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Biomedical subjects

R Handgretinger

Publications and source records attributed to R Handgretinger.

At least 19 recordsLinked to original sources
PubMed2007

Autologous transplantation of CD133 selected hematopoietic progenitor cells for treatment of relapsed acute lymphoblastic leukemia.

A 21-year-old white male with relapsed acute lymphoblastic leukemia (ALL) developed an invasive Zygomycosis infection 3 weeks after beginning re-induction chemotherapy. Because of the high risk of fatal recurrence of the fungal infection, neither long-term maintenance chemotherapy nor allogeneic hematopoietic stem cell transplant (HSCT) was considered appropriate. Because his ALL blasts expressed CD34 but lacked CD133, he received a CD133 selected autologous graft following high-dose consolidation chemotherapy. The patient survives in remission 19 months after HSCT.

AC133 Antigen↗
PubMed2006

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS: As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS: We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION: Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Antigens, Differentiation↗
PubMed2006

Determination of residual T- and B-cell content after immunomagnetic depletion: proposal for flow cytometric analysis and results from 103 separations.

BACKGROUND: T- and B-cell depletion of apheresis products is an attractive alternative to standard stem cell enrichment in haplo-identical transplantation. Thorough T- and B-cell depletion is necessary for prevention of acute GvHD and T-cell depletion-associated lymphoproliferative disorders. However, the large number of non-T and -B cells in the graft requires special protocols for the determination of extremely low frequencies of residual T cells. METHODS: Apheresis products from healthy donors were T- and B-cell depleted by the CliniMACS system using CD3 and CD19 Ab reagents and the LS tubing set. The recovery of cells and degree of depletion were determined. A four-color multigating strategy was used for enumeration of residual T and B cells. RESULTS: One-hundred and three separations were performed, with a mean cell recovery of 38+/-12%, CD34 recovery of 61+/-16% and CD56 recovery of 63+/-33%. T and B cells were depleted by log 4.15+/-0.46 and log 3.64+/-0.63, respectively. Four-color multigating flow cytometry allowed the detection of single T cells. DISCUSSION: Combined T- and B-cell depletion is a feasible method for obtaining stem cell grafts with acceptable stem cell recovery, profound T- and B-cell depletion and a very high amount of NK cells and monocytes. However, analysis of residual T cells is challenging and requires special protocols.

Acute Disease↗
PubMed2006

Isolation, molecular cloning and in vitro expression of rhesus monkey (Macaca mulatta) prominin-1.s1 complementary DNA encoding a potential hematopoietic stem cell antigen.

Human prominin-1 (CD133 or AC133) is an important cell surface marker used to isolate primitive hematopoietic stem cells. The commercially available antibody to human prominin-1 does not recognize rhesus prominin-1. Therefore, we isolated, cloned and characterized the complementary DNA (cDNA) of rhesus prominin-1 gene and determined its coding potential. Following the nomenclature of prominin family of genes, we named this cDNA as rhesus prominin-1.s1. The amino acid sequence data of the putative rhesus prominin-1.s1 could be used in designing antigenic peptides to raise antibodies for use in isolation of pure populations of rhesus prominin-1(+) hematopoietic cells. To the best of our knowledge, there has been no previously published report about the isolation of a prominin-1 cDNA from rhesus monkey (Macaca mulatta).

AC133 Antigen↗
PubMed2005

Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation.

Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitt's lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+ CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

Animals↗
PubMed2004

Antiviral activity against CMV-infected fibroblasts in pediatric patients transplanted with CD34(+)-selected allografts from alternative donors.

Human cytomegalovirus (HCMV) remains a cause of serious infectious complications after allogeneic transplantation of hematopoietic stem cells, especially in recipients of T-cell-depleted grafts. Here we investigated the antiviral activity of natural killer (NK) cells from healthy donors (n = 8) as well as of mononuclear cells (MNC) from transplanted pediatric patients (n = 11) who had received CD34(+) selected (and thus T-cell-depleted) stem cells from unrelated and mismatched related donors. Allogeneic human fibroblasts infected with HCMV laboratory strain AD169 for 5 days were used as targets in a 2-h cytotoxicity assay. Downregulation of human leukocyte antigen class I and upregulation of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) were observed after infection. In this experimental setting, NK cells from healthy donors exerted no specific lysis. However, antibody-dependent cellular cytotoxicity (ADCC) mediated by human anti-CMV IgG (cytoglobin) as well as stimulation with low-dose interleukin-(IL)-2 or IL-15 enhanced lysis markedly. MNC from two thirds of the patients (7/11) were capable of lysing infected targets without stimulation. Here also, lytic activity was significantly increased by IL-2 or IL-15, used in combination with ADCC. In contrast, 4/11 patients exerted no lysis. The observed antiviral activity may contribute to the low incidence of CMV DNAemia (29% at day 100, detected by polymerase chain reaction) in the whole group of our patients who have been transplanted with CD34(+)-selected allografts since 1995. Furthermore, our data suggest a potential benefit of using low-dose IL-2 or IL-15, also combined with anti-CMV immunoglobulinG, for immune modulation in CMV disease.

Adolescent↗
PubMed2004

Transplantation of highly purified peripheral-blood CD34+ progenitor cells from related and unrelated donors in children with nonmalignant diseases.

Transplantation of allogeneic stem cells is currently the only curative treatment for some nonmalignant pediatric diseases. We investigated whether transplantation of purified CD34(+) stem cells prevents acute and chronic GvHD and reduces transplant-related mortality. A total of 25 pediatric patients with nonmalignant diseases underwent allogeneic transplantation from 26 donors (matched related n=4, matched or partially matched unrelated n=14, mismatched related n=8). All grafts were purified peripheral-blood CD34(+) stem cells mobilized with G-CSF. Patients received a median of 12.9 x 10(6) CD34(+) progenitor cells with a median of 6.1 x 10(3) contaminating T-lymphocytes per kilogram of body weight. No post transplant immunosuppressive drugs were given for prophylaxis of GvHD. Engraftment was seen in 21 patients. Three patients engrafted after a second transplant and one patient failed to engraft. Two patients had autologous reconstitution 1.5 years post transplant and one of them was successfully retransplanted. No acute GvHD >grade II was seen, and only two patients developed limited, chronic GvHD. In all, 22 patients (88%) are alive with a median follow-up of 3.7 years. In total, 19 patients (76%) are free of disease or of progression. Transplantation of highly purified peripheral-blood CD34(+) stem cells is associated with low toxicity in patients with nonmalignant diseases.

Adolescent↗
PubMed2004

Effective donor lymphohematopoietic reconstitution after haploidentical CD34+-selected hematopoietic stem cell transplantation in children with refractory severe aplastic anemia.

Peritransplant toxicity and a delay in effective immune reconstitution have limited the utility of alternate donor transplantation for children with refractory severe aplastic anemia. We have assessed the effectiveness of infusing large numbers of highly purified haploidentical CD34+ cells after immunoablative conditioning in three patients who had failed intensive immunosuppression, lacked unrelated donors, and had active or recent serious infections. One patient rejected the first infusion, but engrafted after a second infusion from the same donor. This patient died 4 months after hematopoietic stem cell transplantation with no evidence of lymphoid reconstitution. Two patients experienced mixed chimerism requiring treatment with antibodies and/or donor lymphocyte infusion. Both currently survive more than 1 year after transplantation with normal blood counts, 100% donor engraftment, effective lymphoid reconstitution, and no chronic graft-versus-host disease. We observed functional thymopoiesis as measured by lymphocyte immunophenotyping, T cell receptor excision circles and T cell receptor Vbeta spectratyping complexity analysis. Further study is required to validate the initial promise of these preliminary observations.

Anemia, Aplastic↗
PubMed2004

Increasing mixed chimerism defines a high-risk group of childhood acute myelogenous leukemia patients after allogeneic stem cell transplantation where pre-emptive immunotherapy may be effective.

Children with leukemias and increasing mixed chimerism (increasing MC) after allogeneic stem cell transplantation have the highest risk to relapse. Early immunological intervention was found to be effective in these cases. To substantiate this on a defined group of pediatric acute myelogenous leukemia (AML) patients, we performed serial analysis of post transplant chimerism and pre-emptive immunotherapy in patients with increasing MC. In total, 81 children were monitored, 62 patients revealed complete chimerism (CC), low-level MC or decreasing MC. Increasing MC was detected in 19 cases. Despite early immunological intervention relapse was still significantly more frequent in patients with increasing MC (9/19) than in patients with CC, low-level or decreasing MC (8/62, P<0.005). The probability of 3-year event-free survival (EFS) was 52% for all patients (n=81), 59% for patients with CC, low-level MC, 60% for patients with decreasing MC (n=62), and 28% for patients with increasing MC (n=19, P<0.005). Patients with increasing MC who received early immunological intervention showed a significantly enhanced probability for event-free survival (pEFS 36%, n=15) compared to patients with increasing MC without intervention (pEFS 0%, n=4, P<0.05). These results prove that pediatric AML patients with increasing MC are at highest risk for relapse and that early immunological intervention can prevent relapse in these patients.

Adolescent↗
PubMed2004

Encephalopathy in pediatric patients after allogeneic hematopoietic stem cell transplantation is associated with a poor prognosis.

SUMMARY: Encephalopathy is a poorly characterized complication of hematopoietic stem cell transplantation (HSCT). No comprehensive report of encephalopathy exists for children, and the literature contains only a few for adults. We analyzed a large cohort of 405 pediatric patients who underwent allogeneic HSCT during a 10-year period and identified 26 patients (6.4%) who experienced encephalopathy. Identifiable causes of encephalopathy included infection (n=5), single or multiorgan failure (n=4), medication-related complications (n=3), nonconvulsive seizures (n=4), acute disseminated encephalomyelitis (n=2), thrombotic thrombocytopenic purpura (n=2), and stroke (n=1). We were unable to identify the etiology of encephalopathy in five (19%) patients. The prognosis for pediatric patients with encephalopathy was poor: only four (15%) experienced complete neurologic recovery, and 10 (38%) patients experienced partial recovery with residual neurologic deficits. Nine (35%) patients with complete or partial recovery survive long term. A total of 17 patients died; one died of progressive encephalopathy, and 16 died of either relapse of primary disease or toxicity. MRI, CSF analysis including molecular testing for infectious pathogens, and brain biopsy were helpful in obtaining a diagnosis in most of our patients. However, a standardized approach to accurate and timely diagnosis and treatment is needed to improve outcome in these patients.

Adolescent↗
PubMed2004

A one-step large-scale method for T- and B-cell depletion of mobilized PBSC for allogeneic transplantation.

BACKGROUND: The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. METHODS: T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. RESULTS: The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19x10(10) (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log(10) depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log(10) depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34(+) stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2x10(8) (range 2-10x10(8)). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. DISCUSSION: Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft.

Animals↗
PubMed2004

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha.

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.

Adult↗
PubMed2004

Glycosylation and lectins-examples of immunesurveillance and immune evasion.

Cell surface proteins are posttranslationally modified by tightly regulated enzymes of glycosylation. Typical patterns of glycosylation may signal pathological situations to the immune system. Here, carbohydrate receptors on the surface of cells in the immune system are involved in regulation of effector cells. Moreover, some lectins are circulating in the plasma and take part in host defense. The code of carbohydrate modifications is impaired in malignant cells and yet they are not eliminated. In this review, we focus on recent experimental evidence for regulatory functions of lectins and carbohydrate derivatives in the immune system and tumours.

Animals↗
PubMed2003

Long-term results and risk profiles of patients in five consecutive trials (1979-1997) with stage 4 neuroblastoma over 1 year of age.

During the last two decades new diagnostic and therapeutic tools have been utilized to improve the poor survival chances of children with stage 4 neuroblastoma. This study reviews the risk profiles and the long-term outcome of patients from five consecutive German neuroblastoma trials. A total of 96% of all German patients registered at the German childhood cancer registry with neuroblastoma stage 4 over 1 year of age at diagnosis entered one of the trials during 1979-2001. Eight hundred and twenty-eight consecutive children were analyzed retrospectively. In spite of having significantly improved diagnostic tools like bone marrow superstaging and mIBG scintigraphy the stage 4 incidence did not increase after reaching completeness of the registry (5.4 cases/100,000 children at 1-14 years of age; P=0.52). The distribution of the primary tumors and of metastases was constant over the periods. The amount of bone marrow infiltration did not change with time. The risk factors lactate dehydrogenase, ferritin and MYCN, and the clinical risk groups 4A, 4B, 4C also remained constant over the trials with a few exceptions for NB97. The 5-year event free survival increased from 0.01+/-0.01 (NB79) to 0.14+/-0.03 (NB85), 0.16+/-0.04 (NB82), 0.27+/-0.02 (NB90), and 0.33+/-0.04 (NB97). The overall survival rates improved similarly from 0.04 (NB79) to 0.44 (NB97). In conclusion, the improved survival was associated with better treatment and not caused by lower risk profiles in stage 4 neuroblastoma patients.

Adolescent↗
PubMed2003

Tumour cell contamination of autologous stem cells grafts in high-risk neuroblastoma: the good news?

We analysed the effect of graft-contaminating tumour cells on the long-term survival of 24 patients with high-risk neuroblastoma and found that patients whose grafts contained detectable neuroblastoma cells had a significantly higher probability of survival than did patients with no detectable tumour cells. Estimated contamination of the graft by more than 2000 tumour cells was associated with a significantly higher probability of survival than contamination with fewer tumour cells. We hypothesise that the presence of a critical number of graft-contaminating neuroblastoma cells can elicit a protective antitumour immune response after autologous transplantation.

Antigens, CD34↗