Therapeutic Role of Bone Marrow-Derived Mesenchymal Stem Cells in Controlling Prognosis of Hepatocellular Carcinoma in a Murine Model.

OBJECTIVES: Hepatocellular carcinoma is the fourth leading cause of cancer deaths in the world. Conven - tional methods of cancer therapy are either invasive or have undesirable side effects. Therefore, exploring new therapeutic strategies to control the progression of hepatocellular carcinoma, such as cell-based therapies, is a key issue for prolonging patient survival. In this study, we aimed to evaluate tumor suppressive effects of mesenchymal stem cells on the in vivo pro - gression of hepatocellular carcinoma in murine model. MATERIALS AND METHODS: Hepatocellular carcinoma was induced in 40 rats with diethylnitrosamine. Rats were divided into 4 groups: 1 group injected with diethylnitrosamine only, 1 group injected with diethylnitrosamine and 1 dose of rat bone marrowderived mesenchymal stem cells, 1 group injected with diethylnitrosamine and 2 doses of rat bone marrowderived mesenchymal stem cells, and 1 group was injected with diethylnitrosamine and 3 doses of rat bone marrow-derived mesenchymal stem cells. Rats were killed after 1 month of dose 3. Liver specimens were histopathologically examined, and serum samples were examined for liver function and cytokines. RESULTS: Histopathological examination revealed that mesenchymal stem cell transplant induced liver regeneration. It also improved liver function as revealed by decreased levels of alanine and aspartate aminotransferase. Mesenchymal stem cells also repaired the immunopathology of the liver environment, as it decreased levels of interleukin 2 and 10, tumor necrosis factor α, and interferon γ. CONCLUSIONS: Mesenchymal stem cell infusion significantly enhanced hepatic structure and function of livers in a rat hepatocellular carcinoma model.

Toxicity, inflammatory and antioxidant genes expression, and physiological changes of green synthesis silver nanoparticles on Nile tilapia (Oreochromis niloticus) fingerlings.

The rapid increase of incorporating silver nanoparticles (Ag-NPs) in different anthropogenic and industrial activities increased the discharge of these particles in the aquatic ecosystem. The environmental impact of Ag-NPs, especially the green synthesized is still not completely understood on fish. Therefore, this study aimed to investigate the effects of exposure to graded series of starch-mediated Ag-NPs at levels of 0, 3.31, 6.63, 13.25, and 26.50 mg L-1 representing 0, 6.25, 12.5, 25, and 50% of LC50 on Nile tilapia (O. niloticus), respectively. Fish with initial weight 37.63 ± 0.41 g were maintained in 70 L glass aquaria and exposed to starch-mediated Ag-NPs (average particle size 40 nm) for 28 days. The results revealed that starch-mediated Ag-NPs induced severe changes in the mRNA levels of toxicity (CYP1A and Hsp70) and inflammatory (TNF-α and TGF-ß) genes. The expression of antioxidant genes (SOD and CAT) was significantly suppressed, and the activities of their enzymes were inhibited significantly upon exposure. Simultaneously, the malondialdehyde level increased significantly with increasing the exposure levels of starch-mediated Ag-NPs. The red blood cells, hemoglobin, hematocrit and white blood cell values were decreased significantly with doses over 3.31 mg L-1 of Ag-NPs. In addition, the total protein and globulin decreased significantly with increasing Ag-NPs in a dose-dependent manner. The liver function enzymes and kidney function indicators revealed severe toxicity with Ag-NPs exposure. In conclusion, the effect of starch-mediated Ag-NPs in doses over 3.31 mg L-1 induced obvious toxicity in the molecular and proteomic levels in Nile tilapia fingerlings.

Ascitic Fluid Cytokines in Chronic Liver Disease: A Possible Prognostic Tool.

INTRODUCTION: Malignant ascites results from imbalance between protein in the peritoneal cavity and absorption of fluids via the lymphatic system. More than 20 interleukins (ILs) are known to play an important role in the protection against tumors. MATERIALS AND METHODS: Ascitic fluid IL-1B, IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ levels were assessed in 45 patients with liver cirrhosis and ascites as judged by histopathological and ultrasonographic findings. They were divided into 2 groups according to presence of hepatic focal lesions. Ten patients with focal hepatic lesions were randomly selected and subjected to analysis of serum levels of IL-2 and IL-10. RESULTS: Ascitic fluid IL-4, IL-6 and IL-10 levels were found to be significantly higher in patients with hepatocellular carcinoma (HCC) than patients with cirrhosis. TNF-α, and IFN-γ were also found to be higher in HCC than patients with cirrhosis but with no significance. On the other hand, there was no significant difference in levels of IL-1B and IL-2 between the 2 groups. Ascitic fluid IL-2 and IL-10 levels were found to be higher in ascitic fluid than in serum of the same patients. CONCLUSION: Ascitic fluid levels of IL-4, IL-6 and IL-10 are higher in HCC patients than patients with cirrhosis alone. Levels of ascitic fluid IL-2 and IL-10 proved to be a better prognostic tool than their levels in sera of the same patients. To conclude, patients with cirrhosis may be subjected to scheduled examination of ascitic fluid cytokines to predict transformation into HCC.

Analogues of a Cyclic Antimicrobial Peptide with a Flexible Linker Show Promising Activity against Pseudomonas aeruginosa and Staphylococcus aureus

The emergence of multi-drug resistant bacteria is becoming a major health concern. New strategies to combat especially Gram-negative pathogens are urgently needed. Antimicrobial peptides (AMPs) found in all multicellular organisms act as a first line of defense in immunity. In recent years, AMPs have attracted increasing attention as potential antibiotics. Naturally occurring antimicrobial cyclic lipopeptides include colistin and daptomycin, both of which contain a flexible linker. We previously reported a cyclic AMP BSI-9 cyclo(Lys-Nal-Lys-Lys-Bip-O2Oc-Nal-Lys-Asn) containing a flexible linker, with a broad spectrum of activity against bacterial strains and low hemolytic activity. In this study, improvement of the antimicrobial activity of BSI-9, against the European Committee on Antimicrobial Susceptibility Testing (EUCAST) strains of S. aureus, E. coli, A. baumannii, and P. aeruginosa was examined. This led to synthesis of eighteen peptide analogues of BSI-9, produced in four individual stages, with a different focus in each stage; cyclization point, hydrophobicity, cationic side-chain length, and combinations of the last two. Specifically the modified compound 11, exhibited improved activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC of 4 µg/mL and 8 µg/mL, respectively, compared to the original BSI-9, which had an MIC of 16-32 µg/mL.

Sleep disordered breathing induced by cervical spinal cord injury and effect of adenosine A1 receptors modulation in rats.

Sleep-disordered breathing (SDB) is very common after spinal cord injury (SCI). The present study was designed to evaluate the therapeutic efficacy of adenosine A1 receptor blockade (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on SDB in a rodent model of SCI. We hypothesized that SCI induced via left hemisection of the second cervical segment (C2Hx) results in SDB. We further hypothesized that blockade of adenosine A1 receptors following C2Hx would reduce the severity of SDB. In the first experiment, adult male rats underwent left C2Hx or sham (laminectomy) surgery. Unrestrained whole body plethysmography (WBP) and implanted wireless electroencephalogram (EEG) were used for assessment of breathing during spontaneous sleep and for the scoring of respiratory events at the acute (~1 wk), and chronic (~6 wk) time points following C2Hx. During the second experiment, the effect of oral administration of adenosine A1 receptor antagonist (DPCPX, 3 times a day for 4 days) on SCI induced SDB was assessed. C2Hx animals exhibited a higher apnea-hypopnea index (AHI) compared with the sham group, respectively (35.5 ± 12.6 vs. 19.1 ± 2.1 events/h, P < 0.001). AHI was elevated 6 wk following C2Hx (week 6, 32.0 ± 5.0 vs. week 1, 42.6 ± 11.8 events/h, respectively, P = 0.12). In contrast to placebo, oral administration of DPCPX significantly decreased AHI 4 days after the treatment (159.8 ± 26.7 vs. 69.5 ± 8.9%, P < 0.05). Cervical SCI is associated with the development of SDB in spontaneously breathing rats. Adenosine A1 blockade can serve as a therapeutic target for SDB induced by SCI.NEW & NOTEWORTHY The two key novel findings of our study included that 1) induced cervical spinal cord injury results in sleep-disordered breathing in adult rats, and 2) oral therapy with an adenosine A1 receptor blockade using DPCPX is sufficient to significantly reduce apnea-hypopnea index following induced cervical spinal cord injury.

Cord blood-derived mesenchymal stem cells with hepatogenic differentiation potential ameliorate chronic liver affection in experimental models.

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.

Ameliorative effect of bone marrow-derived stem cells on injured liver of mice infected with Schistosoma mansoni.

The technique of stem cells or hepatocytes transplantation has recently improved in order to bridge the time before whole-organ liver transplantation. In the present study, unfractionated bone marrow stem cells (BMSCs) were harvested from the tibial and femoral marrow compartments of male mice, which were cultured in Dulbecco's modified Eagle's medium (DMEM) with and without hepatocyte growth factor (HGF), and then transplanted into Schistosoma mansoni-infected female mice on their 8th week post-infection. Mice were sacrificed monthly until the third month of bone marrow transplantation, serum was collected, and albumin concentration, ALT, AST, and alkaline phosphatase (ALP) activities were assayed. On the other hand, immunohistopathological and immunohistochemical changes of granuloma size and number, collagen content, and cells expressing OV-6 were detected for identification of liver fibrosis. BMSCs were shown to differentiate into hepatocyte-like cells. Serum ALT, AST, and ALP were markedly reduced in the group of mice treated with BMSCs than in the untreated control group. Also, granuloma showed a marked decrease in size and number as compared to the BMSCs untreated group. Collagen content showed marked decrease after the third month of treatment with BMSCs. On the other hand, the expression of OV-6 increased detecting the presence of newly formed hepatocytes after BMSCs treatment. BMSCs with or without HGF infusion significantly enhanced hepatic regeneration in S. mansoni-induced fibrotic liver model and have pathologic and immunohistopathologic therapeutic effects. Also, this new therapeutic trend could generate new hepatocytes to improve the overall liver functions.

Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool.

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 IgG(2a) 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.

CD133(+) human umbilical cord blood stem cells enhance angiogenesis in experimental chronic hepatic fibrosis.

The in vivo angiogenic potential of transplanted human umbilical cord blood (UCB) CD133(+) stem cells in experimental chronic hepatic fibrosis induced by murine schistosomiasis was studied. Enriched cord blood-derived CD133(+) cells were cultured in primary medium for 3 weeks. Twenty-two weeks post-Schistosomiasis infection in mice, after reaching the chronic hepatic fibrotic stage, transplantation of stem cells was performed and mice were sacrificed 3 weeks later. Histopathology and electron microscopy showed an increase in newly formed blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand factor. Few hepatocyte-like polygonal cells showed positive expression of human vascular endothelial growth factor and inducible nitric oxide synthase. The transplanted CD133(+) human stem cells primarily enhanced hepatic angiogenesis and neovascularization and contributed to repair in a paracrine manner by creating a permissive environment that enabled proliferation and survival of damaged cells rather than by direct differentiation to hepatocytes. A dual advantage of CD133(+) cell therapy in hepatic disease is suggested based on its capability of hematopoietic and endothelial differentiation.

Does granulocyte-colony stimulating factor administration induce damage or repair response in schistosomiasis?

AIM: To introduce Granulocyte-colony stimulating factor (G-CSF) as a new therapeutic modality for schistosomiasis through stem cell mobilization, immunomodulation or fibrosis remodeling. METHODS: In this study, a 5 d course of human recombinant G-CSF (100 µg/kg sc) was applied to Schistosoma mansoni-infected mice at different stages of disease (5 d before infection as well as 3, 5 and 7 wk post-infection). The animals were sacrificed at 10 d as well as 4, 6 and 8 wk post infection. Mice were examined for: (1) Total leukocyte count which is an accepted surrogate marker for the stem cell mobilization into the circulation; (2) Egg count in intestine and liver tissue to assess the parasitic load; and (3) Histopathological changes in Hx/E and Masson trichrome stained sections as well as collagen content in Sirius red-stained liver sections to determine the severity of liver fibrosis. RESULTS: Mice developed leukocytosis. The egg load and the number of granulomas were not affected by the G-CSF treatment but there was an obvious change in the composition of granulomas towards an increased cellularity. Moreover, fibrosis was significantly decreased in treated groups compared to untreated animals (collagen content either preinfection or at 3 and 5 wk post infection: 5.8 ± 0.5, 4.7 ± 0.5, 4.0 ± 0.7 vs 8.2 ± 0.9; P ≤ 0.01). CONCLUSION: Although G-CSF did not cause direct elimination of the parasite, it enhanced granulomatous reaction and reduced the fibrosis. Further investigation of the underlying mechanisms of these two actions is warranted.