Treatment of recurrent heart rejection with mycophenolate mofetil (RS-61443): initial clinical experience.

Mycophenolate mofetil (RS-61443), a derivative of mycophenolic acid, is a new immunosuppressive agent that inhibits de novo purine synthesis in activated lymphocytes. In a clinical trial of mycophenolate mofetil in the treatment of recurrent or persistent heart rejection, 17 patients 0.6 to 104 months (median 5.4 months) after transplantation received a daily oral dose of mycophenolate mofetil of 3000 mg, with seven patients increasing to 3500 mg daily. Azathioprine was routinely discontinued at the start of mycophenolate mofetil treatment. One patient in shock from acute rejection required retransplantation before starting mycophenolate mofetil and died 68 days later of cytomegalovirus sepsis. Another patient died 72 days after mycophenolate mofetil of protracted multisystem failure (present before mycophenolate mofetil). One patient required early cessation of mycophenolate mofetil, and the other 14 patients were alive and well 5 to 10 months after initiating mycophenolate mofetil treatment. Three patients required transient dose reduction and one patient required discontinuation of mycophenolate mofetil because of nausea, diarrhea, or abdominal cramps. No other clinical side effects were noted. Frequency of rejection decreased from 0.67 rejection episodes per patient per month before mycophenolate mofetil to 0.27 rejection episodes per patient per month after mycophenolate mofetil (p < 0.0001). Frequency of infection was unchanged after mycophenolate mofetil (p = 0.9). Renal function was not affected by mycophenolate mofetil (creatinine clearance 1.8 mg/dl before mycophenolate mofetil vs 1.7 mg/dl after mycophenolate mofetil; p = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

The use of mycophenolate mofetil (RS-61443) in human heart transplant recipients.

Mycophenolate mofetil is a potent inhibitor of de novo guanine nucleotide synthesis that selectively blocks lymphocyte proliferative responses. In animal models, mycophenolate mofetil has been shown to prolong allograft survival, reverse ongoing rejection, and induce strain-specific tolerance. To assess the safety and efficacy of mycophenolate mofetil in cardiac transplantation, 30 recipients with mild rejection were enrolled in an 8-week phase I trial. Mycophenolate mofetil in doses from 500 to 3000 mg/day orally was substituted for azathioprine, while baseline cyclosporine levels and corticosteroid doses were maintained. Rejection resolved in the majority of patients, with a significant decrease in mean biopsy score. By protocol, mycophenolate mofetil was discontinued in 4 patients due to persistent mild rejection, and in 4 patients due to progression to moderate rejection. The rate of progression to moderate rejection compared favorably with that observed in patients with mild rejection maintained on azathioprine without augmentation of immunosuppression. Significant increases were observed in hematocrit, total white blood cell count, and absolute neutrophil count. Absolute lymphocyte count remained unchanged. No nephrotoxicity or hepatotoxicity was observed. Gastrointestinal side effects prompted discontinuation of mycophenolate mofetil in one patient. Two major infections occurred. Mycophenolate mofetil remained well tolerated during long-term maintenance immunosuppression, with a rate of rejection similar to that in patients receiving azathioprine. We conclude that mycophenolate mofetil is safe and well tolerated in cardiac transplant recipients, is less myelosuppressive than azathioprine, and appears to be at least equipotent to azathioprine.

Experience with mycophenolate mofetil (RS61443) in renal transplantation at a single center.

OBJECTIVE: Mycophenolate mofetil (MM) is a new immunosuppressive agent that reversibly inhibits guanine nucleotide synthesis and DNA replication. Its activity is highly selective for T and B lymphocytes. Two open-label multicenter trials of MM in renal transplantation have been performed. This report summarizes the results from one center involved in these two trials. METHODS AND RESULTS: The initial trial of MM was an open-label dose-ranging trial in primary cadaveric renal transplantation. Mycophenolate mofetil was included in the maintenance immunosuppression regimen from the day after transplantation. Of the 21 patients enrolled in this trial, one (5%) was withdrawn for side effects. There was one graft loss due to recurrent renal disease and two patients were withdrawn for difficulty with follow-up. Mean follow-up is 26 months, and patient and graft survival at 2 years are 100 and 95% respectively. The second trial was designed to study the efficacy of mycophenolate in reversing refractory renal allograft rejection. Patients enrolled in the trial had biopsy-proven acute rejection and had previously received at least one course of high-dose corticosteroids and/or OKT3. Of the 26 patients enrolled in this trial, one (4%) was withdrawn for side effects. There were two deaths. Mean follow-up is 20 months, and patient and graft survival at 12 months was 91 and 54%. The incidence of infections in the two groups was 38% and there were no deaths in either group attributable to infection. CONCLUSIONS: The results of these two studies indicate that mycophenolate mofetil could be administered safely to renal allograft recipients for periods up to 2 years. It appears to be effective in reversing acute rejection in a high percentage of patients refractory to other forms of therapy.

RS-61443 (mycophenolate mofetil). A multicenter study for refractory kidney transplant rejection.

RS-61443 (mycophenolate mofetil) inhibits a key enzyme of the de novo synthesis of purine nucleotides in T and B lymphocytes. The purpose of this study was to evaluate the efficacy of RS-61443 in patients with refractory renal allograft rejection. Patients eligible for the study had previously undergone anti-rejection therapy with high-dose steroids or OKT3 monoclonal antibody. All rejection episodes were proven by renal biopsy. Successful rescue was achieved in 52 (69%) patients. Rescue was more successful when patients were entered with a creatinine of 4 mg/dL or lower (79%), versus a 52% rescue rate in patients entered with a creatinine of 4 mg/dL or above. Major side effects were predominantly gastrointestinal, but there was no overt nephrotoxicity, hepatotoxicity, or bone marrow suppression. The overall infection rate was 40%, with the spectrum of infections characteristic for the highly immunocompromised patient. The conclude that this pilot study suggests that RS-61443 is effective in refractory kidney allograft rejection. Based on this study, prospectively randomized multi-center trails have been planned and are in progress.

RS-61443--a phase I clinical trial and pilot rescue study.

RS-61443, a morpholinoethyl ester of mycophenolic acid, inhibits the synthesis of guanosine monophosphate, which plays a pivotal role in lymphocyte metabolism. The drug blocks proliferative responses of T and B lymphocytes, and inhibits antibody formation and the generation of cytotoxic T cells. In vivo, RS-61443 prolongs the survival of islet allografts in mice, heart allografts in rats, and kidney allografts in dogs. Reversal of ongoing acute rejection was demonstrated in rat heart allografts and kidney allografts in dogs. Preliminary evidence suggests that the drug prevents chronic rejection. The purpose of this study was to test the safety and tolerance in patients receiving primary cadaver kidneys. RS-61443 in doses from 100 mg/day p.o. to 3500 mg/day p.o. was given to patients in combination with cyclosporine and prednisone. Further study goals were to evaluate the pharmacokinetics of RS-61443, watch for the occurrence of opportunistic infections and acute rejection, and establish dosages for further clinical trials. Forty-eight patients were entered, with six patients in each dose group. RS-61443 was well tolerated in all dose groups, with only one adverse event possibly related to the drug (hemorrhagic gastritis). There was a statistically significant correlation between rejection episodes and dose (P = 0.022), patients with rejection episodes versus dose (P = 0.038), and number of OKT3/prednisone courses versus dose (P = 0.008). There was no overt nephrotoxicity or hepatotoxicity. Preliminary results of a rescue trial in 20 patients with kidney transplants will also be presented.

RS-61443: successful rescue therapy in refractory renal rejection.

RS-61443, the morpholinoethyl ester of mycophenolic acid (mPA), is a potent, noncompetitive, reversible inhibitor of eucaryotic inosine monophosphate (IMP) dehydrogenases. Because of the importance of the guanosine and deoxyguanosine nucleotides in activating phosphoribosyl pyrophosphate (PRPP) synthesis and ribonucleotide reductase, respectively, it was postulated that depletion of GMP (and consequently GTP and GDP) would have antiproliferative effects on lymphocytes. Furthermore, since lymphocytes rely on de novo pruine synthesis whereas other cell types do not, antiproliferative effects produced in this way are more selective for lymphocytes than other cell types.

Are there antiendometrial antibodies in sera of women with endometriosis?

STUDY OBJECTIVE: To identify and characterize the antiendometrial tissue specific antibody response in endometriosis patients. DESIGN: Retrospective. SETTING: Industrial research laboratory. PATIENTS: Twenty untreated women with laparoscopically diagnosed endometriosis, 10 healthy women without symptoms of endometriosis, 12 women in whom laparoscopy failed to yield evidence of endometriosis, 10 healthy men, and 4 individuals with elevated titers of serum antinuclear antibodies. RESULTS: Several immunological methods have been employed including immunofluorescence, hemagglutination, enzyme-linked immunoabsorbent assays, and protein blotting. Contrary to reports in the literature, results of these analyses have failed to demonstrate the presence of detectable levels of antiendometrial immunoreactivity in sera from patients with endometriosis. CONCLUSION: The association of endometrial autoimmunity with endometriosis remains to be established.

Use of a direct fluorescent antibody test for detecting Chlamydia trachomatis cervical infection in women seeking routine gynecologic care.

We determined the sensitivity, specificity, and predictive value of a direct fluorescence test for Chlamydia trachomatis infection compared with culture of the endocervix in women seeking routine gynecologic care. Of 527 patients seen in a hospital-based practice, 23 (4.4%) had a positive culture for C. trachomatis. The overall sensitivity of the direct test was 70%, and the specificity was 98%. When five or more endocervical cells were present on the direct test slide, the sensitivity increased to 92%, and the specificity decreased to 96% (P less than .05). When the presence of any columnar epithelial cells, five or more elementary bodies, or both was used as the criteria for accepting specimens, the sensitivity and specificity of the direct test were 80% and 96%, respectively. However, 44% of the specimens would be rejected if these criteria were used. The overall probability that an individual with a positive direct test would have a positive culture was 62%.

Cytolytic T-cell mediated lysis of reovirus-infected cells: requirements for infectious virus, viral particles, and viral proteins in infected target cells.

The virological requirements for the recognition of infected target cells by cytolytic T lymphocytes (CTL), using reovirus, a nonenveloped, icosahedral virus has been investigated. Using mouse L cells infected at the nonpermissive temperature with ts (temperature-sensitive) mutants of reovirus in complementation groups C and G, it has been shown that the production of complete viral particles is not necessary for efficient lysis of infected cells by CTL. In addition, adsorption of purified viral particles and viral top component (TC), empty capsids lacking genome ds-RNA, to L cells just prior to use in cytolytic T cell assays is sufficient to produce target cells capable of being lysed, though target production is less efficient than with L cells infected with reovirus. Membrane fluorescence analysis of cells infected with reovirus ts mutants at the nonpermissive temperature and with adsorbed viral particles revealed the presence of the viral sigma 1 protein on the cell surface. For adsorbed particles, the degree of membrane fluorescence paralleled the capacity of CTL to lyse target cells. It is concluded that cells infected with icosahedral, nonenveloped viruses, like cells infected with enveloped viruses, express viral antigens on the cell surface even in the absence of the production of complete viral particles; adsorbed viral particles can be incorporated into the cell membrane in a manner sufficient for recognition and lysis by CTL, in the absence of actual infection of the cells.

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells.

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state.

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.

Cell receptors for the mammalian reovirus. II. Monoclonal anti-idiotypic antibody blocks viral binding to cells.

A monoclonal anti-idiotypic antibody directed against an idiotypic determinant on a monoclonal anti-reovirus type 3 hemagglutinin antibody with viral neutralization activity is capable of binding to the surface of lymphoid cells (BW5147, R.1.1, and R1.E) and of inhibiting viral binding to cell surface receptors. The inhibition of viral binding was specific for the anti-idiotype antibody; viral binding was not inhibited by antibodies bound to the H-2k and Thy-1.2 antigens on the surface. Viral binding to idiotype-negative cells (P-815, EL-4) was not inhibited by the anti-idiotypic antibody, although these cells are susceptible to type 3 reovirus infection. These data suggest that there are at least two structural classes of type 3 reovirus receptors on murine cells. It is probable that anti-idiotypic antibodies of this type will be useful in studying the structure and regulation of viral receptors on cell surfaces and for the purification of these receptors, and may provide a way to block viral infection.

Determinants of reovirus interaction with the intestinal M cells and absorptive cells of murine intestine.

Reovirus type 1 penetrates the gastrointestinal tract in suckling mice via specialized epithelial cells, designated membranous cells, or M cells, located in the epithelium overlying Peyer's patches. We have examined whether the interaction of reovirus with murine mucosa of in situ closed ileal loops is influenced by mouse age or strain or reovirus serotype. Neither mouse age (suckling or adult), strain (C3H/HeJ or Balb/cJ), nor reovirus serotype (types 1 and 3) affected reovirus adherence to and transport through M cells. In all conditions, reovirions adhered to the M-cell surface and were transported across M cells in endocytic vesicles. The adherence to and endocytosis by M cells of type 1 reovirus and reassortants with the viral hemagglutinin of type 1 were selective in suckling mice; type 1 virus was not adherent to nor endocytosed by absorptive cells. In adult mice, type 1 reovirions adhered to the surface of a minority of absorptive cells but were never seen within absorptive cell cytoplasm. In contrast, type 3 reovirus and reassortants with the viral hemagglutinin of type 3 adhered to and were endocytosed not only by M cells but also by absorptive cells of suckling mice. Virions accumulated within lysosomelike bodies in absorptive cells but transport of virions across absorptive cells was not observed. These studies indicate that (a) adherence of reovirus to the apical surface of and transcellular transport by M cells is independent of viral serotype or viral surface proteins, (b) adherence of reovirus to and transcellular transport by M cells is independent of mouse age after 9 days and comparable in two mouse strains, and (c) adherence of reovirus to and their endocytosis by absorptive cells of suckling mice is determined by the viral hemagglutinin (sigma 1 protein).

The sigma 1 protein determines the extent of spread of reovirus from the gastrointestinal tract of mice.

After intragastric inoculation of adult mice, type 1 reovirus was initially concentrated in Peyer's patches over the first 4 hr after inoculation, then spread sequentially to the mesenteric lymph nodes and spleen. For type 3 reovirus, however, initial entry into Peyer's patches in adult mice was followed by loss of viral infectivity so that by 4 hr after inoculation virtually no infectious virus was detected in the intestine, and spread to extraintestinal tissues did not occur. In 10-day-old mice, type 3 was capable of spread to the mesenteric lymph nodes but not the spleen. Thus, as animals aged there was a greater restriction of the spread of type 3 from the intestine. Studies using a field isolate of type 3 reovirus that is resistant to intestinal proteases, and genetic studies utilizing type 1 x type 3 viral reassortants, revealed that the viral sigma 1 protein determined the capacity of reovirus to spread from the intestine in both adult and 10-day-old mice. Thus, the interaction of reovirus with host defense mechanisms, and the age-dependent restriction of spread of type 3 reovirus from the intestine are mediated by the viral sigma 1 protein.

An adherent cell lyses virus-infected targets: characterization, activation, and fine specificity of the cytotoxic cell.

We have previously reported that mice with defective T lymphocyte function clear a primary reovirus type 1 infection in a normal fashion. The present studies were initiated to determine what cells may play a role in the clearance of this primary infection. We show that macrophages from unprimed mice are capable of lysing reovirus type 1-infected target cells in the absence of specific antibody. Macrophages from nu/nu mice have higher levels of this lytic activity than macrophages from nu/+ and normal mice. In addition, PEC from endotoxin nonresponsive C3H/HeJ mice have virtually no anti-viral lytic activity, while PEC from C3H/FeJ mice have high levels of such activity. Incubation of PEC from C3H/HeJ mice overnight in Con A supernatants restores this lytic activity. PEC are capable of lysing reovirus type 1-infected target cells but not those infected with type 3 reovirus. Using intertypic recombinant viruses, we show this striking target cell specificity to be a property of the sigma 1 protein, the viral hemagglutinin.

CT hybridomas: tumor cells capable of lysing virally infected target cells.

By fusing primed murine lymphocytes with a syngeneic T cell lymphoma, we have been able to select for H-2-restricted, virus-specific cytotoxic T cell hybridomas (CTH). These T cell hybrids, which replicate in ordinary tissue culture medium or in ascites, are capable of lysing virally infected target cells, and their activity is facilitated by the presence of lectins in the assay medium. Unlike cells mediating lectin nonspecific lysis, these hybridomas are H-2 restricted and specific for single viral proteins. The ability to maintain these cells in culture for over 18 mo and to pass them in vivo without loss of activity or specificity indicates that they will provide sufficient material for the analysis of surface proteins and genetic information required for the recognition and lysis of virally infected cells by killer T cells.

T lymphocyte immunity to reovirus: cellular requirements for generation and role in clearance of primary infections.

The cellular requirements for the development of an immune response to reovirus type 1 and the role of such a response in the clearance of a primary infection with that virus were explored. An Ia-bearing antigen-presenting cell requirement is demonstrated for the in vitro generation of secondary anti-reovirus cytolytic T lymphocytes (CTL). It is then shown that mice whose spleens are depleted of Ia-bearing adherent cells by exposure in vivo to ultraviolet (UV) radiation exhibit depressed priming for reovirus-specific T lymphocyte function-CTL generation, delayed-type hypersensitivity reactivity, and T cell proliferative responsiveness. These UV-irradiated mice clear primary systemic reovirus infections as readily as normal mice. Further, athymic 'nude' mice show no defect in their ability to clear a reovirus infection. The implications of these findings for our understanding of the role of virus-specific T cell function in the clearance of systemic viral infections are discussed.