HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Undetectable HCV-RNA at treatment-week 8 results in high-sustained virological response in HCV G1 treatment-experienced patients with advanced liver disease: the International Italian/Spanish Boceprevir/Peginterferon/Ribavirin Name Patients Program.

In many countries, first-generation protease inhibitors (PIs)/peginterferon/ribavirin (P/R) still represent the only treatment option for HCV-infected patients. Subjects with advanced disease and previous failure to P/R urgently need therapy, but they are under-represented in clinical trials. All treatment-experienced F3/4 Metavir patients who received boceprevir (BOC)+P/R in the Italian-Spanish Name Patient Program have been included in this study. Multivariate logistic regression analysis (MLR) was used to identify baseline and on-treatment predictors of SVR and adverse events (AEs). Four hundred and sixteen patients, mean age 57.7 (range 25-78 years), 70% males, 69.5% (289/416) F4, 14% (41/289) Child-Pugh class A6, 24% (70/289) with varices and 42% (173/416) prior null responders to P/R, were analysed. Overall, SVR rate (all 381 patients who received one dose of BOC) was 49%, (58% in F3, 45% in F4, 61% in relapsers, 51% in partial, 38% in null responders, and 72% in subjects with undetectable HCV-RNA at treatment-week (TW)8. Among patients with TW8 HCV-RNA ≥ 1000 IU/L, SVR was 8% (negative predictive value = 92%). Death occurred in 3 (0.8%) patients, while decompensation and infections were observed in 2.9% and 11%, respectively. At MLR, SVR predictors were TW4 HCV-RNA ≥ 1log10 -decline from baseline, undetectable TW8 HCV-RNA, prior relapse, albumin levels ≥3.5 g/dL and platelet counts ≥100 000/µL. Metavir F4, Child-Pugh A6, albumin, platelets, age and female gender were associated with serious and haematological AEs. Among treatment-experienced patients with advanced liver disease eligible for IFN-based therapy, TW8 HCV-RNA characterised the subset with either high or poor likelihood of achieving SVR. Using TW8 HCV-RNA as a futility rule, BOC/P/R appears to have a favourable benefit-risk profile.

Bone marrow-derived cell mobilization by G-CSF to enhance osseointegration of bone substitute in high tibial osteotomy.

PURPOSE: To evaluate granulocyte colony-stimulating factor (G-CSF) efficacy in accelerating bone regeneration following opening-wedge high tibial valgus osteotomy for genu varum. METHODS: A phase II trial was conducted for evaluating the preoperative administration of G-CSF given at 10 µg/kg/day for 3 consecutive days with an additional half-dose 4 h before the opening-wedge high tibial valgus osteotomy. Overall, 12 patients (Group A) received G-CSF treatment, and the subsequent 12 patients (Group B) underwent surgery without G-CSF. The osteotomy gap was filled by a bone graft substitute. Bone marrow cell (BMC) mobilization was monitored by CD34+ve cell and clonogenic progenitor cell analysis. All patients underwent a clinical (Lysholm Knee Scale and SF-36) and radiographic evaluation preoperatively, as well as at given intervals postsurgery. RESULTS: All patients completed the treatment program without major side effects; G-CSF was well tolerated. BMC mobilization occurred in all Group A patients, with median peak values of circulating CD34+ve cells of 110/µL (range 29-256). Circulating clonogenic progenitors paralleled CD34+ve cell levels. A significant improvement in Lysholm Knee Scale was recorded at follow-up in Group A compared to Group B. At the radiographic evaluation, there was a significant increase in osseointegration at the bone-graft junction in Group A at 1, 2, 3 and 6 months postsurgery compared to Group B. The computerized tomography scan of the grafted area at 2 months postsurgery showed no significant difference in the quality of the newly formed bone between the two Groups. CONCLUSIONS: Although the limited number of patients does not allow firm conclusions, the study suggests that G-CSF can be safely administered preoperatively in subjects undergoing opening-wedge high tibial valgus osteotomy; in addition, the clinical, radiographic and CT monitoring indicate that G-CSF and/or mobilized BMCs may hasten bone graft substitute osseointegration. LEVEL OF EVIDENCE: I.

Coracoid tunnels in open and arthroscopic treatment of acromioclavicular dislocation: an experimental cadaveric study.

PURPOSE: Treatment of acromioclavicular joint (ACJ) dislocation is not encoded uniquely. Type I and II injuries are usually treated conservatively, while types IV, V and VI surgically. Controversy still exists over the treatment of type III injuries. In the operative approach, there is no agreement on the best surgical technique. Our purpose is to compare the mini-open and arthroscopic approach focusing on the evaluation of the anatomical precision of the coracoid drilling. METHODS: This is a controlled laboratory study. Ten fresh-frozen cadaveric shoulders were randomly assigned to the two techniques in order to compare them. We performed essential surgical gestures to drill the tunnel using MINAR® System (mini-open) and Dog-Bone® (ARTHREX, arthroscopic). The anatomical specimens were then subjected to CT-scan investigation. We statistically evaluated the precision of these two techniques analyzing DICOM files using two parameters. Parameter 1 evaluates the tunnel entry area on the superior side of the coracoid. Parameter 2 describes the orientation of the tunnel. RESULTS: There are no statistically significant differences (95% confidence level) between arthroscopic and mini-open approach about the precision in the location of the coracoid hole, regarding the entry area (p = 1.00) and the orientation (p = 0.196). CONCLUSION: The evidences collected enable the orthopedic surgeon to choose equally between the two techniques in the treatment of AC joint dislocation toward precision.

Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen-associated (SCCA) variants' serum levels in anti-HCV positive cirrhotic patients.

Aberrant squamous cell carcinoma antigen (SCCA) expression is an early event in hepatocarcinogenesis, and increasing serum levels of SCCA variants IgM immune complexes (SCCA-IgM IC) have been found in cirrhotic patients developing hepatocellular carcinoma (HCC). We longitudinally evaluated a cohort of cirrhotic patients with hepatitis C virus infection (HCV) who underwent pegylated interferon (PEG-IFN) and ribavirin treatment. SCCA-IgM IC levels were assessed in the sera of 33 cirrhotic patients with HCV (21 males, median age 57 years) before, at the end and at 6-month and 1-year follow-up after treatment with PEG-IFN and ribavirin. SCCA-IgM IC serum levels (arbitrary units/mL, AU/mL) were evaluated according to treatment outcome: sustained virological response (SVR) vs nonresponse (NR). Overall, 15 patients obtained a SVR to antiviral therapy (45%). There was no significant difference in baseline SCCA-IgM IC serum levels between SVR and NR patients. When compared to baseline (451.2 AU/mL), SVR patients showed a significant decrease in median SCCA-IgM IC serum levels at the end of treatment (186.8 AU/mL, P = 0.013) and at both 6-month (96.8 AU/mL, P < 0.001) and 1-year follow-up (52.4 AU/mL, P < 0.001), while no significant modification was observed in NR patients. In patients with HCV-related liver cirrhosis, successful antiviral therapy is associated with a dramatic and significant decrease in SCCA-IC serum levels. Because of the pathophysiological correlation between SCCA and liver carcinogenesis, it is hypothesized that in patients with liver cirrhosis, SVR may be accompanied by a decreased proliferative stimulation.

Serious obstetric complications interact with hypoxia-regulated/vascular-expression genes to influence schizophrenia risk.

The etiology of schizophrenia is thought to include both epistasis and gene-environment interactions. We sought to test whether a set of schizophrenia candidate genes regulated by hypoxia or involved in vascular function in the brain (AKT1, BDNF, CAPON, CHRNA7, COMT, DTNBP1, GAD1, GRM3, NOTCH4, NRG1, PRODH, RGS4, TNF-alpha) interacted with serious obstetric complications to influence risk for schizophrenia. A family-based study of transmission disequilibrium was conducted in 116 trios. Twenty-nine probands had at least one serious obstetric complication (OC) using the McNeil-Sjostrom Scale, and many of the OCs reported were associated with the potential for fetal hypoxia. Analyses were conducted using conditional logistic regression and a likelihood ratio test (LRT) between nested models was performed to assess significance. Of the 13 genes examined, four (AKT1 (three SNPs), BDNF (two SNPs), DTNBP1 (one SNP) and GRM3 (one SNP)) showed significant evidence for gene-by-environment interaction (LRT P-values ranged from 0.011 to 0.037). Although our sample size was modest and the power to detect interactions was limited, we report significant evidence for genes involved in neurovascular function or regulated by hypoxia interacting with the presence of serious obstetric complications to increase risk for schizophrenia.

Oxytocin receptor mRNA expression in dorsolateral prefrontal cortex in major psychiatric disorders: A human post-mortem study.

There is growing interest in oxytocin as a putative treatment for various psychiatric disorders including major depressive disorder, bipolar disorder and schizophrenia/schizoaffective disorder. However, potential alterations in the endogenous brain oxytocin system in these disorders are poorly characterized. Brain expression of oxytocin and its receptor genes in patients with these psychiatric disorders has not been well studied outside the hypothalamus. We measured expression of mRNA for oxytocin and its receptor in the dorsolateral prefrontal cortex of postmortem brains using quantitative polymerase chain reaction in a total of 581 individuals. These individuals either were diagnosed with major depressive disorder (n = 135), bipolar disorder (n = 57), schizophrenia/schizoaffective disorder (n = 169), or were control subjects, defined as individuals with no lifetime history of any of these disorders (n = 220). Diagnoses of major depressive disorder and bipolar disorder were associated with significantly increased oxytocin receptor mRNA levels in the dorsolateral prefrontal cortex. This finding is discussed in light of the extant literature on the dysregulation of oxytocin signaling in individuals with major psychiatric disorders.

Regional cerebral blood flow and cerebrovascular reactivity in IDDM.

OBJECTIVE: To investigate both rCBF and cerebrovascular reactivity, evaluated as pre- and post-ACZ rCBF differences in a group of IDDM patients with differences in duration of disease and severity of complications. RESEARCH DESIGN AND METHODS: rCBF was measured by the 133Xenon inhalation method in 20 IDDM patients and in 15 healthy control subjects before and after an intravenous injection of ACZ, a carbonic anhydrase inhibitor commonly used to assess cerebrovascular reactivity. RESULTS: Basal global CBF (the mean of 32 regional values) was within the normal range in all patients but 1, who showed slight hyperperfusion; moreover, in 3 patients with long-lasting disease, some hypoperfused regions were found. ANOVA showed an inverse correlation between basal global CBF (P < 0.01) and duration of diabetes, but no correlation with Hb, MABP, serum glucose concentration, or GHb. Compared with control subjects, the percentage of global CBF increment after ACZ administration was significantly impaired in 4 patients and gave a borderline response in 2 patients; 4 of these poor ACZ responders had retinopathy, and 1 had suffered from a TIA. Duration of diabetes, Hb, MABP, serum glucose concentration, and GHb did not correlate with the percentage of post-ACZ global CBF changes, and did not differ among the 6 poor ACZ responders and the other diabetic patients or control subjects. CONCLUSIONS: These results confirm that global CBF is within the normal range in most IDDM patients, although it is significantly influenced by the duration of diabetes; pathophysiological correlates of the altered cerebrovascular reactivity need to be further investigated. rCBF measurements, before and after ACZ administration, seem to represent a safe and reliable tool for assessing cerebrovascular function in IDDM.

Reduction of cerebral blood flow in subclinical hepatic encephalopathy and its correlation with plasma-free tryptophan.

Cerebral blood flow (CBF), measured by the noninvasive xenon-133 inhalation method, EEG, and plasma levels of ammonia (NH3) and free tryptophan were determined in 18 hospitalized cirrhotic patients affected with subclinical hepatic encephalopathy, as diagnosed by the Kurtz test. CBF results were significantly lower (p less than 0.001) in the patients' group as compared with a sex- and age-matched normal control population, although seven patients had values in the normal range. NH3 was increased only in six, while free tryptophan was increased in all but two patients. A significant negative correlation (p = 0.02) between CBF and free tryptophan was found, even though it appears to be difficult to interpret. We suggest that CBF impairment in some cirrhotic patients with subclinical hepatic encephalopathy may be related to the systemic metabolic derangement caused by the liver disease; free tryptophan could have some implication in producing CBF reduction.

Regional cerebral blood flow and anxiety: a correlation study in neurologically normal patients.

Regional CBF (rCBF) was evaluated by the 133Xe inhalation method in 60 neurologically normal patients (30 men and 30 women) and hemispheric and regional values were correlated with anxiety measurements collected by a self-rating questionnaire before and after the examination. Statistically significant negative correlations between rCBF and anxiety measures were found. rCBF reduction for high anxiety levels is in line with results previously reported by others and could be related to lower performance levels for moderately high anxiety scores as those reported in the present population. This could perhaps be explained by rearrangement of flow from cortical zones to deeper areas of the brain, classically known to be implicated in the control of emotions. However, these results should be interpreted cautiously, since they were obtained in patients and not in normal subjects.

PET study of competition between intravenous cocaine and [11C]raclopride at dopamine receptors in human subjects.

OBJECTIVE: Animal data suggest that the strong euphoriant effects of cocaine are related to the drug's enhancement of available dopamine at the synaptic cleft. The authors' goal was to determine whether this mechanism is the same in humans because the development of putative pharmacological agents for treatment of cocaine dependence depends on this knowledge. METHOD: Positron emission tomography with [11C]raclopride was used to examine the effects of the intravenous administration of 48 mg of cocaine (a typical "street" dose) on the occupancy of dopamine 2 receptors in the putamen of 11 self-identified intravenous drug abusers. RESULTS: All 11 subjects reported subjective stimulation and euphoria in response to cocaine administration. Radioligand occupancy at dopamine receptors was decreased significantly after cocaine administration, suggesting that higher dopamine concentrations were competing at the receptor site. CONCLUSIONS: These results support the concept of dopamine system involvement in human cocaine abuse.

Effects of beta-adrenoreceptor antagonists on cerebral blood flow of cirrhotic patients with portal hypertension.

The current study evaluated the effect of two beta adrenergic-blocking agents, propranolol (PRP) and atenolol (ATN), versus placebo on cerebral blood flow (CBF) of three homogeneous groups of cirrhotic patients with portal hypertension. CBF was measured by the noninvasive 133-Xenon inhalation method at rest and 1 hour after a single oral dose of PRP (40 mg), or ATN (100 mg), or placebo. Blood pressure and heart rate (HR) were measured at the beginning of each examination, and end-tidal pCO2(PeCO2) was monitored. The HR decreased significantly in both the PRP and ATN groups (P less than .01), whereas no changes were recorded for both PeCO2 and mean arterial blood pressure (MABP). The comparisons of the CBF differences among groups (ANOVA with the significance levels adjusted by the Bonferroni's correction) showed a significant increase in CBF after ATN as compared with both placebo (P less than .02) and PRP (P less than .01), whereas no significant differences were seen after PRP as compared with placebo. Our results confirm that PRP does not significantly affect CBF, whereas ATN induces an increase in CBF, although the underlying mechanism is difficult to explain.

Comparison of functional MR and H2 15O positron emission tomography in stimulation of the primary visual cortex.

PURPOSE: To locate spoiled gradient-echo functional MR signal changes in relation to brain parenchyma. METHODS: The region of the primary visual cortex was evaluated using functional MR and H2 15O positron emission tomography in each of six male subjects who were being visually stimulated by means of red light-emitting diode flash goggles. RESULTS: The positron emission tomography technique demonstrated substantially greater relative signal change with visual stimulation than did the functional MR technique. Furthermore, the functional MR signal changes were concentrated in loci around the periphery of brain parenchyma exhibiting increased radiotracer activity, as opposed to being collocated. CONCLUSIONS: Signal changes found using functional MR based on gradient-echo techniques reflect primarily phenomena occurring within small veins and underrepresent activity intrinsic to brain parenchyma, thus introducing potential inaccuracies in locating regions of activated brain tissue. Positron emission tomography, however, directly measures changes in metabolically related activity within the parenchyma.

Regional cerebral blood flow during the Wisconsin Card Sorting Test in normal subjects studied by xenon-133 dynamic SPECT: comparison of absolute values, percent distribution values, and covariance analysis.

We studied regional cerebral blood flow (rCBF) by xenon-133 dynamic single photon emission computed tomography (SPECT) in 17 normal volunteers who were performing the Wisconsin Card Sorting Test (WCST), a task that is particularly sensitive to disturbance of the prefrontal cortex, and a simple matching-to-sample task (BAR) as a sensorimotor control. Three methods for statistical analysis of regional "subtraction" data were used: absolute rCBF values, percent distribution values, and means adjusted for global CBF changes (covariance analysis). The absolute values had high variance, due to the combination of interindividual differences in global flow and intra-individual variation, and showed no statistically significant regional changes. This variation was greatly reduced by percent values and covariance analysis, which had quite similar outcomes. With both methods, significant increases of rCBF during the WCST as compared with the BAR were seen in the right anterior dorsolateral prefrontal and left occipital cortices, and reduction of rCBF in the left pararolandic region. Moreover, significant correlations with performance were found in the medial regions of the frontal lobes, with opposite trends for the right and left hemisphere. The posterior dorsolateral prefrontal region showed a negative correlation with sensory-motor frequency, an index related to the task's difficulty. These results are consistent with previous findings using other rCBF techniques and confirm the statistical advantage of normalization and covariance methods, which yield practically identical results, at least in this analysis based on regions of interest.

A comparison of xenon-133 and xenon-127 for the determination of regional cerebral blood flow measured by dynamic SPECT.

Phantom studies and cerebral blood flow (CBF) measurements in 11 normal subjects at rest were performed by single photon emission tomography (SPECT) with Xe-133 (16 mm full-width at half-maximum [FWHM] collimation) and Xe-127 (16 mm, 12 mm, and 9 mm FWHM collimation). The phantom results clearly illustrated the feasibility of Xe-127 studies and the advantage of Xe-127 over Xe-133 for equivalent patient dose exposures. CBF values obtained with Xe-127 were comparable to those of Xe-133 for the 16 mm collimator, although higher flow values were found with the better resolution, probably because of reduced partial volume effects. The correlations between the various groups of examinations were high, except for the Xe-133 and Xe-127 16 mm collimator groups. Xe-127 allows a considerable increase in the resolution of the images, while exposing the patient to a lower radiation dose. Potential limitations because of higher energy penetrating photons from Xe-127 were not observed in this specially shielded equipment.

Brain functional imaging in senile psychopathology.

Quantified electroencephalogram (EEG) and regional cerebral blood flow (rCBF) measurements are reliable and currently employed techniques in the functional exploration of the aging brain; they can be routinely employed, since discomfort to the patient is minimal. Topographical analysis of EEG and rCBF results is performed in our laboratory by a fully automated mapping system, which also enables statistical comparisons in real time. The goal of our study is to ascertain if there are systematic modifications in the topographic distribution of rCBF and EEG parameters in normal aging, dementia, cerebrovascular disease and in conditions of increased risk for cerebral pathology (e.g. hypertension). Dementias and cerebrovascular pathologies present characteristic brain functional abnormalities, which can be detected by comparing the patient data to an age-matched normal population by the appropriate statistical tests; therefore, the accurate selection of healthy aged controls appears as a crucial issue in order to improve the sensitivity of statistics.

Acute effects of acetyl-L-carnitine on regional cerebral blood flow in patients with brain ischaemia.

Ten male patients with brain ischaemia were studied. Regional cerebral blood flow (rCBF) was measured in the resting condition and 1 h after intravenous infusion of 1500 mg acetyl-L-carnitine. rCBF values were compared to those of a control population of the same age decade by a computer mapping system of our own design, which enables rapid visual inspection of cerebral blood flood values distribution and of statistical significance of differences. Beneficial effects of the drug were observed in at least four patients, especially in the affected hemisphere, in another four, rCBF elevations in the areas which were lowest on resting examination were observed, which was paralleled by reductions in surrounding zones. In two patients no modification of rCBF values after acetylcarnitine was seen.

Judo and choking: EEG and regional cerebral blood flow findings.

Ten judoka were examined at rest by EEG and regional Cerebral Blood Flow (rCBF) (133-Xenon inhalation method); in seven of them a syncope was induced by choking and EEG was recorded during the loss of consciousness while rCBF was measured immediately after recovery. Baseline EEG and rCBF examinations were normal. During choking, EEG showed diffuse 2-3 Hz. high voltage waves, predominantly in the anterior regions, then the EEG gradually recovered to normal. After choking, rCBF decreased in all but one subject, slightly in four and more markedly in two. Our findings suggest that there is no evidence of permanent CNS functional changes due to judo practice and choking. The variable rCBF features soon after choking-induced syncope may reflect a different timing of recovery from cerebral ischemia for each subject.