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H Waldmann

Publications and source records attributed to H Waldmann.

At least 55 records · Page 3Linked to original sources
PubMed2001

The role of CD4+ T-cell subsets in determining transplantation rejection or tolerance.

Peripheral tolerance to allogeneic organ grafts can be induced in rodents by treating with non-depleting CD4 and CD8 monoclonal antibodies. This tolerance is maintained by CD4+ T cells with a potent capacity to induce tolerance in further cohorts of T cells (i.e. infectious tolerance). We have cloned CD4+ T-cell subsets against the male transplantation antigen in vitro and find, in contrast to Th1 or Th2 clones that elicit rejection, that there is a distinct population of CD4+ T cells that suppress rejection by adoptive transfer (here called Treg). In order to identify molecular markers associated with tolerance and gain insights into the mechanisms of action of Treg cells, we carried out serial analysis of gene expression. We identified genes overexpressed in Treg compared to Th1 and Th2 cultures and found that some of these correlated in vivo with CD4-induced transplantation tolerance rather than rejection. The genes overexpressed in Treg cultures and within tolerated skin grafts were primarily expressed by mast cells (e.g. tryptophan hydroxylase and FcepsilonR1alpha), suggesting that regulatory cell activity and this form of tolerance may be associated with a localised but non-destructive form of Th2-like activation and a recruitment of mast cells.

Animals↗
PubMed2001

Comparison of allogeneic T cell-depleted peripheral blood stem cell and bone marrow transplantation: effect of stem cell source on short- and long-term outcome.

We report the results of a retrospective single-center study comparing engraftment, acute and chronic GVHD, relapse and survival in patients with malignant hematological disorders transplanted with allogeneic peripheral blood stem cells (alloPBSCT, n = 40) or bone marrow cells (alloBMT, n = 42). All transplants were T cell depleted by in vitro incubation with the Campath-1 monoclonal antibody. Primary graft failure occurred in none of the patients receiving an alloPBSCT compared with 3/42 of the recipients of an alloBMT. In addition, two patients in the alloBMT group showed no platelet engraftment. Recipients of PBSC had a more rapid recovery of neutrophils (median 14 days) compared to BM transplant recipients (median 32 days). Platelet recovery was also accelerated in PBSC recipients compared to BM recipients (11 vs 38 days). There was an increase in the incidence of grade II acute GVHD and chronic GVHD in patients after alloPBSCT (18% and 23%, respectively) compared to patients receiving alloBMT (5% and 8%, respectively). The 2-year cumulative incidence of relapse was similar in both groups (47%). At 6 months after transplantation, transplant-related mortality (TRM) was lower in PBSCT recipients than in BMT recipients. However, at a follow-up of 3 years TRM was similar in both groups. The disease-free survival rate at 3 years after transplantation did not differ between the groups (42% for PBSCT and 41% for BMT recipients). Our results indicate that T cell-depleted alloPBSCT compared to alloBMT is associated with a more rapid hematopoietic reconstitution and a decreased TRM at 6 months follow-up after transplantation. However, at a follow-up of 3 years, no sustained survival benefits were observed.

Adult↗
PubMed2001

Excessive T cell depletion of peripheral blood stem cells has an adverse effect upon outcome following allogeneic stem cell transplantation.

We evaluated the outcome of two modes of T cell depletion for HLA-identical sibling stem cell transplants in 34 consecutive adult patients: group A (n = 11) received PBSC post CliniMACs immuno-magnetic enrichment of CD34(+) cells and group B (n = 23) received bone marrow following in vitro incubation with CAMPATH-1M and complement. All patients received an identical conditioning regimen which consisted of in vivoCAMPATH-1H 20 mg over 5 days, thiotepa 10 mg/kg, cyclophosphamide 120 mg/kg and 14.4 Gy TBI. No additional graft-versus-host disease prophylaxis was given. The mean T cell dose administered was 0.02 +/- 0.05 x 10(6)/kg for group A and 2.8 +/- 2.8 10(6)/kg for group B (P < 0.001). With a median follow-up of 28 months overall survival was 36.4% for group A at 12 months compared to 78.3% for group B (P = 0.001). Transplant-related mortality in group A at 12 months was 63.6% as compared to 18.0% in group B (P = 0.003). Most of the procedure-related deaths in group A occurred secondary to infection. These results suggest that extensive in vitro T cell depletion of peripheral blood stem cells in combination with in vivo T cell depletion may have profound effects upon the incidence of infections following allogeneic stem cell transplantation and this may adversely effect transplant-related mortality.

Adult↗
PubMed2001

The effect of treatment with Campath-1H in patients with autoimmune cytopenias.

We describe 21 patients with severe and life-threatening autoimmune cytopenias resistant to standard immunosuppression who were treated with the monoclonal antibody Campath-1H. Four patients had autoimmune neutropenia, four had autoimmune haemolytic anaemia, four had pure red cell aplasia, one had immune thrombocytopenia purpura (ITP), three had autoimmune haemolytic anaemia and ITP (Evan's syndrome), three had autoimmune pancytopenia (ITP, autoimmune neutropenia and autoimmune haemolytic anaemia), one had ITP (associated with acquired Glanzmann's disease) and autoimmune neutropenia, and one had ITP and red cell aplasia. Campath-1H was administered at a dose of 10 mg/d as an intravenous infusion for 10 d. Responses were seen in 15 patients, which were sustained in six. Relapse occurred in eight patients after Campath-1H treatment. Patients entering the study later, received cyclosporine after Campath-1H in an attempt to reduce the incidence of relapse. Three patients received a second course of Campath-1H; all responded but later relapsed. Fourteen patients are alive at a median of 12 months (range 4-61) after Campath-1H. Campath-1H represents an alternative therapeutic option for severe, refractory autoimmune cytopenias.

Adolescent↗
PubMed2001

CAMPATH-1 antibodies in stem-cell transplantation.

BACKGROUND: CAMPATH-1 (CD52) Abs have been used in stem-cell transplants for the prevention of GvHD and graft rejection. These complications can effectively be prevented by depletion of T lymphocytes from both donor and recipient. However, donor lymphocytes might contribute an anti-leukemia effect and lymphocyte depletion may exacerbate problems with immune reconstitution. There is a fine balance between the risks of GvHD and host-versus-graft reactions, relapse and infection. METHODS: Clinical outcomes for 4264 patients were reported to a central registry and analyzed by univariate and multivariate methods to determine the superior protocols. Various protocols of lymphocyte depletion were tested, using either CAMPATH-1M (IgM) plus complement or CAMPATH-1G (IgG2b) to treat the donor BM ex vivo and CAMPATH-1G in vivo to treat the recipient. The humanized antibody CAMPATH-1H has recently replaced CAMPATH-1G. A meeting of the clinical collaborators was convened to discuss the results and to review the experiences of individual centers. RESULTS: Interest focused on the use of mobilized PBSC for transplantation and on the use of reduced-intensity conditioning regimens ('mini' or 'non-Correspondence myeloablative' transplants). These approaches are likely to become increasingly important in the future and will allow transplant procedures to be used for relatively older patients. The use of CAMPATH-1G or CAMPATH-1H was associated with a low incidence of GvHD or rejection, though there were some differences that might be related to the longer half-life of the humanized antibody. An unexpected and apparently paradoxical effect of post-transplant CYA was observed - it appeared to reduce the risk of dying from infection after 6 months. Although part of the benefit could be explained by a reduction in GvHD, the effect was still evident when patients with GvHD or graft rejection were excluded from analysis. DISCUSSION: CAMPATH-1H appears to have a useful role in the prevention of graft rejection and GvHD, particularly in patients who are at high risk of these complications. It can equally well be used by admixture with the infused stem cells, or by administration to the patient prior to the transplant. Future studies will seek to understand the mechanism of the CYA effect and to improve the quality of immune reconstitution.

Alemtuzumab↗
PubMed2001

In vivo CAMPATH-1H prevents GvHD following nonmyeloablative stem-cell transplantation.

BACKGROUND: We have investigated a novel nonmyeloablative conditioning regimen in 44 patients with hematological malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. METHODS: Recipient conditioning consisted of CAMPATH-1H 20 mg/day on Days -8 to -4, fludarabine 30 mg/m(2) on Days -7 to -3 and melphalan 140 mg/m(2) on Day -2. Thirty-six recipients received unmanipulated G-CSF mobilized PBSC from HLA identical siblings and eight received unmanipulated BM from MUD. GvHD prophylaxis was with CYA alone for 38 patients and CYA plus MTX for six sibling recipients. RESULTS: Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite PCR indicate that 18 of 31 patients studied were full donor chimeras, while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range, 3-29 months) 33 patients remain alive in CR, or with no evidence of disease progression. Seven patients relapsed or progressed post-transplant and four of them subsequently died. Four patients died from regimen-related complications. There were no cases of Grades III-IV acute GvHD. Only two patients developed Grade II acute GvHD and only one had chronic GvHD. The estimated probability of non-relapse mortality at 1 year was 11%.Results: Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity and low incidence of GvHD.

Adolescent↗
PubMed2001

Approaching tolerance in transplantation.

Basic research has provided substantial encouragement that tolerance processes may be harnessed to the benefit of organ transplants. The goal of achieving mixed chimerism to ensure a robust tolerance, however elegant, may yet prove to be too complex and, consequently, risky as a procedure to compensate for the breadth of genetic differences, and prior immunological experiences of donor and host. Tolerance through regulation may prove to be easier to generate, but insufficiently robust to maintain. In the end the chosen protocol will be one that is simple, cheap and can guarantee patient compliance. Pragmatically, such a protocol need not be one aimed at clear-cut drug-free tolerance, but rather one which is trouble free. This could end up as a combination of partial tolerance (where tolerance processes have been harnessed) induced through a short-term treatment, in conjunction with easily tolerated maintenance therapy with select immunosuppressive drugs. Perhaps to the patient, the holy grail of tolerance is not as important as the complete assurance that the graft will survive and continue to function in all circumstances.

Animals↗
PubMed2001

The chemical biology of Ras lipidation.

In this review, we summarize the successful interplay between three disciplines, organic synthesis, biophysics and cell biology, in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in molecular detail. Highlighting the example of the Ras proteins, we show how the development of new synthetic methodologies paved the road to the synthesis of lipidated peptides and--by a combination of chemical and molecular biological techniques--lipidated proteins as molecular tools. We further give an overview of the results of the biophysical properties and biological activities of the molecules synthesized by means of this interdisciplinary approach. This successful combination of different disciplines led to a better understanding of the selective targeting of Ras and related lipoproteins to the plasma membrane.

Animals↗
PubMed2000

Directed differentiation of dendritic cells from mouse embryonic stem cells.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

Animals↗
PubMed2000

Solid-phase synthesis and biological evaluation of a teleocidin library--discovery of a selective PKCdelta down regulator.

Protein kinaseC (PKC) is linked to the signal-induced modulation of a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis, and tumor development. The design and synthesis of small molecules that regulate these different cellular signaling systems is at the forefront of modern drug design. Herein we report a) an efficient method for the synthesis of indolactamV (6), a PKC activator, and its N13-des(methyl) analogues (19) using a regioselective organometallic transformation, a convenient aminomalonate derivative (10) to introduce the appropriate functionality and an enantiospecific enzymic hydrolysis as key steps; b) the use of this method in the first solid-phase synthesis of a teleocidin library modifying the N-13, C-12 and C-7 alkyl chains, and, therefore, producing a library of potential activators and/or inhibitors of PKC of the general structure (32); c) the activation of PKC by selected members of the library using a MARCKS translocation in vivo assay system; d) the observation that some of these analogues are nearly as effective as the natural PKC activators phorbol dibutyrate and (-)-indolactam V (6), and e) the observation that some of these analogues have different potential to induce down-regulation of members of the PKC gene family after chronic stimulation.

3T3 Cells↗
PubMed2000

Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance.

Nondepleting anti-CD154 (CD40 ligand) mAbs have proven effective in inducing transplantation tolerance in rodents and primates. In the induction phase, anti-CD154 Ab therapy is known to enhance apoptosis of Ag reactive T cells. However, this may not be the sole explanation for tolerance, as we show in this study that tolerance is maintained through a dominant regulatory mechanism which, like tolerance induced with CD4 Abs, manifests as infectious tolerance. Therefore, tolerance induced with anti-CD154 Abs involves not only the deletion of potentially aggressive T cells, but also a contagious spread of tolerance to new cohorts of graft-reactive T cells as they arise.

Adoptive Transfer↗
PubMed2000

Enzyme-labile protecting groups in peptide synthesis: development of glucose- and galactose-derived urethanes.

The development of the tetra-O-acetyl-D-glucopyranosyloxycarbonyl (AGlOC) and tetra-O-acetyl-beta-D-galactopyranosyloxycarbonyl (AGalOC) protecting groups, which are fully enzyme-labile, carbohydrate-derived urethanes, is described. The protected amino acids were easily synthesized and subsequently converted into a series of model dipeptides through classical peptide couplings. Cleavage of an alpha/beta-anomeric mixture of a model AGlOC dipeptide was achieved with a "one-pot" procedure in good yield. To gain a better understanding of the enzymatic deprotection reaction, the AGalOC group was removed through a two step biotransformation (lipase catalyzed deacetylation, followed by beta-galactosidase catalyzed glycosidic bond fragmentation). Under these very mild reaction conditions (aq. buffer pH7.0, 37 degrees C), the desired N-terminal, unprotected dipeptide conjugates were obtained. The methodology was further utilized for the synthesis of an advanced tetrapeptide model system.

Acetylation↗
PubMed2000

Phosphoisoprenoid binding specificity of geranylgeranyltransferase type II.

Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Linking the fields--the interplay of organic synthesis, biophysical chemistry, and cell biology in the chemical biology of protein lipidation.

Research in the biological sciences has undergone a fundamental and dramatic change during the last decades. Whereas biology was more phenomenologically oriented for a long time, today many biological processes are investigated and understood in molecular detail. It has become evident that all biological phenomena have a chemical basis: Biology is based on chemical principles. In the past, this insight had led to the development of biochemistry, molecular biology, and modern pharmacology. Today it increasingly determines the manner in which various biological phenomena are studied. The tools provided by classical biological techniques often are not sufficient to address the prevailing issues in precise molecular detail. Instead, the strengths of both chemical and biological methodology have to be used. Several recent research projects have proven that combining the power of organic synthesis with cell biology may open up entirely new and alternative opportunities for the study of biological problems. In this review we summarize the successful interplay between three disciplines-organic synthesis, biophysics, and cell biology-in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in precise molecular detail. This interplay is highlighted by using the Ras protein as a representative example. The development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of their biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and Ras proteins in cell biological experiments are addressed. The successful combination of these three disciplines has led to a better understanding of the factors governing the selective targeting of Ras and related lipid-modified proteins to the plasma membrane.

Biophysical Phenomena↗
PubMed2000

In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation.

A novel nonmyeloablative conditioning regimen was investigated in 44 patients with hematologic malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. Recipient conditioning consisted of CAMPATH-1H, 20 mg/day on days -8 to -4; fludarabine, 30 mg/m(2) on days -7 to -3; and melphalan, 140 mg/m(2) on day -2. Thirty-six recipients received unmanipulated granculocyte colony-stimulating factor-mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. GVHD prophylaxis was with cyclosporine A alone for 38 patients and cyclosporine A plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite polymerase chain reaction indicate that 18 of 31 patients studied were full-donor chimeras while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range 3 to 29 months), 33 patients remain alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and 4 of them subsequently died. Four patients died of regimen-related complications. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of nonrelapse mortality was 11%. Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD.

Adolescent↗
PubMed2000

IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.

IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at -89 to -77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.

5' Untranslated Regions↗
PubMed2000

Posttranscriptional regulation of IL-10 gene expression through sequences in the 3'-untranslated region.

IL-10 is an 18-kDa immunoregulatory cytokine the transcription of which is controlled by the ubiquitously expressed transcription factors Sp1 and Sp3. Although many cell types express IL-10 mRNA, not all make detectable amounts of protein, and levels of protein expression vary enormously. We show here that much of this variation can be accounted for by posttranscriptional mechanisms. Multiple copies of potential mRNA destabilizing motifs AUUUA and related sequences can be found to the 3'-untranslated region (UTR) of IL-10 mRNA distributed through three potential regulatory regions. Evidence of RNA-destabilizing activities in all three regions was deduced from luciferase reporter assays. The half-life of RNA containing the 3'-UTR of IL-10 mRNA was quite short in both nonstimulated (t1/2 = 1 h), and PMA-stimulated EL-4 cell (t1/2 = 3 h). In contrast, the half-life of RNA lacking the 3'-UTR was much longer (t1/2 = >12 h) whether cells were stimulated or not. This suggests that many cells are poised to secrete IL-10 and will do so if they receive appropriate posttranscriptional signals.

3' Untranslated Regions↗