PubMed Health⌕ Search

Biomedical subjects

K Nadeau

Publications and source records attributed to K Nadeau.

At least 19 recordsLinked to original sources
PubMed2001

Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients.

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

Animals↗
PubMed2000

Inhibition of CD40 ligand (CD154) in the treatment of factor VIII inhibitors.

The development of persistent, high titer inhibitors represents a serious complication of the treatment of patients with severe hemophilia A. Elimination of these inhibitory antibodies is usually attempted through repeated administration of high doses of factor VIII. Such regimens are costly, time-consuming and often fail when the inhibitor is of very high titer or of longstanding duration. A potential alternative approach to inhibit the production of antifactor VIII antibodies is blockade of the T-cell/B-cell collaboration that is required to generate humoral responses. One cognate receptor pair that is required for T-cell-dependent B-cell activation consists of CD40, which is expressed on B-lymphocytes and other antigen presenting cells, and CD40 ligand (CD40L, CD154), which is transiently expressed on activated T-cells. To determine whether blockade of the CD40-CD40L pathway can inhibit the production of anti-factor VIII antibodies, a clinical study has been designed in which patients with hemophilia A and a high titer inhibitor (> 10 BU) receive monthly exposures to factor VIII in the presence of a humanized mouse monoclonal antibody to human CD40L (hu5c8*). Subjects must be between the ages of 5 and 60 years old and be HIV seronegative. To date, three subjects have received at least three doses of hu5c8 at the initial protocol dose of 10 mg/kg. Preliminary results suggest that anti-CD40L inhibition may be effective in blocking anamnestic responses to factor VIII in some patients. It remains to be determined whether this effect will persist and whether patients may eventually become tolerant to factor VIII in the absence of hu5c8 co-administration.

Adolescent↗
PubMed1998

Cloning and characterization of the two enzymes responsible for trypanothione biosynthesis in Crithidia fasciculata.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (gamma-Glu-Cys-Gly) and spermidine to form trypanothione (N1,N8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.

Amide Synthases↗
PubMed1998

Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection.

Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We used the fusion protein CTLA4Ig to block CD28-B7 T cell costimulation late after the onset of initial graft injury. Using the F334 into LEW rat model of chronic renal allograft rejection, transplant recipients were treated with a 10-d course of cyclosporine, and a subgroup received a single injection of CTLA4Ig at 8 wk after transplant. Functionally, CTLA4Ig administration prevented development of progressive proteinuria (14.3+/-4.1 mg/24 h versus 41.0+/-12.0 mg/24 h at 24 wk after transplant, P < 0.05). Histologically, graft mononuclear cell infiltration, glomerular hypertrophy, focal and segmental glomerulosclerosis, and intimal vascular hyperplasia were all attenuated in CTLA4Ig-treated animals. Lastly, reverse transcriptase-PCR and immunohistologic studies showed a significant reduction in the intragraft expression of key products of T cell and macrophage activation, and upregulation of what have recently been termed as "protective" genes, including the bcl family members, Bcl-2 and Bcl-xL, and hemoxygenase. Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection.

Abatacept↗
PubMed1998

Ciprofloxacin overdose: acute renal failure with prominent apoptotic changes.

Acute renal failure due to ciprofloxacin has been well described. The previously reported cases have been consistent both clinically and pathologically with tubulointerstitial nephritis (TIN). We report a case of ciprofloxacin overdose leading to acute renal failure characterized by acute tubular necrosis (ATN). The outstanding features in the patient's renal biopsy were those of distal nephron apoptosis, best seen in plastic-embedded sections and electron microscopy. This report shows that the distal nephron can be singularly involved in acute renal failure and that electron microscopy or plastic-embedded sections may be necessary to define its involvement.

Acute Kidney Injury↗
PubMed1997

Cellular and molecular predictors of chronic renal dysfunction after initial ischemia/reperfusion injury of a single kidney.

BACKGROUND: Initial ischemia/reperfusion injury occurring secondary to organ retrieval, storage, and transplantation has been associated with late renal allograft deterioration and failure. In addition, there is an apparent synergy, reported in several clinical series, between the initial injuries of ischemia/reperfusion and acute rejection; the long-term results of graft survival are significantly deceased after both events in combination as compared with either alone or if no such episodes occur. METHODS: In the present study, we examined patterns of proteinuria, cellular infiltration, cytokine expression, and glomerulosclerosis over time in Lewis and Fischer 344 rats after 45 min of warm ischemia of a single kidney and with or without contralateral nephrectomy. Both early (4 hr to 7 days) and late (2-52 weeks) events were studied serially in the affected kidneys morphologically, by immunohistology and by reverse transcriptase polymerase chain reaction. RESULTS: Intercellular adhesion molecule 1, endothelin, and major histocompatibility complex class II expression were up-regulated within 2 to 5 days after injury; T cells and macrophages increased transiently. Proteinuria developed after approximately 8 weeks only in animals bearing a single injured kidney, and not in those with a retained native organ. Progressive morphological changes occurred after 16 weeks, including glomerulosclerosis, arterial obliteration, and interstitial fibrosis. After a period of relative quiescence, expression of intercellular adhesion molecule 1 again increased in relation to progressive macrophage infiltration and their associated products, particularly, interleukin 1, tumor necrosis factor-alpha, transforming growth factor-beta, and inducible nitric oxide synthase. Monocyte chemotactic protein 1 was intensely up-regulated by 24 weeks, coincident with a dramatic rise in this infiltrating population. These changes remained virtually at baseline in animals with a retained native kidney. CONCLUSIONS: These data imply that chronic injury after significant initial ischemia and reperfusion occurs when there is already a 50% renal mass reduction, but not when two kidneys remain in place. Permanent nephron loss resulting from such an insult could account for this phenomenon. Early ischemia and reperfusion, if severe enough in a single kidney, may be an important antigen-independent risk factor for later renal deterioration and failure. In the context of a renal allograft, it may contribute to chronic rejection.

Animals↗
PubMed1997

Nephron mass modulates the hemodynamic, cellular, and molecular response of the rat renal allograft.

Functioning nephron mass has recently been implicated as a risk factor for development of chronic "rejection" of kidney allografts. Reductions in nephron number below 50% may induce glomerular hypertension and hyperfiltration in surviving units, which in turn lead to graft injury. In the present study, which extends and amplifies our previous investigations, cellular and molecular characteristics of single allografts from F344 donors in bilaterally nephrectomized LEW recipients, our standard experimental model of chronic renal allograft dysfunction, were compared with allografts from recipients where total renal mass was reduced (by ligating branches of the graft renal artery) or restored to normal levels by transplanting or retaining a second kidney. Our findings in this study confirm that progressive proteinuria and structural injury in recipients of single allografts were accentuated in grafts with reduced mass but virtually absent in rats with increased kidney mass. A striking observation was that patterns of cell surface molecule expression, cellular infiltration, and expression of all T cell- and macrophage-associated products studied were all markedly modulated by changes in renal mass. Moreover, several molecules that are up-regulated before evidence of graft injury are down-regulated by providing increased renal mass. These data show that the quantity of functioning renal mass is not only an important independent determinant of the tempo and intensity of chronic renal allograft failure, but also a potent modulator of fundamental cellular and molecular components of a complex process. This phenomenon involves antigen-dependent and antigen-independent elements that ultimately result in chronic allograft failure.

Animals↗
PubMed1996

Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection.

Blocking CD28-B7 T-cell costimulation by systemic administration of CTLA4Ig, a fusion protein which binds B7 molecules on the surface of antigen-presenting cells, prevents rejection and induces tolerance in experimental acute allograft rejection models. We tested the effect of CTLA4Ig therapy on the process of chronic renal allograft rejection using an established experimental transplantation model. F344 kidneys were transplanted orthotopically into bilaterally nephrectomized LEW recipients. Control animals received low dose cyclosporine for 10 days posttransplantation. Administration of a single injection of CTLA4Ig on day 2 posttransplant alone or in addition to the low dose cyclosporine protocol resulted in improvement of long-term graft survival as compared with controls. More importantly, control recipients which received cyclosporine only developed progressive proteinuria by 8-12 weeks, and morphological evidence of chronic rejection by 16-24 weeks, including widespread transplant arteriosclerosis and focal and segmental glomerulosclerosis, while animals treated with CTLA4Ig alone or in addition to cyclosporine did not. Competitive reverse transcriptase-PCR and immunohistological analysis of allografts at 8, 16, and 24 weeks showed attenuation of lymphocyte and macrophage infiltration and activation in the CTLA4Ig-treated animals, as compared with cyclosporine-alone treated controls. These data confirm that early blockade of the CD28-B7 T-cell costimulatory pathway prevents later development and evolution of chronic renal allograft rejection. Our results indicate that T-cell recognition of alloantigen is a central event in initiating the process of chronic rejection, and that strategies targeted at blocking T-cell costimulation may prove to be a valuable clinical approach to preventing development of the process.

Abatacept↗
PubMed1994

Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90.

Deoxyspergualin (DSG), a spermidinyl, alpha-hydroxyglycyl, 7-guanidinoheptanoyl peptidomimetic, shows immunosuppressive activity. In confirmation of a recent report that immobilized methoxyDSG selectively retains the heat shock protein Hsc70, we report here quantitative binding of DSG and analogs to both Hsc70 and the 90-kDa heat shock protein Hsp90. We have utilized affinity capillary electrophoresis to obtain Kd values for DSG and analogs, and stimulation of the ATPase activity of Hsc70 to obtain Km values for DSG, that are comparable and corroborative. Kd values are 4 microM for DSG binding to Hsc70 and 5 microM for DSG binding to Hsp90. Two active analogs, methoxy- and glycylDSG, bind with similar affinities. Glyoxylylspermidine and des(aminopropyl)DSG, two inactive metabolites, have much reduced affinity for Hsc70 and Hsp90. These data validate binding of these novel immunosuppressant agents to these molecular chaperones, at concentrations in the range of pharmacologically active doses, and indicate that further characterization of Hsc70 and/or Hsp90 as potential targets for DSG is warranted.

Adenosine Triphosphatases↗
PubMed1993

Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases.

Heat shock proteins of the 82-90 kDa class (hsp82 and hsp90) are abundant, conserved, and ubiquitous from prokaryotes to eukaryotes. Although proposed to be chaperones, they had not been reported to possess enzymatic activity until our recent observation that pure trypanosomatid hsp83 had potent ATPase activity (Nadeau, K., Sullivan, M., Engman, D., and Walsh, C. T. (1992) Protein Sci. 1, 970-979). We have now purified the hsp90 homolog from Escherichia coli (HtpG) and from Saccharomyces cerevisiae (hsp82) to homogeneity and observe ATPase activity with kcat values of 3 min-1 and 140 min-1. In addition, examinations of purified rat hsp90 and human hsp90 detect ATPase activity with a kcat of 0.6 min-1 and 10 min-1. Each of these hsp90s undergoes autophosphorylation on serine or threonine residues. In prokaryotes and eukaryotes, the induction of hsps during heat shock is controlled, respectively, by the binding of an alternate sigma 32 or a transcriptional activator (heat shock factor or HSF) at heat shock promoter elements. Here we show that E. coli HtpG immobilized to Affi-Gel beads selectively retains sigma 32 while the yeast hsp90 and rat hsp90 retain HSF. The peptidyl prolyl isomerase hsp59 of the FK506 binding class is known to bind to hsp90. We also detect binding of the other family of PPIases, the cyclophilins, to immobilized hsp90, consistent with a functional convergence of protein foldases.

Adenosine Triphosphatases↗
PubMed1992

Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.

Amide Synthases↗
PubMed1992

83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.

Adenosine Triphosphatases↗
PubMed1991

Molecular studies on trypanothione reductase, a target for antiparasitic drugs.

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.

Animals↗