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Objectives: Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. Design: We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. Methods: Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. Results: Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. Conclusion: Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.

It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in complete elimination of the virus, or whether small quantities of virus persist. Our study group comprised 17 patients with chronic HCV who had sustained virological response (SVR) after interferon/ribavirin treatment. Serum and peripheral blood mononuclear cells were collected 2 to 3 times at 3- to 6-month intervals starting 40 to 109 months (mean, 64.2 ± 18.5 months) after the end of therapy. In addition, lymphocyte and macrophage cultures were established at each point. In 11 patients, frozen liver tissue samples were available from follow-up biopsies performed 41 to 98 months (mean, 63.6 ± 16.7 months) after therapy. Presence of HCV RNA was determined by sensitive reverse-transcriptase polymerase chain reaction, and concentration of positive and negative strands was determined by a novel quantitative real-time reverse transcriptase polymerase chain reaction. Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed compartments. HCV RNA was detected in macrophages from 11 patients (65%) and in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3 of 11 livers and in sera from 4 patients. Viral replicative forms were found in lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our results suggest that in patients with SVR after therapy, small quantities of HCV RNA may persist in liver or macrophages and lymphocytes for up to 9 years. This continuous viral presence could result in persistence of humoral and cellular immunity for many years after therapy and could present a potential risk for infection reactivation. (HEPATOLOGY 2005;41:106–114.)

It is unclear whether patients who test positive for anti-hepatitis C virus (HCV) antibodies and have normal alanine aminotransferase (ALT) levels remain infected with the virus. Eleven patients who tested positive for anti-HCV antibodies, had persistently normal ALT levels, and tested negative for HCV RNA by commercial test were studied. Serum and peripheral blood mononuclear cells (PBMCs) were collected 2-3 times at 3-6-month intervals, and PBMCs were cultured with phytohemagglutinin and pokeweed mitogen. HCV RNA was detected in serum samples from 6 (55%) and in PBMCs from 11 (100%) patients. Our results suggest that, in asymptomatic patients who test positive for anti-HCV antibodies, small quantities of HCV RNA commonly persist, even in patients who test negative for HCV RNA in serum by commercial tests.

Hepatitis C virus (HCV) has been reported to replicate in monocytes/macrophages in infected patients. However, it is unclear whether macrophages are susceptible to infection in vitro and whether such an infection is consequential. Sera from 26 HCV-infected patients were incubated with primary human macrophages collected from healthy donors. Virus negative strand was detected by a Tth enzyme-based strand-specific assay and virus sequences were analysed by single strand conformation polymorphism (SSCP) and sequencing. Concentrations of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12p70 were measured in culture supernatants and respective mRNAs were analysed in cell extracts by quantitative RT-PCR. For 15 sera, HCV RNA was detectable in 2- and 3-week cultures from at least one donor. Virus negative strand was detected in 29 % of macrophage samples in this group. In four cases, HCV RNA sequences amplified from macrophages differed from those amplified from sera suggesting evolution during infection. Concentrations of TNF-alpha and IL-8 were found to be significantly higher in supernatants from HCV-infected cultures. In conclusion, these preliminary data suggest that primary human macrophages are susceptible to HCV infection in vitro and this infection is associated with the induction of cytokines TNF-alpha and IL-8.

Romano-Ward syndrome is an unusual and potentially devastating disorder manifested by prolongation of the QT interval. Persons with this syndrome are at risk for sudden death from ventricular dysrhythmis. A case of a gravid woman with the disorder is presented

Background. The dynamics of hepatitis C virus (HCV) quasi species in the E2 region may correlate with the course of infection after orthotopic liver transplantation (OLT).Methods. Thirty-four patients who underwent transplantation for HCV-related cirrhosis were studied. Serum and liver samples were available before OLT and at 1 week, 4 months, and 1 year after OLT. Patients were divided into group 1 (Knodell/Ishak fibrosis stage [FS] at 1 year

Patients with chronic hepatitis C frequently report tiredness, easy fatigability, and depression. The aim of this study is to determine whether hepatitis C virus (HCV) replication could be found in brain tissue in patients with hepatitis C and depression. We report two patients with recurrent hepatitis C after liver transplantation who also developed severe depression. One patient died of multiorgan failure and the other, septicemia caused by Staphylococcus aureussis. Both patients had evidence of severe hepatitis C recurrence with features of cholestatic fibrosing hepatitis. We were able to study samples of their central nervous system obtained at autopsy for evidence of HCV replication. The presence of HCV RNA–negative strand, which is the viral replicative form, was determined by strand-specific Tth-based reverse-transcriptase polymerase chain reaction. Viral sequences were compared by means of single-strand conformation polymorphism and direct sequencing. HCV RNA–negative strands were found in subcortical white matter from one patient and cerebral cortex from the other patient. HCV RNA–negative strands amplified from brain tissue differed by several nucleotide substitutions from serum consensus sequences in the 5' untranslated region. These findings support the concept of HCV neuroinvasion, and we speculate that it may provide a biological substrate to neuropsychiatric disorders observed in patients with chronic hepatitis C. The exact lineage of cells permissive for HCV replication and the possible interaction between viral replication and cerebral function that may lead to depression remain to be elucidated. ( Liver Transpl 2002;8:1014-1019. )

Hepatitis C virus (HCV) sequences were detected in cerebrospinal fluid (CSF) in 8 of 13 HCV-positive patients. In four patients harboring different virus strains in serum and peripheral blood mononuclear cells (PBMC), CSF-derived virus was similar to that found in PBMC, which suggests that PBMC could carry HCV into the brain.

Patients with chronic hepatitis C are more likely to have significant changes in their physical and mental well-being than patients with liver disease of other etiology, and hepatitis C virus (HCV) has been occasionally implicated in diseases of the central nervous system. We analyzed the presence of the HCV negative-strand RNA sequence, which is the viral replicative intermediary, in autopsy brain tissue samples from six HCV-infected patients. Negative-strand HCV RNA was searched for by a strand-specific Tth -based reverse transcriptase PCR, and viral sequences amplified from brain tissue and serum were compared by single-strand conformational polymorphism analysis and direct sequencing. HCV RNA negative strands were detected in brain tissue in three patients. In two of these patients, serum- and brain-derived viral sequences were different and classified as belonging to different genotypes. In one of the latter patients, HCV RNA negative strands were detected in lymph node and, while being different from serum-derived sequences, were identical to those present in the brain. The results of the present study suggest that HCV can replicate in the central nervous system, probably in cells of the macrophage/monocyte lineage.

9022 Background: We recently validated a GEP test that provides accurate prediction of metastatic risk for CM cases. The GEP of 31 genes provides a binary outcome of Class 1 (low risk of metastasis...

Background. We have previously studied hepatitis C (HCV)-infected recipients of livers from HCV-infected donors and found that either the donor’s strain or the recipient’s strain predominate in serum. The current study was undertaken to determine whether these changes are complete and whether they are reflected in the population of virus associated with peripheral blood mononuclear cells (PBMCs). Methods. We analyzed HCV ribonucleic acid from sequential serum and PBMC samples from 11 and 8 patients, respectively. The relatively stable NS5 region was chosen for analysis because it allowed for dependable identification of donor and recipient strains. Viral sequences were analyzed by direct sequencing and by sensitive strain–specific polymerase chain reaction assays. These assays were capable of detecting the minor sequence present at a concentration 1:104–10−7 below that of the major sequence. Results. Five patients retained their original infecting strain; the donor strain was detected only transiently. In the remaining six patients, recipient strain was detected for the first few weeks, after which only the donor strain was consistently present. However, in one patient the second nondominant strain was detected from the background of the major strain on a single occasion 8 months after transplantation. All changes in serum were closely paralleled by those occurring in PBMCs. Conclusions. Viral population changes in the setting of liver transplantation from HCV-infected donors to HCV-infected recipients occur simultaneously in PBMCs and serum. The takeover of one strain by another in PBMC- and serum-derived viral populations seemed to be complete and long lasting.

We have analyzed three cases of hepatitis C virus (HCV)-infected recipients who received blood from HCV-infected donors. Two recipients were exposed to two different HCV RNA-positive donors, and one was exposed to a single donor. All parental genomes from the actual infecting units of blood and the recipients were defined, and their presence in the follow-up serum samples was determined using sensitive strain-specific assays. The strain from one of the donors was found to predominate in all recipients' serum samples collected throughout the follow-up period of 10 to 30 months. In two recipients exposed to two infected donors, the strain from the second donor was occasionally found at very low level. However, the original recipients' strains were not detected. Our observations show that HCV-infected individuals can be superinfected with different strains, and this event may lead to eradication or suppression of the original infecting strain. Furthermore, our findings demonstrate that simultaneous exposure to multiple HCV strains may result in concomitant infection by more than one strain, although a single strain could rapidly establish its dominance. The results of the present study suggest the existence of competition among infecting HCV strains which determines the ultimate outcome of multiple HCV exposure.

PurposeThymoma represents one of the rarest of all malignancies. Stage and completeness of resection have been used to ascertain postoperative therapeutic strategies albeit with limited prognostic accuracy. A molecular classifier would be useful to improve the assessment of metastatic behaviour and optimize patient management.MethodsqRT-PCR assay for 23 genes (19 test and four reference genes) was performed on multi-institutional archival primary thymomas (n = 36). Gene expression levels were used to compute a signature, classifying tumors into classes 1 and 2, corresponding to low or high likelihood for metastases. The signature was validated in an independent multi-institutional cohort of patients (n = 75).ResultsA nine-gene signature that can predict metastatic behavior of thymomas was developed and validated. Using radial basis machine modeling in the training set, 5-year and 10-year metastasis-free survival rates were 77% and 26% for predicted low (class 1) and high (class 2) risk of metastasis (P = 0.0047, log-rank), respectively. For the validation set, 5-year metastasis-free survival rates were 97% and 30% for predicted low- and high-risk patients (P = 0.0004, log-rank), respectively. The 5-year metastasis-free survival rates for the validation set were 49% and 41% for Masaoka stages I/II and III/IV (P = 0.0537, log-rank), respectively. In univariate and multivariate Cox models evaluating common prognostic factors for thymoma metastasis, the nine-gene signature was the only independent indicator of metastases (P = 0.036).ConclusionA nine-gene signature was established and validated which predicts the likelihood of metastasis more accurately than traditional staging. This further underscores the biologic determinants of the clinical course of thymoma and may improve patient management.

7605 Background: Thymomas and thymic carcinomas (TC) are rare epithelial tumors derived from the thymic gland in the anterior mediastinum. Although all histological types of thymomas, albeit with different frequencies, can give rise to metastases, TC have a more aggressive behavior and metastasize earlier and more frequently than thymomas. We previously developed a prognostic gene signature able to accurately determine metastatic behavior of thymomas (Gökmen-Polar et al. ASCO 2012). The signature is currently used in clinical practice to identify patients at high or low risk for metastatic disease. In the current study, we sought to evaluate the utility of this signature for determining risk from TC tumors. Methods: FFPE tissue sections were macrodissected from 35 primary TC. RNA was isolated and RT-PCR was performed to assess the expression of 23 genes (19 test and four reference genes). Predictive modeling was performed using Radial Basis Machine (RBM) software from JMP Genomics (SAS), and survival analysis was done using the Kaplan-Meier method. Results: Samples from the TC cohort ranged from stage II through IVB, with a median age of 54 years. 26 samples had evidence of metastatic progression, while nine samples did not. Prediction of metastasis, based upon comparison to the previously developed thymoma training set and using a 19-gene signature, yielded an ROC = 0.66. Independent analysis of the TC cohort with the thymoma 19-gene signature resulted in a predictive model with an ROC = 0.97 (overall accuracy = 87%, sensitivity = 73% and specificity=100%). Further modeling and gene set reduction revealed a separate ten-gene signature able to segregate metastatic from non-metastatic cases with 100% accuracy. All the cases classified as high risk (n= 26) developed metastasis within 5 years while none of the cases categorized as low-risk (n= 9) had any events at 5 years of follow-up. Conclusions: A ten-gene signature was established that appears to predict metastatic behavior of TC with a high degree of accuracy; however, validation in an independent cohort is necessary. Our data suggests that the biologic determinants of the clinical course of TC maybe distinct from thymoma and could be used to improve patient management.

9022 Background: Wide variability exists in metastatic rates of primary cutaneous melanoma (CM), even within TNM stage groupings. We developed an RT-PCR based gene expression profile (GEP) test to predict distant metastases in early stage CM. Methods: RNA was isolated from formalin-fixed, paraffin embedded biopsies or wide excisions of primary CM from patients with stage 1-4 CM (87% stage 1-2) converted to cDNA and analyzed using RT-PCR. All analyses were done using JMP Genomics and WinSTAT. Radial Basis Machine (RBM) modeling was used to predict metastasis-free survival (MFS) for the training set of 149 samples. Independent validation was performed on an additional 107 CM samples. RBM reports a binary Class 1 (low risk) and Class 2 (high risk). Results: Analysis of the training set resulted in a model with 85% receiver operating characteristic (ROC) and sensitivity of 82%. Prediction of metastatic risk for the validation set resulted in a 90% ROC and sensitivity of 89%. 30 of 33 stage 1-3 cases with a known metastatic event were accurately called Class 2. Kaplan-Meier analysis showed 5-year MFS rates of 88% and 25% for predicted Class 1 and Class 2, respectively, in the training set (p

A product form approximation, based on the principle of maximum entropy (ME), is characterised for arbitrary open discrete time queueing network models (QNMs) of shared buffer ATM switches under the departures first (DF) buffer management policy. Traffic entering and flowing in the network is assumed to be bursty and is modelled by a Compound Bernoulli Process (CBP) with geometrically distributed bulk sizes. Entropy maximisation implies decomposition of the network into individual shared buffer switches which are analysed to obtain cell loss probabilities and mean delays. The ME queue length distribution of a single shared buffer queue under DF policy, together with closed form expressions for the first two moments of the effective flow, play the role of building blocks in the solution process. Typical numerical results are included to demonstrate the utility and computational efficiency of the ME procedure. Comments on current work, involving discrete time finite capacity queues with space priority and correlated traffic, are included.

It is the observation of this author that a majority of US patients who seek and receive care for urogynecologic disorders are Caucasian and have insurance or other means to pay for their care. There are, of course, many exceptions. However, the impression remains that urogynecologic disorders are largely addressed in women who have the luxury of time to do so, access to urogynecologic services and the ability to pay. Unlike cancer, urinary incontinence and prolapse are usually not life threatening. Unlike Obstetrics, the disorder does not have a self-limited course. Urogynecologic conditions only uncommonly warrant urgent attention. However, these disorders have a significant impact on the quality of life of women of all races and social strata. African American women have been found to have greater rates of detrusor overactivity. Latina women have been found to have higher rates of prolapse. There is little or no data on Native American women. Medical insurance coverage is less common among minorities in the US, but quality of life issues undoubtedly transcend one’s race and socioeconomic status. So what can be done to address yet another disparity in our health care system? It begins with the recognition that our profession selects for a certain type of patient. It continues with the willingness to accept patients in to our practice with limited or no resources. Then, we must advocate for them in their efforts to secure public assistance or other health care coverage including charity care within our hospital systems. National organizations should recognize the issue and advocate for public coverage for patients who are otherwise disabled by the effects of their urogynecologic disorders, just as one might be disabled by her “bad back”. Underserved women also have more difficulty finding qualified physicians to address their problems because we are largely driven to practice in high resource settings. With this in mind, we have developed a model for delivery of urogynecologic services in one hospital on the Navajo reservation. This may be applicable to other underserved populations, especially those with public funding. Native women with federal medical coverage who have urinary incontinence or prolapse often have no access to urogynecologic services. They must either suffer or seek referral to facilities which are sometimes hundreds of miles away. Sometimes their referrals are approved, sometimes not. Most of these women do not have the resources to travel back and forth to the referral institution for an initial evaluation, followup visit, pre-operative visits, surgery etc. Once or twice yearly, we visit the reservation to perform surgery on patients with prolapse and/or incontinence. These patients are prescreened according to strict criteria and confirmed as suitable candidates prior to the procedure. These visits serve to both help individual patients and teach the Indian Health Service physicians new techniques. The Indian hospital saves money on referrals and reimburses us for expenses. Over time, we have developed a trusting and professional relationship with the IHS physicians which ultimately nourishes us all. There are many ways that physicians in urogynecology and related fields can address the needs of the medically underserved. It is incumbent on our specialty to devise innovative ways to care for patients of all races and socioeconomic statuses. We must be tireless advocates for these patients until the time when all patients are able to receive equal, high quality care. Int Urogynecol J (2006) 17:429 DOI 10.1007/s00192-006-0182-y