FTY720/cyclosporine regimens in de novo renal transplantation: a 1-year dose-finding study.

FTY720 is a novel immunomodulator being investigated for rejection prophylaxis in renal transplantation when combined with full-dose cyclosporine (CsA; FDC). This 1-year phase II study compared FTY720 plus FDC (Neoral) with FTY720 plus reduced-dose CsA (RDC) and mycophenolate mofetil (MMF) plus FDC in de novo renal transplant patients. Patients were randomized 2:2:2:1 to FTY720 5 mg plus RDC (n = 72); FTY720 2.5 mg plus RDC (n = 74); FTY720 2.5 mg plus FDC (n = 76); or MMF plus FDC (n = 39) for 12 months. CsA exposure in the RDC group was reduced on average by 50% as assessed by C(2) monitoring. The primary efficacy endpoint was the composite incidence of biopsy-proven acute rejection (BPAR), graft loss, death or premature study discontinuation. The incidences for this composite endpoint were 24% and 22%, respectively, for FTY720 5 mg plus RDC and FTY720 2.5 mg plus FDC versus 39% for MMF plus FDC. Patients receiving FTY720 2.5 mg plus RDC were discontinued from treatment due to risk of under-immunosuppression. FTY720 2.5 mg plus FDC and FTY720 5 mg plus RDC were safe and effective in de novo renal transplant patients over 12 months.

Differential use of cardiac troponin T versus I in hemodialysis patients.

BACKGROUND: Cardiac troponin T (cTnT) is frequently elevated in asymptomatic hemodialysis (HD) patients and predicts increased cardiovascular morbidity and mortality. Compared to cTnT, cardiac troponin I (cTnI) has a shorter half-life. How this influences its diagnostic reliability in chronic HD patients is only partially known. PATIENTS AND METHODS: First, in a cross-sectional study cardiac troponins were measured in 31 asymptomatic HD patients. A third-generation cTnT assay was used. The rate of false positive tests and the intraindividual variability were determined. Second, in a retrospective analysis over 12 months all acute events with clinical suspicion for acute coronary syndrome (ACS) were analysed in the same patients to determine the diagnostic power of cTnT by receiver-operating curve (ROC) plot. RESULTS: Cross-sectional study: 9 of 52 (17%) cTnT and 0/52 cTnI (0%) tests were positive in asymptomatic HD patients with a low intraindividual variability. Retrospective analysis: 16 acute clinical events with determination of cTnT were recorded, and in 4/16 an ACS was diagnosed. Using a cut-off level of 0.1 microg/l, the cTnT test reached a sensitivity of 100%, a specificity of 42%, a positive predicitive value of 36% and a negative predictive value of 100%, using a cut-off level of 0.2 microg/l the corresponding values were 75%, 58%, 38% and 88%. CONCLUSIONS: Cardiac TnT, but only rarely cTnI, is elevated in a significant number of asymptomatic HD patients. For diagnosis of ACS in HD patients, a combination of cTnT and cTnI may be used, since the former has higher sensitivity and the latter higher specificity. A higher threshold value for cTnT in HD patients could further increase its diagnostic accuracy.

CD28-B7-mediated T cell costimulation in chronic cardiac allograft rejection: differential role of B7-1 in initiation versus progression of graft arteriosclerosis.

Provision of adequate T cell costimulation is critical for the development of acute and chronic allograft rejection. We have previously reported that early blockade of CD28-B7 T cell costimulation prevents the development of graft arteriosclerosis, in the LEW into F344 rat cardiac transplant model. In this study, we used the same model to examine the requirement for CD28-B7-mediated T cell costimulation in the progression of established chronic rejection and examined the individual roles of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules. Late blockade of CD28-B7 T cell costimulation by the fusion protein CTLA4Ig, which binds both CD80 and CD86, attenuated the development of transplant arteriosclerosis, mononuclear cell infiltration, and parenchymal fibrosis in this model. Selective blockade of CD80 using the mutant fusion protein Y100F was as effective as CTLA4Ig in this regard. In contrast to CTLA4Ig, blockade of CD80 alone by Y100F was ineffective at preventing early graft loss and prolonging graft survival when given early after transplantation. This study is the first to demonstrate that late blockade of CD28-B7 T cell costimulation interrupts chronic cardiac allograft rejection, and it indicates the importance of continued T cell activation in this process. This study further defines functional differences between CD80 and CD86 costimulatory molecules in vivo.

In vitro and in vivo immunomodulatory effects of RDP1258, a novel synthetic peptide.

Peptides derived from certain regions of human class I MHC molecules are known to have immunomodulatory effects. In particular, amino acid residues 75-84 of the HLA-B7 and HLA-B2702 molecules have demonstrated allele nonspecific immunosuppression in several animal transplant models. There is evidence that these effects are mediated by binding to intracellular heat shock proteins, including heme oxygenase-1. A new derivative of these peptides, RDP1258, was developed using a novel computer-assisted rational design technique. In vitro, RDP1258 peptide inhibited rat heme oxygenase activity in a dose-dependent manner. Similar to observations made with other in vitro heme oxygenase inhibitors, in vivo administration of RDP1258 peptide to naive rats resulted in upregulation of splenic heme oxygenase activity. The effects of the peptide on alloimmune responses were then tested. Addition of RDP1258 to rat and human mixed leukocyte reactions inhibited proliferation in a dose-dependent manner. In a rat renal transplantation model, peptide therapy combined with a sub-therapeutic dose of cyclosporin A significantly prolonged allograft survival. These data provide further evidence that modulation of the heat shock protein heme oxygenase by rationally designed peptides affects immune effector functions and may allow the development of novel immunomodulatory strategies in organ transplantation.

Immunomodulatory functions of low-molecular weight hyaluronate in an acute rat renal allograft rejection model.

Low molecular weight hyaluronate (LMW-HA) blocks interactions between T lymphocyte CD44 and hyaluronate (HA), a heteropolysaccharide that is expressed on the surface of endothelial cells and ubiquitously in the extracellular matrix. This study was undertaken to assess the ability of LMW-HA to modify the course of experimental acute renal allograft rejection. Lewis (LEW) rats were bilaterally nephrectomized and received an orthotopic, fully MHC-mismatched, Wistar-Furth (WF) kidney transplant. Animals received either no treatment, low doses of cyclosporin A (CsA) on days 0 to 5, LMW-HA on days 0 to 5, or CsA plus LMW-HA on days 0 to 5 after transplantation. With no treatment, CsA monotherapy, or HA monotherapy, animals rejected their allografts at a median of 15, 13, and 7.5 d, respectively (P = NS). In contrast, combined CsA plus LMW-HA therapy prevented acute rejection and significantly prolonged graft survival (P = 0.008) to a median of 49.0 d. CsA/LMW-HA-treated grafts also demonstrated better preservation of renal function at day 30 (serum creatinine level, 1.38+/-0.8 mg/dl), compared with surviving animals treated with CsA alone (2.9+/-0.55 mg/dl, P<0.05). Histologic graft analysis of CsA/LMW-HA-treated animals at day 7 after transplantation showed minimal rejection and leukocyte infiltration, compared with all other groups. Intragraft gene expression analysis, using semiquantitative reverse transcription-PCR, at the same time point showed reductions of CD4, CD8, and interferon-gamma transcript levels in the combined-treatment group. This is the first study demonstrating the immunomodulatory functions of LMW-HA in vivo in the setting of organ transplantation. Defining the exact mechanisms that underlie this immunomodulation may provide the rationale to develop novel strategies for use in clinical transplantation.

Immunomodulatory functions of hyaluronate in the LEW-to-F344 model of chronic cardiac allograft rejection.

BACKGROUND: CD44 is an important leukocyte cell surface glycoprotein with diverse functions including cell adhesion, homing, migration, and activation. METHODS: Because administration of the principal ligand of CD44, hyaluronate (HA), in soluble form, can inhibit CD44-HA interaction, we tested the effects of HA in vivo in an established model of chronic allograft rejection. Control F344 recipients of LEW hearts received either no treatment or low-dose cyclosporine (CsA) for 30 days from the day of transplantation. Experimental animals received 30 days of CsA in combination with 30 or 90 days of low molecular weight HA (LMW-HA). RESULTS: CsA therapy alone resulted in approximately 40% long-term (>100 days) graft survival, whereas CsA + LMW-HA (30-day and 90-day protocols) significantly increased long-term graft survival to 60% and 92%, respectively. Light microscopy and immunohistology of CsA-treated and CsA + LMW-HA-treated grafts harvested at day 30 after transplantation demonstrated that LMW-HA + CsA therapy decreased mononuclear cell infiltration and afforded better preservation of myocardial architecture. In addition, LMW-HA + CsA-treated grafts exhibited decreased expression of interferon-gamma and the growth factors transforming growth factor-beta, platelet-derived growth factor, and fibrogenic growth factor-beta. Long-term surviving grafts were assessed for arteriosclerosis, the sine qua non of chronic rejection in this model. Using a standardized scoring system, significantly less arteriosclerosis was seen in grafts from LMW-HA + CsA-treated animals at 120 days after transplantation compared with CsA alone-treated grafts. This difference was not significant, however, in grafts harvested at >150 days. CONCLUSION: This is the first report indicating that CD44-HA interactions play an important role in chronic allograft rejection.

Serum hippuric acid concentration in renal allograft rejection, ureter obstruction, and tubular necrosis.

Plasma from 35 renal allograft recipients (21 males and 14 females) was sampled daily and analyzed for hippuric acid (HA) by high-performance liquid chromatography (HPLC) and serum creatinine. Twelve of these patients experienced an acute renal allograft rejection or a ureter obstruction as proven by clinical signs and biopsy, as well as by radiography or ultrasound, respectively. Two patients suffered from tubular necrosis followed by rejection during the postoperative period. Mean serum HA increased by 39.9 mumol/l from baseline (range 20.4-115.5 mumol/l) in patients with acute rejection 3 days after an initial increase that was observed 24 h before the mean serum creatinine increased by 107.1 mumol/l (range 21-193 mumol/l). In cases of ureter obstruction, HA rose by 1.6 mumol/l (range 1-8.2 mumol/l), significantly less than elevations due to rejection. The increase in creatinine, however, amounted to 65.3 mumol/l (range 22-140 mumol/l) and was not different from the change in rejecting patients. Successful antirejection treatment coincided with a decrease in serum HA starting 24 h earlier than the decrease in the serum creatinine concentration. Of special interest was the observation of a parallel decrease in HA with creatinine concentration in patients with tubular necrosis after allotransplantation; HA increased in cases of an additional rejection. Our data suggest that HA, which is excreted by tubular secretion and glomerular filtration, could be a sensitive and early marker of acute allograft rejection. Furthermore, it seems to discriminate between acute renal allograft rejection and ureter obstruction. It might, therefore, be of value in the diagnosis of rejection complicating tubular necrosis after transplantation.

Cytotoxicity, differentiating activity and metabolism of tiazofurin in human neuroblastoma cells.

The IMP dehydrogenase inhibitor, tiazofurin (TR)-2-beta-D-ribofuranosylthiazole-4-carboxamide, which exhibited oncolytic activity in patients with chronic myelogenous leukaemia (CML) in blast crisis was found to inhibit the growth of human neuroblastoma SK-N-SH cells with an IC50 of 4.2 microM. TR treatment of cells perturbed nucleic acid and catecholamine pathways. As biochemical markers of TR action decreased cellular GTP pools, increased inosine and hypoxanthine concentrations and depleted dopamine content were found. Incubation of tumour specimens obtained from paediatric patients with grade-IV neuroblastoma with TR resulted in the formation of the active metabolite, thiazole-4-carboxamide adenine dinucleotide, in concentrations sufficient to inhibit tumour growth. Cytotoxic and biochemical effects of TR were enhanced by combining it with allopurinol (an inhibitor of xanthine dehydrogenase), and hypoxanthine (an alternate substrate for hypoxanthine-guanine phosphoribosyltransferase). Induction of transdifferentiation of SK-N-SH cells from a neuroblast to an epitheloid, substrate-adherent phenotype was more pronounced with TR than with all-trans-retinoic acid. Transdifferentiating treatment with TR resulted in a 2-fold-enhanced sensitivity towards adriamycin. However, differentiation with all-trans-retinoic acid rendered the cells more resistant to adriamycin. Our results suggest that TR might be a promising agent for the treatment of children suffering from neuroblastoma.

Purine metabolism of human glioblastoma in vivo.

The aim of this study was to identify targets for rational chemotherapy of glioblastoma. In order to elucidate differences in the biochemistry of tumor and normal human brain, in vivo pool sizes of purine nucleotides, nucleosides, and nucleobases and of purine metabolizing enzymes in biopsy material from 14 grade IV astrocytomas and 4 normal temporal lobe samples were analyzed. Specimens were collected during surgery using the freeze-clamp sampling technique and analyzed by high pressure liquid chromatography. Total purine nucleotides, adenylates, and guanylates in the tumors were 2186, 1865, and 310 nmol/g (wet weight), respectively, which corresponds to 61, 60, and 71% of normal brain tissue concentrations. Relative to normal brain the tumors had significantly lower ATP and GTP levels, essentially normal pool sizes of purine nucleosides and bases, unchanged activities of the salvage enzymes hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and adenosine kinase (659, 456, and 98 nmol/h/mg protein, respectively) and 4-fold higher activities of IMP dehydrogenase (11.6 nmol/h/mg protein); the latter is the rate limiting enzyme for guanylate de novo synthesis. IMP pools in the tumors were 64% of values in normal brain. Modulation of the guanylate pathway in glioblastoma by inhibition of IMP dehydrogenase with tumor specific agents such as tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) appears to be a rational therapeutic approach. Preliminary in vitro experiments with normal and malignant tissue specimens from 2 additional patients revealed that significant amounts of the active metabolite thiazole-4-carboxamide adenine dinucleotide are formed from tiazofurin. At a concentration of 200 microM this drug was able to deplete guanylate pools in the tumors to a median of 54% of phosphate buffered saline treated controls. Flux studies with [14C]formate showed that tiazofurin strongly inhibited de novo synthesis of guanylates in glioblastoma to an average of 10% of controls. This effect was more pronounced in the tumors as compared to normal brain. No inhibition of salvage of [14C]guanine by tiazofurin could be observed in normal and malignant tissues. Supportive measures have to be considered to inhibit the highly active salvage enzyme hypoxanthine-guanine phosphoribosyltransferase that can partly antagonize a tiazofurin induced decrease in guanine nucleotides.

[Permanent tracheostomy in rats]. / Permanente Tracheostomie bei der Ratte.

For bronchoalveolar lavages (BAL) after lung transplantation in the rat two different tubes for creation of a permanent tracheostoma were devised and tested. Typ I was of plastic material and required closure of the trachea oral of the tube thus excluding the upper respiratory tract for breathing. All these animals deprived of their sense of smell and direction died within 50 hours despite optimal husbandry. Typ II was a T-shaped metall tube allowing normal nasal breathing. This tube was well tolerated by the animals and facilitated repeated BAL. BAL on day 3 following tracheotomy with 5% lymphocytes, 2% polymorphonuclear leukocytes and 95% alveolar macrophages was considered to be normal when compared with human findings. An increase in polymorphonuclear leucocytes and epithelial cells together with bacteria on day 6 was believed to be due to tracheobronchitis. This assumption was supported by the fact, that both cell types and the number of bacteria significantly decreased 3 days later. A steady increase of lymphocytes seems to be associated with repeated BAL.