Higher Serum Transaminase Levels Research Articles

Four-And-A-Half LIM-Domain Protein 2 (FHL2) Deficiency Aggravates Cholestatic Liver Injury.

Cholestasis occurs in different clinical circumstances and leads to severe hepatic disorders. The four-and-a-half LIM-domain protein 2 (FHL2) is a scaffolding protein that modulates multiple signal transduction pathways in a tissue- and cell context-specific manner. In this study, we aimed to gain insight into the function of FHL2 in cholestatic liver injury. FHL2 expression was significantly increased in the bile duct ligation (BDL) model in mice. In Fhl2-deficient (Fhl2-ko) mice, BDL caused a more severe portal and parenchymal inflammation, extended portal fibrosis, higher serum transaminase levels, and higher pro-inflammatory and pro-fibrogenic gene expression compared to wild type (wt) mice. FHL2 depletion in HepG2 cells with siRNA resulted in a higher expression of the bile acid transporter Na+-taurocholate cotransporting polypeptide (NTCP) gene. Furthermore, FHL2-depleted HepG2 cells showed higher expression of markers for oxidative stress, lower B-cell lymphoma 2 (Bcl2) expression, and higher Bcl2-associated X protein (BAX) expression after stimulation with deoxycholic acid (DCA). In hepatic stellate cells (HSCs), FHL2 depletion caused an increased expression of TGF-β and several pro-fibrogenic matrix metalloproteinases. In summary, our study shows that deficiency in FHL2 aggravates cholestatic liver injury and suggests FHL2-mediated effects on bile acid metabolisms and HSCs as potential mechanisms for pronounced hepatocellular injury and fibrosis.

Lutein mitigates cyclophosphamide induced lung and liver injury via NF-κB/MAPK dependent mechanism

This study targeted to test the potential protective role of lutein against lung and liver damage associated with cyclophosphamide (CP) administration. Lutein was given orally for 5days at two different doses both before and after CP injection. Results have shown that CP administration caused marked pulmonary and hepatic injurious effects in mice. Lung damage was evident through increased lung wet/dry ratio, elevated inflammatory cells infiltration into the pulmonary tissues, increased total protein content and lactate dehydrogenase (LDH) activity in the broncho-alveolar lavage fluid. Estimation of high levels of serum transaminases, alkaline phosphatase and LDH in serum revealed hepatic injury. Histopathological examination of both organs confirmed the biochemical analysis. Elevation of oxidative stress along with depressed anti-oxidant status of lung and liver were evident in CP-intoxicated animals. Furthermore, CP induced elevation of inflammatory cytokines (NOx, TNF-α, IL-6) contaminant with activation of nuclear factor kappa-B (NF-κB) and p38 mitogen activated protein kinase (p38-MAPK). On the other side, lutein treatment successfully protected the lung and the liver as indicated by improvement of the biochemical and histopathological parameters. These results suggest that lutein can ameliorate CP-induced pulmonary and hepatic oxidative injurious effects via inhibition of reactive oxygen species (ROS)/NF-κB/MAPK pathway.

Asymptomatic acute hepatitis E in a female patient with ulcerative colitis

We present a 60-year-old female patient with asymptomatic acute hepatitis E that was fortuitously detected during the course of ulcerative colitis (UC). She was admitted to hospital on October 30, 2015. Endoscopy and histological examination of the colon showed typical findings of UC. All parameters of liver function tests were normal on this date. Combination therapy with oral prednisolone and mesalazine was started and intravenous administration of infliximab once every 8weeks was added later. Her symptoms gradually improved after these treatments, and she was discharged on February 7, 2016. In a periodic check-up on July 7, 2016, high levels of serum transaminases were detected in liver function tests. Although drug-induced liver injury was first suspected, anti-hepatitis E virus (HEV) immunoglobulin A was positive. The genotype and subgenotype of this HEV are 3 and 3a, respectively, although the infectious route of the HEV was unclear. Within 2weeks after the onset of acute liver injury, the HEV viremia disappeared and her liver function tests improved. Examination of serum anti-HEV immunoglobulin A should be added at the time of abnormal liver function tests in patients with UC receiving immunosuppressive and biological drugs.

Predictors of poor outcome in gastrointestinal bleeding in emergency department

To determine the prognostic risk factors of gastrointestinal bleeding in emergency department cases. The trial was a retrospective single-center study involving 600 patients over 18-years-old and carried out with approval by the Institutional Ethics Committee. Patient data included demographic characteristics, symptoms at admission, past medical history, vital signs, laboratory results, endoscopy and colonoscopy results, length of hospital stay, need of intensive care unit (ICU) admission, and mortality. Mortality rate was the principal endpoint of the study, while duration of hospital stay, required interventional treatment, and admission to the ICU were secondary endpoints. The mean age of patients was 61.92-years-old. Among the 600 total patients, 363 (60.5%) underwent upper gastrointestinal endoscopy and the most frequent diagnoses were duodenal ulcer (19.2%) and gastric ulcer (12.8%). One-hundred-and-fifteen (19.2%) patients required endoscopic treatment, 20 (3.3%) required surgical treatment, and 5 (0.8%) required angiographic embolization. The mean length of hospital stay was 5.21 ± 5.85 d. The mortality rate was 6.3%. The ICU admission rate was 5.3%. Patients with syncope, higher blood glucose levels, and coronary artery disease had significantly higher ICU admission rates (P = 0.029, P = 0.043, and P = 0.002, respectively). Patients with low thrombocyte levels, high creatinine, high international normalized ratio, and high serum transaminase levels had significantly longer hospital stay (P = 0.02, P = 0.001, P = 0.019, and P = 0.005, respectively). Patients who died had significantly higher serum blood urea nitrogen and creatinine levels (P = 0.016 and P = 0.038), and significantly lower mean blood pressure and oxygen saturation (P = 0.004 and P = 0.049). Malignancy and low Glasgow coma scale (GCS) were independent predictive factors of mortality. Prognostic factors for gastrointestinal bleeding in emergency room cases are malignancy, hypotension on admission, low GCS, and impaired kidney function.

Investigation of Hepatitis B and Hepatitis C Virus Infections by Serological and Molecular Methods in Hemodialysis Patients

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are significant causes of morbidity and mortality in hemodialysis patients, since those patients are highly susceptible to infections due to immune suppression. The aims of this study were to investigate the presence of HBV and HCV infections in chronic hemodialysis patients by serological and molecular methods, and to determine the rate of occult HBV infection and the viral genotypes. A total of 201 patients who were under hemodialysis due to end-stage renal disease, were retrospectively evaluated. The study involved the patients at three different centers in Antalya, Turkey during 2006. HBV and HCV markers in the patients' sera were screened by ELISA method, viral nucleic acids were investigated by real-time polymerase chain reaction (PCR) in patients' plasma and viral genotypes were determined by DNA sequence analysis. Detection of at least one of the HBV markers HBsAg, anti-HBc total, and HBV DNA, was accepted as HBV infection, and detection of anti-HCV and/or HCV RNA was accepted as HCV infection. HBsAg positive patients with negative HBV DNA were considered as occult HBV infection. Of the patients 80 (40%) were female, 121 (60%) were male and the mean age was 51.16 ± 16.28 (range 17-93) years. In our study, sole anti-HBs positivity due to HBV vaccination, was detected in 89 (44.3%) patients. One hundred (50%) patients were found positive in terms of HBV infection and 40 (20%) were positive for HCV infection, while 24 (12%) patients had HBV and HCV co-infections. Eighty-five (42.3%) patients had no HBV and HCV infection. Among the 5 (2.5%) patients who were HBsAg positive, four were also HBV DNA positive. Occult HBV infection was detected in 1 (0.5%) patient. Anti-HCV and HCV RNA were found positive in 37 (18.4%) and in 24 (12%) patients, respectively. Among the HCV-RNA positive patients, 3 (12.5%) were anti-HCV negative. ALT and AST levels were found normal in all of the HBV DNA positive patients, and 62.5% (15/24) of HCV RNA positive patients. All of the HBV isolates were identified as genotype D and HCV isolates as genotype 1b. No statistically significant correlation was detected between the HBV infection and patients' age, duration of hemodialysis and elevation of serum transaminase levels (p> 0.05). On the other hand, HCV infection was seen to increase with age (p= 0.047). HCV infection showed a statistically significant increase with the duration of hemodialysis. HCV infection risk was increased in patients who were under hemodialysis for ≥ 25 months (p< 0.001, OR: 0224, 95% CI= 0089-0562). There was also a statistically significant correlation between the presence of HCV infection (anti-HCV and/or HCV RNA positive) and high levels of serum transaminases (p< 0.001). However, in two of the three cases who were anti-HCV negative and HCV RNA positive, serum transaminase levels were normal while the viral loads were high. Therefore to follow-up HCV infection in the hemodialysis patients, anti-HCV and serum transaminase levels may not be sufficient alone and these patients should be evaluated periodically for HCV RNA. In addition, the detection of occult HBV infection in one of the study patients, indicated that HBV DNA should also be investigated at regular intervals in the hemodialysis patients.

Presence of antibodies against self human leukocyte antigen class II molecules in autoimmune hepatitis.

Autoimmune hepatitis (AIH) can arise de novo after liver transplantation (LT) for non-autoimmune liver diseases. Considering the identical features of de novo AIH after LT and classical AIH, as well as the importance of anti-human leukocyte antigen (HLA) antibodies in graft rejection, we investigated the presence of circulating anti-HLA class II antibodies in the sera of 35 patients with AIH, 30 patients with primary biliary cirrhosis (PBC), and 30 healthy donors using fluorescent dye-impregnated beads bound to HLA molecules. We then investigated the allele specificity of the antibodies and identified the HLA alleles in each patient using DNA-based HLA typing. We also examined HLA class II expression in liver samples using immunohistochemistry. Anti-HLA class II antibodies were detected significantly more frequently in the patients with AIH (88.1%) than in the patients with PBC (33.3%) or in the healthy donors (13.3%) (both P <0.01). We confirmed that the anti-HLA class II antibodies in the AIH patients showed specificity for several HLA class II alleles, including self HLA class II alleles. Moreover, positive reactivity with anti-self HLA class II antibodies was associated with higher serum transaminase levels. In conclusion, we demonstrated, for the first time, that antibodies against self HLA class II alleles were detectable in patients with AIH. Our results suggest that an antibody-mediated immune response against HLA class II molecules on hepatocytes may be involved in the pathogenesis or acceleration of liver injury in AIH.

Endogenous A1 adenosine receptor protects mice from acute ethanol-induced hepatotoxicity

Previous studies have indicated a critical role of adenosine and its receptors in the pathogenesis of liver diseases. The aim of this study was to determine the contribution of A1 adenosine receptor (A1AR) to acute ethanol-induced hepatotoxicity. Wild-type (WT) and A1AR−/− mice were intragastrically administered with ethanol (5g/kg), and hepatic injury was evaluated 6h thereafter. Mice lacking A1AR were more susceptible to ethanol-induced liver damage than WT mice, as evidenced by higher serum transaminase levels and increased extent of histopathological changes. Ethanol induced triglycerides accumulation in the serum and liver, and this accumulation was augmented in A1AR−/− mice. Analysis of gene expression in the liver revealed up-regulated mRNA levels of genes related to lipogenesis (including: FAS, SCD1, ACC1, DGAT2, and PPARγ) in A1AR−/− mice after ethanol treatment. In addition, lack of A1AR aggravated lipid peroxidation and superoxide dismutase depletion caused by acute ethanol exposure. A subsequent study revealed that, pretreatment with A1AR antagonist DPCPX increases the sensitivity of mice to ethanol-induced liver injury. In conclusion, these results indicated that endogenous A1AR activation protects mice against acute ethanol-induced liver injury by reducing oxidative stress and decreasing lipid accumulation.

Serum transaminase elevation in children with rotavirus gastroenteritis: Seven years’ experience

There are no studies on clinically significant transaminase elevation due to rotavirus gastroenteritis in the literature. Also, there are significant discrepancies among previous studies regarding the prevalence of increased serum transaminase levels in rotavirus infection. Patients investigated for rotavirus by stool antigen testing, who were followed between January 2005 and May 2012, were retrospectively enrolled in this study. Patients were divided into 2 groups according to their rotavirus results: rotavirus-positive acute gastroenteritis (RPAG) and rotavirus-negative acute gastroenteritis (RNAG) groups. A total of 4317 children who presented with acute gastroenteritis were assessed. The study was completed with 642 patients who met the inclusion criteria. In the RPAG group (n = 272), elevated alanine aminotransferase (ALT) was found in 42 (15.4%) patients and elevated aspartate aminotransferase (AST) in 69 (25.4%), while in the RNAG group (n = 370), these numbers were 25 (6.8%) and 44 (11.9%), respectively. The elevated ALT and AST levels were found to be significantly higher in the RPAG group than in the RNAG group (both p < 0.001). The prevalence of elevated transaminase levels was found to be similar with respect to gastroenteritis severity score (p > 0.05). The high serum transaminase levels normalized uneventfully in all patients in the RPAG and RNAG groups during follow-up. In this study, our results clearly signify a liver influence in rotavirus infections. Therefore, rotavirus infections should be kept in mind when evaluating the aetiology of transaminase elevation in patients with acute gastroenteritis.

Donation after cardiac death liver transplantation: Lose a bit on each one and make it up in volume

See Article on Page 771 DBD, donation after brain death; DCD, donation after cardiac death. Liver transplant surgeons are engaged in a race to reduce waiting-list death rates, and this increasingly is requiring the expanded use of organs with a higher risk of graft failure. Declining rates of young brain-dead donors have resulted in national and international efforts to increase the donor pool, often at the expense of posttransplant outcomes. Nowhere is this more true than donation after cardiac death (DCD) liver transplantation. In multiple single-center and registry analyses, DCD liver transplantation has been demonstrated to increase the risk of graft loss from ischemic biliary injury, and retransplantation is often required.1 The impact of DCD allografts on overall patient survival remains controversial, with some centers reporting equivalent patient survival after transplantation but many demonstrating increased rates of death and retransplantation.2, 3 Although the clinical impact of DCD liver transplantation continues to be debated, it is now well established that DCD transplantation is associated with dramatically greater costs during the posttransplant period in comparison with donation after brain death (DBD) transplantation.4, 5 In this issue of Liver Transplantation, Broomhead et al.6 report a matched case series analysis comparing early posttransplant events in recipients of organs from DBD and DCD donors. They demonstrate a higher risk of perioperative events, more postoperative complications, and increases in resource utilization (including longer intensive care unit stays) among recipients of DCD organs. Among their important observations is the impact of DCD transplantation on intraoperative coagulopathy and hemodynamic instability. Compared with the DBD recipients, the DCD recipients had prolonged heparin effects (which led to increased transfusion requirements and thus increased use of expensive blood products, even though this was not a statistically significant finding), extended periods of vasopressor support, and greater evidence of allograft injury (manifested by higher serum transaminase levels). Postoperatively, the DCD patients also needed prolonged mechanical ventilator support and renal replacement therapy. Finally, the rate of graft survival at 1 year was markedly lower in the DCD liver group (75% versus 100%, P = 0.03). Despite the dire need to increase the number of organs available for transplantation, the use of DCD liver transplantation in the United States appears to have leveled off.7 Currently, 6% of liver transplants nationally use DCD organs, and this rate appears to have been unchanged for the past 4 years. There is marked regional variation in the utilization of these organs, which is driven by ongoing disparities in access to deceased donor organs. Highly competitive in access regions 1, 5, 9 continue to use the majority of the DCD organs nationally. Despite a leveling-off of the demand for DCD livers, the proportion of DCD donors in the overall donor pool increased from 2% in 2000 to 11.5% in 2009. Whether this reflects economic incentives at donor hospitals to shorten the intensive care unit length of stay and limit the opportunity for patients to progress to brain death, changes in neurosurgical or critical care treatment plans, or educational efforts and donor management by the organ procurement organizations is unclear. What is certain is that the number of donors who have progressed to brain death is diminishing. As aptly demonstrated by Broomhead et al.,6 the outcomes of DCD liver transplantation are inferior to the outcomes of DBD liver transplantation. In comparison with DBD liver transplantation, patients may survive end-stage organ failure only to have a more difficult intraoperative course and a recovery characterized by repeated intrahepatic abscesses, early graft loss, and higher utilization of costly resources. Each DCD transplant represents an unpredictable risk of long-term morbidity that might have been avoided had the donor progressed to brain death. As a result, a growing tension exists between the well-intentioned efforts of the organ donation and transplantation collaborative of the Health Resources and Services Administration to expand access to transplantation through the goal of “every organ, every time” and the day-to-day realities facing transplant centers. Unfortunately, our current reimbursement and public reporting mechanisms may be inadequate to mitigate the economic and outcome risks that DCD organs pose to transplant centers. While we are waiting for improved methods to protect organs from the deleterious impact of warm ischemic injury during the agonal period, we would be well advised to focus our attention on strategies promoting donor support to brain death if possible. Unlike business, if you lose a bit of benefit from each transplant, you cannot make it up in increased volume.

Hepatocellular Carcinoma in 2 Young Adolescents With Chronic Hepatitis C

Hepatocellular Carcinoma in 2 Young Adolescents With Chronic Hepatitis C

Chronic Pericardial Constriction Induced Severe Ischemic Hepatitis Manifesting as Hypoglycemic Attack

Ischemic hepatitis, otherwise known as "shock liver", is characterized by a massive, but transient increase in serum transaminase levels, usually associated with cardiac failure. A patient who did not have a predisposition to hypoglycemia was discovered at home with disturbed consciousness caused by hypoglycemia. She had been diagnosed as having constrictive pericarditis several years earlier and had developed ischemic hepatitis. Though the high serum transaminase levels were rapidly normalized, severe jaundice gradually developed and the patient finally died of multiple organ failure. Hypoglycemia, which is considered secondary to reduced gluconeogenesis in the exhausted liver, is a rare complication of constrictive pericarditis.

Comparison of Recurrence of Hepatocellular Carcinoma After Resection in Patients with Cirrhosis to Its Occurrence in a Surveilled Cirrhotic Population

The presence of cirrhosis is the only risk factor that is advocated for recurrence of hepatocellular carcinoma (HCC) 2 years after hepatic resection compared with noncirrhotic control subjects; however, data for cohorts of exclusively patients with cirrhosis are lacking. This study was designed to assess risk factors and annual incidence of early (<2 years) and late (>2 years) recurrence after resection of cirrhosis and to compare these findings with those of patients with cirrhosis enrolled in HCC surveillance programs (HCC occurrence). Data from 204 patients with cirrhosis resected for HCC and 150 surveilled for cirrhosis were retrospectively collected and compared using propensity score matching to overcome biases of nonrandomized study. Risk factors for early recurrence (incidence = 21.8%/year) were higher serum alpha-fetoprotein (AFP) levels, poorly differentiated tumor, and presence of microvascular invasion (P < 0.05). Risk factors for both late recurrence (18.4%/year) and HCC occurrence (3.3%/year) were male gender, older age, and higher serum transaminase levels; multiple primary tumors and higher AFP were additional risk factors for late recurrence and HCC occurrence respectively (P < 0.05). After propensity adjustment, resected patients with less than two risk factors for late recurrence showed an annual incidence of HCC (6.2%/year) similar to that of surveilled patients with > or =2 risk factors (5.8%/year; P = 0.898). Early and late recurrence of HCC for patients with cirrhosis after resection have distinct risk factors. Annual incidence of HCC 2 years or more after resection may be similar to that of general patients because the same risk factors are involved; assessment of these characteristics could be useful in tailoring clinical management.

Fatal amiodarone-induced hepatotoxicity: a case report and literature review

To report a fatal case of amiodarone-induced acute hepatotoxicity after intravenous amiodarone administration and similar fatal cases review. A 72-year-old woman with a history of hypertension, prior cardiovascular disease, atrial fibrillation and diabetes mellitus was admitted to the hospital with acute pyelonephritis and transferred to the intensive care unit due to cerebral infarction. An antidiabetic drug, a low dose of aspirin and intravenous amiodarone therapy was started. After receiving a second dose of amiodarone (1,200 mg; injection rate 1 mg/min), the woman developed ascites, jaundice, high levels of serum transaminases, decreased prothrombin time, and finally became unconscious. Immediately after treatment was discontinued, her extremely high hepatic parameters returned to normal. According to the Naranjo probability scale, this adverse reaction was highly probable. The occurrence of acute liver damage after intravenous amiodarone is rare but harmful. It can be induced by polysorbate 80, a solubilizer, by immunomediated centrilobular necrosis, or by the presence of a functional PPAR-I+/- gene. Amiodarone is an effective antiarrhythmic agent for preventing and treating atrial and ventricular arrhythmias. The molecular mechanism causing acute hepatic damage after amiodarone treatment is not clear. Therefore, amiodarone must be administered with care, and liver function should be monitored closely in patients treated with this drug.

Hepatocyte-Specific IKKγ/NEMO Expression Determines the Degree of Liver Injury

NEMO is the regulatory subunit of the I kappa B kinase (IKK) complex and is involved in controlling nuclear factor kappaB (NF-kappaB) activation. NEMO knockout mice die during embryogenesis due to massive hepatocyte apoptosis. Here we investigated the role of NEMO-dependent signaling in hepatocytes during acute liver injury. We generated conditional hepatocyte-specific NEMO knockout mice using the loxP system with the Cre recombinase under the control of the albumin promoter (NEMODeltaLPC). In these mice, we studied mechanisms of tumor necrosis factor (TNF)- and ischemia/reperfusion-dependent liver cell damage. In adult NEMODeltaLPC animals, NEMO is specifically deleted in hepatocytes and no differences in survival, growth, and fertility were found when compared with wild-type (NEMO(f/f)) mice. TNF stimulation of NEMODeltaLPC mice resulted in high serum transaminase levels and massive hepatocyte apoptosis, which were associated with lack of I kappa B alpha degradation, inhibition of NF-kappaB activation, and target gene transcription. Additionally, ischemia/reperfusion resulted in higher nonparenchymal cell-dependent induction of oxidative stress and stronger inflammation in NEMODeltaLPC mice. This led to massive hepatocyte apoptosis and death of the animals, while NEMO(f/f) mice survived with significantly lesser liver damage, showing mainly necrotic cell death. Thus, complete inhibition of NF-kappaB activation in hepatocytes, in contrast to attenuation in hepatocyte-specific IKK2(-/-) mice, determines the type of liver cell damage during ischemia/reperfusion injury and is associated with a poor prognosis. Our results show that understanding of the fine tuning of NF-kappaB modulation during liver injury is essential to develop new therapeutic strategies.

Liver injury is associated with enhanced regulatory T‐cell activity in patients with chronic hepatitis B

Chronic hepatitis B virus (HBV) infection is associated with impairment of HBV-specific immune responses. Recently, it has been shown that regulatory T (Treg) cells downregulate HBV-specific immune responses but their role in chronic hepatitis B is still controversial. We hypothesized that liver injury enhances the influence of Treg cells on HBV-specific immune responses. The frequency of Treg cell and the in vitro expansion of HBV-specific CD8+ T cell detected by the tetramer method were investigated in 79 patients with chronic hepatitis B. Thirty-three healthy volunteers were enrolled to measure the frequency of Treg cell as controls. The results showed that in chronic hepatitis B cases, the frequency of Treg cells in peripheral blood was significantly higher than that in normal volunteers. The higher level of serum transaminase was associated with higher frequency of Treg cells, which both had a linear correlation relationship. HBV-DNA level, HBe status, age and sex had no statistical association with Treg cell frequency. Furthermore, in patients with higher serum transaminase levels, the expansion of HBV-specific CD8+ T cells was higher after removal of Treg cells when compared with patients with lower serum transaminase levels. In conclusion, our data indicate a significant association between serum transaminase level and frequency/activity of Treg cells. Based on this observation, we propose that liver-injury enhances Treg cell frequency/activity in chronic hepatitis B patients.

Title: Obstetric Cholestasis Presenting With Unusually High Levels Of Serum Transaminases

Obstetric cholestasis is typically suspected when a pregnant woman presents with generalized pruritus without a rash. Biochemically, only a mild abnormality of liver function tests is seen without any systemic signs and symptoms. The patient is usually treated with Ursodeoxycholic acid which improves both pruritus and liver function tests. We present a case where a pregnant woman presented with pruritus and was found to have unusually high levels of serum transaminases. The diagnosis of obstetric cholestasis was made only by exclusion of other causes of hepatobiliary dysfunction. Fortunately the condition responded well to the standard treatment and both mum and baby had a favourable outcome.

Cell-Specific Viral Gene Therapy of a Hurthle Cell Tumor

We evaluated the effectiveness of a replication-defective adenovirus-transducing thymidine kinase (TK) gene under the control of the rat Tg (rTg) promoter (AdrTgtk) in therapy of a human Hurthle cancer (XTC-1 cell) in vitro and in vivo. The ganciclovir (GCV) sensitivity of infected XTC-1 cells was assessed in vitro by H3-thymidine incorporation assay and Trypan-blue exclusion, and by an in vivo tumor development assay. Proliferation was strongly inhibited by adding GCV into the culture medium of infected cells, but not uninfected cells, proving cell infection and expression of TK in the XTC-1 cells. AdrTgtk, and also viruses that have the noncell-specific cytomegalovirus (CMV) promoter-directing expression of TK (AdCMVtk), or luciferase (AdCMVLuc), were used to transduce XTC-1 cells to evaluate killing effects. After infection with AdCMVtk or AdrTgtk, followed by GCV treatment, 70% of infected cells were killed in the presence of GCV, compared with less than 20% of cells infected by AdCMVLuc and treated with GCV. In vivo toxicity was studied in BALB/c mice. When adenovirus is given iv, liver is the major organ infected. No significant changes of the serum transaminase levels and no histological abnormalities were found in animals treated with AdrTgtk/GCV given iv, compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer’s cell prominence, and extensive single-cell hepatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by the noncell-specific CMV promoter. XTL-1 cells (2 × 106) were injected sc into BALB/c-severe combined immunodeficient mice (BALB/c-SCID), and the mice developed tumors after 3 wk. After intratumoral injection of AdrTgtk and treatment with GCV, tumors stabilized in 15 of 17 mice within 3 wk, 9 tumors remained stabilized after 5 wk of treatment, and 2 disappeared during observation. In AdCMVLuc/GCV-treated control mice, almost all tumors grew continuously. The average tumor size in AdrTgtk-treated mice was significantly smaller than that of control animals after 2 wk of treatment. Our data confirm the effectiveness and specificity of an adenovirus using rTg promoter to express TK, and support its future application to thyroid cancer gene therapy in humans.

Cell-specific viral gene therapy of a Hurthle cell tumor.

We evaluated the effectiveness of a replication-defective adenovirus-transducing thymidine kinase (TK) gene under the control of the rat Tg (rTg) promoter (AdrTgtk) in therapy of a human Hurthle cancer (XTC-1 cell) in vitro and in vivo. The ganciclovir (GCV) sensitivity of infected XTC-1 cells was assessed in vitro by H(3)-thymidine incorporation assay and Trypan-blue exclusion, and by an in vivo tumor development assay. Proliferation was strongly inhibited by adding GCV into the culture medium of infected cells, but not uninfected cells, proving cell infection and expression of TK in the XTC-1 cells. AdrTgtk, and also viruses that have the noncell-specific cytomegalovirus (CMV) promoter-directing expression of TK (AdCMVtk), or luciferase (AdCMVLuc), were used to transduce XTC-1 cells to evaluate killing effects. After infection with AdCMVtk or AdrTgtk, followed by GCV treatment, 70% of infected cells were killed in the presence of GCV, compared with less than 20% of cells infected by AdCMVLuc and treated with GCV. In vivo toxicity was studied in BALB/c mice. When adenovirus is given iv, liver is the major organ infected. No significant changes of the serum transaminase levels and no histological abnormalities were found in animals treated with AdrTgtk/GCV given iv, compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer's cell prominence, and extensive single-cell hepatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by the noncell-specific CMV promoter. XTL-1 cells (2 x 10(6)) were injected sc into BALB/c-severe combined immunodeficient mice (BALB/c-SCID), and the mice developed tumors after 3 wk. After intratumoral injection of AdrTgtk and treatment with GCV, tumors stabilized in 15 of 17 mice within 3 wk, 9 tumors remained stabilized after 5 wk of treatment, and 2 disappeared during observation. In AdCMVLuc/GCV-treated control mice, almost all tumors grew continuously. The average tumor size in AdrTgtk-treated mice was significantly smaller than that of control animals after 2 wk of treatment. Our data confirm the effectiveness and specificity of an adenovirus using rTg promoter to express TK, and support its future application to thyroid cancer gene therapy in humans.

Adenoviral-Mediated Gene Therapy for Thyroid Carcinoma Using Thymidine Kinase Controlled by Thyroglobulin Promoter Demonstrates High Specificity and Low Toxicity

A replication defective adenovirus transducing thymidine kinase (TK) gene under the control of the rat thyroglobulin (rTg) promoter (AdrTgtk) was developed to evaluate its cell-specific killing activity in gene therapy. We also developed adenoviruses containing the TK gene driven by the cytomegalovirus (CMV) promoter (AdCMVtk), and luciferase (Luc) gene driven by the rTg or CMV promoter (AdrTgLuc or AdCMVLuc). Luc activity in FRTL-5, HepG2, COS1, rMTC, hMTC, Hela, GH3, T98G, and CA77 cells was measured after infection with AdrTgLuc or AdCMVLuc. FRTL-5 cells produce thyroglobulin (Tg), whereas all other cells are non-Tg-producing cell lines. Transduction by AdCMVLuc caused high Luc activity in all cell lines. However, infection with AdrTgLuc induced Luc activity only in FRTL-5 cells. AdCMVtk or AdrTgtk was used to transduce various cell lines to evaluate the different killing effect. After infection with AdCMVtk vector followed by ganciclovir (GCV) treatment, cell growth was strongly suppressed in all cell lines compared both to noninfected cells and to cells infected by AdCMVLuc in the presence of GCV. When FRTL-5 cells were infected with AdrTgtk followed by GCV treatment, more than 90% were killed, but only a minimal effect was observed in other cell lines, indicating that the Tg promoter transduced TK expression only in Tg-producing cells. When adenovirus is given intravenously, liver and spleen are the major organs infected. A high Luc activity was found in liver and spleen of AdCMVLuc treated animals. No Luc activity was found in liver and spleen of AdrTgLuc-treated animals, indicating that rTg does not transduce Luc expression in non-Tg-producing tissues in vivo. No significant changes of the serum transaminase levels and histologic abnormalities were found in animals treated with AdrTgtk/GCV compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer's cell prominence, and extensive single cell hypatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by a noncell-specific promoter. These results indicate that transfer of TK gene driven by the rTg promoter has thyroid cell-specific killing ability in the presence of GCV, little in vivo toxicity, and should be useful in the future for treating thyroid Tg-producing cancers.

Long-Term Therapy of Chronic Hepatitis B With Lamivudine

Lamivudine therapy induces improvements in chronic hepatitis B in a high proportion of patients, but prolonged therapy is limited by the development of viral resistance. We analyzed clinical responses and virologic resistance in 27 patients treated continuously with lamivudine for 2 to 4 years. Serum transaminases, hepatitis B virus (HBV) DNA by both branched DNA (bDNA) signal amplification and quantitative polymerase chain reaction were monitored at 4- to 8-week intervals. Virologic resistance to lamivudine was confirmed by the presence of mutations in the YMDD motif of the polymerase gene by restriction fragment-length polymorphism analysis. Serum HBV-DNA levels decreased rapidly in all treated patients, falling by 4 to 5 logs within 1 year. Transaminase levels also decreased and were normal in 70% of patients at 1 year, at which point liver histology had improved in 81% of patients. Viral resistance began to emerge after 8 months of therapy, eventually developing in 14 patients, including 76% of hepatitis B e antigen (HBeAg)-positive patients but only 10% of HBeAg-negative patients. Lamivudine withdrawal led to reappearance of wild-type HBV species, but retreatment led to more rapid reappearance of the mutant virus. Clinical, serum biochemical, and histologic improvements were maintained in the 13 patients who did not develop resistance. Thus, long-term therapy with lamivudine resulted in maintained improvements in virologic, biochemical, and histologic features of disease in most patients with HBeAg-negative chronic hepatitis B and in the subgroup of HBeAg-positive patients with high serum transaminase levels. A high rate of resistance limited efficacy, particularly in patients who remained HBeAg positive on therapy.(Hepatology 2000;32:828-834.)