Long-term responders without eradication of hepatitis C virus after interferon therapy: characterization of clinical profiles and incidence of hepatocellular carcinoma.

AIMS/BACKGROUND: The aim of this study was to determine the characteristics of patients with chronic hepatitis C showing long-term normalization of alanine aminotransferase (ALT) without eradication of HCV RNA, as well as to investigate the incidence of hepatocellular carcinoma (HCC) in such patients. METHODS: Four hundred and nineteen patients with histologically-proven chronic hepatitis C who had received interferon (IFN) therapy were studied. Complete response (CR) was defined as persistent normalization of ALT levels with eradication of serum HCV RNA (n= 126). Long-term biochemical response with positive HCV RNA (HCV-positive BR) was defined as a normal ALT level at 6 months after IFN therapy with further persistent normalization of ALT levels for 2 or more years without eradication of serum HCV RNA (n=49). All other patterns were classified as non-response (NR, n=244). RESULTS: Mean follow-up periods of CR, HCV-positive BR and NR groups were 4.9, 5.2 and 4.9 years, respectively. The HCV-positive BR group had significantly higher serum HCV RNA levels and a higher rate of HCV serological group 1 classification than the CR group. The other characteristics of the HCV-positive BR group were lower histologic activity, lower ALT levels, and a higher rate of females when compared with both the CR and NR groups. Histologic staging in the HCV-positive BR group was significantly lower than that in the NR group. Cumulative incidences of HCC estimated by the Kaplan-Meier method in both the CR and HCV-positive BR groups were significantly lower than those in the NR group (log-rank test, CR vs NR p<0.001, HCV-positive BR vs NR p=0.026). CONCLUSION: The patients with HCV-positive BR were virologically different from those with CR, and had lower ALT levels and histologic activity when compared to those with CR and NR.

Changes in serum hepatic fibrosis markers in biochemical responders to interferon therapy for chronic hepatitis C.

Serum hepatic fibrosis markers (7s domain of type IV collagen, N-terminal peptide of type III procollagen, and hyaluronate) were determined during and after a 6-month interferon treatment of patients with chronic hepatitis C. Changes in these markers were compared among the patients who showed a sustained normalization of serum alanine transaminase (ALT) levels with and without eradication of serum hepatitis C virus RNA (complete responders and biochemical responders) and nonresponders. In the case of complete responders, the serum 7s domain of type IV collagen and the N-terminal peptide of type III procollagen levels decreased at the end and 24 weeks after the end of the treatment. Hyaluronate levels were significantly decreased 24 weeks after the end of the treatment, as compared with those prior to the treatment. During and after interferon treatment, changes in these markers in the case of biochemical responders were nearly the same as those in the complete responders. These results suggest that serum hepatic fibrosis markers decrease in patients with chronic hepatitis C who show a sustained normalization of ALT after interferon treatment, even if serum hepatitis C virus RNA fails to be eradicated.

Injection of mitomycin-C-treated spleen cells induces donor-specific unresponsiveness to cardiac allografts in rats.

BACKGROUND: In this study, preoperative mitomycin-C- (MMC) treated donor-specific transfusion (DST) was examined for its ability to induce unresponsiveness to cardiac allografts in rats. METHODS: DA (RT1a) rats were used as donors, BUF (RT1b) or WS (RT1k) rats as recipients, and Lew (RT1l) rats as third party donors. BUF or WS rats were given i.v. injection of DA spleen cells (SPCs) suspension (5x10(7)/l ml) with or without MMC treatment 10 days before cardiac transplantation. Delayed-type hypersensitivity and complement-dependent cytotoxicity assays were carried out in these animals separately to examine in vivo immunosuppressive effect. Suppressor assay was also examined to determine in vitro immunosuppressive effects in allogeneic mixed leukocyte culture. RESULTS: In the full allogeneic DA-to-BUF rat strain combination, preoperative i.v. administration of MMC-treated donor SPCs led to a significant prolongation of graft survival over the control (110+/-66 versus 7.2+/-0.8 days: P<0.01), although administration of nontreated donor SPCs did not (9.3+/-1.0 days). This beneficial effect of MMC treatment was also seen in the DA-to-WS rat combination (31+/-16 days versus donor-specific transfusion alone; 11+/-1.5 days or untreated control; 12+/-1.5 days; P<0.05). However, injection of third party DA SPCs in the Lew-to-BUF combination induced no significant prolongation of cardiac allograft survival compared with the untreated control (11+/-0.6 versus 11+/-2.0 days; NS), indicating that this prolongation effect was induced in an antigen-specific manner. The immunosuppressive effect was also secured for both delayed-type hypersensitivity response and anti-donor cytotoxic antibody production. Moreover, addition of MMC-treated SPCs to mixed lymphocyte culture led to antigen-specific suppression. CONCLUSIONS: Preoperative i.v. injection of MMC-treated donor SPCs is promising for inducing unresponsiveness in rat cardiac allograft model.

Relation of interferon therapy and hepatocellular carcinoma in patients with chronic hepatitis C. Osaka Hepatocellular Carcinoma Prevention Study Group.

BACKGROUND: The effect of interferon therapy on the incidence of hepatocellular carcinoma in chronic hepatitis C is poorly defined. OBJECTIVE: To compare the incidence of hepatocellular carcinoma in interferon-treated patients with chronic hepatitis C to that of historical controls and to examine whether response to therapy is related to incidence of hepatocellular carcinoma in patients with chronic hepatitis C. DESIGN: Retrospective cohort study. SETTING: One university hospital and seven university-affiliated hospitals. PATIENTS: 419 consecutive patients with chronic hepatitis C who started interferon therapy between January 1992 and December 1993 (interferon group) and 144 patients with chronic hepatitis C who had liver biopsy between January 1986 and December 1989 and did not receive interferon (controls). INTERVENTION: Patients in the interferon group received human lymphoblastoid interferon, recombinant interferon-alpha2a, or recombinant interferon-alpha2b for 6 months. MEASUREMENTS: The end point was development of hepatocellular carcinoma on abdominal ultrasonography or computed tomography. Sustained response was defined as persistent normalization of alanine aminotransferase (ALT) levels during interferon therapy and follow-up. Relapse was defined as a normal serum ALT level at the end of treatment with an increase to an abnormal level after cessation of treatment. Nonresponse included all other ALT patterns. RESULTS: Median follow-up in the interferon and control groups was 47.6 and 46.8 months, respectively. During follow-up, hepatocellular carcinoma was found in 28 interferon-treated patients and 19 controls. Cox proportional hazards regression analysis that included all patients revealed that interferon therapy (P=0.041), older age (P=0.003), greater histologic activity (P=0.029), and higher histologic stage (P=0.049) were independent factors associated with the development of hepatocellular carcinoma. The risk ratios for development of hepatocellular carcinoma in patients with sustained response, relapse, and nonresponse were 0.06 (95% CI, 0.01 to 0.46), 0.51 (CI, 0.20 to 1.27), and 0.95 (CI, 0.48 to 1.84), respectively, compared with controls. CONCLUSIONS: The incidence of hepatocellular carcinoma was lower in patients with sustained response to interferon therapy than historical controls and nonresponders. Interferon therapy may decrease the risk for hepatocellular carcinoma in patients with chronic hepatitis C.

Indefinite survival of skin allografts in adult thymectomized, antilymphocyte serum-treated mice given bone marrow and thymus grafts of donor origin: tolerance induction by donor bone marrow and thymus.

BACKGROUND: Perigraft immunosuppression with antilymphocyte serum (ALS) and postgraft infusion of donor bone marrow cells (BMC), which induces allograft unresponsiveness in a class I major histocompatibility complex (MHC)-disparate mouse combination, is much less effective in class I and II MHC-disparate strain combinations. Because the thymus plays a central role in the maturation, differentiation, and education of T lymphocytes, which are the principal mediator of allograft responses, we examined whether transplantation of donor-specific thymus combined with adult thymectomy of recipients enhances the tolerogenic effect of ALS and donor BMC infusion in a strongly histoincompatible class I and II MHC-disparate DBA/2-to-B6AF1 mouse strain combination. METHODS: Adult thymectomy was performed 4 weeks before grafting. B6AF1 mice received ALS (days -1 and 2), skin allografts (day 0), and BMC and/or thymus grafts (ThyTx) (day 7). Flow cytometric analysis was used to detect donor cells in tolerant mice. Limiting dilution assay was used to calculate the frequencies of precursor cytotoxic T lymphocytes against donor and third-party alloantigens. The presence of suppressor cells was determined by in vivo adoptive transfer assay and in vitro coculture mixed lymphocyte culture. RESULTS: When adult thymectomy and postgraft donor ThyTx were combined with ALS and donor BMC, DBA/2 allograft survival was prolonged with all grafts surviving for >122 days. Third-party (DBA/1) or recipient ThyTx was not effective. The tolerant mice accepted the second donor skin allografts but acutely rejected the third-party grafts. No significant chimerism was detected. Marked reduction of precursor cytotoxic T lymphocyte frequencies continued only against donor alloantigens after second donor and third-party skin grafting. No suppressor cell activity was detected. Immunopathological analysis revealed that the cells in the ThyTx of tolerant mice are of donor origin. CONCLUSION: Donor ThyTx combined with donor BMC infusion in adult thymectomized, ALS-treated mice induced clonal deletion of donor-reactive T cells.

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Superiority of sirolimus (rapamycin) over cyclosporine in augmenting allograft and xenograft survival in mice treated with antilymphocyte serum and donor-specific bone marrow.

BACKGROUND: Sirolimus is a potent immunosuppressive agent with great therapeutic potential. The objective of our study was to evaluate the efficacy of sirolimus versus cyclosporine in augmenting the unresponsiveness induced by an antilymphocyte serum (ALS)/donor-specific bone marrow (BM)-based regimen across three levels of histoincompatibility: class I and II disparate (DBA/2 to B6AF1), complete mismatch (AKR to C57BL/6), and xenograft (ACI rat to B6AF1). METHODS: Full-thickness skin grafts were taken from donors and placed on recipients in standard fashion. Seven groups of recipient mice (n=10-28) received various combinations of the following treatment protocols: sirolimus, 1.5 mg/kg (3.0 mg/kg for xenografts) every other day from day 0 to day 12; cyclosporine, 50 mg/kg every other day from day 10 through 22; ALS, 0.5 ml on days -1 and 2 for allografts and days -1, 2, and 4 for xenografts; and BM, 25 million donor-specific cells IV on day 7. RESULTS: The administration of ALS or ALS/BM resulted in modest but significant prolongation of skin graft survival in all combinations tested. Cyclosporine combined with ALS or ALS/BM significantly extended allograft survival compared with ALS or ALS/BM alone (P<0.05) but had no effect on xenograft survival. In contrast, the combination of sirolimus with ALS or ALS/BM resulted in a two- to threefold increase in allograft survival and over a fourfold increase in xenograft survival when compared with the comparable cyclosporine-based regimen. Additionally, lymphocytes isolated from class I and II incompatible mice with skin grafts surviving >100 days demonstrated markedly reduced interleukin 2 and interferon-gamma secretion in response to irradiated donor-specific lymphocytes in culture. CONCLUSIONS: In the regimens tested, sirolimus was superior to cyclosporine in augmenting donor BM-induced skin graft prolongation in ALS-treated mice across all levels of histoincompatibility.

A protocol with FK 506 for inducing unresponsiveness to murine islet allografts.

BACKGROUND: The most favorable protocol for transplantation is inducing unresponsiveness before operation by means of nondangerous modalities. This would permit discontinuance of long-term use of immunosuppressants. In this study we developed a potential protocol for inducing unresponsiveness to islet allografts by preoperative donor spleen cell inoculation (DSI) and a single injection of FK 506. METHODS: BALB/c (H-2d) and C57BL/6 (H-2b) mice were used as islet donors and recipients, respectively. The streptozocin-induced diabetic mice that had been given DSI at a dose of 1 x 10(7) or 1 x 10(4) and a single injection of FK 506 (10 mg/kg intramuscularly) at different schedules (on day 1, 3, 5, or 7 relative to DSI on day 0) were subjected to islet allografting on day 10. RESULTS: All islet recipients returned to normoglycemia within a few days with no toxic effect of FK 506 treatment. DSI at a dose of 1 x 10(7) alone led to shortening of the mean survival time to 10.1 +/- 4.1 days, as compared with 13.5 +/- 6.3 days for the untreated animals. In contrast, marked prolongation of graft survival was induced when FK 506 was given on day 3 (> 84 +/- 27.3 days, p < 0.0001) or on day 5 after DSI (> 50.9 +/- 46.0 days, p < 0.05). Five of seven allografts given FK 506 on day 3 and three of seven allografts given FK 506 on day 5 survived indefinitely. Other time schedules of DSI and FK 506 treatment (on day 1 or 7 after DSI) or FK 506 treatment alone had no significant effect on mean survival time. With the same protocol, third-party islet allografts (C3H) were immediately rejected (10.6 +/- 2.6 days). CONCLUSIONS: Prolongation of islet allograft survival was induced by certain doses of DSI and preoperative FK 506 treatment. This modality prevents an adverse effect of FK 506 on grafted islets and can induce unresponsiveness to islet allografts, offering a protocol for successful clinical islet transplantation.

A potential immunosuppressive effect of anti-lymphocyte function-associated antigen-1 monoclonal antibody on islet transplantation.

The immunosuppressive potentials of mAbs to lymphocyte function-associated antigen-1 (LFA-1) and CD2 molecules were examined in murine islet transplantation. Crude digested islets from BALB/c (H-2d) mice were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. The rat mAbs of KBA (anti-LFA-1) and RM2-1 (anti-CD2) were given intraperitoneally immediately after transplantation and on the first day after grafting at a dose of 0.1 mg/mouse/day. In nontreated animals, the islet allografts were acutely rejected with a mean survival time (MST) of 19.6 +/- 8.3 days. Control isotype-matched anti-CD18 treatment did not prolong the MST of 12.8 +/- 1.6 days. Anti-LFA-1 treatment alone produced indefinite survival in 5 of 10 recipients with MST of 72.2 +/- 33.4 days. Anti-CD2 treatment failed to do so, although MST was marginally prolonged to 32.8 +/- 20.5 days. When both mAbs were given together, additional benefit with anti-CD2 treatment was not observed (MST: 77.4 +/- 31.1 days). In spite of the unresponsiveness to islet allografts, the animals did not suffer from any severe infectious disease. Mice bearing long-term functioning islets rejected third-party skin grafts as well as islet donor strain skin grafts. The long-term surviving islet allografts were also rejected coincidentally. These results indicate that a perioperative short course of anti-LFA-1 mAb treatment can induce unresponsiveness to islet allografts, although it is not systemic, and that costimulatory signals through these adhesion molecules play a central role in inducing an immune response leading to rejection of the allografted islets.

Low temperature collagenase digestion for islet isolation from 48-hour cold-preserved rat pancreas.

We examined the efficacy of relatively low temperature collagenase digestion at 20 degrees C on the yield and viability of islets after long-term cold preservation. Wistar rat pancreases were distended with University of Wisconsin solution via a pancreatic duct at the time of harvesting to which collagenase and 2.5 mM calcium chloride were added. The pancreases were cold-preserved at 4 degrees C for 24 or 48 hr. After storage, they were incubated for collagenase digestion at 37 degrees C or 20 degrees C for various incubation periods to obtain the peak yield. At 20 degrees C, in vitro collagenase activity measured by the FALGPA method was one fourth of that at 37 degrees C, and pancreases were well digested with a prolonged digestion period (60-90 min vs. 15-20 min for the 37 degrees C group). In vitro insulin secretion of islets isolated from freshly removed pancreases was maintained at 20 degrees C for 120 min in University of Wisconsin solution as compared with 30 min at 37 degrees C. Therefore, the preserved pancreases used in this study were incubated either at 37 degrees C or 20 degrees C at various times in order to obtain peak islet yields. The islet yields from 24-hr cold-preserved pancreases at 37 degrees C and 20 degrees C digestion were 573 +/- 59/rat (n = 6) and 497 +/- 84/rat (n = 11), respectively, and those from 48-hr cold-preserved pancreases were 395 +/- 113/rat (n = 6) and 414 +/- 75/rat (n = 6), respectively. The yields from 24- and 48-hr cold-preserved pancreases were significantly low compared with 635 +/- 52/rat for fresh pancreases (n = 15), but there was no significant difference between the 2 methods. The viability of the isolated islets, which was examined by transplantation to streptozotocin-induced diabetic C57BL/6 mice, showed a significant difference in the capacity to ameliorate diabetes. The functional success rate of islet transplantation after 24-hr cold preservation was equally good (8/8 for 37 degrees C group vs. 9/10 for 20 degrees C group), but the rate for those from 48-hr cold-preserved pancreases was significantly better with digestion at 20 degrees C than at 37 degrees C (1/8 for 37 degrees C group vs. 7/8 for 20 degrees C group, P < 0.05). We concluded that viable islets can be isolated from 48-hr cold-preserved pancreases with the low temperature collagenase digestion method, which shows promise as a modality for successful clinical islet transplantation.