Enhancing ex vivo expansion of cord blood-derived unrestricted somatic stem cells for clinical applications.

OBJECTIVES: To study effects of serum-containing medium (SCM) versus serum-free medium (SFM) and influence of seeding density, on rate of expansion of cord blood (CB) unrestricted somatic stem cells (USSCs), as a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. MATERIAL AND METHODS: Isolation, propagation and characterization of USSCs from CB samples were performed and followed by their passage 3 culture in SCM and SFM, at cell densities of 5, 50, 500 and 5000 cells/cm(2) . RESULTS: The cells were CD44(+) , CD90(+) , CD73(+) , CD105(+) , CD34(-) , CD45(-) , and HLA-DR, with Oct4 & Sox2 gene expression; they were differentiated into osteoblasts and adipocytes. USSCs cultured in SCM had significantly higher population doubling levels (P < 0.01) than those cultured in SFM. Those cultured in SCM at 5 cells/cm(2) and those cultured in SFM at 50 cells/cm(2) had significantly higher population doubling (P < 0.01) levels than those cultured at higher cell densities. CONCLUSIONS: For scaling up of USSCs from 106 (?) to 1012 (?) in 6 weeks, culturing of CB-derived cells of early passage (≤P3) in SCM at low cell seeding density (5 cells/cm(2) ) is suggested for increasing cell count with lower passaging frequency, followed by culture of expanded USSCs at 50 cells/cm(2) in SFM, to avoid undesirable effects of bovine serum in clinical applications.

Human schistosomiasis haematobium: effective diagnosis of active infection using a pair of monoclonal antibodies against soluble egg antigen.

The present study was designed to prepare monoclonal antibodies (MAbs) against Schistosoma haematobium soluble egg antigen (SEA) with immunodiagnostic potential for urinary schistosomiasis. From a panel of MAbs, a pair of IgG1 MAbs (2D/11C and 10B/2C) specific for S. haematobium SEA was selected. Both MAbs recognized one band with a 42-kDa molecular weight by western blots. The pair of MAbs was employed in sandwich ELISA for the detection of circulating schistosome antigen (CSA), one as antigen-capturing antibody and the other as peroxidase-conjugated antigen-detecting antibody. The lower detection limit of the assay was 1 ng/ml of S. haematobium SEA. The assay was performed on sera of 65 S. haematobium-infected patients, 25 patients infected with other parasites (Fasciola hepatica, Echinococcus granulosus), and 20 noninfected individuals. CSA was demonstrated in 89% of the S. haematobium-infected group. However, CSA was negative in the sera of healthy individuals and patients infected with other parasites, giving an overall specificity of 100% for the CSA assay. A positive correlation (r=0.37, p<0.01) was detected between the number of S. haematobium eggs excreted in 10 ml urine and the CSA level detected in the sera of S. haematobium-infected patients. Our data show that the use of anti-S. haematobium MAbs for the detection of CSA provides a sensitive and specific method for the immunodiagnosis of active S. haematobium-infected patients. Moreover, CSA assay using this anti-S. haematobium MAb/ELISA system was proven to correlate with intensity of infection and hence morbidity assessment.

Stability and reproductive fitness of Schistosoma mansoni isolates with decreased sensitivity to praziquantel.

These studies are focused on schistosomes derived from human infections not cured by three successive doses of praziquantel that also produced infections in mice that were significantly more difficult to cure than infections with control worms. Half (three of six) of these isolates retained their decreased response to praziquantel after multiple passages through the life-cycle in the absence of therapeutic pressure. Two of the isolates, including the one initially least sensitive to praziquantel; reverted, to a sensitivity not significantly different from controls. For example, the EE6 isolate initially required 680 mg/kg praziquantel to affect a 50% reduction in worm load in murine infections, but after only six passages through the life cycle over 5 years this was reduced to 113 mg/kg, not different from control infections. The stability of some of the isolates and the reversion of others indicates that the biological or genetic factors conferring decreased praziquantel response varies among the isolates. The three isolates that retained decreased sensitivity to praziquantel all showed compromises in reproductive fitness in the laboratory, expressed most frequently as a decreased cercarial production from snails infected with those isolates compared to controls. For example, the total cercarial production of snails infected with the EE10 isolate was only 57% that of controls. The reversion of some of the isolates to a praziquantel sensitive state and the decreased reproductive fitness of those that did not revert suggest that there is some biological cost associated with the relative praziquantel insensitivity of these worms, which could help limit the impact of such isolates in the field. Infections with the less sensitive isolates also produced significantly less circulating schistosomal antigen in mice, suggesting that a decrease in the host immune response elicited by these worms could be one of the factors contributing to the diminished praziquantel efficacy.

Correlation between infection intensity, serum immunoglobulin profile, cellular immunity and the efficacy of treatment with praziquantel in murine schistosomiasis mansoni.

This study was conducted to evaluate the efficacy of praziquantel (CAS 55268-74-1, EMBAY 8440, Biltricide) in different grades of Schistosoma mansoni infection. Moreover, the relationship between the post treatment worm burden, hepatic granuloma volume, and serum immunoglobin profile was also investigated. Four groups of Swiss albino mice infected with Schistosoma mansoni cercariae were used: Highly infected untreated control mice (infected with 120 Schistosoma mansoni cercariae) and their corresponding praziquantel treated group. Lightly infected untreated control mice (infected with 60 Schistosoma mansoni cercariae) and their corresponding praziquantel treated group. Praziquantel was given seven weeks post infection in a dose of 500 mg/kg body weight for two consecutive days. Animals were sacrificed two weeks post treatment. Praziquantel achieved better cure rates in mice with heavy infection than in less intensely infected animals. The drug reduced the hepatic granuloma in animals with light intensity infection. This reduction was more accentuated in highly infected animals. The serum immunoglobulin profile (immunoglobulin G and immunoglobulin M) showed a higher level in highly infected treated mice (1.2 +/- 0.6 optical density unit and 1.1 +/- 0.5 optical density unit, respectively) and was reduced in animals with low intensity infection (1.18 +/- 0.6 optical density unit and 0.7 +/- 0.6 optical density unit, respectively). This study may be of value in tropical regions, where schistosomiasis with low worm burden is a common occurrence.

A monoclonal antibody against Schistosoma haematobium soluble egg antigen: efficacy for diagnosis and monitoring of cure of S. haematobium infection.

A monoclonal antibody (mAb), 2F/11F, raised against Schistosoma haematobium soluble egg antigen (SEA) was found to be nonreactive with S. mansoni SEA or other parasite antigens (Fasciola hepatica, Echinococcus granulosus). This IgG1 mAb recognized a repetitive epitope on S. haematobium SEA in the molecular-weight regions of 70, 42, and 35 kDa. It was employed as both an antigen-capture and a biotinylated detection antibody in a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of circulating schistosome antigen (CSA) and had a detection limit of <1 ng S. haematobium SEA/ml. CSA levels were measured in serum and urine samples from 116 S. haematobium-infected rural students before therapy and at 4, 8, and 12 weeks after praziquantel treatment. Serum and urine samples from 50 S. mansoni -infected patients, 15 patients harboring other parasites, and 30 noninfected individuals were also assessed. CSA was detected in 90.5% of serum samples and 94% of urine samples from S. haematobium-infected patients. CSA was undetectable in serum from the 15 patients harboring other parasites and in 94% of serum samples and 84% of urine samples from S. mansoni-infected patients. In the S. haematobium-infected group a positive correlation was detected between CSA levels in serum and urine samples and the egg load per 10 ml urine. A significant reduction in CSA levels was detected in serum and urine samples after praziquantel therapy. CSA was undetectable in 87% of serum samples and 81.5% of urine samples from schistosomiasis haematobium patients at 12 weeks post-treatment. These data demonstrate that the use of mAb 2F/11F for detection of CSA provides a sensitive method for the immunodiagnosis and monitoring of cure of schistosomiasis haematobium.

Effect of early treatment with praziquantel on serum connective tissue metabolite markers in children and adolescents with intestinal schistosomiasis mansoni.

This work was designed to assess the reflection of early treatment by praziquantel (CAS 55268-74-1, EMBAY 8440, Biltricide) on serum connective tissue metabolite markers (hyaluronic acid and procollagen III peptide) in patients with active intestinal schistosomiasis. Children and adolescent subjects from primary and secondary schools in an endemic area of schistosomiasis mansoni were included. Age-matched subjects from an urban area served as normal controls. All subjects were examined clinically and parasitologically. Detection of hepatitis B seromarkers was also done. The infected subjects were treated with praziquantel at a dose of 60 mg/kg of body weight which was repeated after 4 weeks. Serum hyaluronic acid and procollagen III peptide were measured by radioimmunoassay. High hyaluronic acid was encountered in infected subjects when compared to their respective age-matched controls. Significant decrease of 4 and 8 weeks post-treatment was noted when compared to ist level before treatment. There was no significant change in serum procollagen III peptide on comparing infected subjects to their controls, whereas a significant increase was observed in its level after 4 and 8 weeks post-treatment compared to that before treatment. This work suggests that early treatment of intestinal schistosomiasis with specific chemotherapy (praziquantel) decreases serum hyaluronic acid and increases procollagen III peptide probably via downregulation of granulomatous inflammatory cell reaction and activation of collagenase enzymes, respectively.

Detection of circulating schistosome antigens in serum and urine of schistosomiasis patients and assessment of cure by a monoclonal antibody.

From a panel of monoclonal antibodies (MAb), an IgM monoclonal antibody (7F1/6B) reactive with repetitive epitopes on S. mansoni soluble egg antigen was selected. This MAb was employed both as antigen capture and detection antibody in a sandwich ELISA and had a detection limit < 1 ng S. mansoni SEA/mi. Serum and urine samples were collected from rural students who had S. mansoni (169 subjects) or mixed S. mansoni and S. haematobium (64 subjects) infections. Samples were collected before and at 4, 8 and 12 weeks after praziquantel therapy. Circulating schistosome antigens (CSA) were demonstrated in 90% of sera and 97% of urine samples of S. mansoni group and in 91% of sera and 100% of urine samples of mixed infection group. All sera from 29 uninfected individuals, 30 patients with other parasites and 70% of 55 S. haematobium-infected subjects were negative in this assay. CSA level in serum and urine samples correlated positively with the number of S. mansoni eggs/g stool in both groups. A significant reduction in CSA level was observed in serum and urine samples after praziquantel therapy. By 12 weeks post-treatment, negativity was 98% in sera and 97% in urine of S. mansoni-infected group and 98% in sera and 91% in urine of mixed infection group. The data demonstrate that the use of MAb 7F1/6B for the detection of CSA provides a sensitive method for immunodiagnosis of schistosomiasis and monitoring of cure.

Evaluation of specific Fasciola antigen in the immunodiagnosis of human fascioliasis in Egypt.

Enzyme-linked immunosorbent assay (ELISA) and enzyme linked immunoelectrotransfer blot technique (EITB) were employed for the detection of circulating Fasciola antibodies in infected human sera using a specific Fasciola antigen, prepared by immunoaffinity purification of homogenates of Fasciola hepatica adult worms. Ninety two individuals diagnosed clinically and parasitologically were classified into: Fascioliasis group (21 patients), schistosomiasis group (21 patients) and subjects harbouring other parasitic infections (50 patients). Eighteen healthy individuals served as normal controls. ELISA was 100% sensitive and 93% specific with 96.5% diagnostic efficacy, whereas EITB was 100% sensitive and specific with 100% diagnostic efficacy. Our data revealed that ELISA can be used as a good screening test while EITB can serve as a confirmatory test for immunodiagnosis of fascioliasis.

Purification and characterization of a specific Fasciola antigen.

Specific Fasciola antigen was prepared from homogenates of Fasciola hepatica adult worms. The homogenate was ultracentrifuged and the supernatant containing crude Fasciola antigen was then passed over a cyanogen bromide activated sepharose 4B column coupled with antiserum against Schistosoma mansoni adult worm surface antigen. The specific, Schistosoma-free Fasciola antigen was tested for its specificity by immunodiffusion. Characterization of the specific Fasciola antigen was done by gradient poly-acrylamide gel electrophoresis and immunoblotting technique. The electrophoresis migration pattern of specific Fasciola antigen, stained with Coomassie blue, showed 7 bands in the 12-54 kDa regions. Using the immunoblotting technique, a batch of positive fascioliasis sera recognized two specific bands at the 33 and 54 kDa regions.