Veysel Tahan

Hepatic differentiation of amniotic epithelial cells.

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell–derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte‐like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α‐hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)‐treated SCID/beige mice, naïve hAECs differentiated into hepatocyte‐like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. Conclusion: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation. (HEPATOLOGY 2011;)

Isolation of amniotic mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteocytes, chondrocytes, and adipocytes and possess immunomodulatory properties. Amniotic membrane from human term placenta is a potential source of multipotent MSCs that could be useful for regenerative medicine. This unit describes a detailed and simple protocol for the isolation of amniotic mesenchymal cells. We also introduce a simple density separation technique for the purification of this cell type from possible contamination with amniotic epithelial cells.

Melatonin reduces dimethylnitrosamine-induced liver fibrosis in rats

Abstract:  Increased deposition of the extracellular matrix components, particularly collagen, is a central phenomenon in liver fibrosis. Stellate cells, the central mediators in the pathogenesis of fibrosis are activated by free radicals, and synthesize collagen. Melatonin is a potent physiological scavenger of hydroxyl radicals. Melatonin has also been shown to be involved in the inhibitory regulation of collagen content in tissues. At present, no effective treatment of liver fibrosis is available for clinical use. We aimed to test the effects of melatonin on dimethylnitrosamine (DMN)‐induced liver damage in rats. Wistar albino rats were injected with DMN intraperitoneally. Following a single dose of 40 mg/kg DMN, either saline (DMN) or 100 mg/kg daily melatonin was administered for 14 days. In other rats, physiologic saline or melatonin were injected for 14 days, following a single injection of saline as control. Hepatic fibrotic changes were evaluated biochemically by measuring tissue hydroxyproline levels and histopathogical examination. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione (GSH) and superoxide dismutase (SOD) levels were evaluated in blood and tissue homogenates. DMN caused hepatic fibrotic changes, whereas melatonin suppressed these changes in five of 14 rats (P < 0.05). DMN administration resulted in increased hydroxyproline and MDA levels, and decreased GSH and SOD levels, whereas melatonin reversed these effects. When melatonin was administered alone, no significant changes in biochemical parameters were noted. In conclusion, the present study suggests that melatonin functions as a potent fibrosuppressant and antioxidant, and may be a therapeutic choice.

Vitamin E has a dual effect of anti-inflammatory and antioxidant activities in acetic acid-induced ulcerative colitis in rats.

BACKGROUND Increased free radical production, decreased antioxidant capacity and excessive inflammation are well-known features in the pathogenesis of inflammatory bowel disease. Vitamin E is a powerful antioxidant and a scavenger of hydroxyl radicals, and it has been shown to have anti-inflammatory activities in tissues. We investigated the effects of vitamin E on inflammatory activities using an acetic acid (AA)-induced ulcerative colitis model in rats. METHODS Wistar rats were divided into 4 groups. Acetic acid was given to 2 groups of animals to induce colitis while the other 2 groups received saline intrarectally. One AA-induced colitis group and 1 control group received vitamin E (30 U/kg/d) intraperitoneally and the pair groups received saline. After 4 days, we evaluated colonic changes biochemically by measuring proinflammatory cytokine levels in tissue homogenates and by histopathologic examination. RESULTS Acetic acid caused colonic mucosal injury, whereas vitamin E administration suppressed these changes in the AA-induced colitis group (p < 0.001). Administration of AA resulted in increased levels of tumour necrosis factor-α, interleukin-1β, interleukin-6, myeloperoxidase and malondialdehyde, and decreased levels of glutathione and superoxide dismutase; vitamin E reversed these effects (all p < 0.001). CONCLUSION Our study proposes that vitamin E is an effective anti-inflammatory and antioxidant and may be a promising therapeutic option for ulcerative colitis.

Prevalence of Hepatic Granulomas in Chronic Hepatitis B

An increasing frequency of hepatic granulomas, up to 10%, in chronic hepatitis C patients is reported, and their presence is considered to be a predictor of treatment success. However, there is only one prevalence study on granuloma in chronic hepatitis B, and its significance for treatment outcome is unknown. We aimed to determine the prevalence of hepatic granulomas in a larger group of chronic hepatitis B patients and to compare their presence with the response to interferon therapy. Biopsy specimens of chronic hepatitis B patients were reevaluated for the presence of hepatic granulomas. All patients with hepatic granuloma were screened for other granulomatous diseases by tuberculin skin test, chest X-ray and computed tomography, venereal disease research laboratory, Brucella agglutination tests, and exposure to hepatotoxic agents. We screened 663 cases of chronic hepatitis B. Hepatic granulomas were found in 10 cases (1.5%). The granulomas could not be ascribed to any other reason. Of the 10 patients with hepatic granulomas, 4 responded to interferon therapy, 2 dropped out, and 4 were nonresponders. We conclude that hepatic granuloma is a rare finding in chronic hepatitis B and its presence does not seem to predict the response to interferon therapy.

Melatonin ameliorates methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis in rats.

Abstract:  Nonalcoholic steatohepatitis (NASH) may progress to advanced fibrosis and cirrhosis. Mainly, oxidative stress and excessive hepatocyte apoptosis are implicated in the pathogenesis of progressive NASH. Melatonin is not only a powerful antioxidant but also an anti‐inflammatory and anti‐apoptotic agent. We aimed to evaluate the effects of melatonin on methionine‐ and choline‐deficient diet (MCDD)‐induced NASH in rats. Thirty‐two male Wistar rats were divided into four groups. Two groups were fed with MCDD while the other two groups were fed a control diet, pair‐fed. One of the MCDD groups and one of the control diet groups were administered melatonin 50 mg/kg/day intraperitoneally, and the controls were given a vehicle. After 1 month the liver tissue oxidative stress markers, proinflammatory cytokines and hepatocyte apoptosis were studied by commercially available kits. For grading and staging histological lesions, Brunt et al.’s system was used. Melatonin decreased oxidative stress, proinflammatory cytokines and hepatocyte apoptosis. The drug ameliorated the grade of NASH. The present study suggests that melatonin functions as a potent antioxidant, anti‐inflammatory and antiapoptotic agent in NASH and may be a therapeutic option.

Changes in malondialdehyde levels in bronchoalveolar fluid and serum by the treatment of asthma with inhaled steroid and beta2‐agonist

Oxidative stress plays an important role in the pathogenesis of asthma. Recent data suggest that clinical indices of the patients with asthma may not correlate with the underlying inflammatory process. We aimed to measure the level of malondialdehyde (MDA), which is a marker of lipid peroxidation, a free radical‐mediated process, before and after a well‐accepted treatment of asthma.

Acute hepatitis C: prevention and treatment

HCV can cause acute or chronic hepatitis and is a health problem all over the world. It is one of the leading causes of cirrhosis and hepatocellular carcinoma, and is a common indication for liver transplantation. Unrecognized patients with HCV infection may transmit the virus to uninfected people. The acute form of the disease leads to chronic hepatitis in the majority of cases. Since the success rate of treatment given in the chronic phase is much lower than that given in the acute phase, recognizing acute hepatitis is critical. Although HCV is less prevalent since 1990s in the Western world after improved blood-donor screening programs, needle-exchange facilities and education among intravenous drug users, it is still endemic in some regions, including African countries, Egypt, Taiwan, China and Japan. Acute HCV infection may be a challenge for the clinician; since it is often asymptomatic, detection and diagnosis are usually difficult. After an incubation period of 7 weeks (2–12 weeks), only a minority of patients (10–15%) report symptoms. The spontaneous clearance of the virus is more frequent primarily during the first 3 months of clinical onset of the disease, but may occur anytime during the 6 months of acute infection. This spontaneous resolution seems to be more frequent in symptomatic cases. Viremia persisting more than 6 months is accepted as chronic infection. The virus is transmitted more frequently through infected blood or body fluids. Detection of antibodies against HCV is not a reliable method of diagnosing acute HCV infection since the appearance of antibodies against HCV can be delayed in up to 30% of patients at the onset of symptoms. Thus, the diagnosis of acute hepatitis C relies on the qualitative detection of HCV RNA, which may appear as early as 1–2 weeks after exposure quickly followed by highly elevated alanine aminotransferase. After a follow-up period of 8–12 weeks for allowing spontaneous resolution, treatment should be initiated. Pegylated interferon monotherapy for 24 weeks seems effective, and the therapy can be individualized according to the characteristics of the patient.

Tuberculous lymphadenopathy in adults: a review of 35 cases.

Abstract We retrospectively reviewed clinical, diagnostic, therapeutic and prognostic features of 35 patients (25 female, 10 male, mean age: 33 years, range: 16–70) with tuberculous lymphadenopathy (TB LAP) which had been followed since 1980. The diagnosis was established by tissue sampling in 32 cases (caseating granulomatous adenitis in 89%) or presence of acid-fast bacilli (AFB) in the aspirate in 2 cases and in the drainage in 1 case. Paraffin-embedded granulomatous tissues were stained by Ehrlich-Ziehl-Neelsen (EZN) and also Mycobacterium tuberculosis DNA was studied by polymerase chain reaction (PCR) (n = 21). The patients were admitted with enlarging LAP (34%), draining LAP (9%), and both systemic complaints and enlarging LAP (57%). Cervical lymph nodes were the most frequently involved site (77%). Pathologic findings on chest X-ray were seen in 23%. Erythrocyte sedimentation rate (ESR) was higher than 100 mm/hour in 25% and associated with systemic complaints. Tuberculin skin test was positive in 91%. AFB could not be seen in any granulomatous tissue (n: 21), but PCR study was positive in 33% (7/21). All patients were given anti-TB treatment (INH, RMP, EMB and/or PZA). Surgical excision of draining LAP with surrounding inflammatory tissues in addition to the medical treatment was needed in 2 cases. Clinical improvement was obtained within 3 months of the treatment and ESR returned to normal within 5 months. After completion of the treatment, 22 patients were followed-up; mean duration was 3 years, and none relapsed. In conclusion, a patient with TB LAP generally presents with a few small, painless, cervical lymph nodes, which are slowly enlarging. For exact diagnosis, excisional biopsy for histologic and microbiologic studies is essential. Use of anti-TB drugs is the main therapeutic option.

Placental stem cell correction of murine intermediate maple syrup urine disease

There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme‐proficient liver‐engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latters safety and ethical concerns and differentiate to hepatocyte‐like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α‐keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. Conclusion: Placental‐derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver‐based metabolic diseases. (HEPATOLOGY 2013)