Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: implications for tissue engineering and wound healing.

Vascular endothelial cell growth factor (VEGF) has strong stimulating effects on vascularization. Though very potent, VEGF is rapidly degraded due to its short half-life and when administrated by uncontrolled and nonspecific methods; however, its systemic administration in large doses can cause harmful side effects. Controlled release technology would allow delivering desired levels of bioactive VEGF within extended periods and permit examination of the in vivo effects of the compound in a broader way. The objective of this study was to determine the in vitro release behavior of VEGF from calcium alginate microspheres and the potency of this controlled release system in promoting localized neovascularization at the subcutaneous site of the rat model. In vitro release of human VEGF165 (2 and 4 microg/cm3 microsphere) was studied for 3 weeks under static conditions at 25 degrees C, and daily hormone release was measured using a competitive enzyme immunoassay. Following an uncontrolled release within the first 4 days, a quite constant zero-order VEGF release of 50 to 90 and 70 to 120 ng/day was achieved from 2 and 4 microg/cm3 polymer loaded microspheres respectively. In vivo angiogenesis was studied for a period of 8 weeks and evaluated using immunoperoidase staining and histopathological measurements. In vivo studies with rats (n = 24) showed a considerable level of capillary network formation at the epigastric groin fascia of VEGF microsphere-implanted rats starting from the first week. The most extensive neovascularization was observed in the group with 3 week postimplanted 4 microg VEGF containing microspheres; this level of vascularization was quite similar after 8 weeks. While the control group showed no evidence of angiogenesis, the difference in VEGF-induced neovascularization is statistically significant (p < 0.03). Immunostaining of the specimens showed a strong relationship between the release of human VEGF and neovascularization. The controlled VEGF release system described here promotes vigorous angiogenesis and has applicability for tissue engineering and wound healing studies.

Hepatitis C screening strategies in hemodialysis patients.

Hepatitis C virus (HCV) infection is common in patients undergoing chronic hemodialysis, with an estimated yearly incidence of 0.2% and prevalence between 8% and 10%. Although a screening strategy based on alanine aminotransferase (ALT) values is currently recommended, this strategy has not been evaluated for cost-effectiveness compared with other potential screening strategies. A comparison therefore was made using a decision-analysis model of a simulated cohort of 5,000 hemodialysis patients followed up for 5 years. Using direct medical costs, three strategies were evaluated, including: (1) ALT values with confirmatory testing (biochemical), (2) serial enzyme-linked immunosorbent and strip immunoblot assay testing (serological), and (3) polymerase chain reaction (viral). Under baseline assumptions, the per-patient cost of screening hemodialysis patients for HCV was $378 for biochemical-based testing, $195 for serological-based testing, and $696 for viral-based testing. Our model was robust when varying the costs of testing, as well as the incidence and prevalence of HCV infection. Results of sensitivity analysis by varying costs, HCV incidence, and HCV prevalence indicated that serological-based screening was less costly than biochemical testing. Biochemical testing was in turn less costly than viral-based screening. Serological-based testing was also more effective in the diagnosis of de novo HCV infection, with a likelihood ratio of 85, in contrast to the likelihood ratio of 44 with biochemical-based testing using viral-based screening as the gold standard. A serological-based screening strategy is less costly and more effective than biochemical-based screening in the diagnosis of de novo HCV infection. Serological-based screening should be considered for HCV screening in hemodialysis populations.

Automated RIBA HCV strip immunoblot assay: a novel tool for the diagnosis of hepatitis C virus infection in hemodialysis patients.

Hemodialysis (HD) patients remain a high-risk group for hepatitis C virus (HCV) infection. Serological assays (enzyme-linked immunosorbent assays, ELISAs) are the only tests currently approved by the Food and Drug Administration in the United States for the diagnosis of HCV. The RIBA HCV Strip Immunoblot Assay (SIA) is an established method for supplemental testing of repeat reactive hepatitis C ELISA patients on HD. However, the current manual procedure is labor intensive, requiring subjective band scoring and result interpretation. Recently, the automated CHIRON RIBA HCV Processor System has been designed to perform RIBA supplemental testing. The CHIRON RIBA HCV Processor System consists of a bench-top instrument that provides objective evaluation of the RIBA immunoblot strips, by measuring the light differentially reflected from the developed bands and white background, creating a density of reflectance. The CHIRON RIBA HCV Processor System assesses the intensity of each of the reactive bands in relation to the intensity of the internal control bands on each RIBA HCV strip. Comparison between processor and manual protocols was performed using a large (n = 200) cohort of ELISA 3.0 HCV negative and positive patients on maintenance HD. The test characteristics of RIBA HCV 3.0 SIA were identical with manual and automated runs. The relative intensity values of antigenic bands by the CHIRON RIBA HCV 3.0 Processor System between anti-HCV positive and negative patients were significantly different; only 15 of 784 (1.9%) antigenic bands had borderline reactivities. The correlation of test results between manual and automated runs was very high (kappa value 0.989). Among positive results by RIBA HCV 3.0 SIA, there was a strong concordance between manual and automated runs with regard to the pattern of reactivity (kappa value 0.943). The discordant results between manual and automated protocols were attributable to increased variability of antigen scores close to the cutoff value for both tests. In conclusion, the CHIRON RIBA HCV 3.0 Processor System is capable of performing RIBA HCV 3.0 SIA in the HD population accurately with minimal operator involvement. The test characteristics of RIBA HCV 3.0 SIA were identical by manual and automated runs. There was a strong correlation between the results of the manual and automated runs; the few discordant results between the two procedures were mostly due to increased variability of antigen scores close to the cutoff value for both tests. The Centers for Disease Control and Prevention in the USA have recently included chronic HD patients among those persons for whom routine HCV testing is recommended; HCV-infected patients on HD often have a high rate of indeterminate results by manual RIBA technology which is operator dependent for band scoring and result interpretation. The CHIRON RIBA HCV 3.0 Processor System may be very useful for supplemental anti-HCV testing of ELISA repeat reactive specimens in clinical practice within dialysis units.

Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: A National Heart, Lung, and Blood Institute collaborative study.

Persons with non-A, non-B hepatitis (cases) identified in 5 transfusion studies in the early 1970s have been followed ever since and compared for outcome with matched, transfused, non-hepatitis controls from the same studies. Previously, we reported no difference in all-cause mortality but slightly increased liver-related mortality between these cohorts after 18 years follow-up. We now present mortality and morbidity data after approximately 25 years of follow-up, restricted to the 3 studies with archived original sera. All-cause mortality was 67% among 222 hepatitis C-related cases and 65% among 377 controls (P = NS). Liver-related mortality was 4.1% and 1.3%, respectively (P =.05). Of 129 living persons with previously diagnosed transfusion-associated hepatitis (TAH), 90 (70%) had proven TAH-C, and 39 (30%), non-A-G hepatitis. Follow-up of the 90 TAH-C cases revealed viremia with chronic hepatitis in 38%, viremia without chronic hepatitis in 39%, anti-HCV without viremia in 17%, and no residual HCV markers in 7%. Thirty-five percent of 20 TAH-C patients biopsied for biochemically defined chronic hepatitis displayed cirrhosis, representing 17% of all those originally HCV-infected. Clinically evident liver disease was observed in 86% with cirrhosis but in only 23% with chronic hepatitis alone. Thirty percent of non-A, non-B hepatitis cases were unrelated to hepatitis viruses A,B,C, and G, suggesting another unidentified agent. In conclusion, all-cause mortality approximately 25 years after acute TAH-C is high but is no different between cases and controls. Liver-related mortality attributable to chronic hepatitis C, though low (<3%), is significantly higher among the cases. Among living patients originally HCV-infected, 23% have spontaneously lost HCV RNA.

Serum alanine aminotransferase in hepatitis c screening of patients on hemodialysis.

It is recommended that patients on hemodialysis (HD) therapy undergo regular screening for hepatitis C virus (HCV) infection by using alanine aminotransferase (ALT) values. However, the utility of using ALT values in this setting is unknown. The aim of this prospective study at the University of California Los Angeles Hepatitis Screening Program is to determine the sensitivity, specificity, and predictive values of an elevated ALT level for the diagnosis of HCV infection in HD patients. We screened 2,440 HD patients from 39 dialysis centers for viral infection by using hepatitis antibody serological testing and ALT values. We found the sensitivity and specificity of a newly elevated ALT level for acute HCV infection to be 83% and 90%, respectively. According to Bayes' theorem, the positive predictive value was 4% and the positive likelihood ratio was 8.74. For chronic HCV infection, the sensitivity of a newly elevated aminotransferase level was 21%, and specificity was 91%. The positive predictive value was 16% (according to Bayes' theorem), and the positive likelihood ratio was 2.47. The negative predictive value of a newly elevated aminotransferase value was 99% for acute HCV infection and 94% for chronic HCV infection. Our results indicate that although a newly elevated aminotransferase level is sensitive and specific for acute HCV infection, its positive predictive value is inadequate. A newly elevated aminotransferase level was neither sensitive nor positively predictive of chronic infection. Therefore, an elevated ALT level is an ineffective method for screening for HCV infection in HD patients.

Alginate microbead release assay of angiogenesis.

Recently, the acceleration (and retardation) of blood vessel growth has been an increasingly frequent subject of study. With its potential application to a wide range of clinical disease processes, investigation certainly remains essential and promising. While in vitro investigation is traditional, well-controlled, and objective, studying angiogenesis in vivo can be quite difficult for a number of reasons. One major reason is the inherent tissue differences associated with blood vessel growth. Because all tissues are different, certain tissues tend to be inherently more vascular than others. As such, the growth (and concentration) of blood vessels occurs at different rates and proportions depending on that specific tissue. In the past several years, most in vivo angiogenesis work has been performed in the sclera as it allows for relatively easy access and the possibility of repeated observation. The sites to which investigation of angiogenesis might be applied, however, are invariably quite different and therefore additional tissues such as solid organs, fascia, muscle, and skin need to be studied as well. How can this be performed?

Serotyping strip immunoblot assay for assessing hepatitis C virus strains in dialysis patients.

Recent accumulated evidence shows that dialysis patients are a high-risk group for hepatitis C virus (HCV) infection. Assessment of HCV genotype distribution among dialysis patients may be important because specific viral genotypes are associated with different clinical manifestations, disease progression, and response to antiviral therapy. However, polymerase chain reaction-based methods are cumbersome and unsuitable for analyzing large cohorts of dialysis patients with HCV. Instead, this information can be obtained by using a novel recombinant immunoblot assay (RIBA) recently developed for determining HCV serotype. The RIBA HCV serotyping strip immunoblot assay (SIA; Chiron Corporation, Emeryville, CA), is based on an immunoblot strip with five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS4) and core regions of the genomes of HCV types 1, 2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS4 serotype-specific HCV peptide band in relation to the internal control band (human immunoglobulin G) intensity on each strip. HCV core peptide reactivity is used only in the absence of NS4 reactivity. We compared RIBA HCV serotyping SIA with genotyping using sera from a large (n = 107) cohort of HCV-infected patients undergoing chronic hemodialysis (HD). We successfully serotyped 79 of 107 patients (74%) undergoing HD. We found a remarkable concordance (65 of 70 results; 93%) between RIBA HCV serotyping SIA and genotyping (line probe assay [LiPA]) techniques (kappa = 0.786) with sera from viremic patients infected with a known genotype. Only 5 of 70 patients (7%) had apparently discordant results. In a subset of patients (28 of 107 patients; 26%) not typed by RIBA HCV serotyping SIA, most (24 of 28 patients; 86%) were successfully genotyped by LiPA technology. It was possible to assess serotype reactivity in some patients (9 of 107 patients; 7%) who could not be genotyped. The distribution of HCV serotypes was associated with the antibody response against HCV proteins and the patterns of reactivity by RIBA HCV 2.0 SIA. In conclusion, (1) we found good agreement between serotyping and genotyping methods in our large cohort of dialysis patients infected with HCV; (2) the impaired immunocompetence conferred by uremia may limit serotyping analysis in some HCV-infected patients undergoing HD; (3) RIBA HCV serotyping SIA may be useful in tracking transmission routes for HD patients who cleared the virus and have only anti-HCV antibody; and (4) the distribution of HCV serotypes was associated with the antibody response against HCV proteins and the patterns of reactivity by RIBA HCV 2.0 SIA. Assessment of HCV strains appears to be very useful in the routine clinical activity of nephrologists within HD units because consistent biological differences among HCV strains exist. RIBA serotyping SIA is a simple, inexpensive, and highly reproducible assay to obtain information about HCV types in the HD setting.

Biological dynamics of viral load in hemodialysis patients with hepatitis C virus.

The biological dynamics of hepatitis C virus (HCV) viremia in uremic patients with chronic infection have not been fully characterized. We prospectively studied fluctuations of HCV-RNA in sera from 52 patients with end-stage renal disease who were undergoing maintenance hemodialysis (HD) and had chronic HCV infection. We measured HCV viremia monthly over the course of 13 months with the branched-chain DNA (bDNA) signal amplification assay and prospectively analyzed liver function, expressed by monthly serum aspartate (AST) and alanine aminotransferase (ALT) determinations. We observed three different patterns of HCV viremia: (1) patients persistently positive by bDNA assay (persistent viremia; 23 of 52 patients; 44%), (2) individuals with alternatively positive and negative results (intermittent viremia; 17 of 52 patients; 33%), and (3) patients persistently negative by bDNA assay (12 of 52 patients; 23%). The HCV viral load over the follow-up was greater among patients with persistent compared with intermittent viremia (persistent, 31.7 x 10(5) Eq/mL; range, 6.3 x 10(5) to 16.03 x 10(6) Eq/mL versus intermittent, 10.4 x 10(5) Eq/mL; range, 1.1 x 10(5) to 9.4 x 10(6) Eq/mL; P = 0.0001). In addition, patients with persistent viremia had over time greater AST and/or ALT activities than the intermittent group (AST: persistent, 26.5 IU/L; range, 9.6 to 73.7 IU/L versus intermittent, 21.3 IU/L; range, 8 to 56.8 IU/L; P = 0.001 and ALT: persistent, 14.7 IU/L; range, 3.7 to 57.9 IU/L versus intermittent, 10.9 IU/L; range, 2.3 to 52.1 IU/L; P = 0.001). In the group with persistent viremia, the mean difference between maximum and minimum values of HCV-RNA observed in each individual patient was 2.09 +/- 0.7 natural logarithm (Log(n)) and in intermittent viremic patients, 1.55 +/- 1 Log(n) (P = 0.045). The HCV load at study entry (19.4 x 10(5) Eq/mL) was rather low and did not change versus the end of follow-up in all patients (P = not significant [NS]). In the entire group, the fluctuations in HCV-RNA levels over time between and within individuals were not significant (P = NS). No difference in variability of HCV-RNA values over time between patients infected with different HCV genotypes was seen. In conclusion, three different patterns of HCV viremia in HD over time were assessed; one third of viremic patients had intermittent viremia, and those patients had less HCV-RNA, enzyme-linked immunosorbent assay, and aminotransferase activity than did patients with persistent HCV load. Larger fluctuations in HCV RNA levels occurred in patients with persistent than with intermittent HCV viremia. However, the viremic HCV load was low and relatively stable over a 13-month follow-up in our population. Studies with longer observation periods are warranted to understand fully the natural history of HCV in these immunosuppressed individuals.

Epidemiology and natural history of hepatitis G virus infection in chronic hemodialysis patients.

UNLABELLED: Patients on chronic hemodialysis (HD) have recently been identified as having a high prevalence of hepatitis G virus (HGV) infection. The clinical significance of HGV in this population remains unclear, with no data available as to the acquisition and natural history of HGV infection in this group. AIMS: To assess the prevalence and risk factors of HGV in a large cohort of chronic HD patients, and to evaluate the incidence and clinical consequences of HGV over time in this population. METHODS: Paired sera from 292 patients undergoing chronic HD treatment in four units in the Los Angeles area were tested for HGV RNA before and after they had been on HD for a mean period of 9.7 +/- 1.9 months. HGV was tested by a single-step RT-PCR using two couples of primers located in two different portions (5'UTR, NS5a) of the genome. The amplified products were detected by hybridization with 5' biotin-labeled probes specific for each region. RESULTS: At study entry there were 50 HGV RNA-positive patients, thus the HGV prevalence was 17% (50/292). The multivariate analysis by ordinal logistic regression model showed association (p = 0.0013) between HGV RNA and the location of patients among the HD units. No other significant associations were observed. Three (3/50 = 6%) HGV RNA-positive patients at study entry and 3 (3/41 = 7%) at the end of the follow-up showed a mild increase of alanine aminotransferase (ALT) activity in absence of other apparent causes of liver damage. 35 (70%) out of 50 HGV viremic patients had persistently detectable viremia during the study period; 15 (30%) had non-persistently detectable HGV RNA in the second serum specimen. There was no significant difference between the patients with persistently detectable HGV RNA and those who showed non-persistently detectable HGV viremia with regard to demographic, clinical or virological features. Six patients without detectable HGV viremia at the start of the study showed de novo HGV infection during the follow-up, thus the HGV incidence was 3.07% per year. These individuals did not simultaneously acquire HBV or HCV markers; de novo HGV infection was not associated with other demographic, clinical or virological features. One (16.7%) out of 6 individuals with HGV acquisition had persistently raised ALT levels and chronic HBsAg positivity. The prevalence of HGV was 14% (41/292) at the end of the observation period. CONCLUSIONS: The prevalence of HGV in our HD population was high; HGV positivity was strongly associated with the location of HD patients among the units; some HD individuals with current HGV infection showed biochemical signs of liver disease without other apparent causes. De novo acquisition of HGV occurred within HD units in the absence of evident parenteral risk factors for HGV other than their presence in the HD environment. A large portion of HGV viremic patients showed non-persistently detectable HGV viremia during the study. Acquisition of HGV was not associated with a rise in ALT activity unlike prior experience with de novo HCV in HD patients. Further investigations are warranted to explain the modes of HGV acquisition and the clinical significance of HGV in th HD population.

Epidemiology of Helicobacter pylori in chronic haemodialysis patients using the new RIBA H. pylori SIA.

BACKGROUND: There are few data concerning the epidemiology of H. pylori in patients on chronic haemodialysis (HD) treatment. These surveys concerned small populations and were made with ELISA technique. However, ELISA-based assays do not differentiate between strains of H. pylori that are associated with ulcers. Recent literature reports that formation of ulcers correlates strongly with the expression of cytotoxin-associated protein (CagA) and vacuolating cytotoxin (VacA) of H. pylori. METHODS: A novel serological test (RIBA H. pylori strip immunoblot assay (SIA)) has been recently introduced, it uses the H. pylori lysate (Lys) along with two additional purified recombinant antigens derived from CagA and VacA of H. pylori. AIM: To study the epidemiology of H. pylori using RIBA H. pylori SIA among chronic HD patients and blood donors as a control group. In addition, the activity of H. pylori was analysed by immunoblot technique in a group of patients with documented ulcers and normal renal function. RESULTS: The prevalence of antibody towards H. pylori among HD patients, blood donors, and patients with documented ulcers was 56% (127/228), 53% (84/158), and 100%, (21/21) respectively; the difference was significant (P=0.0001). The frequency of anti-H. pylori-positive individuals was significantly higher in patients with documented ulcers than HD patients and blood donors, 21/21 (100%) vs 211/386 (55%), P=0.0001. The frequency of antibody to H. pylori in the HD population was significantly associated with race (P= 0.005); no relationship between anti-H. pylori antibody and numerous demographic, biochemical, and clinical features of patients was seen. The frequency of antibodies against virulent strains of H. pylori in HD patients and blood donors with H. pylori was 60% (76/127) and 61% (51/84) respectively; it was 86% (18/21) among individuals with documented ulcers. No significant difference among these three groups occurred. CONCLUSIONS: The frequency of antibody towards H. pylori by RIBA H. pylori SIA was high both in HD patients and blood donors; patients with documented ulcers and normal renal function had significantly higher frequency of anti-H. pylori antibody. The anti-H. pylori antibody rate among HD patients was strongly associated with race. The prevalence of antibody against virulent strains of H. pylori did not change among HD patients and control groups. Studies in large cohorts of HD patients with documented peptic ulcer disease are in progress.

Detection of de novo hepatitis C virus infection by polymerase chain reaction in hemodialysis patients.

Patients on chronic hemodialysis (HD) treatment have been identified by serological testing, including second- and third-generation enzyme-linked immunosorbent assay (ELISA), as a high-risk group for hepatitis C virus (HCV) infection. Previous studies have shown that de novo cases of HCV may occur in HD units in the absence of other parenteral exposures, which suggests the spread of HCV between patients. In addition, the reverse-transcription polymerase chain reaction (RT-PCR), which directly detects HCV virus, has identified HCV infection in chronic HD patients who are seronegative. The aim of this study was to determine the incidence of HCV infection detected by RT-PCR technology in a large cohort of chronic HD patients. One hundred and twenty chronic HD patients, HCV-negative by serological assays (second-generation ELISA) and molecular techniques (branched DNA and RT-PCR), were observed for a mean period of 9.5 months. They were tested monthly for serum alanine aminotransferase levels (ALT) and by second-generation ELISA. At the end of the follow-up period, they were again evaluated by branched DNA and RT-PCR testing. HCV RNA was detected in patients' sera by RT followed by PCR using two separate primer sets from the 5'-untranslated region of the HCV genome. Southern blot was performed using a digoxigenin-labeled probe. Two patients who had HCV RNA detectable by RT-PCR at the end of the follow-up period remained branched-DNA-negative. Thus, the incidence of de novo acquisition of HCV infection in the current investigation was 2.1% per year. In 1 patient RT-PCR positivity and anti-HCV ELISA seroconversion occurred. The 2nd patient remained anti-HCV ELISA-negative, although viremic. In both patients, the onset of positivity by RT-PCR was associated with a rise of ALT levels into the 'abnormal range' in our laboratory. In these 2 patients, de novo acquisition of HCV infection was observed in the absence of obvious parenteral risk factors other than their presence in the HD environment. In conclusion, de novo acquisition of HCV infection may be undetected by ELISA and branched-DNA assays. The need to monitor chronic HD patients by serial ALT testing is emphasized. RT-PCR should be incorporated into diagnostic testing for HCV infection in chronic HD patients. RT-PCR technology can identify HCV in HD individuals with raised ALT activity.

Xenotransplantation of fetal porcine hepatocytes in rats using a tissue engineering approach.

Hepatocytes can be successfully transplanted into highly vascular sites such as the spleen, liver, and lungs. Subcutaneous sites lack adequate vascularization to nutritionally support transplanted hepatocytes. We recently reported that matrix-immobilized angiogenic growth factors, e.g., endothelial cell growth factor (ECGF), can induce a high degree of neovascularization. Using this technique, we explored the possibility of transplanting isolated fetal porcine hepatocytes to create liver tissue organoids at a specific subcutaneous site. We evaluated chitosan as a scaffold biomaterial because of its structural similarity to glycosaminoglycans; glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. Freshly isolated fetal porcine hepatocytes (FPH) (viability greater than 97%) were cultured on modified chitosan scaffolds and transplanted into rat groin fat pads with or without ECGF-induced neovascularization. Cell density and attachment kinetics on chitosan were examined by scanning electron microscopy (SEM) and quantified using a flavianic acid binding assay. Hepatocyte viability and liver organoid formation were examined immunohistochemically. FPH transplanted without prior neovascularization died within 1 day post-transplantation. When transplanted after ECGF-induced neovascularization, FPH thrived for at least 2 weeks and formed liver tissue like structures. Immunohistochemical analysis revealed the presence of hepatocyte-specific cytokeratin staining as well as the presence of alpha-fetoprotein. Light microscopy and SEM revealed that FPH did not change their morphology after attachment to the chitosan surfaces. Thus, chitosan-based biomaterial surfaces have good hepatocyte attachment properties. However, extensive neovascularization is essential for hepatocyte survival and organoid formation. In the future, chitosan-based biomaterials may be useful as scaffolds for creating liver tissue organoids.

Quantitative assessment of HCV load in chronic hemodialysis patients: a cross-sectional survey.

UNLABELLED: Recent evidence has been accumulated showing that chronic hemodialysis (HD) patients have a very high prevalence of antibodies to hepatitis C virus (HCV). In contrast, there is little information addressing the virological characteristics of HCV infection in this population. AIM: To measure HCV viral load and to correlate this with demographic, biochemical, and clinical features of a large cohort of HCV-infected patients on chronic HD. METHODS: 394 chronic HD patients were tested by branched-DNA signal amplification assay, anti-HCV enzyme-linked immunosorbent assay 2.0, and on the basis of the aspartate aminotransferase/alanine aminotransferase (AST/ALT) activity. Multivariate analysis by ordinal logistic regression model was performed: age, gender, race, time on HD, allocation of the patients among the HD units, etiology of end-stage renal disease, HBsAg status, anti-HCV positivity, HCV genotype, and AST/ALT levels were independent factors, and viremic levels of HCV in serum were assumed as dependent variables. RESULTS: 88 (22.3%) patients showed serological and/or virological signs of HCV infection. 59 (15%) out of 394 had detectable HCV RNA in serum, the mean HCV load was 19.4 x 10(5) (95% CI, 6.06 x 10(7) to 6.2 x 10(4)) Eq/ml. According to the criteria suggested by others [J Infect Dis 1994;169:1219-1225], there were 8 (13.5%) individuals with high-titer viremia (>1 x 10(7) Eq/ml) in the subset of viremic patients. A small subset (8/394 or 2%) of individuals was seronegative, but viremic; 29 (7%) out of 394 were seropositive without detectable HCV RNA in serum. Univariate analysis showed that the frequency of anti-HCV positivity was significantly higher in viremic patients as compared with individuals with no detectable HCV viremia: 51/59 (86%) vs. 29/335 (8.6%), p = 0.0001. Serum AST and ALT levels were significantly higher in viremic patients than in individuals with no detectable HCV RNA in serum: 23.8 (95% CI 60.8-9.3) vs. 17.1 (95% CI 50.4-5.8) U/l (p = 0.009) and 14.4 (95% CI 48.9-4.3) vs. 9.8 (95% CI, 37.3- 2. 5) U/l (p = 0.008). Logistic regression analysis showed an association between HCV viremia and anti-HCV positivity (p = 0. 00001) and ALT activity (p = 0.01). CONCLUSIONS: Hepatitis C virus infection is highly prevalent in the HD population; the viral load is relatively low, and it was associated with elevated hepatic enzyme levels and anti-HCV positivity. No other clinical characteristics were associated with HCV RNA levels. Seronegative but viremic patients were also found. Longitudinal studies with long follow-up periods are necessary to evaluate the course of HCV load over time in this population.

Hepatocyte attachment on biodegradable modified chitosan membranes: in vitro evaluation for the development of liver organoids.

Extracellular matrix structures including glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. We evaluated chitosan ([1-->4] linked 2-amino-2-deoxy-beta-D-glucan) as a biomaterial for hepatocyte attachment because of its structural similarity to glycosaminoglycans. Freshly isolated rat and fetal porcine hepatocytes were seeded on chitosan membranes that had been previously blended with collagen, gelatin, or albumin to improve biocompatibility and surface roughness. The optimal cell density and attachment kinetics were quantified. The metabolic activity was investigated by measuring daily urea and total protein secretion by the cells for 2 weeks. While collagen blended-chitosan membranes provided a good attachment surface for rat hepatocytes, albumin and gelatin blended chitosan membranes were superior for fetal porcine hepatocyte attachment. The optimal attachment was maintained with membranes of medium molecular weight (Mr = 750,000 daltons) chitosan, at 3-4 x 10(4) cells/cm2 after 3 h of incubation. In vitro experiments demonstrated that fetal porcine hepatocytes survived at least 14 days when seeded on the chitosan-albumin matrix, demonstrating that this biomaterial can provide suitable cell attachment scaffolds for creating liver tissue organoids.

Acquisition of hepatitis C virus in hemodialysis patients: a prospective study by branched DNA signal amplification assay.

Serological data indicate that hepatitis C virus (HCV) infection is very common among chronic hemodialysis (HD) patients. Circumstantial evidence suggests that hemodialysis per se is an important risk factor for this infection. We used a novel methodology, the branched DNA (bDNA) signal amplification assay, which is capable of detecting HCV RNA and of quantifying HCV viral load in serum, to prospectively determine the rate of acquisition of HCV infection in 274 anti-HCV-negative patients undergoing HD treatment in four hemodialysis units. Moreover, we used bDNA testing to analyze the dynamics of HCV acquisition among HD patients, a high-risk group for HCV infection with immune compromise conferred from uremia. Two patients were identified with de novo acquisition during 1 year of prospective bDNA testing. Thus, the HCV incidence was 0.73% per year. De novo acquisition of HCV infection was observed in the absence of identifiable parenteral risk factors. Both patients showed the same pattern of HCV acquisition: they underwent an initial viremic phase that was associated with an increase in alanine transaminase (ALT) activity and that preceded the anti-HCV seroconversion. This was followed by HCV RNA clearance and normalization of ALT activity. Anti-HCV positivity occurred 1 and 2 months after the ALT increase in the first and second patients, respectively. Although HCV incidence was low (0.73%), further research is warranted to set the optimal policy for eliminating the risk of nosocomial transmission of HCV in the HD setting. Our findings show the pattern of HCV acquisition in chronic HD patients and emphasize the need to screen the HD population for ALT measurement combined with anti-HCV testing for detecting hepatitis C. HCV RNA testing can identify HCV before seroconversion in individuals with deranged liver function tests. The acquisition of HCV in HD patients without identifiable risk is confirmed.

Automated RIBA hepatitis C virus (HCV) strip immunoblot assay for reproducible HCV diagnosis.

A comparison between the CHIRON RIBA hepatitis C virus (HCV) processor and manual systems was performed by using 88 specimens repeatedly reactive by the second-generation HCV enzyme-linked immunosorbent assay (ELISA) (HCV 2.0 ELISA) and 111 random specimens from volunteer donors. For the second-generation RIBA HCV strip immunoblot assay (SIA) (RIBA HCV 2.0 SIA), test results correlated strongly between the manual and the automated runs (kappa value, 0.937). For the RIBA HCV 3.0 SIA, the correlation of the test results was also high (kappa value, 0.899). Among the specimens with positive results by RIBA HCV 2.0 and 3.0 SIAs, there was a very strong concordance of the test results between the manual and the automated runs with regard to the reactive bands. Nine samples had discordant results between the manual and the automated runs; this was probably attributable to increased variability in antigen scores close to the cutoff values for both tests. Run-to-run and within-run testing by the CHIRON RIBA HCV Processor System showed a very low rate of conflicting values. In conclusion, the CHIRON RIBA HCV Processor System is capable of performing RIBA HCV 2.0 and 3.0 SIAs accurately with minimal operator involvement. In addition, the CHIRON RIBA HCV Processor System shows excellent reproducibility, with the potential for operator-to-operator and site-to-site variability being greatly reduced. Our data indicate that this novel methodology may be very useful for supplemental anti-HCV testing of specimens repeatedly reactive by ELISA in routine clinical assessments and epidemiologic evaluations.

Hepatitis C: controversies, strategies and challenges.

Risk factors for hepatitis C infection include I.V. drug use (42%); history of blood transfusion (6%); exposure to multiple heterosexual partners (6%); exposure to a household contact (3%); health care employment (2%); or hemodialysis (1%). Forty percent of patients have no identifiable risk factors. The HCV is a single-stranded, positive-sense RNA virus. Six major genotypes have been identified; each contains a series of subtypes. In the U.S., prevalences are type 1 (74%); type 2 (15%); type 3 (6%); and type 4 (1%). Within an infected individual, HCV also exists as a spectrum of closely related genotypes referred to as a quasispecies, and more complex quasispecies correlate with longer duration of disease, higher levels of viremia, genotype 1 infection, and poorer response to interferon therapy. Diagnosis is made by measuring anti-HCV by EIA, with confirmation by RIBA or HCV RNA. Patients with chronic HCV infection, with or without aminotransferase elevation, have detectable serum RNA by PCR. Standard therapy is interferon alfa 2b (Intron A) at a dosage of 3 million units 3 times a week for 6 months. This results in a 40%-50% complete response at the end of treatment (normal aminotransferases and undetectable HCV RNA), but relapse occurs in 60%-80% of cases over the next six months. Longer (12 month to 18 month) courses are now widely advocated. Better patient selection, e.g., those with low serum HCV RNA levels and absence of cirrhosis, and increased duration of therapy may lead to better response rates. Combination therapy with other antiviral agents, such as ribavirin, has dramatically reduced relapse rates.

The bioartificial liver: state-of-the-art.

The rationale for artificial liver support is based on the hypothesis that if essential liver functions can be restored during the critical phase of liver failure, it should be possible to improve the survival of patients with severe liver disease. In the case of bridge-to-transplantation, it should provide the patient sufficient metabolic support until a donor liver can be found and transplanted. Since the management of acute liver failure requires the replacement of the liver's myriad metabolic functions, the idea of a hybrid bioartificial liver (BAL) support system has been proposed. BAL systems incorporate a biological (hepatocytes) and a synthetic housing component (plastic housing shell and semipermeable membrane) coupled in such a way as to facilitate the delivery of essential liver functions. Of the several BAL designs that have been proposed, only the capillary hollow-fiber based systems have been rapidly developed for clinical trials. Capillary hollow-fiber based BAL devices are basically off-the-shelf artificial kidney membranes that have been modified for use as an artificial liver. However, most capillary hollow-fiber based BAL designs have inherent physical limitations of total diffusion surface area and capacity for hepatocyte mass. We have proposed a novel BAL design using microencapsulated hepatocytes to overcome these physical limitations. This new BAL design (UCLA-BAL) involves the direct hemoperfusion of a packed-bed column of microencapsulated porcine hepatocytes within an extracorporeal chamber. In extensive animal studies using a well-characterized animal model fulminant hepatic failure (FHF), we demonstrated that the UCLA-BAL system had superior diffusion surface area and a higher capacity for hepatocytes compared to conventional capillary hollow-fiber based BAL devices. UCLA-BAL treatment significantly (P<0.001), improved the survival rate of FHF animals and significantly (P<0.01) prolonged the survival time of similar animals with very severe liver injury. BAL treatment was convenient, easy to operate and well tolerated, and did not adversely affect the animal's hemodynamics during treatment. We therefore suggest that the UCLA-BAL is a significant improvement over conventional, first-generation, capillary hollow-fiber BAL systems.

The impact of managed care on gastroenterology.

Managed care has emerged out o pressures to reduce the high cost of healthcare and has brought about unprecedented change and uncertainty for the specialist. To flourish in a managed care environment the subspecialist will need to rely on strategic planning. Four emerging approached to healthcare lend themselves to strategic planning. They are 1) practice-based clinical trials, 2) outcomes research, 3) clinical practice guidelines, and 4) systems-based disease management. This report explains how subspecialists in private practice can participate in and profit from each of these new approaches.

Controlled release of endothelial cell growth factor from chitosan-albumin microspheres for localized angiogenesis: in vitro and in vivo studies.

Endothelial cell growth factor (ECGF) stimulates vascularization, however its relatively short half-life requires this angiogenic factor to be frequently administrated by non-specific and uncontrolled methods. This work describes the use of biocompatible chitosan, a polysaccharide having structural similarity to glycosaminoglycans, -albumin microspheres, as well as its fiber form, as a potential delivery system for the controlled and localized release of ECGF. Chitosan-albumin microspheres (400-600 microns) and fibers, formed in 0.5 M sodium hydroxide-methanol solution were incubated with ECGF. In vitro release was performed in PBS at 37 degrees C, under constant stirring. In vivo experiments were realized by implanting ECGF loaded matrices subcutaneously into rat groin fascia. After an initial ECGF burst of 1.32-1.62 mg (22-27%) within the first 2 hours, a daily release of 120-420 micrograms (2-7%) during the first, and 60-240 micrograms (1-4%) during the second week was observed from M(r) 70.000, 750.000, and 2,000.000 chitosan containing microspheres of 6 mg/ml loading. ECGF release rate of < 30 micrograms (0.5%)/day was maintained during the third week of experiments. By the increase in ECGF loading (12 mg/ml polymer), while the amount of release increased, percent release decreased. Chitosan-albumin fibers gave a ECGF release rate nearly similar to microspheres, and in vivo studies demonstrated a high degree of neovascularization for both types of implants, starting from 7 day-post implantation. Control animals that received ECGF injection did not show any significant neovascularization, after same period of time.