Michelle M. Richardson

Non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling 3 (SOCS-3) in patients with chronic hepatitis C, viral genotype 1

Background: Interferon α (IFN-α) activated cellular signalling is negatively regulated by inhibitory factors, including the suppressor of cytokine signalling (SOCS) family. The effects of host factors such as obesity on hepatic expression of these inhibitory factors in subjects with chronic hepatitis C virus (HCV) are unknown. Objectives: To assess the independent effects of obesity, insulin resistance, and steatosis on response to IFN-α therapy and to determine hepatic expression of factors inhibiting IFN-α signalling in obese and non-obese subjects with chronic HCV. Methods: A total of 145 subjects were analysed to determine host factors associated with non-response to antiviral therapy. Treatment comprised IFN-α or peginterferon alpha, either alone or in combination with ribavirin. In a separate cohort of 73 patients, real time-polymerase chain reaction was performed to analyse hepatic mRNA expression. Immunohistochemistry for SOCS-3 was performed on liver biopsy samples from 38 patients with viral genotype 1 who had received antiviral treatment. Results: Non-response (NR) to treatment occurred in 55% of patients with HCV genotypes 1 or 4 and 22% with genotypes 2 or 3. Factors independently associated with NR were viral genotype 1/4 (p<0.001), cirrhosis on pretreatment biopsy (p = 0.025), and body mass index ⩾ 30 kg/m2 (p = 0.010). Obese subjects with viral genotype 1 had increased hepatic mRNA expression of phosphoenolpyruvate carboxy kinase (p = 0.01) and SOCS-3 (p = 0.047), in comparison with lean subjects. Following multivariate analysis, SOCS-3 mRNA expression remained independently associated with obesity (p = 0.023). SOCS-3 immunoreactivity was significantly increased in obesity (p = 0.013) and in non-responders compared with responders (p = 0.014). Conclusions: In patients with chronic HCV viral genotype 1, increased expression of factors that inhibit interferon signalling may be one mechanism by which obesity reduces the biological response to IFN-α.

Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis

The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV‐infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki‐67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases. (HEPATOLOGY 2005;41:809–818.)

Comparison of Drug Dosing Recommendations Based on Measured GFR and Kidney Function Estimating Equations

BACKGROUND Kidney disease alters the pharmacokinetic disposition of many medications, requiring dosage adjustment to maintain therapeutic serum concentrations. The Cockcroft-Gault (CG) equation is used for pharmacokinetic studies and drug dosage adjustments, but the Modification of Diet in Renal Disease (MDRD) Study equation is more accurate and more often reported by clinical laboratories than the CG equation. STUDY DESIGN Diagnostic test study. SETTINGS & PARTICIPANTS Pooled data set for 5,504 participants from 6 research studies and 4 clinical populations with measured glomerular filtration rate (GFR). INDEX TEST Estimated kidney function using the MDRD Study and CG equations incorporating actual (CG) or ideal body weight (CG(IBW)) and standardized serum creatinine concentrations. REFERENCE TEST Measured GFR assessed by using iodine-125-iothalamate urinary clearance. OUTCOME Concordance of assigned kidney function categories designated by the Food and Drug Administration (FDA) Guidance for Industry for pharmacokinetic studies and recommended dosages of 15 medications cleared by the kidneys. RESULTS Concordance of kidney function estimates with measured GFR for FDA-assigned kidney function categories was 78% for the MDRD Study equation compared with 73% for the CG equation (P < 0.001) and 66% for the CG(IBW) equation (P < 0.001). Concordance between the MDRD Study equation and CG and CG(IBW) equations was 78% and 75%, respectively (P < 0.001). Concordance of kidney function estimates with measured GFR for recommended drug dosages was 88% for MDRD Study equation compared with 85% for the CG equation (P < 0.001) and 82% for the CG(IBW) equation (P < 0.001), with lower concordance when dosing recommendations for drugs included narrow GFR ranges. Concordance rates between the CG and CG(IBW) equations and MDRD Study equation were 89% and 88%, respectively (P < 0.05). LIMITATIONS Results based on simulation rather than pharmacokinetic studies. Outcome was drug dosage recommendations, rather than observed drug efficacy and safety. CONCLUSIONS The MDRD Study equation can also be used for pharmacokinetic studies and drug dosage adjustments. As more accurate GFR-estimating equations are developed, they should be used for these purposes.

Steatosis and liver cell apoptosis in chronic hepatitis C: a mechanism for increased liver injury.

Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis C; however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl‐2, Bcl‐xL, Bax, and tumor necrosis factor alpha (TNF‐α) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r = 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl‐2 mRNA levels and an increase in the proapoptotic Bax/Bcl‐2 ratio (r = −0.32, P = .007; and r = 0.27, P = .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r = 0.26, P = .11; r = 0.06, P = .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r = 0.35, P = .047; r = 0.33, P = .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF‐α mRNA levels and active caspase‐3 (r = 0.54, P = .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis. (HEPATOLOGY 2004.39:1230‐1238.)

A combination of genetic polymorphisms increases the risk of progressive disease in chronic hepatitis C

Background: There is increasing interest in the influence of host genetic factors on hepatic fibrosis, and whether genetic markers can reliably identify subjects at risk of developing severe disease. We hypothesised that hepatitis C virus (HCV) infected subjects with progressive fibrosis, classified using strict criteria based on histology at biopsy in addition to disease duration would be more likely to inherit several genetic polymorphisms associated with disease progression compared with subjects with a low rate of disease progression. Methods: We examined polymorphisms in eight genes that have been reported to have an association with hepatic fibrosis. Results: Associations between polymorphisms in six genes and more rapidly progressing fibrosis were observed, with individual adjusted odds ratios ranging from 2.1 to 4.5. The relationship between rapidly progressing fibrosis and possession of ⩾3, ⩾4, or ⩾5 progression associated alleles was determined and the adjusted odds ratios increased with increasing number of progression associated alleles (9.1, 15.5, and 24.1, respectively). Using logistic regression analysis, a predictive equation was developed and tested using a second cohort of patients with rapidly progressing fibrosis. The predictive equation correctly classified 80% of patients in this second cohort. Conclusions: This approach may allow determination of a genetic profile predictive of rapid disease progression in HCV and identify patients warranting more aggressive therapeutic management.

Temporal Trends in Health-Related Quality of Life among Hemodialysis Patients in the United States

BACKGROUND AND OBJECTIVES Health-related quality of life (HRQOL) is a measure of the well being of hemodialysis patients and an independent prognostic predictor. Our aim was to determine whether HRQOL among hemodialysis patients has changed over time. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We retrospectively analyzed data collected by Dialysis Clinic, Inc. from adult patients starting hemodialysis between January 1, 1997 and May 31, 2006. The primary outcome was HRQOL assessed by Short Form 36, 6 to 18 months after and closest to the 1-year anniversary of starting hemodialysis. Secular trends were analyzed by linear regression for continuous variables and logistic regression for categorical ones. Year of starting dialysis was the predictor. A five-point difference on a 0 to 100 scale was considered clinically significant. RESULTS Short Form 36 scores were available for 11,079 patients. Role physical, general health, vitality, social functioning, and physical component summary scores were unchanged among patients over the study period. Statistically significant (P < 0.05) but clinically insignificant changes were observed in physical functioning (-0.2 points/yr), bodily pain (+0.2 points/yr), mental health (+0.15 points/yr), and mental component summary scores (+0.13 points/yr). Only role emotional showed clinically significant improvement. Trends were unchanged after adjusting for age, gender, race, diabetes, hemoglobin, phosphorous, Kt/V, and albumin. CONCLUSIONS Most HRQOL domains showed either no statistically significant change or statistically but not clinically significant change over almost a decade. These results suggest that, despite important developments in hemodialysis care since 1997, little progress was made in improving HRQOL of hemodialysis patients.

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL‐12p70, through which they play a direct role in the development of IFN‐γ‐secreting Th1 cells, costimulation of CTL differentiation and NK‐cell activation. In contrast, IL‐10, which is also produced by DC, negatively regulates IL‐12 production. IL‐12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL‐12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL‐12p70 secretion by monocyte‐derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL‐12p70, presence of the IL10 high genotype restricted IL‐12p70 production in these individuals. These observations provide a further genetic control of IL‐12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

Recovery and identification of bacterial DNA from illicit drugs

Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug users as well as enabling forensic links between seizures to be examined.

Testicular germ cell tumors exhibit evidence of hormone dependence.

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone‐dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin‐releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real‐time RT‐PCR analysis was used to determine the expression profiles of hormone pathway‐associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real‐time RT‐PCR analysis of follicle‐stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone‐related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model.

Precision versus Approximation: The Trade Off in Assessing Kidney Function and Drug Dosing

Aristotle(384–322BC)Caringforpatientsrequiresdata. Somedataderivefrommeasurement(i.e.,precision),somefrom estimates (i.e., an approximation), andsomefromexperienceorjudgment. Whengiventhechoicebetweenameasurementandanestimate,clinicianspreferthemeasuredvalue. However,itisoftennotfeasibletoobtainameasurement. Inthosecases,anestimateorapproximationispre-ferredtofaultyinformationortonoinformationat all. These principles apply to evaluatingkidneyfunction.Thegoldstandardtoevaluatekidneyfunctionhaslongbeentomeasureglomerularfiltrationrate(GFR)usingafiltrationmarker(e.g.,inulin,iothalamate). However,measuringGFRisnotclinically feasible. It requires expertise andspecialtrainingtoperform,istimeconsuming,andisexpensive. Surrogatemarkershavebeenusedtocompensatefortheinherentdifficultiesin measuring GFR. Serum creatinine (S