Effects of Different Cold Preservation Solutions on the Functions of Cultured Isolated Human Hepatocytes.

BACKGROUND: Hepatocyte transplantation using isolated human hepatocytes is an alternative source that can be used for the treatment of metabolic diseases and acute liver failure as a time bridge to liver transplantation. These cells can also be used for bioartificial liver systems and in vitro study of drug toxicity. OBJECTIVE: To determine which cold preservation solution is better maintain the liver function. METHODS: We prepared 4 cold preservation solutions made of different combination of antioxidants, chelating, membrane protective, and anti-apoptotic agents as well as inhibitor of cyclophilin D. For hepatocyte isolation, we used livers obtained from unused deceased donor livers and the liver of patients with Crigler-Najjar syndrome who were candidates of partial liver transplantation. After culture and cold preservation, the level of albumin, and urea production were measured as indices of liver functionality. RESULTS: We found that albumin production significantly decreased after cold preservation in solution 1. There was no significant difference in urea production after cold preservation in solution 1 compared with control 24 h. No significant differences in albumin production were found after cold storage in solution 2 and solution 4 compared with control 24 h. Urea production significantly decreased after cold storage in solutions 2 and 4 compared with control 24 h. As a whole albumin and urea production were significantly decreased after cold preservation. Although albumin and urea production were decreased after cold preservation, but the results of albumin production of two solutions were not significantly different from that of the control group (p=0.109 and 0.951). CONCLUSION: Cold preservation of cultured human hepatocytes in solution 2 and solution 4 could maintain the function of albumin production better than other cold preservation solutions in our experiments; solution 1 was more effective on urea production of cultured human hepatocytes at 4 °C for 24 h. To determine if these hepatocytes are suitable candidates for transplantation, further studies should be performed.

The Effects of Cold Preservation Solutions Supplemented with UDCA and α-Lipoic Acid on the Viability and Function of Isolated Human Hepatocytes.

BACKGROUND: Liver transplantation is the only treatment for end-stage and genetic liver diseases. The main burden of this treatment is the shortage of both living and cadaveric liver donors. An alternative treatment is using liver cell transplantation, which can be obtained from unused livers for transplantation. These hepatocytes should be kept ready in viable and functional situation in a frozen state to be instantly used when they would be needed. In our previous experience, we had isolated hepatocytes from unused livers. OBJECTIVE: To find a preserving solution for increasing viability and function of the isolated hepatocytes that are stored to be transplanted. METHODS: 9 cadaveric donor livers, which were not used for transplantation due to various causes such as severe steatosis, were selected to isolate hepatocytes. Various cold storage solutions were tried to find the best temperature for more viability and functionality for preservation of hepatocytes. University of Wisconsin (UW) solution and Williams E media were used as control media. 2 anti-apoptotic and anti-oxidative solutions, i.e., α-lipoic acid and ursodeoxycholic acid (UDCA), were used as cold preservatives solutions. The numbers of viable hepatocytes were estimated by trypan blue method; the functionality was assessed by the cells ability to produce urea. RESULTS: The highest number of viable and functional hepatocytes was obtained from freshly isolated cells. However, after preservation, the number of these viable hepatocytes and their functionality were not significantly different in cold storage solutions comparing to the control media used. Functionality of the isolated hepatocytes stored in UW with and without UCDA solution was similar to freshly isolated hepatocytes. CONCLUSION: Preservatives with anti-apoptotic and antioxidant activity could not increase the number of viable hepatocytes. Functionality of cold storing hepatocytes could be preserved similar to freshly isolated hepatocytes by UW solution with and without UCDA.

Comparison of Human Mesenchymal Stem Cells Derived from Various Compartments of Human Adipose Tissue and Tunica Adventitia Layer of the Arteries Subsequent to Organ Donation.

BACKGROUND: Mesenchymal stem cells are one of the most interesting cell sources used in regenerative medicine. OBJECTIVE: In the present study, we isolated and characterized the mesenchymal stem cells from various compartments of human adipose tissue and tunica adventitia layer of the arteries. METHODS: Tissue explant culture was done from various compartments of the human adipose tissue and tunica adventitia layer of the arteries, including adipose tissue far from the vessels, perivascular tissues that are completely attached to the vessels, and tunica adventitia layer of the arteries. After the cell culture, characterization of the cells was determined at 3rd-5th passages. Flow cytometry was performed for antigen expression analysis of CD34, CD45, CD44, CD90, CD29, CD73, and CD105. For the evaluation of cell differentiation potential, adipogenic and osteogenic differentiation was conducted under appropriate protocols. RESULTS: The cells were positive for CD44, CD90, CD29, and CD73 and negative for CD34, CD45, and CD105. Adipogenic and osteogenic differentiation potentials were different among the cells from various compartments. The cells derived from perivascular tissue demonstrated better adipogenic and osteogenic differentiation. CONCLUSION: It is essential to characterize the cells from different tissues and compartments for different purposes in regenerative medicine.

Comparison between the effect of kartogenin and TGFß3 on chondrogenesis of human adipose- derived stem cells in fibrin scaffold.

BACKGROUND: Due to very sluggish turnover at the molecular and cellular level, the healing of chondral damages has been considered difficult. In the current study, the effects of the Kartogenin, a small heterocyclic molecule on chondrogenic differentiation of stem cells was compared to TGF-ß3. METHODS: Human Adipose-Derived Stem Cells were extracted during an elective surgery. Cell viability was estimated by MTT assay, differentiated cells evaluated by histological and immunohistochemical techniques. Expression of cartilage specific genes (SOX9, Aggrecan, type II and X collagens) assessed by real-time PCR. RESULTS: The real-time PCR assay has revealed the expression of gene marker of chondrogenesis, SOX9, Aggrecan and type II collagen, both in Kartogenin and TGFß3 groups compared to the control group, significantly (p < 0.05). A low expression level of collagen type X as a hypertrophic marker was seen in cartilage produced by using Kartogenin. Meanwhile, the level of type X collagen protein in Kartogenin group was significantly decreased (p > 0.05) compared to TGF-ß3 group. CONCLUSION: Kartogenin was suitable for successful chondrogenic differentiation of human adipose- derived stem cells and a suppressor of the consequent hypertrophy (Tab. 1, Fig. 5, Ref. 31).

Phase 1b randomized, double-blind study of namilumab, an anti-granulocyte macrophage colony-stimulating factor monoclonal antibody, in mild-to-moderate rheumatoid arthritis.

BACKGROUND: Namilumab (AMG203) is an immunoglobulin G1 monoclonal antibody that binds with high affinity to the GM-CSF ligand. This was a phase 1b, randomized, double-blind study (PRIORA) to assess namilumab in active, mild-to-moderate rheumatoid arthritis (RA). The primary outcome was the safety and tolerability of repeated subcutaneous injections of namilumab in patients with mild-to-moderate RA. METHODS: Adults with mild-to-moderate RA on stable methotrexate doses for ≥12 weeks were eligible. Patients received three subcutaneous injections of namilumab 150 or 300 mg, or placebo on days 1, 15, and 29, with 12 weeks' follow-up. Primary objective was safety/tolerability. RESULTS: Patients in cohort 1 were randomized to namilumab 150 mg (n = 8) or placebo (n = 5). In cohort 2, patients were randomized to namilumab 300 mg (n = 7) or placebo (n = 4). Incidence of treatment-emergent adverse events (TEAEs) was similar across the three groups (namilumab 150 mg: 63%; namilumab 300 mg: 57%; placebo: 56%). TEAEs in ≥10% of patients were nasopharyngitis (17%) and exacerbation/worsening of RA (13%). No anti-namilumab antibodies were detected. The pharmacokinetics of namilumab were linear and typical of a monoclonal antibody with subcutaneous administration. In a post hoc efficacy, per protocol analysis (n = 21), patients randomized to namilumab showed greater improvement in Disease Activity Score 28 (erythrocyte sedimentation rate and C-reactive protein [CRP]), swelling joint counts and tender joint counts compared with placebo. Difference in mean DAS28-CRP changes from baseline between namilumab and placebo favored namilumab at both doses and at all time points. In addition area under the curve for DAS28-CRP was analyzed as time-adjusted mean change from baseline. A significant improvement in DAS28-CRP was shown with namilumab (150 and 300 mg groups combined) compared with placebo at day 43 (p = 0.0117) and also 8 weeks after last dosing at day 99 (p = 0.0154). CONCLUSIONS: Subcutaneous namilumab was generally well tolerated. Although namilumab demonstrated preliminary evidence of efficacy, patient numbers were small; phase 2 studies are ongoing. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01317797 . Registered 18 February 2011.

First-in-human trial of the safety, pharmacokinetics and immunogenicity of a PEGylated anti-CD40L antibody fragment (CDP7657) in healthy individuals and patients with systemic lupus erythematosus.

OBJECTIVE: The objective of this paper is to investigate the safety, pharmacokinetics (PK) and immunogenicity of CDP7657, a PEGylated anti-CD40L antibody fragment, in healthy individuals and patients with systemic lupus erythematosus (SLE). METHODS: This randomized, double-blind, single-dose, dose-escalation phase I study consisted of two parts. In part 1, 28 healthy individuals received CDP7657 IV (0.004-5 mg/kg) or placebo. In part 2, 17 patients with SLE received CDP7657 IV (5-60 mg/kg) or placebo. The CDP7657:placebo ratio was 3:1. RESULTS: Adverse events (AEs) were reported by 76% of healthy individuals and 100% of patients with SLE treated with CDP7657; most were mild or moderate in intensity. Two healthy individuals reported serious AEs (SAEs), one of which was considered treatment related (infusion-related reaction; 5 mg/kg cohort). One patient with SLE (60 mg/kg cohort) experienced three SAEs, one of which was considered treatment related (herpes zoster infection). No thromboembolic events were reported. CPD7657 exposure increased in a dose-proportional manner. Low anti-CDP7657 antibody titres were detected in the majority of CDP7657-treated participants with no apparent impact on the PK of CDP7657. CONCLUSION: Single doses of CDP7657 showed predictable PK in healthy individuals and patients with SLE and were well tolerated, with no safety signals of concern. These findings support further investigation of CDP7657 as a therapy for SLE.

Effects of human placental serum on proliferation and morphology of human adipose tissue-derived stem cells.

Media used for tissue culture may have significant effects on the growth and morphology of the adipose tissue-derived stem cells (ADSCs). As fetal bovine serum (FBS) may induce an immunological reaction and health risks, this study was designed to evaluate and compare the effects of human placental serum (HPS) on the proliferation and morphology of hADSCs. We cultured hADSCs for at least three passages in four different culture media containing either FBS, HPS, autologous serum (AS) or human allogeneic serum (HAS). Morphological and immunophenotypic characteristics, as well as proliferation rates of the hADSCs were determined. The rates of proliferation of hADSCs seemed as follows: AS≥HPS>HAS>>FBS. Morphologically, hADSCs isolated and expanded in medium containing HPS were similar to those grown in medium containing AS, whereas the morphology of cells cultured in human sera was different in comparison with FBS-ADSCs cultures. The immunophenotypic markers of hADSCs grown up in medium containing placental serum such as CD44+, CD90+ and CD105+, were similar to hADSCs grown up in media containing other sera. These results indicate that medium enriched with HPS provided a better microenvironment for hADSCs in comparison with medium enriched with commercially available FBS, and other human sera.

Future perspectives for hand transplant in iran.

Hand transplant program is a communion of physicians and researchers during the current decade. 72 hands and digits were transplanted in 53 patients over the past 13 years. Unlike a solid organ transplant, hand transplantation involves various tissues, so it is called "composite tissue allotransplantation." This article discusses the plans for performing the first hand transplant in Iran.

The effect of water stress on the antioxidant content, protective enzyme activities, proline content and lipid peroxidation in wheat seedling.

An investigation was conducted to study the effect of water stress on the antioxidant content, protective enzyme activities, proline content and lipid peroxidation in wheat seedlings. Drought stress increases the amount of Reactive Oxygen Species (ROS), leading to metabolic disorders. It is now known that higher levels of activity-protective mechanisms render the cells more enduring against environmental stress including drought. Two widely cultivated cultivars of wheat in Iran, Sab. and N. Sar. were grown up according to the hydroponic method. Having reached the stage of 4-5 leaves growth; the plants were kept under 4, 8 and 12 bars potential resulting from using Polyethylene Glycol 8000 (PEG 8000). Hogland solution was used as the control. Then the amount of ascorbate, glutathione, superoxide dismutase and catalase activity, proline and lipid Peroxidation was measured in cut samples of the leaves. The result indicated an increase in the amount of Ascorbate and Glutathione as the stress was intensified in the case of Sab. Moreover, the reduced form of Ascorbate (ASC) and Glutathione (GSH) were higher in Sab. at 8 and 12 bars. The amount of Proline accumulation was considerably higher in Sab. than N. Sar. SOD activity, on the other hand, diminished at 8 and 12 bar levels. CAT activity is also regarded as a limiting factor. Lipid peroxidation was also geared up as the stress was intensified. These limiting factors rendered N. Sar. cultivar more sensitive to water stress resulting from PEG8000 compared to Sab.

A proinflammatory role of IL-18 in the development of spontaneous autoimmune disease.

Serum from patients with systemic lupus erythematosus (SLE) contained significantly higher concentrations of IL-18 than normal individuals. MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease, also had higher serum levels of IL-18 than wild-type MRL/++ mice. Daily injections of IL-18 or IL-18 plus IL-12 resulted in accelerated proteinuria, glomerulonephritis, vasculitis, and raised levels of proinflammatory cytokines in MRL/lpr mice. IL-18-treated MRL/lpr mice also developed a "butterfly" facial rash resembling clinical SLE. In contrast, MRL/lpr mice treated with IL-18 plus IL-12 did not develop a facial rash. The facial lesion in the IL-18-treated mice showed epidermal thickening with intense chronic inflammation accompanied by increased apoptosis, Ig deposition, and early systemic Th2 response compared with control or IL-12 plus IL-18-treated mice. These data therefore show that IL-18 is an important mediator of lupus-like disease and may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases.

Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis.

IL-18 expression has recently been detected in rheumatoid arthritis (RA) synovial membrane. We investigated the mechanisms by which IL-18-induced collagen-induced arthritis in DBA/1 mice primed intradermally with type II bovine collagen in IFA and boosted i.p. 21 days later with CII in saline. Mice were injected i.p. with rIL-12, rIL-18, or both (100 ng) during days -1 to 4 and again on days 20-24. Control mice received PBS. Mice treated with IL-12 or IL-18 alone developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with IL-12 and IL-18. The cytokine treatments led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls. Cytokine-treated mice produced significantly more IFN-gamma, TNF-alpha, and IL-6 than the controls. Interestingly, IL-18-treated mice produced more TNF-alpha and IL-6, but less IFN-gamma, compared with mice treated with IL-12. Furthermore, splenic macrophages from DBA/1 mice cultured in vitro with IL-18, but not IL-12, produced substantial amounts of TNF-alpha. Mice treated with IL-18 or IL-18 plus IL-12 produced markedly more IgG1 and IgG2a anti-collagen Ab compared with controls, whereas IL-12 treatment only led to an enhanced IgG2a response. Together these results demonstrate that IL-18 can promote collagen-induced inflammatory arthritis through mechanisms that may be distinct from those induced by IL-12.

Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production.

MRL/MP-lpr/lpr (MRL/lpr) mice have a single mutation (lpr) of the fas apoptosis gene. The mutant mice developed significantly smaller lesions than the wild-type mice at the earlier stage of infection with the intracellular protozoan parasite Leishmania major. However, while all the wild-type mice achieved complete lesion resolution, the disease in the mutant mice progressed inexorably. The mutant mice had more IL-12 and nitrite/nitrate in the serum than wild-type mice following infection. Lymphoid cells from infected MRL/lpr mice produced more IFN-gamma but less IL-4 and IL-5 than cells from MRL-+/+ mice. Peritoneal macrophages from the mutant mice also produced more IL-12 and NO after stimulation with LPS. Thus, Fas expression is essential for resistance against leishmaniasis, and Fas-mediated apoptosis may form an integral part of the Th1-mediated microbicidal function.