Mahmoud Mohamed Bahgat

DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity effect induced in mice.

DNA-based vaccine technology was used to induce an immune response in mice against a schistosome cysteine proteinase, asparaginyl endopeptidase (Sm32). The cDNA coding for Sm32 was cloned in a mammalian expression vector under control of the CMV promoter/enhancer and expressed for the first time in transfected mammalian cells as well as in mice immunized with the Sm32-encoding DNA construct. These mice developed antibodies which recognized the native protein not only in homogenates of Schistosoma mansoni worms but also in the gut on cryostat sections of the parasites. This DNA vaccine led to an anti-fecundity effect: female worms of a challenge infection produced 37% less eggs than those growing in naïve mice. The results suggest that Sm32 may be a candidate antigen for the generation of an anti-pathology vaccine against schistosomes.

Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum and Trichobilharzia ocellata

Cercarial secretions from different species of the parasite Schistosoma and from Trichobilharzia ocellata contain a proteolytic activity, cercarial elastase, which was demonstrated by a 30 kDa band in gelatin gels. Sera of patients infected with Schistosoma mansoni, Schistosoma haematobium or Schistosoma japonicum contain immunoglobulin G which react in ELISA with cercarial secretions from all schistosomes and cross‐react among the different parasite species. In Western blots, however, infection sera from patients, as well as heavily infected mice or rabbits, did not react with a 30‐kDa protein. Moreover, when sections from infected snails (Biomphalaria, Bulinus and Lymnaea) were analysed by immunofluorescence using the same infection sera, only the tegument of the developing cercariae was recognized, but not the acetabular glands. In contrast, when antisera against purified cercarial elastase from either S. mansoni or S. haematobium were tested with sections of infected Biomphalaria or Bulinus, fluorescence was strong in the preacetabular glands of the cercariae of either species, but undetectable with the tegument. Cross‐reactivity of both antisera extended to T. ocellata‐infected Lymnaea, but not to S. japonicum‐infected Oncomelania. In conclusion, although immunization with purified cercarial elastase results in antibody production, the enzyme does not induce an apparent antibody response following natural infection.

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.

Enzyme-linked immunosorbent assay with worm vomit and cercarial secretions of Schistosoma mansoni to detect infections in an endemic focus of Burkina Faso

Cercariae and adult Schistosoma mansoni were used to prepare, respectively, cercarial secretions (CS) and worm vomit (WoV). These were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to test the IgG-reactivity of sera obtained in an S. mansoni-endemic area of Burkina Faso. Among the egg-excreting individuals (n = 240), 94.6% reacted positively with WoV, but only 62.9% with CS, thus suggesting a high diagnostic sensitivity of WoV, but not of CS. Among those individuals without detectable eggs in two Kato-Katz thick smears from different stool specimens (n = 215), the respective percentages of positive IgG reactivity were 78.1% and 63.3%. These positive reactions in the absence of detectable eggs are interpreted in terms of limited sensitivity of parasitological stool examinations. Optical density values in ELISA with CS, but not with WoV, correlated negatively with age, which may reflect decreasing exposure to cercariae in older individuals.

Synthesis of new 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine derivatives with an incroporated thiazolidinone moiety and testing their possible serine protease and cercarial elastase inhibitory effects with a possible prospective to block penetration ofSchistosoma mansoni cercariae into the mice skin

Abstract5-Substituted 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine were synthesized by interaction of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonylhydrazide with some aldehydes to give the corresponding Schiff-bases, which after cyclization gave corresponding thiazolidinones. For some of the thiazolidinones, Mannich bases reaction was carried out. All the derivatives were tested for their possible inhibitory effect onSchistosoma mansoni cercarial elastase (CE). Only,N′-(4-methylbenzyledine)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonylhydrazide was found to have potent inhibitory effect on the CE activity with IC50=264 μM. Upon its use as a paint for mice tails before infection with S. mansoni cercariae, the compound formulated in jojoba oil caused a significant reduction (93%; P-value=0.0002) in the worm burden. IgG & IgM in mice sera were measured by using severalS. mansoni antigens by ELISA. Sera from treated infected mice (TIM) 2, 4, and 6 weeks (W) post infection (PI) showed 1.2 folds lower, 1.2 folds higher, 1.7 folds lower IgM reactivity against soluble cercarial antigenic preparation (CAP), respectively, when compared with sera collected from infected untreated mice (IUM). Sera from TIM 2, 4, and 6WPI showed 1.3, 1.6, and 1.7 folds higher IgG reactivity, respectively against CAP than the IgG reactivity from IUM. Sera from TIM 2, 4 and 6WPI showed 1.5, 1.2 folds lower and 1.4 folds higher IgM reactivity, respectively against soluble worm antigenic preparation (SWAP) when compared with sera collected from IUM. Sera from TIM 2, 4, and 6WPI showed 1.4, 1 folds lower and 1 fold higher IgG reactivity, respectivley to SWAP when compared with sera from IUM. Sera from TIM 2, 4, and 6WPI had generaly lower IgM and IgG reactivities against soluble egg antigen (SEA) when compared with sera from IUM.

Species-restricted antibody response against a DNA-construct coding for aspartic proteinase from Schistosoma japonicum

Abstract. DNA-based vaccine technology was used to immunize against the schistosome digestive enzyme, cathepsin D aspartic proteinase. The cDNA coding for Schistosoma japonicum aspartic proteinase was cloned in a mammalian expression vector under control of the CMV promoter/enhancer and expressed for the first time in transfected mammalian cells as well as in mice immunized – by means of intra-ear pinna injection – with the aspartic proteinase-encoding DNA construct. Mice developed antibodies which recognized the native protein in homogenates of S. japonicum worms and reacted with the gut and, to a much lesser degree, with the parenchyma of the parasites in cryostat sections. It was noteworthy that the vaccinated mouse sera did not detectably cross-react with S. mansoni antigens either in homogenates or on cryostat sections. By contrast, infection sera of mice or humans strongly cross-reacted with both schistosome species. We conclude that DNA vaccination can induce species-restricted antibody responses against schistosome proteins. The implications of this previously unrecognized specificity are discussed.

Antibodies induced in mice by a DNA-construct coding for the elastase of Schistosoma mansoni recognize the enzyme in secretions and preacetabular glands of cercariae.

A DNA-construct coding for the elastase of the parasite Schistosoma mansoni was prepared from adult S. mansoni worm RNA which was reverse transcribed into cDNA. The gene coding for the elastase was amplified using primers specific for the sequence of cercarial elastase and was cloned into a mammalian expression vector. Expression of the elastase gene at the transcriptional level was achieved for the first time in transfected mammalian cells (COS-7) and was also successful in muscle tissue of mice injected with the DNA-construct. These mice developed antibodies recognizing in Western blots the elastase from cercarial secretions. Also, these antibodies reacted in immunofluorescence tests with the preacetabular glands of cercariae, i.e. the site of origin for elastase. Thus, the DNA-construct induced the expression of elastase in mice and formation of antibodies that recognized the native antigen.

Immune responses in mice after immunization with antigens from different stages of the parasite Schistosoma mansoni.

Mice responses to immunization with Schistosoma mansoni antigens were investigated. Priming with cercarial antigen preparation (CAP) induced significant (P < 0.05) IgM, IgG, IgG2a, IgG2b, and IgA increases, while booster caused a significant IgG1 increase. A soluble worm antigen preparation (SWAP) caused significant IgG elevation. Priming with soluble egg antigen (SEA) caused significant IgM and IgG2a increases, while booster induced significant IgM, IgG and IgA increases. CAP-immunized mice sera (IMS) recognized CAP peptides ranging from 23 - 78 kDa. SWAP-IMS recognized SWAP peptides ranging from 40 - 75 kDa. SEA-IMS recognized SEA peptides ranging from 33 - 101 kDa. The cross-reactive peptides among the 3 antigens were identified. CAP caused significant increases in mesenteric lymph nodes (MLNs) CD4,8+, B lymphocytes, CD8+ thymocytes, CD4+ T and B splenocytes. SWAP priming caused significant increases in MLNs CD4,8+ thymocytes and B splenocytes. SWAP booster caused significant increases in MLNs CD8+ T and B lymphocytes, CD4,8+ thymocytes and CD4+ T and B splenocytes. SEA caused significant increase in CD4+ T cells.

Detection and characterization of hepatitis A virus circulating in Egypt

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

Early Lymphocyte Loss and Increased Granulocyte/Lymphocyte Ratio Predict Systemic Spread of Streptococcus pyogenes in a Mouse Model of Acute Skin Infection

Background: Group A streptococci may induce lymphopenia, but the value of lymphocyte loss as early biomarkers for systemic spread and severe infection has not been examined systematically. Methods: We evaluated peripheral blood cell indices as biomarkers for severity and spread of infection in a mouse model of Streptococcus pyogenes skin infection, using two isolates of greatly differing virulence. Internal organs were examined histologically. Results: After subcutaneous inoculation, strain AP1 disseminated rapidly to peripheral blood and internal organs, causing frank sepsis. In contrast, seeding of internal organs by 5448 was mild, this strain could not be isolated from blood, and infection remained mostly localized to skin. Histopathologic examination of liver revealed microvesicular fatty change (steatosis) in AP1 infection, and examination of spleen showed elevated apoptosis and blurring of the white pulp/red pulp border late (40 h post infection) in AP1 infection. Both strains caused profound lymphopenia, but lymphocyte loss was more rapid early in AP1 infection, and lymphocyte count at 6 h post infection was the most accurate early marker for AP1 infection (area under the receiver operator curve [AUC] = 0.93), followed by the granulocyte/lymphocyte ratio (AUC = 0.89). Conclusions: The results suggest that virulence of S. pyogenes correlates with the degree of early lymphopenia and underscore the value of peripheral blood indices to predict severity of bacterial infections in mice. Early lymphopenia and elevated granulocyte/lymphocyte ratio merit further investigation as biomarkers for systemic spread of S. pyogenes skin infections in humans and, possibly, related pyogenic streptococci in humans and animals.