the aforementioned humoral and cellular parameters 60 days post infection. The results showed that alum increased the protective efficacy of the exosomes. Immunization with exosome-alum induced both humoral and mixed Th1/Th2 cellular immune responses. Exosome-alum gave higher levels of the humoral and cellular parameters, compared to ESA-alum. After challenge infection, exosome-alum significantly reduced the brain cyst burden by 75 % while ESA-alum gave 42 % reduction and evoked higher humoral and cellular immune responses. Therefore, the possibility of using T. gondii infected cells-derived exosome-alum as a vaccine is a new perspective in toxoplasmosis.

Comparative analysis of the diagnostic performance of crude sheep hydatid cyst fluid, purified antigen B and its subunit (12 Kda), assessed by ELISA, in the diagnosis of human cystic echinococcosis.

The diagnosis of patients with cystic echinococcosis (CE) by means of serology has a limited support in clinical practice due to cross-reactivity with other helminthes leading to overestimation of the parasite's true prevalence. A wealth of reports on the diagnostic performance of antigen B (AgB) has been produced. This study was designed to comparatively assess the diagnostic efficacy of crude sheep hydatid cyst fluid (HCF), AgB and its subunit (12 KDa) to detect IgG or IgG4 antibodies in CE patients' sera using enzyme-linked immunosorbent assay (ELISA).The best diagnostic performance was obtained with anti-HCF IgG ELISA which gave 92.4% sensitivity and 92.6% specificity. Despite the low sensitivity of anti AgB IgG ELISA (84%), it gave the best specificity (94.4%) with less cross-reaction with sera of subjects infected with other parasites. In conclusion, it is recommended to use anti-HCF IgG ELISA for initial screening in large seroprevalence studies. Further analysis of positive serum samples with anti AgB IgG ELISA would allow the confirmation of true positives. Specific IgG4 ELISA may represent a complementary assay, useful as secondary confirmatory tests for patients with suspected CE and negative for total IgG ELISA.

Genetic variability of antigen B among Echinococcus granulosus Egyptian isolates.

Genetic polymorphisms of encoding antigen B2 gene (AgB2) in Echinococcus granulosus were studied using PCR-RFLP and DNA sequencing among 20 Egyptian isolates. Five isolates from different host origins (humans, camels, pigs, and sheep) were collected and used. All examined isolates of each host group gave very similar patterns of PCR-RFLP after restriction enzyme digestion with AluI, with the gene size of approximately 140 bp and 240 bp for sheep and human isolates, and approximately 150 bp and 250 bp for pig and camel isolates. No digestion pattern was obtained after incubation of all studied isolates with EcoRI. These results reveal high intra-group homogeneity. DNA sequence analysis highlighted that human infecting strain showed 100% identity with respect to sheep infecting isolate, 96% and 99% with pig and camel infecting isolates, respectively.

Immunopathogenic role of IgG antibody and RANTES in house dust mite-induced chronic bronchitis.

Based on immunological and clinical examinations, 21 patients were diagnosed as having house dust mite (HDM)-induced chronic bronchitis and classified into three groups according to the clinical presentation of the disease: stable bronchitis, exacerbated bronchitis and asthma on top of bronchitis. Using ELISA, the levels of serum anti-Dermatophagoides farinae and anti-D. pteronyssinus IgG antibodies and plasma RANTES (regulated upon activation, normal T-cell-expressed and secreted; a chemokine with attractive and activator role for eosinophils) were measured in correlation to serum eosinophil cationic protein (ECP, a marker of eosinophil activation and degranulation measured by chemiluminescent immunometric technique). Using immunoblotting, IgG binding components of D. farinae and D. pteronyssinus were determined providing a clue for diagnosis of HDM-induced chronic bronchitis. Significant higher levels of anti-D. farinae and anti-D. pteronyssinus IgG antibodies and RANTES were found in asthmatic group followed by exacerbated chronic bronchitis in comparison to stable bronchitis and control groups. ECP level correlated significantly with IgG and RANTES levels in exacerbated bronchitis and asthmatic groups. The results provided evidence that over expression of IgG and RANTES plays a crucial role, as mediator in immunopathogenesis of HDM-induced chronic bronchitis and as marker of the immunological changes likely responsible for progression of bronchitis to asthma in HDM-sensitive patients yet, RANTES seemed to be an early indicator. Definition of the immunopathogenic role of IgG and RANTES in HDM-induced bronchitis should enable the manipulation of the critical immune response in the hope of establishing new therapies. D. farinae and D. pteronyssinu antigenic bands at > 205 and 205 KDa, respectively, considered together showed 71.4% sensitivity in diagnosis of HDM induced chronic bronchitis and 100% specificity by immuno-blotting.

Avidity of immunoglobulin G antibody response to the different antigenic fractions of soluble Schistosoma mansoni adult worm antigen preparation (SWAP) using avidity immunoblotting assay.

The detection of IgG antibodies against the different SWAP antigenic fractions plus the determination of their avidity in avidity immunoblotting assay using 6 M urea wash, presents a novel alternative for the characterization of optimal antigenic markers for acute and chronic phases of Schistosoma mansoni infection and for vaccine development. Human serum samples from 25 acute schistosomiasis patients, 20 chronic cases and 15 normal healthy controls were analysed by IgG avidity enzyme linked immunosorbent assay (ELISA) and IgG avidity immunoblotting assay. Using avidity ELISA, a pronounced overlap of avidity index values was observed between acute and chronic infections with a range of uncertainty (0.86-1) which was encountered in both groups. Using avidity immunoblotting assay, antigenic bands at >116, 84, 48, 40 & 34 KDa were exclusive for the acute phase. From these bands, 34 KDa was recognized mostly by low-avidity antibodies and showed a high sensitivity (96%) and specificity (100%) making it an optimal marker for the acute phase. 40 KDa band was recognized mostly by high-avidity antibodies even during acute infection. Bands of 80, 70, 42, 36, 30 & 26 KDa were exclusive for the chronic phase. Only 70 KDa band was recognized by high-avidity antibodies yet, with low sensitivity (35%), that limits its use as an optimal marker for the chronic infection. Meanwhile, 70 and 40 KDa bands, recognized by high-avidity antibodies, are considered as potential vaccine antigen candidates.

Application and assessment of a dipstick assay in the diagnosis of hydatidosis and trichinosis.

The aim of the present work was to apply and evaluate a dipstick assay for the serodiagnosis of human hydatidosis as well as human and experimental trichinosis using camel hydatid cyst fluid (HCF) and Trichinella spiralis muscle larval (TSML) antigens, respectively, and compare this to enzyme-linked immunoelectrotransfer blot (EITB) and Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA). Sera samples were collected from patients with confirmed hydatidosis and trichinosis and with other parasitic diseases as well as from normal healthy individuals. Also, sera samples were collected from mice experimentally infected with T. spiralis which were sacrificed at different time points post-infection (PI). HCF and TSML antigens were used in EITB after separation and characterization of their antigenic components using 5-22.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing condition. For the diagnosis of hydatidosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100, 91.4 and 95.1% while those of FAST-ELISA were 96.2, 100 and 98.4%, respectively. For the diagnosis of human trichinosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100% while those of FAST-ELISA were 85.7%. FAST-ELISA proved to be more sensitive in the early diagnosis of experimental T. spiralis infection (100% sensitivity from the second week PI) than the dipstick and EITB (100% sensitivity from the third week PI). All tests retained their sensitivity till the 12th week PI. Since the dipstick assay is extremely easy to perform with a visually interpreted result within 15 min, in addition to being both sensitive and specific, the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment and the technological expertise needed for EITB and FAST-ELISA.

Evaluation of an IgG cystatin capture enzyme-linked immunosorbent assay for the detection of anti-cysteine proteinase antibodies in asymptomatic trichomoniasis patients.

All isolates of T. vaginalis release cysteine proteinases proteolytic enzymes that are shed into the vagina or culture medium. Cystatin has been used successfully as a capture agent in ELISA to detect cysteine proteinase antibodies without the need for purified proteinases. The ELISA was evaluated in comparison to wet mount microscopy and culture techniques. IgG cystatin capture ELISA proved to be a sensitive and highly specific (100%) assay that could rapidly detect anti-cysteine proteinase antibodies in both vaginal washouts and sera of asymptomatic patients with a sensitivity of 100% and 86.7%, respectively. A defined discrimination between sero-positive and sero-negative individuals was markedly observed for ELISA-vaginal washouts providing a more conclusive diagnosis by this technique. The results demonstrated that in trichomoniasis patients (52 cases) whether symptomatic or asymptomatic. T. vaginalis infection was detected in 19 (36.5%), 49 (94.2%), 50 (96.1%) and 48 (92.3%) by wet mount, culture, cystatin capture ELISA-vaginal washouts and ELISA-sera, respectively. The assay was reliable also as a test of cure with a specificity of 94.4% in the vaginal washouts and 83.3% in sera.

Immunological indicators of morbidity in human schistosomiasis mansoni: role of vascular endothelial growth factor and anti-soluble egg antigen IgG4 in disease progression.

Based sonographic examinations of 90 schistosomiasis mansoni, they were divided into five groups: lightly infected, heavily infected, intestinal, early hepatosplenic and periportal fibrosis. Using ELISA, the levels of circulating vascular endothelial growth factor (VEGF) and anti-soluble egg antigen (SEA) IgG4 were measured in their sera. Compared to normal controls, VEGF levels were significantly raised in all schistosomiasis patients groups except lightly infected and intestinal patients were insignificantly elevated. The level of VEGF correlated with disease progression from lightly infected to periportal fibrosis patients. It also correlated with sonographic indicators of portal hypertension; presence of portosystemic collaterals, portal vein dilatation and splenomegaly. Serum IgG4 was significantly raised in only periportal fibrosis and portal hypertension patients. The results provided evidence that circulating levels of VEGF can serve as a new indicator of progression of schistosomiasis mansoni reflecting angiogenesis that regulates the granuloma and fibrosis development in liver while IgG4 seemed to be an indicator of only fibrosis development. Both are potential sensitive markers for effectiveness of treatment in periportal fibrosis patients. The understanding of the key role of VEGF in schistosomiasis mansoni pathogenesis may provide a new pharmacological target.