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Biomedical subjects

O Christ

Publications and source records attributed to O Christ.

At least 19 recordsLinked to original sources
PubMed2005

Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients' samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-beta2microglobulin-/- mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL+/Ph+ cells. The regenerated leukemic cells included an initial, transient population derived from CD34+CD38+ cells as well as more sustained populations derived from CD34+CD38- progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34+) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.

Animals↗
PubMed2005

BCR-ABL-transduced human cord blood cells produce abnormal populations in immunodeficient mice.

In this study, we describe the successful use of a gene transfer approach to demonstrate the ability of forced BCR-ABL expression to deregulate the growth and differentiation of primitive naive human hematopoietic cells after their transplantation into immunodeficient mice. Human CD34+ cord blood cells were exposed to an MSCV retrovirus containing a BCR-ABL-IRES-GFP (P210) cassette and then injected immediately into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) or NOD/SCID-beta2microglobulin-/- mice. P210- and control-transduced (GFP+) human hematopoietic cells were produced in the bone marrow of the mice at similar levels until termination of the experiments 5-6 months later. However, the P210-transduced cells produced a markedly different spectrum of progeny, with an increased ratio of myeloid to B-lymphoid cells and a frequently prolonged increase in erythroid and megakaryocytic cells. After 5 months, several of the mice transplanted with P210-transduced cells developed an increased WBC count and/or splenomegaly due to an expansion of the human GFP+ population. These findings demonstrate that forced expression of BCR-ABL in primitive transplantable human hematopoietic cells is sufficient to cause a rapid and persistent deregulation of their growth and differentiation in vivo with occasional evidence after several months of progression to an early stage of disease.

Animals↗
PubMed2001

Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination.

Clinical success in tumor vaccination frequently does not reach expectation. Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-beta-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms. BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated Salmonella typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the beta-galactosidase protein or mice were vaccinated with both DNA and protein. Although all regimens led to a prolongation of survival time, oral vaccination with transfected S. typhimurium followed by i.v. transfer of protein-loaded DC provided the optimal schedule. In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-beta-gal. As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4(+) and CD8(+) cells and 2) CD8(+) CTL are most strongly activated by vaccination with transformed Salmonella, whereas vaccination with protein-loaded DC is superior for the activation of Th. The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms. The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.

Animals↗
PubMed2001

Tumour-induced suppression of immune response and its correction.

Immunosuppressive features of tumour cells are a major obstacle for immunotherapy of cancer. We recently noted that RENCA cells effectively interfere with the in vivo activation of RENCA-specific T cells. To unravel the underlying mechanism, we evaluated the influence of RENCA cells on a mixed-lymphocyte/ tumour reaction as well as an allogeneic mixed-lymphocyte reaction. We observed that RENCA cells were not directly immunosuppressive. Instead, they initiated deviation of an immune response in at least two independent directions: (i) expansion of a population of NK1.1+/CD3+ cells, which was accompanied by elimination of mainly CD4+ lymphocytes, and (ii) production of a leukocyte-derived inhibitory factor. Expression of the costimulatory molecule B7.1 by RENCA cells prevented induction of anergy, while expression of MHC class II molecules prevented expansion of NK1.1+ cells, which was accompanied by a significant decrease in cell death. Hence, an unimpaired response was observed only when RENCA cells expressed B7.1 plus MHC class II molecules. Thus, even if a tumour itself is not immunosuppressive, it can induce a strong deviation of the immune response. It is concluded that the first contact between elements of the immune system and the tumour cell can confer a severe bias on immunoregulatory circuits.

Animals↗
PubMed2001

Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

OBJECTIVE: Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. MATERIALS AND METHODS: Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. RESULTS: Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. CONCLUSION: Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

Animals↗
PubMed2001

Gene delivery by attenuated Salmonella typhimurium: comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination.

Using the murine B16F1 melanoma, we compared a CTL- versus helper T cell (TH)-directed vaccination approach. Mice were either orally vaccinated with attenuated Salmonella typhimurium (SL) or subcutaneously with dendritic cells (DCs) loaded with gp100 peptides predicted to bind to H2-Kb/H2-Db molecules. SL were transformed with the murine gp100 cDNA (SL-gp100) or with a fusion construct of gp100 and a fragment of invariant chain cDNA (SL-gp100/Ii). Transcription of these genes in vivo has been readily observed in monocytes and DC. Retardation of B16F1 growth was more efficiently achieved by vaccination with SL-gp100 than with DC. Vaccination with SL-gp100/Ii aiming at preferential presentation by MHC II molecules provided some further improvement due to a stronger expansion of TH and CTL. The importance of help was further sustained by a prolongation of the survival time when mice concomitantly received IL2. Notably, prophylactic, compared to therapeutic, vaccination had no additional impact on survival time/rate. This was due to a striking decrease in frequencies of gp100-specific TH, CTL, and cytokine-expressing cells during tumor growth. Thus, the efficacy of vaccination was limited by tumor-induced immunosuppression. Our data demonstrate the oral route of vaccination via Salmonella as a most convenient transfer regimen and confirm the superiority of protocols aiming at preferential activation of TH.

Animals↗
PubMed2001

Importance of CD44v7 isoforms for homing and seeding of hematopoietic progenitor cells.

The adhesion molecule CD44 consists of many isoforms of which particularly CD44v7 is of major importance in hematopoietic progenitor cell homing. An increase of progenitor cells in the periphery was observed after treating mice with a CD44v7-specific antibody, concomitant with a substantially augmented marrow-repopulating ability (MRA). Because CD44v7 is expressed on a fraction of bone marrow cells (BMC) as well as on long-term bone marrow culture-derived stromal cells, we aimed to differentiate between the functional relevance of CD44v7 on either cell type by transferring CD44v7+/+ BMC into CD44v7-/- mice and vice versa. CD44v7+/+ BMC homed poorly in the bone marrow of CD44v7-/- mice and their MRA was severely impaired. CD44v7-/- BMC, instead, exhibited an improved MRA when transferred into the CD44v7+/+ host, although their MRA remained below that of CD44v7+/+ BMC. Thus, it is predominantly, but not exclusively, expression of CD44v7 on stromal cells which supports progenitor cell homing.

Animals↗
PubMed2001

Efficacy of local versus systemic application of antibody-cytokine fusion proteins in tumor therapy.

Application of immunocytokines [fusion proteins (FuPs)] where the cytokine has been coupled to an antibody may not produce the severe side effects frequently observed during systemic application of cytokines in cancer therapy. However, it has not been explored whether FuPs are sufficient for intratumoral activation of leukocytes or whether intratumoral versus systemic application may be of greater efficacy. Interleukin 2 (IL2) or tumor necrosis factor (TNF) coupled to an anti-epidermal growth factor receptor monoclonal antibody (IL2-FuP or TNF-FuP) were tested in SCID mice bearing a human epidermal growth factor receptor-positive melanoma transplant and being reconstituted with human HLA-matched peripheral blood leukocytes. Whole-body autoradiography revealed larger accumulation and prolonged retention of i.v. or intratumorally applied IL2-FuP or TNF-FuP compared with the antibody. Even with low doses of FuP, tumor growth was significantly retarded, with the survival time being further prolonged by the intratumoral application. Furthermore, outgrowth of the tumor was prevented in approximately 50% of mice as long as they received weekly injections of peripheral blood leukocytes concomitantly with the FuPs, which confirmed that it was the donor leukocytes activated in vivo that retarded tumor growth. An in vitro analysis revealed that the IL2-FuP supported mainly proliferation and lymphokine-activated killer cell activity, whereas TNF-FuP stimulated cytokine production and cytotoxic activity of monocytes and, to a low degree, of T cells. Both TNF-FuP and IL2-FuP significantly accumulated in the tumor, which led to retardation of tumor growth. The therapeutic effect was improved by intratumoral application. Importantly, the efficacy of both IL2-FuP and TNF-FuP depended on the induction of an immune response in vivo.

Animals↗
PubMed2001

Interleukin 2-antibody and tumor necrosis factor-antibody fusion proteins induce different antitumor immune responses in vivo.

Two fusion proteins, composed of interleukin 2 (IL-2) or tumor necrosis factor (TNF) coupled to an antibody [fusion protein (FuP); IL-2-FuP or TNF-FuP], were capable of retarding growth of a human malignant melanoma in the severe combined immunodeficient mouse depending on the concomitant application of human peripheral blood leukocytes. Here we have analyzed the mechanisms that determine the therapeutic effect. Tumor-bearing severe combined immunodeficient mice received once per week an i.v. injection of HLA-matched peripheral blood leukocytes and twice per week i.v. or intratumoral injections of FUPS: Leukocyte recovery and their activation state were monitored. The number of draining lymph node cells (LNCs) and tumor-infiltrating leukocytes increased continuously, and the yield of draining LNCs was improved significantly when the FuPs were applied locally. In IL-2-FuP-treated mice, the majority of draining LNCs and tumor-infiltrating lymphocytes expressed T-cell activation markers and IL-2, thus being classified as T helper type 1 cells. These cells displayed strong proliferative activity and initiated activation of lymphokine-activated killer cells and CTLS: TNF-FuP supported activation of CTLs and of monocytes as revealed by TNF expression and cytostatic activity. Neither the antibody, nor IL-2, nor TNF, nor the mixture of antibody and cytokines exhibited the full-fledged activational potency of the FUPS: Notably, activation of immune effector mechanisms was much stronger when the FuPs were applied intratumorally. This is the first report to show that FuPs are efficient immunostimulants in vivo for native leukocytes. Although IL-2-FuP induced a T helper type 1 response with recruitment of LAK and CTL, TNF-FuP efficiently recruited and activated monocytes and, in a less pronounced manner, CTLS:

Animals↗
PubMed2000

In vitro and in vivo induction of a Th cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program.

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4(+) cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-gamma expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.

Adoptive Transfer↗
PubMed2000

Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells.

The use of anti-CD3 x antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 x CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 x CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

Animals↗
PubMed2000

Mathematical modeling of the hydrolysis of anaerobic processes.

In the recently published dynamic simulation model for mesophilic digestion of sewage sludge the hydrolysis constant refers to the total dry solid content without regarding their composition. To apply these models to the digestion of municipal solid waste, the hydrolysis constants of the various fractions must be considered. The major constituents of organic waste were identified as carbohydrates, proteins and lipids. For these three constituents the hydrolysis constants for thermophilic digestion were determined. The implementation of these constants into the existing dynamic models allowed a reasonable characterization of the digestion of municipal organic waste.

Bacteria, Anaerobic↗
PubMed1979

Pharmacokinetics of single and multiple doses of clobazam in humans.

1. The pharmacokinetics of clobazam and its biotransformation product N-desmethylclobazam were investigated after single and multiple doses in normal subjects. 2. The relevant physicochemical properties of clobazam were measured and are presented. Different assay methods (radiochemical, fluorimetric and gas chromatographic) were applied and the results correlated. 3. After single doses the pharmacokinetic profile of clobazam includes time to peak levels 1--4 h after dosing, peak levels increasing linearly with the logarithm of dose, and terminal half-lives of about 18 hours. At least 87% of an oral dose is absorbed, as indicated by urinary recovery of labelled material. 4. In multiple-dose studies unchanged clobazam levelled off at minimum steady-state concentrations within one week of dosing. During 28 d of medication N-desmethylclobazam accumulated to near steady-state levels about eight times higher than those of the unchanged compound. 5. No pharmacokinetic interactions were discovered between clobazam and the antidepressant nomifensine.

Anti-Anxiety Agents↗