Assessment of lung ventilation of premature infants with bronchopulmonary dysplasia at 1.5 Tesla using phase-resolved functional lung magnetic resonance imaging.

BACKGROUND: The most common chronic complication of preterm birth is bronchopulmonary dysplasia (BPD), widely referred to as chronic lung disease of prematurity. All current definitions rely on characterizing the disease based on respiratory support level and do not provide full understanding of the underlying cardiopulmonary pathophysiology. OBJECTIVE: To evaluate a rapid functional lung imaging technique in premature infants and to quantitate pulmonary ventilation using 1.5 Tesla magnetic resonance imaging (MRI). MATERIALS AND METHODS: We conducted a prospective MRI study of 12 premature infants in the neonatal intensive care unit (NICU) using the phase resolved functional lung MRI technique to calculate pulmonary ventilation parameters in preterm infants with and without BPD grade 0/1 (n = 6) and grade 2/3 (n = 6). RESULTS: The total ventilation defect percentage showed a significant difference between groups (16.0% IQR (11.0%,18%) BPD grade 2/3 vs. 8.0% IQR (4.5%,9.0%) BPD grade 0/1, p = 0.01). CONCLUSION: Phase-resolved functional lung MRI is feasible for assessment of ventilation defect percentages in preterm infants and shows regional variation in localized lung function in this population.

Characterization of nuclease-resistant ribozymes directed against hepatitis B virus RNA.

Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.

A genotypic association implicates myeloperoxidase in the progression of hepatic fibrosis in chronic hepatitis C virus infection.

The hepatitis C virus (HCV) is a major cause of liver disease and the complications of cirrhosis. Liver biopsies, performed prior to the development of liver cirrhosis, characteristically show an inflammatory cell infiltrate with varying degrees of fibrosis. Precisely how HCV infection induces hepatic fibrogenesis is unknown. Recent studies suggest the release of oxidants, cytokines and proteases from the host immune system are key to the development of fibrosis. Macrophages and neutrophils, cells heavily represented in the inflammatory cell response, contain the oxidant generating enzyme myeloperoxidase (MPO). Cellular levels of MPO can be influenced by a functional promotor polymorphism, -463G/A, which precedes the MPO gene. We examined the relationship between this MPO promotor genotype and the degree of fibrosis in 166 patients with chronic HCV infection. All patients had previously participated in clinical drug trials for the treatment of chronic HCV infection. The MPO genotype was determined from cryo-preserved lymphocytes obtained from patients prior to treatment. The degree of fibrosis was estimated from liver biopsy specimens obtained prior to treatment. We found that patients with the MPO GA/AA genotype were more likely to have advanced fibrosis scores compared with those with the GG genotype: Of the patients with GG genotype, 78% (79 of 102 cases) had lower Knodell Fibrosis scores of 0 or 1, compared to 56% (37 of 64 cases) of patients with GA/AA genotype (P < 0.05). The mechanism(s) by which MPO contributes to fibrosis progression remains to be determined.

Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNA.

We have recently shown that the replication of an HCV-poliovirus (PV) chimera that is dependent upon the hepatitis C virus (HCV) 5' untranslated region (UTR) can be inhibited by treatment with ribozymes targeting HCV RNA. To determine the antiviral effects of anti-HCV ribozyme treatment in combination with type 1 interferon (IFN), we analysed the replication of this HCV-PV chimera in HeLa cells treated with anti-HCV ribozyme and/or IFN-alpha2a, IFN-alpha2b, or consensus IFN. The anti-HCV ribozyme, or any of the IFNs alone have significant inhibitory effects on HCV-PV replication compared to control treatment (> or = 85%, P < 0.01). The maximal inhibition due to IFN treatment (94%, P < 0.01) was achieved with > or = 50 U/ml for either IFN-alpha2a or IFN-alpha2b compared to control treatment. A similar level of inhibition in viral replication could be achieved with a 5-fold lower dose of IFN if ribozyme targeting the HCV 5' UTR was given in combination. For consensus IFN, the dose could be reduced by > 12.5-fold if ribozyme targeting the HCV 5' UTR was given in combination. Conversely, the dose of ribozyme could be reduced 3-fold if given in combination with any of the IFN preparations. Moreover, treatment with low doses (1-25 U/mL) of IFN-alpha2a, IFN-alpha2b, or consensus IFN in combination with anti-HCV ribozyme resulted in > 98% inhibition of HCV-PV replication compared to control treatment (P < 0.01). These results demonstrate that IFN and ribozyme each have a beneficial antiviral effect that is augmented when given in combination.

Surveillance for hepatocellular carcinoma in patients with chronic viral hepatitis in the United States of America.

BACKGROUND: Measurement of serum alpha-fetoprotein (AFP) and abdominal ultrasound (US) examination are used for the early detection of hepatocellular carcinoma (HCC) in chronic liver disease patients. However, the accuracy and usefulness of these tests in a clinical setting in the United States of America have not been clarified. METHODS: We conducted a 7-year prospective surveillance study by using both AFP and US to detect HCC in 602 patients with chronic viral hepatitis. Our main goal was to determine the optimal test for detection of early HCC. We also assessed the clinical outcome of HCC patients identified during this time period. RESULTS: Thirty-one cases of HCC were detected. Serum AFP levels were elevated in 74% of HCC patients, but was also high in 10% of patients who did not develop HCC. The positive predictive value for AFP to detect HCC was only 12% or less for all AFP cut-off values, and the maximum joint sensitivity and specificity as determined by receiver operator characteristic analysis was approximately 65 and 90%, respectively. Abdominal US identified all 31 cases of HCC. The positive predictive value for US examinations to detect HCC was 78%, while the sensitivity and specificity was 100 and 98%, respectively. After detection of HCC, 24 (77%) patients died within a mean of 16.7 +/- 19.4 months. CONCLUSIONS: Our study indicates that US examination was more accurate in detecting HCC. Because of its poor predictive value and low sensitivity, serum AFP should not be used as the only test for screening and surveillance for HCC.

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon.

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

Measurement of serum hyaluronic acid in patients with chronic hepatitis C and its relationship to liver histology. Consensus Interferon Study Group.

BACKGROUND AND AIMS: Chronic hepatitis C is a slowly progressing inflammatory disease of the liver that can lead to cirrhosis and its complications. Assessment of liver damage in hepatitis C has been primarily via histological evaluation. Liver biopsy, while useful in determining the extent of liver damage, has associated costs and places patients at a small but finite risk of bleeding. Studies in small patient populations have identified serum markers shown to correlate with liver histology, including procollogen III peptide and hyaluronic acid (HA). To determine whether serum HA was a reliable predictor of cirrhosis and fibrosis, we examined serum HA concentrations from 486 chronic Hepatitis C virus (HCV) patients. METHODS AND RESULTS: Patients were anti-HCV and HCV RNA positive, with elevated alanine aminotransferase values and underwent a liver biopsy. Sera were obtained at the baseline for HA using radioimmunoassay methodology. Patients with cirrhosis had significantly higher serum HA concentrations compared with non-cirrhotic patients (382+/-31 vs 110+/-9 microg/L respectively, P< 0.001). Patients with fibrosis had significantly higher mean serum HA concentrations (179+/-11 microg/L) compared with patients without fibrosis (62+/-20 microg/L; P< 0.001). The correlation between HA concentration and the components of the Knodell histological activity index score revealed no strong associations with the exception of fibrosis, which showed moderate correlation (R=0.5421, P<0.001). The clinical value of HA measurement appears to be its ability to exclude cirrhosis. A HA value of < 60 microg/L excluded the presence of cirrhosis or significant fibrosis with a predictive value of 99 and 93%, respectively. CONCLUSIONS: Serum HA measurement may be clinically useful to non-invasively assess the degree of fibrosis and cirrhosis. Further prospective studies are warranted to determine the clinical utility of HA as a non-invasive marker of liver fibrosis.

Pharmacokinetics and tissue distribution of a ribozyme directed against hepatitis C virus RNA following subcutaneous or intravenous administration in mice.

A nuclease resistant ribozyme targeting the 5' untranslated region (5' UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [(32)P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)-labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR-labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C.

Acute toxicology and pharmacokinetic assessment of a ribozyme (ANGIOZYME) targeting vascular endothelial growth factor receptor mRNA in the cynomolgus monkey.

The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.

Assessment of hepatitis C virus RNA and genotype from 6807 patients with chronic hepatitis C in the United States.

Hepatitis C virus (HCV) RNA status and HCV genotype have become important tools in the diagnosis and monitoring of therapy in chronic HCV infection. To establish a database with respect to HCV genotype and serum HCV RNA concentrations in chronic hepatitis C patients in the United States, we analysed 6807 chronic hepatitis C patients who had HCV RNA and HCV genotype tests conducted at a central laboratory. The HCV RNA concentration cut-off for the lower 25th percentile of this population (low titre) was 0.9 x 106 copies ml-1. The median HCV RNA concentration was 3.5 x 106 copies ml-1 and the cut-off for the upper 25th percentile (high titre) was 5 x 106 copies ml-1. Male patients had a median HCV RNA concentration of 3.9 x 106 copies ml-1, which was significantly higher than the median HCV RNA level for females (2.75 x 106 copies ml-1; P < 0.001). HCV genotype 1 was detected in 73% of patients; genotype 2 in 14%; genotype 3 in 8%; mixed genotype in 4%; and genotypes 4, 5 and 6 with a frequency of < 1%. Patients from the Northeast, Southeast and Midwest had significantly (P < 0.001) more infections with genotype 1 than patients from the Western and Southern regions. African-American patients were more likely to be infected with genotype 1 when compared with Caucasian, Hispanic or Asian Pacific Islanders (P < 0.001). Patients infected with HCV genotype 1 and mixed HCV genotypes had significantly higher serum HCV RNA concentrations when compared with HCV genotypes 2 and 3 (P < 0.001 for all comparisons).

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.

The effects of interferon on the expression of human papillomavirus oncogenes.

The effects of interferon (IFN)-alpha, IFN-beta and IFN-gamma on human papillomavirus (HPV) oncogene expression were studied in various cervical carcinoma cell lines containing integrated copies of either HPV type 16 or HPV type 18. The levels of E6 and E7 transcripts were examined 6 h and 30 h after treatment with IFN. In HeLa cells, all three classes of IFNs effected a decrease in the level of HPV-18 E6 and E7 transcripts. On the other hand, none of the IFNs altered the level of these transcripts in C-4II cells. Only IFN-gamma decreased the level of HPV-16 E6 and E7 transcripts in CaSki and HPK1A cells, while IFN-gamma actually increased the level of these transcripts in SiHa cells. This differential IFN regulation of HPV expression in various cervical cancer cell lines may account for the contradictory clinical results observed after treatment of cervical cancer with IFN.

Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers.

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME) targeting the Flt-1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo-controlled, single-dose escalation study, ribozyme was administered as a 4-hour i.v. infusion of 10 or 30 mg/m2 or as a s.c. bolus of 20 mg/m2. Peak ribozyme plasma concentrations of 1.5 and 3.8 micrograms/mL were observed after the 10 and 30 mg/m2 i.v. infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m2. Peak concentrations of 0.9 microgram/mL were observed approximately 3.25 hours after a 20 mg/m2 s.c. bolus of ribozyme. The dose-normalized AUCs obtained after s.c. dosing were compared to the mean dose-normalized AUC after i.v. dosing to estimate an absolute s.c. bioavailability (f) of approximately 69%. An average elimination half-life of 28 to 40 minutes was observed after i.v. administration, which increased to 209 minutes after s.c. administration. Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4-hour i.v. infusion of up to 30 mg/m2 or a single s.c. bolus of 20 mg/m2.

Patients co-infected with human immunodeficiency virus and hepatitis C virus demonstrate higher levels of hepatic HCV RNA.

Serum and liver hepatitis C virus (HCV) RNA levels in patients with hepatitis C have previously been quantified using different techniques. In this work, we used an automated, multicycle, polymerase chain reaction (PCR)-based technique to quantify HCV RNA in 1-2 mm of frozen liver tissue, and in serum, from 70 patients with antibodies to HCV (anti-HCV), with and without human immunodeficiency virus (HIV) co-infection. Stored liver tissue and sera collected at the time of liver biopsy were used for measurement of HCV RNA. Forty-eight HCV patients and 22 HIV/HCV co-infected patients were studied. Co-infected patients had significantly higher median serum and liver HCV RNA (6.7 log copies ml-1 serum and 2.90 log copies microg-1 liver nucleic acids) than patients with HCV alone (6.2 log copies ml-1 serum and 2.19 log copies microg-1 liver nucleic acids). There was only a weak correlation between serum and liver HCV RNA (r = 0.43). There was no correlation between liver and serum HCV RNA and host factors such as duration of disease, CD4 counts, alanine aminotransferase levels or histological score. There was no correlation with HCV genotype. Co-infected patients were more likely to harbour HCV genotype 1 (85%) when compared to patients with HCV alone (58%). An identical genotype was found in liver and serum in 89% of those tested; in 11%, a mixed genotype was present in serum. Patients with HCV genotypes 1 and non-1 had similar histological scores. Hence, an automated PCR-based technique is useful for measuring both liver and serum HCV RNA. Serum HCV genotypes closely paralleled those found in liver tissue. HIV co-infection was associated with higher serum, as well as intrahepatic, HCV RNA levels, by mechanisms not directly related to CD4 counts. The lack of correlation between liver HCV RNA and histology suggests that HCV is not directly cytopathic.

A pilot study of daily subcutaneous interleukin-10 in patients with chronic hepatitis C infection.

The Th1/Th2 cytokine balance is important in persistence of infection and liver injury in chronic hepatitis C. The aim of this study was to administer the anti-inflammatory cytokine, recombinant human interleukin-10 (rHuIL-10), for 28 days in patients with chronic hepatitis C and to assess the safety and measure the effect on alanine aminotransferase (ALT, a marker of hepatic inflammation) levels and serum hepatitis C virus (HCV) RNA values. Three treatment-naive and 13 interferon (IFN) nonresponder patients (total 16 patients) with compensated chronic HCV infection were enrolled in this study. Patients were randomized to receive rHuIL-10 at a dose of 4 or 8 microg/kg/day as a single daily subcutaneous injection for 28 days. ALT values and serum HCV RNA were measured at days 0, 1, 3, 8, 15, 22, and 28 during therapy and at follow-up 2 and 4 weeks after cessation of the 4-week treatment period. ALT values normalized in 9 of 16 patients during therapy and remained normal until the end of treatment in 8 patients. The decreases in ALT values occurred in both the 4 microg and 8 microg dosage groups and were seen in both IFN naive and nonresponder patients. Mean ALT values fell significantly during the study period but usually returned to pretreatment levels by the end of the 4-week follow-up period (p < 0.05). HCV RNA concentrations did not vary significantly during or after therapy. (No patient had either an increase or a decrease in HCV RNA levels of > or =1.5 log during the study.) The drug was well tolerated, with no adverse symptoms noted. Platelet counts fell transiently to 73,000 and 63,000 in 2 patients. No other toxicity was observed, and no patients discontinued therapy. In chronic hepatitis C, short-term therapy with IL-10 was well tolerated and caused transient normalization of ALT values in 50% of patients, which returned to pretreatment levels on cessation of treatment. There were no significant changes observed in serum HCV RNA concentrations during the study. These immunomodulatory effects are similar to those observed with ribavirin monotherapy in chronic hepatitis C. Further study of rHuIL-10 alone or in combination with antiviral agents in chronic hepatitis C is warranted.

A predictive model for the development of hepatocellular carcinoma, liver failure, or liver transplantation for patients presenting to clinic with chronic hepatitis C.

OBJECTIVE: Chronic infection with hepatitis C may lead to the development of cirrhosis, liver failure, and hepatocellular carcinoma. However, not all patients progress to these endpoints. Ideally, clinicians could improve their capability of stratifying the risk and the time frame within which their patients will progress to these endpoints. The purpose of the present study was to construct statistical models predicting disease progression for individual patients. METHODS: Study endpoints were the development of hepatocellular carcinoma, liver transplantation, or death due to liver disease. The study cohort was 256 patients with hepatitis C acquired from either blood transfusion or use of intravenous drugs. During follow-up, 17 patients developed hepatocellular carcinoma, seven received liver transplantation, and 12 died from liver disease. RESULTS: On multivariate analysis a history of decompensation (relative risk [RR] 4.321, 95% confidence interval [CI] 1.777-10.511) and the serum albumin (RR 0.253, 95% CI 0.136-0.474) were independently associated with the study endpoints. Patients without a history of decompensation and with a serum albumin > or = 4.1 mg/dl had a 3.2% chance of developing the study endpoints within 5 yr. Patients with a history of decompensation and a serum albumin < 4.1 mg/dl had a 40% chance of developing a study endpoint within 5 yr. Baseline genotype and quantitative RNA were not associated with development of the clinical endpoints, with the exception of patients coinfected with two or more genotypes. CONCLUSION: Thus, the serum albumin and a history of decompensation are useful for predicting the development of hepatocellular carcinoma, liver transplantation, and death due to liver disease among patients with hepatitis C.

Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection.

The ability to predict accurately a sustained response during therapy in patients with hepatitis C virus (HCV) infection is unavailable. The aim of this study was to differentiate, during therapy, patients who would relapse from those with a sustained response by ultracentrifugation for residual serum HCV RNA. Sixty-one specimens (from 32 patients) collected during interferon therapy were assessed by ultracentrifugation. All were negative using a quantitative polymerase chain reaction (PCR) (detection limit < or = 100 copies/ml). One-milliliter aliquots were ultracentrifuged at 23,000 x g (160 min), and then the nucleic acid pellet was extracted, precipitated, and resuspended. Qualitative PCR was carried out in quadruplicate using two separate 5'UTR primer sets (8 results/specimen). A specimen was positive if > or = 1 gels was positive compared to controls. At weeks 12 and 24, 9/9 (100%) sustained response patients were negative by ultracentrifugation. In the 23 relapse patients at week 12, 7/12 specimens were positive; at week 24, 7/14 were positive. Earlier time points could not differentiate the patients' eventual response to therapy. The predictive value of a positive ultracentrifugation test for relapse at week 12 or 24 was 100%. The predictive value of a negative test for sustained response was 62% and 50% at week 12 and 24, respectively. These preliminary results indicate that patients with an eventual sustained response will have no detectable serum HCV RNA by week 12 or week 24. A positive result is 100% predictive of relapse.

Working with families in reaching end-of-life decisions.

This article provides the reader with a historical perspective of advance directives, the role of surrogate decision making, and the difficulties families encounter surrounding end-of-life decisions. Illustrated with a case study, it includes a framework for advanced practice nurses (APNs) and other health care professionals to assist families in reaching decisions. The APN can be instrumental in initiating an ethical discussion about resuscitation decisions and assist in dealing with conflicts. The APN can strive to make Do Not Resuscitate (DNR) discussions a consistent part of the hospital experience. It is hoped that the use of such a framework will improve care at the end of life.