Expression of inosine monophosphate dehydrogenase type I and type II after mycophenolate mofetil treatment: a 2-year follow-up in kidney transplantation

The objective of the study was to evaluate the effect of mycophenolate mofetil (MMF) on the regulation of inosine monophosphate dehydrogenase (IMPDH) during the first 2 years after renal transplantation. Twelve patients were enrolled, and 10-h time-course evaluations of the effects of MMF were regularly performed during the study. IMPDH activity and gene expression were measured in whole blood and in mononuclear cells, respectively. Type I IMPDH (IMPDH-I) mRNA was increased during the first 3 months following transplantation and reached its maximal level during acute rejection episodes, whereas type II IMPDH mRNA was stable. Furthermore, although no alteration in the predose samples was observed, patients with prolonged MMF treatment exhibited an increase in the induction potency of both IMPDH activity and gene expression. In vitro experiments confirmed that IMPDH-I is inducible, but preferentially in monocytes than in lymphocytes. This finding suggests that the measurement of IMPDH mRNAs may provide reliable information to predict acute rejection.     

Non-radioactive determination of inosine 5'-monophosphate dehydro-genase (IMPDH) in peripheral mononuclear cells.

BACKGROUND: The immunosuppressive activity of mycophenolate mofetil (MMF) is based on the reversible inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). It was the aim of this study to develop a nonradioactive method for specific measurement of IMPDH activity in isolated peripheral mononuclear cells (MNC). METHODS: The procedure is based on the incubation of lysed MNC with inosine 5'-monophosphate (IMP) followed by direct chromatographic determination of produced xanthosine 5'-monophosphate (XMP). IMPDH activity was measured in MNC of MMF-treated patients and nontreated volunteers. RESULTS: The within-run (n = 10) and between-run (n = 20) coefficients of variation (CV) for IMPDH activity were < 8% and < 10%, respectively. IMPDH activity in 60 healthy volunteers (19-63 yr) ranged from 4.72 to 32.92 nmol/h/mg protein (mean = 18.39 +/- 6.24). The IC(50) for in vitro inhibition of IMPDH activity was about 2 to 3 microg/L. Application of a single dose of 1 g MMF in dialysis patients resulted in a significant inhibition (by 47-95%; p < 0.05) of IMPDH activity in lysed MNC. CONCLUSIONS: The proposed assay specifically and reliably measures IMPDH activity in MNC. The procedure is applicable to evaluate pharmacodynamic activity in MMF-treated patients. The observed interindividual variability of IMPDH activity may reflect pharmacodynamic differences in MMF-treated patients.     

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.     

复制缺陷性慢病毒介导的双自杀基因在小鼠异基因骨髓移植中的应用

目的　探讨复制缺陷性慢病毒介导的胞嘧啶脱氨酶 (cytosinedeaminase,CD)和胸苷激酶(thymidinekinase ,TK)双自杀基因在异基因骨髓移植 (allo BMT)时控制移植物抗宿主病 (GVHD)的可行性及其有效性。方法　将携带双自杀基因的复制缺陷性慢病毒通过脂质体转染入T细胞中。将经6 0　Coγ射线照射后的荷瘤小鼠分组进行骨髓移植 ,观察骨髓移植后荷瘤鼠CFU S、CFU GM、Y染色体整合、T细胞中CD TK基因整合、生存率和生存期、体重及病理组织学等指标。结果　复制缺陷性慢病毒载体可将双自杀基因高效稳定转染入T细胞。在allo BMT中 ,除空白对照组外 ,各组小鼠的CFU S的数目和CFU GM产率差异无显著性 ,且均有Y染色体整合。使用前体药物治疗的小鼠未发生明显的GVHD ,其生存率和生存期明显高于其它各组 ;双自杀基因的效果优于单自杀基因。结论　复制缺陷性慢病毒介导的双自杀基因系统可在不影响移植物存活的情况下有效控制GVHD的发生 ,显著提高allo BMT的成功率。     

Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.     

Inosine monophosphate dehydrogenase activity in renal allograft recipients during mycophenolate treatment

OBJECTIVE: Mycophenolic acid (MPA) exerts its immunosuppression by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH), depleting activated lymphocytes of guanine nucleotides and retarding their proliferation. An optimal strategy for monitoring has not been established for mycophenolate mofetil (MMF) in renal transplantation, and clinical investigations of the pharmacokinetic-pharmacodynamic relationship are warranted. MATERIAL AND METHODS: Mycophenolic acid pharmacokinetics and whole blood cell IMPDH activity were investigated in two separate groups of renal allograft recipients. One group was studied within the 12-h dose interval, while the second group was examined by pre-dose samples pre-transplant and then repeatedly during 8 weeks post-transplant. RESULTS: An inverse relationship between plasma MPA and IMPDH activity within the dose interval was demonstrated. Minimum IMPDH activity was a median 8 % of values pre-MMF dose, coinciding with the MPA peak. Six hours post-dose, IMPDH activity had returned to pre-dose values. Patients receiving MMF had a 4.5-fold higher pre-dose enzyme activity than transplanted patients without MMF. During the 8 weeks post-transplant, the median MPA trough concentration was fairly stable. Following an initial decrease during the first 4 days post-transplant, IMPDH activity gradually increased during the 40 days post-transplant, reaching 5-fold the pre-transplant values. CONCLUSIONS: Provided that the changes in IMPDH activity in whole blood cells predict the clinical effect, these pharmacokinetic-pharmacodynamic findings may prove useful in the attempts to identify optimal timing and range for the monitoring of mycophenolate in renal transplantation. The question of whether MPA concentrations or measurements of IMPDH activity per se will be the optimal way of monitoring this immunosuppressant remains open and will only be answered by prospective clinical testing.     

Mycophenolate mofetil, an inhibitor of inosine monophosphate dehydrogenase, causes a paradoxical elevation of GTP in erythrocytes of renal transplant patients

The immunosuppressant MMF (mycophenolate mofetil) has increasingly replaced AZA (azathioprine) in renal transplantation. MMF is a prodrug of MPA (mycophenolic acid), which inhibits lymphocyte IMPDH (inosine monophosphate dehydrogenase), thereby drastically decreasing GTP concentrations essential to lymphocyte proliferation in vitro and in vivo. Erythrocyte GTP concentrations are commonly elevated in severe renal disease, but normalize following successful engraftment. Consequently, elevated GTP in renal transplant recipients might signal impending loss of immunosuppression and graft failure. In the present study, we compared erythrocyte nucleotides and plasma metabolites in two groups of 25 patients after renal transplantation, both receiving prednisolone and cyclosporin A, but one group receiving MMF and the other AZA. No patients had recent allograft biopsy evidence of rejection. Erythrocyte GTP concentrations at MMF commencement were 50.4+/-23.4 micromol/l. An increase occurred during the first 3 months after transplant when MMF was used de novo, stabilizing at 146.7+/-62.9 micromol/l after 4 months. This was significantly higher (P=2.5 x 10(-6)) than erythrocyte GTP (40.4+/-15.9 micromol/l) in the AZA group, which was essentially unchanged from values immediately after successful transplantation. The effect of MMF on erythrocyte GTP levels was reversible, since GTP levels fell when MMF therapy was terminated. The results demonstrate paradoxically high GTP concentrations in erythrocytes of renal transplant patients receiving MMF. MPA may stabilize reticulocyte IMPDH, allowing the protein to persist during erythropoiesis. This behaviour is in marked contrast with the decrease in GTP levels seen in white blood cells of patients on chronic MMF therapy.     

Pharmacodynamic monitoring of mycophenolate mofetil.

The immunosuppressive activity of Mycophenolate Mofetil (MMF) is based on the reversible inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) by mycophenolic acid. Pharmacodynamic monitoring by measurement of IMPDH activity reflects directly the biological response to MMF. For measurement of IMPDH activity in peripheral mononuclear cells we established a modified non-radioactive procedure, based on the incubation of cell lysates with inosine-5'-monophosphate and the chromatographic quantification of produced xanthosine-5'-monophosphate by isocratic ion-pair reversed phase HPLC. The between-run precision and within-run precision were 7% and 5%, respectively. We determined the time course of IMPDH activity in five patients after 1 g MMF and in five healthy subjects without administration of MMF. Additionally, IMPDH activity was determined in a population study of 40 healthy volunteers. In healthy volunteers, we observed a wide range of IMPDH activity (4.7-32.9 nmol/h/mg) with only weak diurnal variation. All patients receiving MMF had a significant reduction of IMPDH activity (65-100%) after administration of the drug. Inhibition persisted for up to 6 hours, and after 11 hours IMPDH activity returned to predose activities. The interindividual variability of IMPDH activity may account for pharmacodynamic differences in MMF-treated patients. Based on pharmacodynamic monitoring better dosing strategies for MMF-treated patients may evolve.     

Assessment of mycophenolic acid-induced immunosuppression: a new approach

BACKGROUND: Mycophenolic acid (MPA), a metabolite of mycophenolate mofetil (MMF), is an immunosuppressive agent that inhibits inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the ex novo synthesis of GTP. We measured IMPDH activity in peripheral blood mononuclear cells (PBMCs) from MMF-treated patients to evaluate the efficacy of MMF in individual patients. METHODS: IMPDH activity was measured by (3)H released from [2,8-(3)H]IMP that had been formed in the cells from added [2,8-(3)H]hypoxanthine in PBMCs of 35 renal transplant recipients treated with cyclosporin A and corticoids plus MMF: 2 g (n = 10), 1.5 g (n = 7), 1 g (n = 10), or 0 g (n = 8) per day. An alternative method, based on the capacity of the patients' sera to inhibit spontaneous proliferation of the CEM cell line, was also analyzed. RESULTS: The IMPDH activity of PBMCs in transplanted patients was highly variable. For the method based on CEM cell line proliferation: (a) cell proliferation was inhibited only in MMF-treated patients; (b) there was a clear postdose increase in inhibition; (c) inhibition was not affected by other immunosuppressants in vitro or in vivo; (d) inhibition from predose to predose sample was correlated; and (e) when the MMF dosage was <20 mg. kg(-1). day(-1), two groups of patients were identified, one that maintained a high inhibitory capacity in all dose intervals, and one with periods of low inhibitory capacity. CONCLUSIONS: Measurement of the inhibition of CEM cell line proliferation by sera from MMF-treated patients may be useful for evaluating the relative efficacy of MMF treatment in individual patients, especially those receiving low doses of MMF.     

Adeno-Associated Viral-Mediated Gene Transfer to Hepatoma: Thymidine Kinase/Interleukin 2 Is More Effective in Tumor Killing in Non-Ganciclovir (GCV)-Treated Than in GCV-Treated Animals

Interleukin 2 (IL-2) enhancement of herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-induced tumor killing was studied by cloning the human interleukin 2 gene into an HSV-TK-bearing adeno-associated viral (AAV) vector (TK/IL-2). The mouse hepatocellular carcinoma cell line Hepa 1-6 was used as a model in this study. We found that TK/IL-2-transduced Hepa 1-6 cells were more susceptible to ganciclovir treatment than tumor cells transduced with only TK in both nude mice and immunocompetent C57L/J mice. TK/IL-2-transduced tumors also showed shrinkage without GCV treatment. The tumor-killing effect of AAV-mediated TK/IL-2 gene transfer was further studied by inoculating animals with TK/IL-2- or TK-transduced tumor cells mixed with unmodified cells with or without GCV treatment. Although tumor growth in each group was inhibited, the best result was obtained from the TK/IL-2-transduced group without GCV treatment. In this group, 10% of the transduced tumor cells could eradicate the whole tumor in 50% of the animals tested as well as provide long-term protection against tumor cell rechallenge. When this group was treated with GCV, the antitumor effect of TK/IL-2 was reduced. We attribute this to the early ablation of transgene-bearing tumor cells by GCV treatment, which thus reduces the duration of IL-2 expression. We conclude that (i) TK/IL-2 plus GCV treatment generates a stronger tumor-killing effect than HSV-TK plus GCV and (ii) tumor killing of TK/IL-2 is more effective in non-GCV-treated animals than in GCV-treated animals.     

Inosine monophosphate dehydrogenase activity in renal allograft recipients during mycophenolate treatment

Objective. Mycophenolic acid (MPA) exerts its immunosuppression by inhibiting inosine 5′‐monophosphate dehydrogenase (IMPDH), depleting activated lymphocytes of guanine nucleotides and retarding their proliferation. An optimal strategy for monitoring has not been established for mycophenolate mofetil (MMF) in renal transplantation, and clinical investigations of the pharmacokinetic‐pharmacodynamic relationship are warranted. Material and methods. Mycophenolic acid pharmacokinetics and whole blood cell IMPDH activity were investigated in two separate groups of renal allograft recipients. One group was studied within the 12‐h dose interval, while the second group was examined by pre‐dose samples pre‐transplant and then repeatedly during 8 weeks post‐transplant. Results. An inverse relationship between plasma MPA and IMPDH activity within the dose interval was demonstrated. Minimum IMPDH activity was a median 8 % of values pre‐MMF dose, coinciding with the MPA peak. Six hours post‐dose, IMPDH activity had returned to pre‐dose values. Patients receiving MMF had a 4.5‐fold higher pre‐dose enzyme activity than transplanted patients without MMF. During the 8 weeks post‐transplant, the median MPA trough concentration was fairly stable. Following an initial decrease during the first 4 days post‐transplant, IMPDH activity gradually increased during the 40 days post‐transplant, reaching 5‐fold the pre‐transplant values. Conclusions. Provided that the changes in IMPDH activity in whole blood cells predict the clinical effect, these pharmacokinetic‐pharmacodynamic findings may prove useful in the attempts to identify optimal timing and range for the monitoring of mycophenolate in renal transplantation. The question of whether MPA concentrations or measurements of IMPDH activity per se will be the optimal way of monitoring this immunosuppressant remains open and will only be answered by prospective clinical testing.     

Mycophenolate mofetil treatment following renal transplantation decreases GTP concentrations in mononuclear leucocytes

MMF (mycophenolate mofetil) has been proven to provide an effective immunosuppression by non-competitive selective reversible inhibition of IMPDH (inosine monophosphate dehydrogenase), the enzyme playing a crucial role in GTP biosynthesis. However, the exact metabolic changes induced by inhibition of IMPDH in target cells of the immune system have been the subject of recent debate. The aim of the present study was to evaluate whether MMF treatment produced sustained changes in the guanosine nucleotide pool of MNLs (mononuclear leucocytes) in vivo. Sixty-two renal failure patients were divided into three groups: chronic renal failure patients undergoing haemodialysis (CRF-HD; n=20) and two groups of patients after renal transplantation, the first on AZA (azathioprine; TN-AZA; n=23) and the second treated with MMF (TN-MMF; n=19). In addition, MNLs from 25 healthy subjects were analysed as controls. Anion-exchange HPLC was used to quantify purine and pyrimidine nucleotides in MNLs. We report a significant decrease in GTP and the total MNL guanine nucleotide pool in the TN-MMF group (P<0.05) compared with control, CRF-HD and TN-AZA groups, although no significant differences were found between any of the other groups. Adenine nucleotide concentrations in MNLs were decreased in the TN-AZA group, but not in the TN-MMF group compared with the CRF-HD group and controls. There were no differences in CTP concentrations, but UTP concentrations were decreased in the CRF-HD, TN-AZA and TN-MMF groups compared with controls. MMF caused a significant and sustained decrease in the guanine nucleotide pool in MNLs from renal transplant recipients. This decrease contrasts with the elevation in GTP reported in erythrocytes of MMF-treated patients.     

A novel 'sort-suicide' fusion gene vector for T cell manipulation

Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.     

IMP dehydrogenase inhibitor mycophenolate mofetil induces caspase-dependent apoptosis and cell cycle inhibition in multiple myeloma cells

Multiple myeloma is an incurable disease for the majority of patients, therefore requiring new biological targeted therapies. In primary myeloma cells, IMP dehydrogenase (IMPDH) was shown to be consistently overexpressed. We therefore tested the IMPDH inhibitor mycophenolate mofetil (MMF) currently available as a clinical therapeutic agent for its antimyeloma activity in vitro. MMF depleted intracellular guanosine 5'-triphosphate (GTP) levels in myeloma cells. We showed apoptosis induction in myeloma cell lines and primary myeloma cells between 1 and 5 mumol/L MMF. MMF was also cytotoxic at this concentration in dexamethasone-resistant and Mcl-1-overexpressed myeloma cell lines shown by the tetrazolium salt XTT assay along with cell survival measured by a modified flow cytometric assay. Apoptosis was not inhibited by the presence of an antioxidant, suggesting that MMF-induced apoptosis is less likely to be associated with reactive oxygen species. However, apoptosis was abrogated by exogenously added guanosine, which activates an alternative pathway for GTP formation, implicating that this effect is directly mediated by IMPDH inhibition. MMF-induced G1-S phase cell cycle arrest and its apoptosis induction mechanism were associated with a caspase-dependent pathway as shown by alteration of mitochondrial membrane potential and cytochrome c release followed by activation of the caspases. MMF-induced apoptosis was also inhibited by a pan-caspase inhibitor Z-VAD-fmk. MMF-treated myeloma cells showed an up-regulation of Bak, which most likely together with Bax resulted in the release of cytochrome c. In summary, MMF attenuates G1-S phase cell cycle progression and activates the pathway of mitochondrial dysfunction, leading to cytochrome c release followed by activation of caspases.     

NOD/SCID repopulating cells contribute only to short-term repopulation in the baboon

We have previously compared the repopulation ability of gene-modified baboon CD34+ cells in an autologous transplantation versus a xenotransplant model in irradiated nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Baboon CD34-selected marrow cells were transduced with a gammaretrovirus vector and infused into irradiated baboons and NOD/SCID mice. A limited integration-site analysis could only detect two common retrovirus integration sites in the NOD/SCID and monkey. Here, we performed locus-specific PCR on 30 clones recovered from NOD/SCID beta2-microglobulin mice reconstituted with transduced baboon CD34+ cells. We identified five common integrants in the baboon early after transplant (2-6 weeks) but none during the long-term follow-up (6 and 12 months). These results confirm that repopulating cells in the NOD/SCID mouse contribute only to short-term repopulation in a clinically relevant large animal model.     

Kinetics of cell death in T lymphocytes genetically modified with two novel suicide fusion genes

Donor lymphocyte infusions (DLI) following allogeneic stem cell transplantation are known to mediate graft-versus-leukemia effect (GVL). A major side effect of these immunotherapies is the development of graft-versus-host diseases (GVHD). One promising approach to prevent GVHD is to genetically modify donor T cells with a suicide mechanism that can be induced in the case of GVHD. Here we report on a retroviral vector containing the death effector domain (DED) of the human Fas-associated protein with death domain (FADD). The DED was fused to two copies of an FKBP506-binding protein and a truncated version of the human low-affinity receptor for nerve growth factor (LNGFR). Activation of the death signal pathway can be triggered upon the addition of chemical inducers of dimerization. This construct was functionally compared to an optimized HSV-TK vector in which a hypersensitive mutant of the herpes simplex virus thymidine kinase gene (TK39) was fused to a cytoplasmic truncated version of the cell surface antigen CD34. A direct comparison between both vectors in primary T lymphocytes showed that the number of T cells transduced with vectors containing the DED was significantly reduced within 24 h of drug administration whereas ganciclovir treatment of TK39-transduced T cells showed a delay in cell death of approximately 3-4 days. Our results indicate that constructs containing the DED may prove to be the most efficient mechanism to quickly eliminate alloreactive T cells.     

Genetically modified donor leukocyte transfusion and graft-versus-leukemia effect after allogeneic stem cell transplantation

Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (ΔLNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.     

Inosine monophosphate dehydrogenase as a probe in antiviral drug discovery

Inosine monophosphate (IMP) dehydrogenase (IMPDH) is a significant enzyme in the purine nucleotide biosynthetic pathway. IMPDH is viewed as an important biological target in the quest for drugs in the antiviral therapeutic area. This review article is focused on the chemistry and biology of IMPDH inhibitors and the use of IMPDH inhibition data as a probe in antiviral drug discovery. Examples of both inosine 5' monophosphate and NAD+ site-directed inhibitors are presented. Correlation of antiviral activities with IMPDH inhibition is discussed.