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Biomedical subjects

H Waldmann

Publications and source records attributed to H Waldmann.

At least 73 records · Page 4Linked to original sources
PubMed2000

Asymmetric steering of the Mannich reaction with phthaloyl amino acids

Mannich-type reactions are powerful methods for the efficient synthesis of beta-amino carbonyl compounds that are valuable intermediates for the construction of natural products, beta-peptides, and peptidomimetics. For the efficient steric steering of Mannich reactions a method was developed that consists in the treatment of imines with N-protected amino acid chlorides to give N-acyliminiumion intermediates that are subsequently attacked with silylketene acetals. The reactions are run best at room temperature and in the absence of any Lewis acid. The highest stereoselectivity is observed if N,N-phthaloyl tert-leucine is employed as chiral auxiliary and if N-aryl,C-aryl Schiffs bases are used that carry two ortho-substituents in either aromatic ring. Under these conditions the Mannich adducts are formed in preparatively useful yields and with excellent stereoselectivity (diastereomer ratio in general >99:1). The chiral auxiliary group is readily removed by 1) cleavage of the phthaloyl imide via reduction with NaBH4 and acid hydrolysis followed by 2) Edman degradation.

Journal Article↗
PubMed2000

Enzymatic protecting group techniques for glyco- and phosphopeptide chemistry: synthesis of a glycophosphopeptide from human serum response factor.

The covalent modification of proteins by phosphorylation and by glycosylation with GlcNAc residues are important regulatory processes which mediate biological signal transduction. For the study of such biological phenomena in molecular detail characteristic peptides which embody both types of modification may serve as efficient tools. However, their synthesis is complicated by their pronounced acid and base lability as well as their multifunctionality. For this purpose the enzyme-labile choline ester was developed. The choline ester can be removed selectively and in high yields from various GlcNAc-glycopeptides and phosphopeptides at pH 6.5 and 37 degrees C. The conditions under which the enzymatic deprotections proceed are so mild that no undesirable side reactions are observed (i.e., no cleavage or anomerization of the glycosidic bonds and no beta-elimination of the phosphate or the carbohydrate occur). The specificity of the biocatalyst guarantees that neither the peptide bonds nor the other protecting groups present are being attacked. When this enzymatic protecting group technique was combined with the enzyme-labile 4-(phenylacetoxy)benzyloxycarbonyl (PhAcOZ) urethane protecting group a complex glycophosphopeptide could be built up. The glycopeptide is equipped with a biotin label by which it can be traced in biological systems. This peptide represents a characteristic partial structure of a glycosylated and phosphorylated sequence from the transactivation domain of serum response factor (SRF), a widely occurring human transcription factor.

Amino Acid Sequence↗
PubMed2000

High dose bone marrow transplantation induces deletion of antigen-specific T cells in a Fas-independent manner.

BACKGROUND: Monoclonal antibody induced tolerance to high doses of multiple lymphocyte stimulating (MLS)+minor mismatched bone marrow has recently been associated with clonal deletion, as reported in fully allogeneic models of bone marrow transplantation. FasL-induced apoptosis has been shown to mediate antigen-specific T cell deletion after antigenic stimulation in wild-type and T cell receptor transgenic mice. Therefore, we investigate a role for the Fas pathway in deletional tolerance to high dose bone marrow. METHODS: Fas mutant and control mice (H-2k, MLS-1b) were tolerized under the cover of monoclonal antibodies to high dose (5 x 10(7) cells) AKR (H-2k, MLS-1a) bone marrow. Tolerance was confirmed by AKR skin grafting after antibody clearance. Antigen-reactive cell deletion was monitored by Vbeta6+ T cell elimination, measured by flow cytometry of peripheral blood throughout the experiment. Donor T cell (Thy1.1+) chimerism was assessed in a similar manner. RESULTS: Fas mutant mice infused with high dose AKR bone marrow under the cover of antibody were tolerant, as demonstrated by indefinite survival of AKR skin grafts. When high levels of donor cell chimerism were established in Fas mutant mice, peripheral deletion of antigen-reactive cells was observed to be independent of signaling through Fas. CONCLUSIONS: Apoptosis mediated by Fas receptor signaling is not the mechanism of clonal deletion of antigen-reactive cells after antibody facilitated high dose marrow transplantation. However, the Fas mutation does impair the development of adequate donor chimerism.

Animals↗
PubMed2000

Dominant tolerance and linked suppression induced by therapeutic antibodies do not depend on Fas-FasL interactions.

BACKGROUND: Nonlytic anti-CD4 monoclonal antibody therapy can be used to induce transplantation tolerance in rodent models. Such tolerance is often associated with dominant regulation, mediated by CD4+ cells, and characterized by infectious tolerance and linked suppression. Understanding the mechanisms by which CD4+ regulatory cells function may improve the manner in which current immunosuppressants are applied and may lead to the development of new tolerance-inducing therapeutics. Fas-mediated apoptosis has been characterized as an important mechanism of peripheral self-tolerance and we here examine whether it has any role in anti-CD4 monoclonal antibody-induced dominant tolerance. METHODS: Tolerance to transplanted skin and bone marrow, mismatched for multiple minor histocompatibility antigens, was induced in Fas mutant and control mice using anti-CD4 and anti-CD8 monoclonal antibodies. To test for linked suppression, animals were transplanted with a second graft-bearing tolerated and third party antigens. The ability of splenocytes from tolerant animals to suppress graft rejection was assessed by transfer into partially immunocompromised recipients. RESULTS: Monoclonal antibody therapy rendered Fas mutant mice tolerant of minor disparate skin and bone marrow. Splenocytes from these and control tolerant animals when transferred into partially immunocompromised Fas mutant or control recipients, induced antigen-specific suppression of graft rejection. Additionally, tolerant Fas mutant mice accepted grafts bearing tolerated and third party antigens. CONCLUSIONS: Signal transduction through the Fas receptor plays no essential role in the induction of tolerance using anti-CD4 and anti-CD8 monoclonal antibodies or its maintenance by active regulation.

Animals↗
PubMed2000

Immune reconstitution after allogeneic bone marrow transplantation depleted of T cells.

BACKGROUND: Immune reconstitution following transplantation in individuals who had received T-cell-depleted marrow from HLA identical siblings was serially documented and correlated with the clinical recovery. METHODS: Patients were preconditioned with radiation containing programs. GvHD prophylaxis was by T-cell depletion with CAMPATH 1G (ex vivo; median dose 20 mg). After transplantation lymphoid development was studied by flow cytometry and serum Ig concentrations were determined. Charts were reviewed to determine the effects of the immune reconstitution on the clinical performance. RESULTS: The mean donor mononuclear cell number infused was 0.89x10(8)/kg. Within 6 months all the patients recovered their blood parameters and only one required therapy for GvHD. However, despite normal blood counts, 15 suffered life-threatening opportunistic infections, developing at a median of 24 weeks post grafting, but occurring even after 11 months. At 8 weeks from marrow infusion when leukocyte values had normalized in 15/20, compared to normal, immunophenotyping of blood cells from BMT revealed a significantly reduced mean lymphocyte count (1.06, SD 0.83x10(9)/l; P = 0.01), cells expressing CD3 (0.7x10(9)/l, SD 0.68; P = 0.05), CD4 (0.13x10(9)/l, SD 0.21; P = 0.0001) and CD19 (0.04x10(9)/l, SD 0.05; P = 0.001). Populations expressing CD8 and CD56 remained within normal range throughout the study. Normalization of cell numbers displaying CD2, CD3 and CD19 was delayed until 52, 52 and 24 weeks respectively, while CD4 counts persisted subnormal even at 72 weeks. Serum IgA levels were significantly decreased for the entire study period. CONCLUSIONS: T-cell depletion with CAMPATH 1G while effectively preventing GvHD, also causes clinically significant and prolonged immunosuppression with apparently important clinical implications.

Blood↗
PubMed2000

Bioorganic synthesis of lipid-modified proteins for the study of signal transduction.

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.

Animals↗
PubMed2000

Extrathymic signals regulate the onset of T cell repertoire selection.

The thymus is the principal site of T cell development, coordinating positive and negative selection of an immunocompetent repertoire. The ability of fetal thymi from C57BL/6 mice to support these selection events in culture is, however, critically dependent on the timing of their excision: whereas thymi from 16 day embryos generate mature CD4+8(-) and CD4(-)8+ thymocytes, 14 day thymi show a profound blockage in positive selection at the CD4+8+ stage. Here we show that this blockage is not due to intrinsic deficiencies in the ability of thymocytes to transduce positive selection signals, suggesting the defect to lie within the thymic microenvironment. Since vascularization of the thymus occurs at day 15 of gestation, we investigated whether the exclusion of plasma proteins from 14 day thymi was responsible. Accordingly, the addition of serum from 16 day embryos to organ cultures of 14 day thymi rescued mature CD4+8(-) and CD4(-)8+ subsets. Activity was found to reside in a low molecular weight fraction of serum, sensitive to proteolysis, which was present only transiently during ontogeny. Our data suggest that repertoire selection is initiated following "priming" of the thymic microenvironment by plasma proteins, thereby ensuring the onset of positive selection to be delayed until the entry of extrathymic proteins to which self tolerance must be established.

Animals↗
PubMed2000

Dendritic cells and prospects for transplantation tolerance.

The past year has witnessed the resolution of some long-standing enigmas surrounding the immunobiology of dendritic cells, illuminating their opposing roles in peripheral tolerance and allograft rejection. Nevertheless these advances have posed many new questions, the answers to which may subtly influence our approach to the treatment of rejection while bringing ever closer the prospect of donor-specific transplanation tolerance.

Animals↗
PubMed2000

Synthesis of a triply phosphorylated pentapeptide from human tau-protein.

Two different strategies for the synthesis of a triply phosphorylated pentapeptide are described. In both cases a monophosphorylated selectively N-deprotected tripeptide is employed as C-terminal fragment. Coupling of this building block with a C-terminally unmasked bis-phosphorylated seryl-dipeptide unexpectedly failed due to decomposition of this peptide upon activation with different coupling reagents. Instead stepwise N-terminal elongation of the peptide chain with serine derivatives and subsequent O-phosphorylation of the serine OH-groups was successful. These results indicate that assembly of multiply phosphorylated peptides from preformed multiply phosphorylated phosphopeptide building blocks in general may be problematic and that a stepwise elongation of the amino acid chain may be preferable.

Humans↗
PubMed2000

The effect of the serotherapy regimen used and the marrow cell dose received on rejection, graft-versus-host disease and outcome following unrelated donor bone marrow transplantation for leukaemia.

Unrelated donor (UD) transplantation is the only potentially curative therapy for many leukaemia patients but is associated with a high mortality and morbidity. We sought to identify factors that could be optimised to improve outcome following UD transplantation in adults. Data was retrospectively analysed on 55 patients sequentially receiving UD transplants for CML or acute leukaemia (AL), all of whom received serotherapy for the prevention of GVHD and rejection. All patients received standard conditioning regimens. The first 28 patients transplanted also received combined pre- and post-transplant serotherapy with Campath 1G (days -5 to +5) and standard dose CsA plus MTX as GVHD prophylaxis (protocol 1). The subsequent 27 patients received a 5-day course of pre-transplant serotherapy alone either with ATG (CML patients) or Campath 1G (AL patients) on days -5 to -1 inclusive, with high-dose CSA plus MTX (protocol 2). The incidence of acute GVHD was low with no patient receiving either protocol developing > grade 2 disease. The use of protocol 2 and the administration of a bone marrow cell dose above the median (2.17 x 10(8)/kg) were the most important factors predicting engraftment (P = 0.03 and P = 0.001, respectively) but this only remained significant for cell dose in multivariate analysis (P = 0.03). Overall survival for the group was 45% at 3 years and was influenced by both age (P = 0.02) and disease status at transplantation (P = 0.001). Receiving a cell dose above the median was also associated with a trend towards better survival (P = 0.08), due primarily to a reduction in the TRM to 8.2% compared with 54.5% in those receiving a lower cell dose (P = 0.002). We conclude that pre-transplant serotherapy alone is highly effective at preventing acute GVHD following UD BMT and that additional post-transplant serotherapy does not confer any benefit. Furthermore, a high marrow cell dose infused has a major effect in reducing transplant related mortality following UD BMT.

Adolescent↗
PubMed2000

CD52 antibodies for prevention of graft-versus-host disease and graft rejection following transplantation of allogeneic peripheral blood stem cells.

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic bone marrow transplantation, but can be avoided by removing T lymphocytes from the donor bone marrow. However, T cell depletion increases the risk of graft rejection. In this study, two strategies are used to overcome rejection: (1) use of high doses of stem cells obtained from peripheral blood (PBSC), (2) admixture with a CD52 monoclonal antibody in order to deplete both donor and residual recipient lymphocytes. Two antibodies are compared: CAMPATH-1G (rat IgG2b) and its humanized equivalent CAMPATH-1H (human IgG1). A total of 187 consecutive patients at six centers received PBSC transplants from HLA-matched siblings between 1997 and 1999. A wide spectrum of diseases, both malignant and non-malignant, was included. The recovery of CD34+ cells after antibody treatment was close to 100%. The risk of acute GVHD (grade 2 to 4) was 11% in the CAMPATH-1G group and 4% in the CAMPATH-1H group (P = NS). The risk of chronic GVHD (any grade) was 11% in the CAMPATH-1G group and 24% in the CAMPATH-1H group (P = 0.03) but the risk of extensive chronic GVHD was only 2%. The overall risk of graft failure/rejection was 2%, not significantly different between the two antibodies. Antibody treatment was equally effective at concentrations between 10 microg/ml and 120 microg/ml and it made no significant difference to the outcome whether the patients received post-transplant immunosuppression or not (87% did not). Transplant-related mortality in this heterogenous group of patients (including high-risk and advanced disease) was 22% at 12 months. It is proposed that treatment of peripheral blood stem cells with CAMPATH-1H is a simple and effective method for depleting T cells which may be applicable to both autologous and allogeneic transplants from related or unrelated donors. Special advantages of this approach are the simultaneous depletion of donor B cells (which reduces the risk of EBV-associated lymphoproliferative disease) and the concomitant infusion of CAMPATH-1H to deplete residual recipient T cells and thus prevent graft rejection.

Alemtuzumab↗
PubMed2000

Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH conditioning: an effective regimen with low procedure-related toxicity.

Autologous transplantation has an established role in the treatment of lymphoproliferative disorders, but allogeneic transplantation remains controversial. In an attempt to reduce the high procedure-related mortality reported with allografting in lymphoma, we have used BEAM (BCNU, etoposide, cytarabine and melphalan), a standard conditioning regimen for autologous transplantation. As BEAM may be insufficiently immunosuppressive to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with the anti-CD52 antibody CAMPATH-1G from day -5 to day -1. Twelve patients (median age 46 years) underwent allogeneic transplantation for lymphoma (n = 11) or chronic lymphocytic leukaemia (n = 1) from HLA-identical (n = 9) or mismatched (n = 3) sibling donors. Cyclosporin A and methotrexate were used as graft-versus-host disease (GVHD) prophylaxis. One patient died of progressive lymphoma at day +12, the remaining 11 patients engrafted rapidly, with eight demonstrating full donor chimerism. One patient had an episode of rejection and received a further stem cell infusion with sustained recovery. Only one patient developed GVHD (grade I). The low incidence of acute GVHD may be in part related to persisting levels of in vivo CAMPATH-IG at the time of transplantation. Of 11 evaluable patients, nine achieved complete remission (CR), and a further patient achieved CR after donor lymphocyte infusion at 5 months. Our preliminary experience is that this regimen was well tolerated with a low risk of GVHD and appears no more toxic than a BEAM autograft. Further follow-up is required to see whether the low incidence of GVHD impacts upon relapse risk.

Adult↗
PubMed2000

Impact on T-cell depletion and CD34+ cell recovery using humanised CD52 monoclonal antibody (CAMPATH-1H) in BM and PSBC collections; comparison with CAMPATH-1M and CAMPATH-1G.

BACKGROUND: Ex vivo T-cell depletion of allogeneic BM (BM) grafts can effectively reduce graft versus host disease (GvHD) and may also apply to transplantation of allogeneic peripheral blood stem cell (PBSC) transplants. METHODS: Here we have evaluated T-cell depletion and progenitor cell recovery by antibody-mediated cells lysis using three CD52 monoclonal antibodies (MAbs) at different concentrations and cell densities. RESULTS: CAMPATH-1M was superior to CAMPATH-1H for T-cell depletion of BM samples. Treatment with CAMPATH-1M resulted in up to 2.55 log depletion of CD3+ cells, with recoveries of >or=45% CD34+ cells, >or=67% CFU-GM and >or=65% BFU-E. CAMPATH-1H treatment resulted in up to 1.64 log depletion of CD3+ cells and similar recoveries of CD34+ cells, CFU-GM and BFU-E as seen with CAMPATH-1M. Depletion of CD19+ cells was similar to that observed for CD3+ cells while natural killer (NK) cells were relatively spared compared with the T and B cell populations. Log depletions of T cells from PBSC, as determined by immunofluorescence studies and limiting dilution analyses, were similar using CAMPATH-1M, -1H, and -1G. There were also no differences in the depletion of CD19+ cells or NK cells using the three MAbs. Similar results were obtained for recoveries of CD34+ cells, CFU-GM and BFU-E using all three MAbs, although the recovery of CD34+ cells using the highest concentration of MAbs was significantly greater in CAMPATH-1H treated samples. Increasing the number of PBSC treated with CAMPATH-1H and -1M had no effect on the log depletion of T, B or NK cells and there were no major differences in the log depletions achieved with CAMPATH-1H or -1M. Likewise, the higher PBSC density had no effect on the recoveries of CD34+ cells or committed progenitors and once again CAMPATH-1H gave similar recoveries to those obtained using CAMPATH-1M. DISCUSSION: Although CAMPATH-1M resulted in greater ex vivo T-cell depletion of BM than CAMPATH-1H, in all other respects, the humanised CAMPATH-1H was just as effective as CAMPATH-1M and -1G.

Alemtuzumab↗
PubMed2000

Campath-1H therapy in refractory ocular inflammatory disease.

BACKGROUND: Standard therapy for severe, immune mediated, ocular inflammation has significant side effects, and may fail to control the disease. T cell directed monoclonal antibody (mAb) therapy can provide long term remission of inflammatory disease in experimental models. The Campath-1H mAb was administered to patients with severe, refractory, ocular inflammation. METHODS: 10 patients with severe, refractory, non-infectious ocular inflammatory disease were treated with Campath-1H mAb. This is a fully humanised mAb which recognises the pan-lymphocyte antigen CD52. RESULTS AND DISCUSSION: Following Campath-1H therapy, all 10 patients showed an initial resolution of their ocular symptoms and signs. Long lasting remissions were achieved in eight patients, in whom baseline immunosuppression could subsequently be reduced to minimal levels. The possible mechanisms of action of Campath-1H therapy are discussed.

Adult↗
PubMed2000

Prope tolerance with induction using Campath 1H and low-dose cyclosporin monotherapy in 31 cadaveric renal allograft recipients.

The last 40 years has been a period of remarkable evolution of organ transplantation from nothing to a well-established form of treatment with good short-term and tolerable long-term results. Nevertheless by ten years approximately 50% of grafts will have been lost due, mainly, to chronic rejection or the side-effects of immunosuppressive therapy. We now have a number of extremely powerful immunosuppressive drugs and antibodies with different mechanisms of action and the stage is set for a move from current continuous high dose immunosuppressive maintenance therapy to low dose or no maintenance immunosuppression. True tolerance can occur in man, examples being successful bone marrow transplantation and patients with liver grafts who have stopped immunosuppression after years of good function. The antibody Campath 1H with a unique target CH52 on T & B lymphocytes and monocytes has been used to eliminate lymphocytes from the blood for a month in patients with renal allografts who have then been maintained on half dose Cyclosporin without any other maintenance drug. The results with a mean two year follow-up have been encouraging, 29 patients having good function without receiving maintenance steroids. It is likely that this protocol could be improved since dosage timing and various minimal maintenance immunosuppressive protocols have not been fully investigated. This almost or "Prope" tolerance could be a major step forward providing a better quality of life for patients and inexpensive maintenance immunosuppression.

Adult↗