Doppler Ultrasonography of the Central Retinal Vessels in Children With Brain Death.

OBJECTIVE: The purpose of this observational study is to explore if bedside Doppler ultrasonography of the central retinal vessels has the potential to become an ancillary study to support the timely diagnosis of brain death in children. DESIGN: Seventeen-month prospective observational cohort. SETTING: Forty-four bed pediatric medical and surgical ICU in an academic teaching hospital. PATIENTS: All children 0-18 years old who were clinically evaluated for brain death at Children's National Health Systems were enrolled and followed until discharge or death. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: All patients had at least one ophthalmic ultrasound within 30 minutes of each brain death examination. The central retinal artery peak systolic blood flow velocity, resistive index, pulsatility index, and Doppler waveforms were evaluated in each patient. Thirty-five ophthalmic ultrasounds were obtained on 13 patients, 3 months to 15 years old, who each had two clinical examinations consistent with brain death. The average systolic blood pressure during the ultrasound examinations was 102 mm Hg (± 28), diastolic blood pressure 65 mm Hg (± 24), mean arterial pressure 79 mm Hg (± 23), heart rate 133 beats/min (± 27), temperature 36°C (± 0.96), arterial CO2 35 mm Hg (± 9), and end-tidal CO2 23 mm Hg (± 6). For all examinations, the average peak systolic velocity of the central retinal artery was significantly decreased at 4.66 cm/s (± 3.2). Twelve of 13 patients had both resistive indexes greater than or equal to 1, average pulsatility indexes of 3.6 (± 3.5) with transcranial Doppler waveforms consistent with brain death. Waveform analysis of the 35 ultrasound examinations revealed 11% with tall systolic peaks without diastolic flow, 17% with oscillatory flow, 29% showed short systolic spikes, and 23% had no Doppler movement detected. A rippling "tardus-parvus" waveform was present in 20% of examinations. CONCLUSION: This study supports that the combination of qualitative waveform analysis and quantitative blood flow variables of the central retinal vessels may have the potential to be developed as an ancillary study for supporting the diagnosis of brain death in children.

Comparative efficacy, pharmacokinetic, pharmacodynamic activity, and interferon stimulated gene expression of different interferon formulations in HIV/HCV genotype-1 infected patients.

The effect of different formulations of interferon on therapeutic response in patients coinfected with HIV and HCV is unclear. In this study, the safety, tolerability, viral kinetics (VK) modeling and host responses among HIV/HCV coinfected patients treated with pegylated-IFN or albinterferon alfa-2b (AlbIFN) with weight-based ribavirin were compared. Three trials treated 57 HIV/HCV coinfected genotype-1 patients with PegIFN alfa-2b (1.5 µg/kg/week) (n = 30), PegIFN alfa-2a (180 µg/week) (n = 10), and AlbIFN (900 µg/q2week) (n = 17) in combination with weight-based ribavirin (RBV). HCV RNA, safety labs, and interferon stimulated gene expression (ISG) was evaluated. Adverse events were documented at all study visits. HCV viral kinetics using a full pharmacokinetic/pharmacodynamic model was also evaluated. Baseline patient characteristics were similar across the three studies. All three formulations exhibited comparable safety and tolerability profiles and efficacy. VK/PK/PD parameters for all three studies as measured by mean efficiency and rate of infected cell loss were similar between the three groups. Host responses (ISG expression and immune activation markers) were similar among the three groups. All three regimens induced significant ISG at week 4 (P < 0.05) and ISG expression strongly correlated with therapeutic response (r = 0.65; P < 0.01). In summary, a comprehensive analysis of responses to three different interferon formulations in HIV/HCV coinfected patients demonstrated similar effects. Notably, interferon-based therapy results in a blunted host response followed by modest antiviral effect in HIV/HCV coinfected patients. This suggests that future treatment options that do not rely on host immune responses such as direct antiviral agents would be particularly beneficial in these difficult to treat patients.

Dysregulation of innate immunity in hepatitis C virus genotype 1 IL28B-unfavorable genotype patients: impaired viral kinetics and therapeutic response.

UNLABELLED: Recent studies have shown that a single-nucleotide polymorphism upstream of the interleukin-28B (IL28B) gene plays a major role in predicting therapeutic response in hepatitis C virus (HCV)-infected patients treated with pegylated interferon (PEG-IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics, IFN pharmacokinetics, IFN pharmacodynamics, and gene expression profiles. Two prospective cohorts (human immunodeficiency virus [HIV]/HCV-coinfected and HCV-monoinfected) completing treatment with IFN/ribavirin were enrolled. Patients were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort, frequent serum sampling was completed for HCV RNA and IFN levels. DNA microarray of peripheral blood mononuclear cells and individual expression of IFN-stimulated genes (ISGs) were quantified on IFN therapy. The IL28B-favorable (CC) genotype was associated with improved therapeutic response compared with unfavorable (CT or TT) genotypes. Patients with a favorable genotype had greater first- and second-phase viral kinetics (P = 0.004 and P = 0.036, respectively), IFN maximum antiviral efficiency (P = 0.007) and infected cell death loss (P = 0.009) compared with unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of natural killer cells, from patients with unfavorable genotypes (P <0.004). Induction of innate immunity genes was also lower in unfavorable genotypes. ISG expression at baseline and induction with IFN was independent of IL28B genotype. CONCLUSION: Carriers of the IL28B-favorable genotype were more likely to have superior innate immune response to IFN therapy compared with unfavorable genotypes, suggesting that the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of ISG expression.

High interferon-stimulated gene ISG-15 expression affects HCV treatment outcome in patients co-infected with HIV and HCV.

Increased baseline expression and lack of induction of interferon-stimulated genes (ISG) are strong negative predictors of therapeutic response to PegIFN/RBV in patients co-infected with HIV and hepatitis C virus (HCV). This study specifically addressed whether ISG-15 expression influences therapeutic responses in 20 HIV/HCV genotype-1 subjects undergoing HCV treatment. Non-responders had significantly higher baseline expression and selective induction of ISG-15 after IFN-α treatment relative to participants with sustained virological response. High baseline levels of ISG-15 were also associated with less induction of ISG with treatment. These results support a role for ISG-15 as a prognostic indicator and resistance factor to IFN-α.

Sofosbuvir and ribavirin for hepatitis C genotype 1 in patients with unfavorable treatment characteristics: a randomized clinical trial.

IMPORTANCE: The efficacy of directly acting antiviral agents in interferon-free regimens for the treatment of chronic hepatitis C infections needs to be evaluated in different populations. OBJECTIVE: To determine the efficacy and safety of sofosbuvir with weight-based or low-dose ribavirin among a population with unfavorable treatment characteristics. DESIGN, SETTING, AND PATIENTS: Single-center, randomized, 2-part, open-label phase 2 study involving 60 treatment-naive patients with hepatitis C virus (HCV) genotype 1 enrolled at the National Institutes of Health (October 2011-April 2012). INTERVENTIONS: In the study's first part, 10 participants with early to moderate liver fibrosis were treated with 400 mg/d of sofosbuvir and weight-based ribavirin for 24 weeks. In the second part, 50 participants with all stages of liver fibrosis were randomized 1:1 to receive 400 mg of sofosbuvir with either weight-based or low-dose 600 mg/d of ribavirin for 24 weeks. MAIN OUTCOMES AND MEASURES: The primary study end point was the proportion of participants with undetectable HCV viral load 24 weeks after treatment completion (sustained virologic response of 24 weeks [SVR24]). RESULTS: In the first part of the study, 9 participants (90%; 95% CI, 55%-100%) achieved SVR24. In the second part, 7 participants (28%) in the weight-based group and 10 (40%) in the low-dose group relapsed after treatment completion leading to SVR24 rates of 68% (95% CI, 46%-85%) in the weight-based group and 48% (95% CI, 28%-69%; P = .20) in the low-dose group. Twenty individuals participated in a pharmacokinetic-viral kinetic substudy, which demonstrated a slower loss rate of infectious virus in relapsers than in participants who achieved SVR (clearance, 3.57/d vs 5.60/d; P = .009). The most frequent adverse events were headache, anemia, fatigue, and nausea. There were 7 grade 3 events including anemia, neutropenia, nausea, hypophosphatemia, and cholelithiasis or pancreatitis. No one discontinued treatment due to adverse events. CONCLUSION AND RELEVANCE: In a population of patients with a high prevalence of unfavorable traditional predictors of treatment response, a 24-week regimen of sofosbuvir and weight-based or low-dose ribavirin resulted in SVR24 rates of 68% and 48%, respectively. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01441180.

HCC surveillance results in earlier HCC detection: results from an Indian cohort.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide, with an increased incidence in South Asia. In order to describe the effect of surveillance for HCC with biannual ultrasound and alpha-fetoprotein (AFP) on diagnosis and survival in an Indian population a retrospective cohort-control study was performed at two liver clinics in India. The medical records of 3,258 patients with cirrhosis who received surveillance for HCC were reviewed, and 100 patients who developed HCC identified. Sixty-four cirrhotic patients diagnosed with HCC during the same time period without a history of surveillance were included and survival, BCLC stage at diagnosis, and treatment were compared. Patients who underwent surveillance were more likely to be diagnosed with potentially curable or treatable BCLC Stage 0/A disease and Stage B/C disease respectively, than late Stage D disease (χ2 = 0.0007). Patients diagnosed at an earlier stage of HCC lived significantly longer after diagnosis than patients diagnosed at a later stage (Stage 0/A: 15.6 ± 14.2 months vs. Stage B/C: 9.43 ± 19.7 months vs. Stage D: 5.59 ± 11.9 months; p = 0.0006). While treatment for HCC improved overall survival, only 28% of eligible patients received treatment, explaining the lack of survival benefit noted in the surveillance group. Surveillance for HCC led to detection of HCC at earlier stages. The impact of surveillance on improved mortality could not be evaluated given the limited number of patients who received treatment. HCC surveillance has the potential to improve survival in South Asian patients with cirrhosis only if improvements in access to appropriate treatment are made.

Short communication: Interferon/ribavirin treatment for HCV is associated with the development of hypophosphatemia in HIV/hepatitis C virus-coinfected patients.

One-third of all HIV-infected individuals in the United States are estimated to be coinfected with the hepatitis C virus (HCV). Treatment of chronic hepatitis C in patients coinfected with HIV is a complex problem associated with toxicities and drug interactions between HIV antiretrovirals and interferon and ribavirin. In recent HCV treatment studies, we observed a previously unreported development of hypophosphatemia in HIV/HCV-coinfected patients treated with interferon/ribavirin (IFN/RBV). To further investigate this observation, we retrospectively reviewed 61 HIV/HCV-coinfected patients on antiretrovirals (ARVs) during treatment with IFN/RBV as well as 154 HIV-infected patients treated with ARVs alone. We found that HIV/HCV-coinfected patients on IFN/RBV therapy were more likely to develop frequent (57% vs. 13%, IFN/RBV-treated patients vs. no IFN/RBV; χ(2)=0.001) and higher-grade hypophosphatemia (67.0% Grade 2, 33.3% Grade 3 vs. 94.7% Grade 2, 5.3% Grade 3, IFN/RBV-treated patients vs. no IFN/RBV; χ(2)<0.001) than untreated patients. In addition, we found that the new onset of hypophosphatemia after IFN/RBV treatment initiation was followed by a diminished frequency of this toxicity upon cessation of IFN/RBV, supporting the idea that a drug-drug interaction may increase the risk of this toxicity. To understand the risks of developing this toxicity, we evaluated the association between individual ARV use and hypophosphatemia incidence. Our data suggest that concomitant tenofovir (TDF) use may be a risk factor for the development of hypophosphatemia in HIV/HCV-coinfected patients treated with IFN/RBV. Although the etiology of this abnormality is likely multifactorial, clinicians should be aware of hypophosphatemia as a potential marker of renal toxicity in HIV/HCV-coinfected patients being treated with IFN/RBV regimens.

Analysis of Notch and TGF-ß Signaling Expression in Different Stages of Disease Progression During Hepatitis B Virus Infection.

OBJECTIVES: CD4+ regulatory T cells (Tregs) seem to have a key role in persistence of hepatitis B virus (HBV) infection. Notch and transforming growth factor (TGF-ß) signaling independently help in the differentiation and regulation of CD4+T cells, including T-helper (T(H)) 1, T(H)2, and Tregs. Whether, the two pathways have modulatory role on different stages of HBV infection and severity of liver disease is not clear. We investigated Notch and TGF-ß families' gene expression in peripheral blood and intrahepatic lymphocytes in patients with different stages of chronic HBV (CHB) infection. METHODS: Peripheral blood mononuclear cells (PBMCs), CD4(+), and CD8(+) T cells were isolated from patients with acute HBV (AVH-B, n=15), CHB (n=16), and controls (HC, n=10). In addition to PBMCs, intrahepatic lymphocytes were obtained from liver biopsies from CHB (n=12), cirrhosis (n=12), hepatocellular carcinoma (HCC, n=5), and healthy livers (n=5). Notch family (Notch1-4, Hes1, Jag1, and NF-kß) and TGF-ß family gene expressions were studied by real-time PCR, flow cytometry, and immunohistochemistry. RESULTS: Relative expression of Notch signaling target genes, Hes1 and NF-kß, was higher in the total PBMCs of AVH-B and CHB patients than that in HC patients (Log relative quantification (RQ); 1.1 AVH-B vs. 0.3 HC, 1.3 CHB vs. 0.3 HC; P=0.02). CD8(+) T cells showed upregulated expression of Hes1 and Notch1 (P=0.02 and 0.01, respectively) in AVH-B than in CHB patients. Also, in AVH-B patients, HBV-specific CD8(+) T-cell proliferation (5.74% vs. 2.7%) and TGF-ß signaling activity were higher. All Notch receptors and ligands were upregulated in the PBMCs in CHB infection (CHB vs. cirrhosis, P=0.001; CHB vs. HCC, P=0.023; and cirrhosis vs. HCC, P=NS). Intrahepatic expression of Notch1 and FoxP3 were significantly higher in cirrhotics and HCCs, and further blockage of Notch signaling reduced the FoxP3 expression. Array data of TGF-ß family showed increased TGF-ß3, TGF-α, SMAD3, SMAD4, SMAD6, and GDF9 expression on intrahepatic lymphocytes in cirrhotic and HCC patients compared with CHB. CONCLUSIONS: Our findings suggest that there is a complementary association between Notch1 and Hes1 in CD8(+)T cells during AVH-B infection. On development of CHB infection, repression of the Notch receptors mediates the regulation of immune response in patients, who progress to cirrhosis and HCC. Finally, HBV infection drives increased Notch1, TGF-ß, and FoxP3 expression on intrahepatic T cells in cirrhosis, resulting in fibrogenesis and disease progression.