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Hans-Peter Kiem

Publications and source records attributed to Hans-Peter Kiem.

At least 37 records · Page 2Linked to original sources
PubMed2004

De novo generation of CD4 T cells against viruses present in the host during immune reconstitution.

T cells recognizing self-peptides are typically deleted in the thymus by negative selection. It is not known whether T cells against persistent viruses (eg, herpesviruses) are generated by the thymus (de novo) after the onset of the infection. Peptides from such viruses might be considered by the thymus as self-peptides, and T cells specific for these peptides might be deleted (negatively selected). Here we demonstrate in baboons infected with baboon cytomegalovirus and baboon lymphocryptovirus (Epstein-Barr virus-like virus) that after autologous transplantation of yellow fluorescent protein (YFP)-marked hematopoietic cells, YFP+ CD4 T cells against these viruses were generated de novo. Thus the thymus generates CD4 T cells against not only pathogens absent from the host but also pathogens present in the host. This finding provides a strong rationale to improve thymopoiesis in recipients of hematopoietic cell transplants and, perhaps, in other persons lacking de novo-generated CD4 T cells, such as AIDS patients and elderly persons.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Duration of immunosuppressive treatment for chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) requires long-term immunosuppressive therapy after hematopoietic cell transplantation. We retrospectively analyzed a cohort of 751 patients with chronic GVHD to identify characteristics associated with the duration of immunosuppressive treatment. Among the 274 patients who discontinued immunosuppressive therapy after resolution of chronic GVHD before recurrent malignancy or death, the median duration of treatment was 23 months. Results of a multivariable model showed that treatment was prolonged in patients who received peripheral blood cells, in male patients with female donors, in those with graft-versus-host HLA mismatching, and in those with hyperbilirubinemia or multiple sites affected by chronic GHVD at the onset of the disease. Nonrelapse mortality was increased among patients with HLA mismatching or hyperbilirubinemia but not among those with other risk factors associated with prolonged treatment for chronic GVHD. Nonrelapse mortality was also increased in older patients and those with older donors, in patients with platelet counts less than 100 000/microL or progressive onset of chronic GVHD from acute GVHD, and in those receiving higher doses of prednisone immediately before the diagnosis of chronic GVHD. After the dose of prednisone was taken into account, progressive onset was not associated with an increased risk of nonrelapse mortality.

Adolescent↗
PubMed2004

Fundamental role of inferior olive connexin 36 in muscle coherence during tremor.

Inferior olive (IO) neurons are electrically coupled by cytosolic pores formed by the neuron-specific connexin 36 (Cx36). Electrical coupling in the IO figures prominently in current views about brain control of movement. However, a role for Cx36 in movement has been questioned and not definitively demonstrated. Previous reports have shown that embryonic deletion of the Cx36 gene resulted in almost complete loss of cytosolic and electrical coupling in the IO without an obvious deficit in movement, possibly due to developmental compensations in ionic conductances that can confound the approach of embryonic gene deletion. We used a replication-incompetent lentiviral vector to stably express a dominant-negative Cx36 mutant in the IO of adult rats. We show that interneuronal cytosolic coupling is severely reduced by the mutant Cx36, without effect on neuron morphology or electrical properties. Multisite electromyography revealed that blocking Cx36 in the IO impaired the coherence of muscle firing during harmaline tremor without affecting its rhythm. The data demonstrate that gap junction coupling within the IO mediated by Cx36 adds 10-20 ms of precision to the fine temporal coordination of muscle firing during movement.

Animals↗
PubMed2004

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.

The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

Animals↗
PubMed2004

Fanconi anemia type C-deficient hematopoietic cells are resistant to TRAIL (TNF-related apoptosis-inducing ligand)-induced cleavage of pro-caspase-8.

OBJECTIVE: The pathophysiology of bone marrow failure in Fanconi anemia (FA) patients is thought to involve excessive apoptosis involving signaling triggered by fas ligation and tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma exposure. We investigated whether a new member of the TNF family, TRAIL (TNF-related apoptosis-inducing ligand), would similarly trigger preferential apoptotic cell death in FA phenotype cells. MATERIAL AND METHODS: Hematopoietic cells from FANCC(-/-) transgenic mice and human FA-C lymphoblasts (HSC536N) as well as their phenotypically corrected counterparts (FANCC(+/+), HSC536/FA-Cneo) were compared for their response to apoptosis induction by TRAIL and fas ligation in the presence or absence of IFN-gamma. Cells were also studied for the protein and gene expression of TRAIL-receptors, caspase-8 and its inhibitory protein, FLIP. RESULTS: TRAIL exposure by itself or in combination with IFN-gamma did not lead to preferential apoptosis induction in human and murine FA-C phenotype hematopoietic cells. This resistance was unrelated to the expression of TRAIL receptors or FLIP isoforms, but correlated with absent cleavage of pro-caspase-8. Results were validated by those from gene expression profiling of relevant genes in the two lymphoblast cell lines. CONCLUSION: TRAIL, in contrast to fas ligation, does not induce preferential apoptosis in FA-C phenotype cells despite shared downstream signaling described in non-FA models. These data provide further insight into the complexity of FA-C-regulated apoptotic signaling.

Animals↗
PubMed2004

Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia.

There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1-7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

Animals↗
PubMed2004

Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.

Animals↗
PubMed2004

Differences in F36VMpl-based in vivo selection among large animal models.

Animal models are indispensable tools for understanding physiological and pathological processes, as well as for developing new therapies. Ultimately, the results of animal experimentation must provide information that can guide the development of therapeutic approaches in humans. Significant differences have been reported comparing a gene therapy approach between different animal models. However, little information exists describing differences among the available large animal models. Here we evaluated, in the hemopoietic cells of baboons, a system of selection that has previously demonstrated activity in mice, in dogs, and in human cells ex vivo. This system employs a derivative of the murine thrombopoietin receptor (F36Vmpl), which is conditionally activated in the presence of a small-molecule drug called a chemical inducer of dimerization (CID). Whereas cultured mouse, human, and, to a lesser extent, dog hemopoietic cells all proliferate in response to the F36Vmpl signal, we observed only a minor and variable response to the F36Vmpl signal in the cultured cells of baboons. Similarly, we have noted significant rises in the frequency of transduced hemopoietic cells in mice and in dogs upon CID administration in vivo; however, here we show that responses to CID administration in three baboons were modest and variable. These findings have general implications for the evaluation and development of new strategies for gene therapy.

Animals↗
PubMed2004

Radiation-induced glioblastoma multiforme in two adult baboons (Papio cynocephalus anubis).

A diagnosis of glioblastoma multiforme (GBM) was made for cerebral masses found at necropsy in two baboons (Papio cynocephalus anubis). Case 1 was an adult (6.18 years old) male baboon that suddenly died during a physical examination as part of a clinical evaluation for a leg lameness. Case 2 was an adult (5.95 years old) female baboon that stopped breathing during anesthesia for an magnetic resonance imaging to evaluate lethargy, weight loss, inappetence, and dilated pupils. Both animals had undergone total body irradiation with cobalt during a research protocol. The incidence of spontaneous brain tumors in nonhuman primates is low, but radiation-induced GBM lesions in rhesus macaques (Macaca mulatta) have been reported. A definitive diagnosis was made in these cases, using histopathologic criteria of cellular pleomorphism, high mitotic rate, regions of coagulation necrosis, and endothelial proliferation.

Animals↗
PubMed2003

Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs.

The optimal stem cell source for stem cell gene therapy has not been defined. Most gene transfer studies have used peripheral blood or marrow repopulating cells collected after administration of granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF). For clinical applications, however, growth factor administration may not be feasible. Thus, in the current study we used a competitive repopulation assay in the dog to directly compare transduction efficiency of steady-state marrow, G-CSF/SCF-primed marrow, and G-CSF/SCF-mobilized peripheral blood. Cells from all three sources were transduced, cryopreserved, and thawed together before infusion into myeloablated dogs. Gene marking in hematopoietic repopulating cells was assessed by polymerase chain reaction. While primed marrow resulted in the highest long-term marking levels, steady-state marrow was transduced at least as efficiently as mobilized peripheral blood in all three dogs. These results suggest that steady-state marrow may be an appropriate source for genetic modification of hematopoietic stem cells.

Animals↗
PubMed2003

Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning.

Lentiviral vectors are increasingly being used for transferring genes into hematopoietic stem cells (HSCs) due to their ability to transduce nondividing cells. Whereas results in in vitro studies and the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) model have been highly encouraging, studies in large animals have not confirmed the superior transduction of HSCs using lentiviral vectors versus oncoretroviral vectors. In contrast to the stable gene marking we have consistently achieved with oncoretroviral vectors in animals that received myeloablative conditioning, we observed the complete disappearance of genetically modified enhanced green or yellow fluorescent protein-expressing cells in 5 baboons that received transplants of HSCs transduced with lentiviral vectors alone or in combination with oncoretroviral vectors. Immune responses to transgene products have been found to be involved in the disappearance of gene-modified cells after nonmyeloablative conditioning. Thus, we examined whether the disappearance of gene-modified cells after ablative conditioning may be due to an immune response. In 4 of 5 animals, cytotoxic T lymphocytes specific for the transgene protein were readily detected, demonstrating that immune reactions were responsible for the disappearance of the gene-marked cells in the animals. In summary, we report the induction of transgene-specific immune responses after transplantation of lentivirally transduced repopulating cells in a myeloablative setting.

Animals↗
PubMed2003

Allogeneic hematopoietic stem cell transplantation for myelofibrosis.

Fifty-six patients, 10 to 66 years of age, with idiopathic myelofibrosis (IMF) or end-stage polycythemia vera or essential thrombocythemia received allogeneic hematopoietic cell transplants from related (n = 36) or unrelated (n = 20) donors. Forty-four patients were prepared with busulfan plus cyclophosphamide and 12 with total body irradiation plus chemotherapy. The source of stem cells was marrow in 33 and peripheral blood in 23 patients. All but 3 patients achieved engraftment. While 50 patients showed complete donor chimerism, 3 patients were found to be mixed chimeras at 26, 48, and 86 months after transplantation, respectively. Two patients died from relapse/progressive disease, and 18 died from other causes. There are 36 patients surviving at 0.5 to 11.6 (median, 2.8) years, for a 3-year Kaplan-Meier estimate of 58% (CI, 43%-73%). Dupriez score, cytogenetic abnormalities, and degree of marrow fibrosis were the most significant risk factors for posttransplantation mortality. Patients conditioned with a regimen of busulfan targeted to plasma levels of 800 to 900 ng/mL plus cyclophosphamide had a higher probability of survival (76% [CI, 62%-91%]) than other patients. Results with unrelated donors were comparable with those with HLA-identical sibling transplants. Thus, allogeneic hematopoietic cell transplantation offers long-term relapse-free survival for patients with myelofibrosis.

Adolescent↗
PubMed2003

Scaffold attachment region-containing retrovirus vectors improve long-term proviral expression after transplantation of GFP-modified CD34+ baboon repopulating cells.

Sustained high-level proviral expression is important for clinical applications of gene therapy. Genetic elements including the beta-interferon scaffold attachment region (SAR) have been shown to improve transgene expression in hematopoietic cells. We hypothesized that SAR elements might improve expression and allow the preselection of successfully transduced cells. Thus, we transplanted green fluorescent protein (GFP)-selected cells, half of which had been transduced with either SAR or non-SAR-containing retrovirus vectors, into 3 animals. All animals showed delayed engraftment compared with historic controls (28 vs 15.5 days). GFP marking was seen at levels up to 8% but declined over the first 6 weeks. Importantly, fluorescence intensity was 2- to 9-fold increased in progeny of SAR versus non-SAR vector-modified cells in all hematopoietic lineages for the duration of follow-up (6-12 months). In conclusion, the use of SAR-containing vectors improved transgene expression in hematopoietic repopulating cells, which may obviate the need for multicopy integration to achieve high-level expression and reduce the risk for insertional mutagenesis.

Animals↗
PubMed2003

Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates.

The nonobese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenotransplantation assay is the most commonly used surrogate assay for the study of human candidate stem cells. In contrast to large animal and human studies, however, it is limited by the short life span of the recipients, the limited proliferative demand placed on the transplanted cells, and the inability to support differentiation into all hematopoietic lineages. In the present study, we directly compared hematopoietic repopulation in NOD/SCID mice with autologous reconstitution in the baboon, a well-established preclinical large animal model for stem cell transplantation. Baboon CD34-enriched marrow cells were retrovirally marked and infused into the irradiated baboon and the NOD/SCID mice. Although the percentage of gene-marked cells was high and remained stable in NOD/SCID mice up to 12 weeks and in those that underwent secondary transplantation, we observed a considerable decline and overall a significantly (10-fold) lower percentage of gene-marked cells in the baboons. In addition, clonal integration site analysis revealed common proviral vector integrants in NOD/SCID repopulating cells and in the baboon at 6 weeks but not at 6 months after transplantation. These results suggest that distinct hematopoietic stem/progenitor cells are responsible for hematopoietic reconstitution in NOD/SCID mice compared with nonhuman primates.

Animals↗
PubMed2003

Interleukin-7 improves CD4 T-cell reconstitution after autologous CD34 cell transplantation in monkeys.

In mice, interleukin-7 (IL-7) hastens T-cell reconstitution and might cause autoimmune diseases, lymphoma, and osteoporosis. We assessed the effect of IL-7 on T-cell reconstitution and toxicity in baboons that underwent total body irradiation followed by autologous transplantation of marrow CD34 cells. Three baboons received placebo and 3 baboons received recombinant human IL-7 (rhIL-7, 75 microg/kg twice a day subcutaneously) between 6 and 10 weeks after transplantation. The mean increase in blood absolute CD4 T-cell counts was 0.9-fold in the placebo-treated animals versus 9.0-fold in those treated with IL-7 (P =.02). The increase observed in the IL-7-treated animals appeared attributable to peripheral expansion rather than de novo generation. The IL-7-treated animals had greater mean increases in the volumes of the spleen (2.0-fold with placebo versus 4.5-fold with IL-7, P =.02) and lymph nodes (1.8-fold with placebo versus 4.1-fold with IL-7, P =.10) but not the thymus (3.4-fold with placebo versus 1.1-fold with IL-7, P =.18). Side effects of IL-7 included thrombocytopenia and possibly neutropenia and hemolytic anemia. One IL-7-treated animal failed to thrive due to a disease resembling graft-versus-host disease. No animals developed lymphoma. Bone density was not decreased. In conclusion, IL-7 raises CD4 T-cell counts in irradiated primates. It remains to be determined whether this is associated with clinical benefit.

Animals↗