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Objective to assess the production of Anti-Schistosoma Monoclonal Antibodies by growing IgM producing hybridoma cell line in serum low medium and to evaluate their diagnostic potential to replace the old conventional method in animal ascetic fluid. Methods Highly reactive and specific IgM MAb secreting hybridoma cell line to S. mansoni SEA was cultured in three different media; serum rich replacement medium (conventional), serum low replacement medium (SLRM) and serum free replacement medium (SFRM). Results Cell count and viability were better in conventional medium and SLRM than SFRM. The lower detection limit of the assay Mab of SLRM culture was 1.1 ng/ml of S. mansoni SEA. SFRM was excluded because of the low concentration and reactivity. MAb was tested against 34 human samples. Sensitivity and specificity of the assay for each were 92% and specificity 90% for SLRM respectively. The diagnostic efficacy of the assay was 94%. Conclusions the production and diagnostic efficacy of antischistosomal MAbs was comparable on usage of either in vitro cell culture supernate in SLRM or conventional media.

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. MSCs were generated from umbilical cord blood. Hepatogenic differentiation was induced on two-dimensional (2D) and three-dimensional (3D) culture system and characterized by morphology, scanning electron microscopy, immunocytochemistry, and gene expression. Albumin and α-1 antitrypsin (AAT) in culture supernatants were measured. Differentiated cells were administered intravenous into a murine model of carbon tetra induced liver cirrhosis. After 12 weeks of injection, liver pathology was examined. The hepatogenic differentiated MSCs stained positively for albumin, alpha fetoprotein, HepPar1, cytokeratin 7 and 18, and OV6 with more mature cells, hexagonal in shape with central nuclei forming large sheets in groups in 3D culture system. AAT secretion and indocyanine green uptake were significantly increased in 3D system. In experimental model, MSC-3D treated group exhibited maximal restoration of liver architecture with absent septal fibrosis and marked improvement of alanine transaminase (ALT) and aspartate transaminase (AST), and mild increase in albumin. Both 3D and 2D culture system are effective in functional hepatogenic differentiation from MSCs and serve as a vehicle in liver tissue engineering. In vivo hepatogenic differentiation is more effective on 3D scaffold, with better functional recovery.

This study was performed to evaluate the effect of two commercially available serum-free culture media; serum free medium (SFM) and chemically defined medium (CDM), on the growth rate, antibody productivity and post adaptation cryopreservation and revival reactivity of hybridoma cells compared to the conventional serum based medium (SBM). In addition, the diagnostic efficacy of MAbs secreted in each culture medium was evaluated by testing their performance in sandwich ELISA for antigen detection. Anti- Schistosoma mansoni soluble egg antigen hybridoma cell line (7A/8F) secreting previously characterized IgG Kappa mAbs, were retrieved and propagated in each of the three aforementioned media. Growth rate and viability were assessed post culturing in each media. The data collected from this study indicated that MAbs secreted from hybridoma cells cultured in SFM were the most abundant, easiest to purify, and the most effective in antigen detection by sandwich ELISA, in comparison to those produced in the other two media. Moreover, combination of fresh and conditioned medium with DMSO 7.5% was the most promising formulation for the cryopreservation of hybridoma cells cultivated in serum independent media (SFM or CDM).

Objective: To develop a new sandwich based lateral flow immunochromatographic strip for rapid detection of circulating Schistosoma mansoni antigen in serum and urine samples of patients with active schistosomiasis. Methods: This lateral flow immunochromatographic strip was prepared by using anti-Schistosoma mansoni soluble egg antigen monoclonal antibody conjugated gold nanoparticles (MAb-AuNPs) as antigen-detecting antibody, while crystalline material (MCM)-41-MAb bioconjugate was immobilized at the test line as antigen-capturing antibody. Both antigen capturing and detecting antibodies formed sandwich complexes with circulating Schistosoma mansoni antigen in the positive samples. Sandwich complexes immobilized at the test line gave distinct red color. The assay reliability was examined by using urine and serum samples of 60 Schistosoma mansoni infected patients, 20 patients infected with parasites other than Schistosoma, and 20 healthy individuals as negative controls. Results were compared with those obtained via sandwich enzyme linked immunosorbent assay (ELISA). Results: The detection limit of circulating Schistosoma mansoni antigen by lateral flow immunochromatographic strip was lower (3 ng/mL) than the detection limit by ELISA (30 ng/mL). The sensitivity and specificity of lateral flow immunochromatographic strip in urine samples were 98.3% and 97.5%, respectively compared to 93.5% and 90.0% by ELISA. In serum samples, they were 100.0% and 97.5%, respectively compared to 97.0% and 95.0% by ELISA. The strip test took approximately 10 min to complete. Conclusions: This new lateral flow immunochromatographic strip offers a sensitive, rapid, and field applicable technique for diagnosis of active schistosomiasis.

Few studies reported the antifibrotic effects of gallic acid (GA) despite its known hepatoprotective and antioxidant activities. Accordingly, this study investigated the antifibrotic effects of GA through clarifying its mechanisms on hepatic stellate cells' (HSCs) activation, proliferation and/or apoptosis. In vitro effects of GA on HSC-T6 activation/proliferation, morphology and safety on hepatocytes were assessed. In vivo, hepatic fibrosis was induced via chronic thioacetamide (TAA)-intoxication. TAA-intoxicated rats were treated with silyamrin or GA. At end of experiment, liver functions, hepatic MDA, GSH, PDGF-BB, TGF-β1, TIMP-1 and hydroxyproline were determined. Histological analysis and Sirius red staining of hepatic sections, expressions of alpha-smooth muscle actin (α-SMA), proliferating cellular nuclear antigen (PCNA) and caspase-3 were examined. In vitro, GA resulted in a concentration and time-dependent inhibition in HSCs activation, proliferation (IC50= 45 and 19 μg/mL at 24 and 48 h respectively); restored the quiescent morphology of some activated HSCs plus its safety on hepatocytes. In vivo, GA reduced ALT, AST, MDA, PDGF-BB levels, collagen deposition and fibrosis score (S1 vs S4); increased caspase-3 expression and restored GSH stores, TGF-β1 level, α-SMA and PCNA expressions. In conclusion, GA counteracted the progression of hepatic fibrosis through reduction of HSCs proliferation/activation mutually with their apoptosis induction., Graphical abstract Image 1

Heterogeneity of Mesenchymal stem cells (MSCs) imposes limitations for their in vitro expansion and accounts for the lack of reproducibility in some clinical studies. So, this study was designed to isolate and enrich clones of multipotent and self-renewing MSCs from cord blood (CB). Enriched clones with higher proliferation and differentiation potential provide regenerative cells suitable for various clinical demands. MSCA and MSCB original (progenitor) cells were isolated from CB samples, and single cells were cloned by limiting dilution method, in mouse embryonic fibroblast conditioned media. Original MSCs and their single-cell derived clones were characterized by identifying their proliferation rate, immunophenotyping of surface antigens, expression of pluripotency and proliferation genes (Oct4, Sox2, Nanog, KLF4, c-Myc, and PDGFRA), and differentiation potential into multiple lineages (osteogenic, adipogenic, and chondrogenic). Some single-cell clones of MSCA showed a higher proliferation rate and greater differentiation potential than their original cells. However, original MSCB cells were of greater proliferation and differentiation potential than their derived single-cell clones, except for one clone which had comparable results. Cloning of MSCs was attainable when cultured in mouse embryonic fibroblast conditioned media. Single clones with higher proliferation and differentiation potential than their original progenitor cells were obtained by cloning of poorly functioning MSCs progenitor cells, enabling the selection of more therapeutically efficacious MSCs with better performance in clinical applications. Moreover, this study draws attention to the importance of CD105 as a possible MSCs biomarker associated with the multilineage commitment of MSCs.

Objectives Liver transplantation is the well-known treatment for chronic liver diseases; however, postoperative complications and lack of donors continue to be limitations with this treatment. Investigating new modalities for treatment of chronic liver illness is a must. In the present study, we aimed to clarify the effects of an in vitro hepatocyte-differentiated human unrestricted somatic stem cell transplant as a new cell-based therapy in an experimental model of chronic liver failure. Materials and methods Human umbilical cord blood-derived unrestricted somatic stem cells were isolated, cultured, propagated, and characterized. Cells were directed to differentiate into hepatocyte-like cells. An animal model of carbon tetrachloride cirrhotic liver failure was prepared, and the human in vitro differentiated unrestricted somatic stem cells were transplanted into the experimental model. Animals that did not receive transplant served as the pathologic control group. Animals were euthanized 12 weeks after transplant, and liver functions and histopathology were assessed. Results Compared with the pathologic control group, the transplant group showed improvements in levels of alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin. Histopathologic examination of the transplant group also showed improvements in hydropic degeneration and fibrosis. Conclusions The use of unrestricted somatic stem cells, isolated and propagated from cord blood and then differentiated into hepatocyte-like cells, improved both fibrosis and normal function of cirrhotic livers. These cells could be considered as a line of cell-based therapy in cases of chronic liver disease.

BACKGROUND The liver is one of the major target organs for which cell-based therapies are very promising. The limitations of various cellular therapies, including bone marrow (BM)-derived mesenchymal stem cells (MSCs), urges the exploration of stem cell sources more suitable for transplantation. Human umbilical cord blood (HUCB) can overcome these drawbacks with a favorable reparative outcome. OBJECTIVES The aim of this study was to evaluate the therapeutic potential of MSCs in 2 groups of chronic liver injury experimental models. MATERIAL AND METHODS Propagation and characterization of MSCs isolated from cord blood (CB) samples were performed and differentiation into osteogenic, adipogenic and hepatogenic lineages was induced. The 1st experimental model group (80 mice) included a negative control, a pathological control and 60 mice infected with Schistosoma mansoni (S. mansoni) and transplanted with MSCs. The 2nd experimental model group (30 hamsters) included 10 healthy hamsters serving as a negative control and 20 hamsters injected with repeated doses of carbon tetrachloride (CCl4) to induce liver fibrosis; 10 of them were treated with an intrahepatic (IH) injection of 3 × 106 MSCs and the other 10 were untreated pathological controls. Mice and hamsters were sacrificed 12 weeks post-transplantation and their liver sections were stained immunohistochemically for the detection of human hepatocyte-like cells. Moreover, the sections were examined for the levels of fibrosis. RESULTS In both models, the transplantation of CB-derived MSCs (CB-MSCs) resulted in the engraftment of the fibrotic livers with newly formed hepatocytes, as evidenced by positive immunohistochemistry staining with human Hepatocyte Paraffin 1 (Hep Par 1), alpha-fenoprotein (AFP), cytokeratin 18 (CK18), cytokeratin 7 (CK7), and OV6 monoclonal antibody. The transplanted liver sections showed markedly reduced hepatic fibrosis with a significantly lower fibrotic index, as well as significantly improved liver functions compared to the pathological control (p < 0.001). CONCLUSIONS This data provides hope that human CB-MSCs can be utilized as multipotent stem cells with unlimited potentiality in regenerative medicine and supports the concept of cellular therapy for the cure of hepatic fibrosis.

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure.Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed.Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group.Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

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/cm2. 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

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.

The ease of culture expansion of unrestricted somatic stem cells (USSCs) represents one of their primary advantages in clinical strategies. However, genetic alterations during culture expansion undoubtedly affect their therapeutic potential. Telomere shortening with aging is another factor that leads to aberrant stem cell functioning, interfering with potential therapeutic designs. This study evaluates the effect of cell density versus passaging number on the proliferation rate of cord blood (CB)-USSCs, reflected on the telomere length, pluripotent transcription factors expression, and differentiation potential. Methodology: CB-USSCs were cultured at seeding densities of 5000, 500, 50, 5 cells/cm2. Cells from different passages of each seeding density were subjected to pluripotency genes (Oct4, Sox2, Nanog, klf4, c-Myc) and PDGFRa gene expression analysis, measurement of absolute telomere length by real-time PCR, and induction of differentiation into osteogenic, adipogenic, and chondrogenic lineages. Proliferation rate was expressed as population doubling (PD) and cumulative PD (CPD). Results: USSCs from earlier passages (P7) cultured at 5000 cells/cm2 showed the highest telomere length with high expression of pluripotency, and proliferation genes which decreased gradually with passaging till reaching their lowest level at P11 Moreover, their PD at P7 was 3 and CPD (P7-P11) was 12.8. USSCs cultured at 5 cells/cm2 showed PD 12.9 at P7, with a higher expression of gene that plays an important role in proliferation (PDGFRa) than that of 5000 cells/cm2 at P7. Differentiation potentiality of 5000 cells/ cm2 at P7 was high with loss of differentiation at P11, while differentiation potentiality of 5 cells/ cm2 at P7 was much lower than that of 5000 cells/ cm2 at same passage. Conclusion: Taken together, the above results suggest the use of USSCs at earlier passages of 5 cells/cm2 cultures if a high expansion rate of CB-USSCs is required in therapeutic strategies, while USSCs culture at 5000 cells/cm2 is the best protocol if the therapeutic target is induction of USSCs differentiation.

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

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

Background This research was carried out to develop a reliable monoclonal antibody (MoAb)-based sandwich enzyme linked immunosorbent assay (ELISA) for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags), a pair (12B/11D/3F and 10A/9D/10G) was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively). Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

In this case report we describe a case of mediastinal ganglioneuroblastoma-secreting vasoactive intestinal peptide (VIP), causing secretory diarrhoea in an 18-month-old child. An 18-month-old girl presented with a 2-month history of diarrhoea, abdominal distension and weight loss. Investigations revealed secretory diarrhoea with hypokalaemia, hyponatraemia and hypochloraemia and metabolic acidosis. Her stool output was 2.5–3.l day −1 with increased stool sodium. VIP levels were strikingly high with normal glucagon and gastrin levels. X-ray of the chest revealed a well-defined mass in the right upper zone with tracheal shift, which was confirmed with computed tomography (CT) of the chest. The mass was resected and the patient became asymptomatic. This case shows that secretory diarrhoea caused by VIP and produced by ganglioneuroblastoma indicates a favourable prognosis, provided it is resectable.

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

OL-021 Echinococcosis of children in Bosnia and Herzegovina A. Bajraktarevic1 *, S. Trhulj Putica1, S. Trninic1, A. Skopljak1, N. Dizdarevic Kreso1, B. Djukic2, A. Hadzimuratovic Jr.3, E. Mujicic Selimovic4, A. Drnda5, Z. Jatic6, A. Hadzimuratovic7, J. Gutic8, M. Ridzal8. 1Public Health Institution of Health Center Sarajevo Pediatrics Department, 2First Medical Aid New Sarajevo, Pediatrics Department, 3Pediatrics Clinic Sarajevo, 4Clinical Medical Center Sarajevo, 5Infective Clinic Sarajevo, 6Medical Faculty of Sarajevo, 7Children Surgery Sarajevo, 8General Hospital Sarajevo, Bosnia and Herzegovina