Gary Gitnick

Non-A, Non-B Post-Transfusion Hepatitis: Looking Back in the Second Decade

Non-A, non-B post-transfusion hepatitis is a sequela commonly associated with the receipt of blood products. In most prospective series conducted before the introduction of surrogate and acquired immunodeficiency syndrome (AIDS) screening, otherwise unexplained abnormalities in serum alanine aminotransferase (ALT) levels developed in about 10% of transfused patients during the 6 months after transfusion [1-3]. Usually, no concomitant serologic evidence of exposure to hepatitis A or B virus could be identified, suggesting that one or more non-A, non-B agents were responsible [4]. The abnormal ALT values persisted for more than 6 months in about 50% of patients [5, 6]. The magnitude of this disease was not appreciated before these prospective studies because of the difficulty in recognizing these patients on the basis of symptoms. During the early phase of the illness, only a minority of patients experience severe fatigue, nausea, and vomiting, and even fewer manifest full-blown clinical symptoms including jaundice [1]. Those who have mild fatigue often blame it, perhaps correctly, on the disease for which they received the transfusion. Very few in whom prolonged abnormalities in serum aminotransferase levels develop complain of any symptoms, although some describe varying degrees of fatigue [7]. Rapidly progressive liver disease and acute symptomatic exacerbations are both uncommon in this form of chronic hepatitis, although the aminotransferase levels may remain abnormal or fluctuate for years. Several diagnostic tools, such as blood tests or biopsies, can detect signs of disease. These signs may exist in the absence of symptoms and may become the focus of attention or of therapy [8]. Disease is perceived by patients, however, if they have symptoms. Signs such as an abnormal ALT level or histologic evidence of inflammation do not themselves interfere with the quality of a persons life. As long as liver function is compensated, a person may remain unaware of the presence of cirrhosis. Thus, the important concern for the patient with non-A, non-B post-transfusion hepatitis is the development of liver failure (or liver cancer) secondary to cirrhosis. Between 1972 and 1980, we identified patients with non-A, non-B post-transfusion hepatitis. We recently evaluated this cohort, focusing on the presence or absence of clinical evidence of liver failure. Methods Between 1972 and 1980, two prospective studies of post-transfusion hepatitis were conducted at the University of California at Los Angeles (UCLA). These studies have been previously described [9]. All patients had normal ALT levels before transfusion and were prospectively identified as having developed non-A, non-B post-transfusion hepatitis when they met the following criteria: 1) the development of at least two consecutive abnormal ALT levels, at least one being twice the upper limit of normal or greater [five times the upper limit of normal or greater in the first study], occurring within 6 months of transfusion; 2) the absence of serologic evidence for recent hepatitis A (negative IgM-specific antibody to hepatitis A) or hepatitis B (negative hepatitis B surface antigen [HBsAg] and negative IgM-specific antibody to hepatitis B core antigen [anti-HBc]); and 3) the absence of any explanation for the abnormalities in the ALT levels other than the presence of presumed viral infection. Patients who developed post-transfusion hepatitis were asked to continue in the prospective evaluation. Those whose ALT values became normal within 6 months of disease onset were considered to have had acute hepatitis and no additional follow-up was sought at that time. Those whose ALT values remained abnormal for longer than 6 months were considered to have developed chronic hepatitis and were asked to continue in an open-ended follow-up. Over the years, many study patients in whom chronic hepatitis developed were lost to follow-up for various reasons (death, move from the area, loss of interest, or lack of desire for continued blood tests). In the fall of 1988, a formal attempt was begun to locate all study patients in whom non-A, non-B post-transfusion hepatitis developed and to evaluate each for the presence or absence of symptoms or signs of hepatitis and evidence of clinical hepatic failure. Symptomatic hepatitis was defined as the presence of one or more of the following: jaundice, loss of appetite, weight loss, or fatigue. (For fatigue to be considered to be significant, it had to interfere with the patients daily lifestyle.) No symptom could be reasonably attributed to a coexistent disease process. Clinical hepatic failure was considered to exist if one or more of the following was found or had occurred: 1) Gastrointestinal bleeding of variceal origin; 2) ascites [not due to noncirrhotic causes]; 3) hepatic encephalopathy; 4) hepatic coagulopathy [abnormal prothrombin time not correctable by the parenteral administration of vitamin K]; 5) hypersplenism (leukopenia or thrombocytopenia not due to noncirrhotic causes; or 6) hypoalbuminemia (a serum albumin level less than the normal range not due to noncirrhotic causes). A telephone interview was conducted to assess current and past health status of all surviving patients who could be located. Questions were designed to identify problems related to hepatic failure. Where pertinent, requests were made to obtain outside medical records. Patients were also asked to submit to a formal history and physical examination and to have blood obtained for the following tests: complete blood count, serum chemistry panel, prothrombin time, and partial thromboplastin time (PT). If the patient agreed to come to UCLA, the examination was conducted by one of the investigators. In a few circumstances, the investigator went to the patients home. If the patient did not agree to these arrangements, medical records or examinations were sought from his or her personal physician. To be considered evaluable between 1989 and 1992, patients had to have completed at least the telephone interview and the laboratory tests. If a patient was dead, a telephone interview was conducted with a family member (or some other knowledgeable contact) to ascertain the state of the patients previous health and the cause of death. In addition, requests were made to obtain pertinent medical records (for example, death certificates or records of terminal hospitalization). Because of the variability in the duration of follow-up, the probability of clinical hepatic failure developing was calculated using life-table analysis [10]. The end point was the appearance of clinical hepatic failure. The time at risk began with the onset of post-transfusion hepatitis and extended until the end point was achieved, until the last medical evaluation, or until death without evidence of clinical hepatic failure. Other statistical comparisons were made by the chi-square test with the Yates correction or by the Student t-test [11]. Between 1972 and 1980, 95 patients with non-A, non-B post-transfusion hepatitis were identified, including the 69 patients previously described [7]. In addition, 20 patients originally classified as having had hepatitis B have since been reclassified. Eighteen had evidence of anti-HBc in the early post-transfusion serum. None of these 18 patients tested positive for IgM-anti-HBc, and the anti-HBc disappeared shortly afterward in all cases. It was presumed that this finding represented passive transfusion of anti-HBc, and the patients were reclassified as having had non-A, non-B disease. Two patients had had post-transfusion increases in the titer of antibody to HBsAg (anti-HBs); the increases in both cases were low-grade and may have represented passive administration of anti-HBs. In retrospect, these patients were also not thought to have had hepatitis B viral infection, and they were also reclassified as having had non-A, non-B-induced disease. Finally, six patients were identified from the last part of the second prospective study; these patients have not been previously described. Of these 95 patients, 5 were excluded from this analysis. In one case, the abnormal ALT level did not reach the biochemical criterion of twice the upper limit of normal until after 6 months of disease. The other four patients had concomitant liver diseases (sclerosing cholangitis [one patient], biopsy-proven cirrhosis before transfusion (one patient), and other potential causes of chronic hepatitis [two patients]), which made it difficult to determine the natural history of the transfusion-associated disease. We therefore evaluated the remaining 90 patients. Serologic tests for antibodies against hepatitis C (anti-HCV) have become available [12]. The stored sera of these patients were tested by a commercial assay that detects antibody to the C-100 HCV antigen (Ortho HCV Enzyme-Linked Immunosorbent Assay [ELISA] Test System, Ortho Diagnostic Systems, Raritan, New Jersey), and those patients who failed to show seroconversion by this assay were tested by a second-generation test (Ortho HCV 2.0 ELISA Test System) [13]. Patients who were anti-HCV negative before transfusion (or, in some cases, before the onset of disease) and who developed anti-HCV in the first year after the hepatitis began were considered to have hepatitis C. (These seroconversions were not considered to be a consequence of passive transfusion of antibody, because the anti-HCV was shown to persist for more than 6 months or because the titer, as reflected by the optical density, increased over time.) In addition, patients who no longer had pretransfusion or predisease sera available were defined as having hepatitis C if anti-HCV was present for at least 6 months in their postdisease sera. The study was conducted in accordance with guidelines established by the Human Subject Protection Committee at UCLA. All potential study participants received an oral explanation of the study as well as an informed consent docume

Non-A, Non-B Posttransfusion Hepatitis—A Decade Later

We have followed up 69 patients who developed non-A, non-B posttransfusion hepatitis in 1972-1978. Chronic hepatitis, defined by biochemical criteria, was observed in 46 patients (67%), the majority of whom subsequently failed to resolve the abnormalities. Chronic hepatitis was a sequela of non-A, non-B posttransfusion hepatitis less often after the blood bank changed to a policy of all volunteer donors. (However, this association may be explained by other coexistent factors.) The alanine aminotransferase level was more likely to be abnormal than the aspartate aminotransferase level during the chronic phase of non-A, non-B posttransfusion hepatitis. By actuarial means it was calculated that the probability of developing normal enzymes after 6-10 yr was 0.47. However, in spite of this high incidence of biochemical disease, virtually all of the patients have remained asymptomatic. Histologic evidence of cirrhosis has been obtained in 4 of these patients, but in only 2 patients at most has clinical evidence of hepatic failure supervened.

Chronic active hepatitis. Who meets treatment criteria

In order to ascertain the proportion of patients with biopsy-proven chronic active hepatitis who meet currently accepted criteria for immunosuppressive treatment, an analysis of 86 patients seen between 1973 and 1978 carrying this diagnosis was undertaken. Only 66 could be confirmed to have this lesion on blind histologic review. Nine of these 66 were on concomitant immunosuppressive therapy, four had inadequate documentation of chronicity, five consumed more than two ounces of alcohol daily, five had concurrent malignancy, two were prepubertal, and one had oxyphenisatin-induced disease. None of the remaining 40 patients met the biochemical criteria for disease activity. The disease was predominantly seen in asymptomatic middle-aged males and was of viral etiology. A small subgroup of elderly female patients was also identified whose disease was apparently nonviral. In conclusion, the vast majority of chronic active hepatitis seen at a large university center occurs in individuals for whom treatment guidelines have not been established.

Contrasting features and responses to treatment of severe chronic active liver disease with and without hepatitis BS antigen.

To determine the clinical implications of HBSAg in severe chronic active liver disease (CALD), patients with HBSAg positive CALD were compared with those chosen by identical clinical, functional, and morphological criteria in whom this test and anti-HBS were negative. HBSAg positive patients were predominantly males over 40 years of age and more frequently failed to respond to conventional treatment programmes with prednisone. HBSAg negative patients were more often female and younger, had a higher incidence of associated immunopathic disease and immunoserological markers in high titre, and more often responded to treatment with full remission of their disease. HBSAg positive patients failing treatment with conventional doses of prednisone often improved with higher doses, but did not reach full remission of their disease. The benefit-risk ratio of both conventional and high doses of prednisone in HBSAg positive severe CALD needs further clarification.

Cryopreserved microencapsulated hepatocytes--transplantation studies in Gunn rats.

Hepatocyte transplantation has been shown to provide significant metabolic support in several animal models of liver diseases. However, for it to be a viable alternative for supplementation of liver function in disease, large quantities of isolated hepatocytes would be necessary. At the present time there are no inexpensive routine methods for cryopreservation of hepatocytes. Existing procedures are cumbersome and require expensive programmable freezers. Hepatocyte cultures are sensitive and easily damaged in handling. By utilizing techniques of microencapsulation and cryopreservation we have attempted to overcome these problems. We have developed a simple, convenient, and inexpensive technique for the long-term storage of hepatocytes. Biological activity of the nonfrozen isolated encapsulated hepatocytes (IEH) and cryopreserved IEH (cIEH) was assessed both in tissue culture and by transplantation in Gunn rats. Significant urea and protein syntheses were detectable during the 10-day culture period even in the 30-day cIEH. Additionally, transplanted IEH and cIEH significantly reduced hyperbilirubinemia in Gunn rats for up to 30 days posttransplantation. Control (empty) microcapsules did not lower serum bilirubin levels. Thus we conclude: (1) cryopreservation of IEH is a convenient and cost-effective method for preserving and storing hepatocytes; (2) cryopreserved IEH function as well as nonfrozen IEH both in vitro and in vivo; (3) microencapsulation may protect hepatocytes from the adverse effects of cryopreservation.

Preliminary report on isolation of mycobacteria from patients with Crohn's disease

Several investigators have recently described the isolation of slow growing mycobacteria from the tissues of patients with Crohns disease (CD). The primary purpose of this study was to culture and identify mycobacteria from the intestines of patients with CD and other intestinal diseases (control tissues). The culture methods were designed to eliminate most rapidgrowing mycobacteria and to enhance the isolation of slow growing mycobacteria. Eighty-two surgically resected intestinal tissue samples were cultured over a four-year period: 27 tissues were from CD patients and 55 from patients with other intestinal diseases. After 4–12 months of culture, five mycobacteria were isolated, but only two have been identified thus far. Both of these organisms appeared to have initially grown as spheroplasts, but revertant bacteria were cultivated after transfer into fresh media. Four of the mycobacteria were from CD tissues, and one isolate was from a control tissue. Two of the isolates have been identified as M. cheloneisubsp. abscessus,strain 390, and M. paratuberculosisstrain 410. This M. paratuberculpsisis similar to the previously identified M. paratuberculosisstrains isolated from other human intestinal tissues from patients with CD. Both strains 390 and 410 were inoculated into neonatal goats, but they failed to reproduce a CD-like disease. The isolation of four mycobacteria from 27 CD tissues and only one from 55 control tissues strengthens the findings of previous investigators and supports the hypothesis that mycobacteria may be etiologically associated with some cases of Crohns disease.

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

Abstract 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 × 10 5 Eq/mL; range, 6.3 × 10 5 to 16.03 × 10 6 Eq/mL versus intermittent, 10.4 × 10 5 Eq/mL; range, 1.1 × 10 5 to 9.4 × 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 × 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.

Post-Transfusion Chronic Liver Disease

To document the sequelae of acute hepatitis among recipients of commercial and volunteer blood and to assess factors influencing the development of chronic hepatitis (CH), 47 patients with post-transfusion hepatitis were followed prospectively from the time they received their transfusions. Twenty-nine had prolongation of at least 2-fold serum glutamic pyruvic transaminase (T) elevations for more than 20 weeks, and were classified as CH. When the patients with CH were compared to those with only acute hepatitis (abnormal T for less that 20 weeks), no difference was found with respect to age, sex, number of units transfused, incubation period, presence or absence of symptoms, occurrence of jaundice, maximum T, receipt or development of hepatitis B surface antigen or antibody, underlying illness, or area of the hospital where the patient was treated. Liver biopsies in 15 of the 29 revealed chronic-active hepatitis in 9, chronic persistent hepatitis in 2, unresolved hepatitis in 4. Five of the 9 patients with chronic active hepatitis were without symptoms. None of these died or have developed cirrhosis. Because chronic liver disease frequently developed after acute post-transfusion hepatitis among multiply transfused hepatitis B surface antigen negative blood recipients, close follow-up, including liver biopsy, is warranted in such patients with prolonged transaminase elevations.

Quantitative Assessment of HCV Load in Chronic Hemodialysis Patients: A Cross-Sectional Survey

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 × 105 (95% CI, 6.06 × 107 to 6.2 × 104) 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 × 107 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.

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.