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P Wernet

Publications and source records attributed to P Wernet.

At least 37 records · Page 2Linked to original sources
PubMed1999

Factors influencing G-CSF-mediated mobilization of hematopoietic progenitor cells during steady-state hematopoiesis in patients with malignant lymphoma and multiple myeloma.

We retrospectively analyzed factors influencing PBPC mobilization during steady-state hematopoiesis in 52 patients with malignant lymphoma (n=35) or multiple myeloma (n=17) who received 77 cycles of G-CSF (12.5-50 microg G-CSF/kg/day). For 15 of these patients, the first mobilization cycle (12.5 microg G-CSF/kg/day) was followed by a second course with an increased dose of G-CSF (25 or 50 microg/kg/day). Leukapheresis was started on day 4, about 2 h after s.c. G-CSF administration, and repeated on 2-5 consecutive days. CD34+ cells were determined by flow cytometry in each apheresis product and in the peripheral blood prior to G-CSF administration, beginning on day 4. Colony assays were performed on cryopreserved samples prior to autografting. In the 15 patients receiving two mobilization cycles the higher G-CSF dose was associated with higher levels of CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful (>2x10(6) CD34+ cells/kg) collections (p=0.058). Patients with limited previous cytotoxic therapy (n=19, up to six cycles of a standard regimen such as CHOP and/or less than 20% marrow irradiation) who received a daily dose of 12.5 microg G-CSF/kg had higher levels of circulating CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful collections (p<0.05) compared with patients previously treated with more intensive radiochemotherapy (n=15). Ten of 20 patients (50%) who failed during the first cycle were successful during subsequent cycles with escalated doses of G-CSF. Trough levels of circulating CD34+ cells on day 4 were predictive for success or failure to achieve >2x10(6) CD34+ cells/kg, especially in heavily pretreated patients. In conclusion, a daily dose of 12.5 microg G-CSF/kg seems sufficient to mobilize PBPC during steady-state hematopoiesis in the majority of patients who have received limited previous radiochemotherapy. Higher doses of G-CSF, up to 50 microg/kg/day, mobilize more PBPC and should be considered for patients previously treated with intensive radiochemotherapy or those failing to mobilize sufficient numbers of CD34+ cells with lower doses of G-CSF.

Adult↗
PubMed1999

Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia.

In the absence of a donor alternative a stem cell transplantation consisting of two cord blood components originating from the haploidentical brother was performed in a 2-year-old girl with c-ALL, early CNS relapse and 7% of blast cells in the BM 14 days before transplantation. Because of various ongoing infectious complications at that time, 1/8 of the immunogenetically acceptable sibling cord blood was ex vivo expanded 10 days before the transplantation date. The total CB consisting of 1.17 x 10(9) NC was cryopreserved in four separate bags. The one containing 1/8 of the total CB with 1.4 x 10(8) NC CliniMACS selected CD34+ cells was expanded in the presence of 100 ng/ml G-CSF, 100 ng/ml TPO and 100 ng/ml flt3-L in 10% autologous CB plasma and X-VIVO 10 medium at day -10 before transplantation. This expanded cell population was sterile and consisted of about 60% granulocytic cells (CD13+, CD15+), about 30% myelomonocytic cells (CD14, HLA-DR+), 5.2% megakaryocytes (CD61+) and 1.2% CD34+ cells. The proportion of T (CD3+), NK cells (CD56+) as well as dendritic cells (CD83+) was below 0.2%. The unseparated CB infused at day 0 and +1 consisted of a total of thawed 4.4 x 10(7) NC/kg BW, 5.8 x 10(4) CFU-GM/kg BW, 1.54 x 10(5) CD34+cells/kg BW and 7. 73 x 10(2) LTC-IC/kg BW. In addition, the 1 x 10(7) NC/kg BW ex vivo expanded cells representing 1.9 x 10(4) CFU-GM/kg BW, 1.13 x 10(5) CD34 cells/kg BW and 4.37 x 10(2) LTC-IC/kg BW, were infused at day +1. At day +2 after transplantation the patient revealed a focal pneumonia on X-ray with generalized sepsis and became catecholamine dependent. From day +4 the patient received 280 microg/m2 G-CSF. At day +5 she developed an erythroderma, which could not be identified as acute GVHD by biopsy. Early engraftment with leukocyte counts at days 8 and 14 were 350 and 700/microl, ANC 310 and 410/microl, respectively. Donor cells determined by chimerism analysis were 97% and 98% in the periphery at this early time. Most importantly, the pneumonia as well as the septicemia subsided within a few days. Notably, as well is the clearly shortened aplastic phase observed after this simultaneous CB cell component transplantation. The patients T cell and NK cell reconstitution could be detected at day +37 with 330 CD3+ cells/microl and 40 CD56+ cells/microl, respectively. The time to reach an absolute platelet count of 20 000 (50 000)/microl was 75 (103) days. The disease-free survival now exceeds 1 year in complete remission without chronic GVHD or any other health problems. These data show that the applicability of ex vivo expanded committed progenitors and LTC-IC, even in high risk leukemia at the time of transplantation, is feasible and can provide sufficient myeloid progenitors resulting in rapid engraftment able to clear bacterial pneumonia and sepsis. In addition, accelerated hematopoietic reconstitution apparently served as a well functioning platform for definitive graft-versus-leukemia activity. This transplantation of defined ex vivo generated components presents a feasible and generally applicable approach and may open a promising new avenue for cell therapy in malignant diseases.

Cell Culture Techniques↗
PubMed1998

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation.

BACKGROUND: The aim of the study was to establish fast methods for postmortem HLA class I and II typing of cornea donors using cadaveric blood. METHODS: The commercially available reagents Lymphokwik MN and Dynabeads were evaluated here to provide an enriched living mononuclear cell (MNC) population and B-cell population for HLA class I and II typing of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted living MNC's, cells were serologically typed by double-fluorescence cytotoxicity assay for HLA class I and II antigens. RESULTS: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was successful and accomplished within 5 h. Results from the serological HLA class I typing were confirmed by the results of HLA class I typing by RNA-based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 148 samples and reverse hybridization of PCR-amplified DNA to immobilized HLA class II specific primers in 270 samples. These data were confirmed by DNA-based sequencing in five cases and by sequence-specific oligonucleotide hybridization in all cases. CONCLUSIONS: These results lead to the following typing strategy: HLA class I typing should be performed by serology. HLA class II typing should be performed by DNA technology because of its relative independence of the quality of the blood sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for ideal HLA matching, increasing the number of available HLA-matched corneas and therefore reducing the number of graft rejections.

Blood Grouping and Crossmatching↗
PubMed1998

The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors.

Assuming a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Düsseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50-70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34+ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34+ CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L plus IL-3 (94.3 +/- 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 +/- 3.4-fold and 5.5 +/- 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 +/- 81.7-fold) than H5100 medium (83.5 +/- 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.

Adult↗
PubMed1998

Congenital infections with human herpesvirus 6.

Cord blood DNA was tested for the presence of human herpesvirus 6 (HHV-6) DNA by the polymerase chain reaction. Specific DNA could be detected in the specimens of 5 (1.6%) of 305 babies born to ostensibly healthy mothers, indicating that intrauterine infection had occurred. These transmissions would not have been detected by serologic methods, because no specific IgM antibody could be found in the fetal sera. These results indicate that, in addition to infections acquired in early childhood, congenital infections may account for the HHV-6 seropositivity in children.

Antibodies, Viral↗
PubMed1998

An eight-fold ex vivo expansion of long-term culture-initiating cells from umbilical cord blood in stirred suspension cultures.

Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation, to overcome a potential limitation due to restricted cell numbers. Therefore, 1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L, SCF and IL-3. At days 0, 7, 10, 14, 21 and 28, the spinner cultures were analyzed for viable cells, colony-forming cells (CFC), including erythroid burst-forming unit (BFU-E), granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold), CFC (maximal at day 14: 45 +/- 36-fold), CFU-GM (maximal at day 14: 88 +/- 85-fold), CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and, thus, it may be possible to improve CB transplantation by ex vivo generated cells.

Antigens, CD34↗
PubMed1998

Volume reduction of cord blood by Hetastarch for long-term stem cell banking.

As of November 1997, a total of 2098 cord blood (CB) units are stored in the CB bank Düsseldorf (mean volume, total nucleated cell count, BFU-E, CFU-GM, CFU-GEMM, CD34+ cells). Considering a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per CB unit, 100%, 67% and 25% of the CB units contain sufficient NC to engraft patients of 10 kg, 35 kg and 50-70 kg, respectively. Within the last 3 years with the majority during 1997, a total of 4000 preliminary searches, 450 extended unit reports were submitted to the CB bank by fax or World Wide Web and 36 units were delivered upon request for transplantation to various centers. 1940 CB units were stored unseparated, frozen in two freezing bags requiring 15 400-l liquid nitrogen tanks for long-term storage. Therefore, the volume reduction method applying Hetastarch (HES) according to Rubinstein's protocol was introduced into the routine processing of the CB units. Until now 158 CB units with a mean recovery of 85 +/- 13% for nucleated cells, 86 +/- 12% and 84 +/- 13% for CFC and CD34+ cells, respectively, were processed and stored with a total volume of 25 ml in one freezing bag.

Blood Banking↗
PubMed1998

NETCORD: a cord blood allocation network.

Several CB-specific prerequisites have to be taken into consideration to deal with the increasing number of stem cell transplants using multiple HLA mismatched umbilical cord blood (CB) specimens. Since CB represents a limited and expensive resource it demands an optimal allocation. Optimal CB allocation can not be optimal based solely on 'first come - first serve' basis, since frequently several CB specimens are simultaneously suitable for more than one patient. Furthermore, the immediate availability of CB gives rise to an above average proportion of urgent search requests for patients who need a transplantation urgently. Thus continuing reservation and release of a CB specimen has considerable influence on the stem cell transplant prospects of other patients. To avoid and/or solve allocation conflicts present in a much higher incidence than in the bone marrow donor setting a global co-ordinated allocation of cord blood specimens is of major advantage for these patients. Therefore, the CB allocation network NETCORD has been founded. Within NETCORD common rules for data representation and logistics were developed to make CB data comparable and interchangeable. The extended need for fast and reliable communication is met by modern communication technology based on Internet standards.

Blood Banks↗
PubMed1998

Functional competence of dendritic cells in human umbilical cord blood.

Dendritic cells (DC) are the main stimulators of primary T cell responses. Very little is known about DC in cord blood (CB), and whether they are involved in the low incidence and severity of GVHD following CB transplantation. Here, CBDC were identified as a HLA-DR+/lineage marker (lin; CD3, CD11b, CD14, CD16, CD19, CD34, CD56 and glycophorin A antigens) negative population, representing 0.3 +/- 0.1% (mean +/- s.d.; n = 15) of CB mononuclear cells. CBDC expressed the CD4, CD11a, CD18, CD45RA, CD50 and CD54 antigens but revealed no expression of the CD1a, CD11c, CD40, CD45R0, CD58, CD83, CD86 and CD102 antigens. Immunomagnetically enriched CBDC showed potent allostimulatory activity for CB T cells. Thus, CBDC are functionally competent and resemble in their immature/resting state CD11c- DC in peripheral blood.

Adult↗
PubMed1997

Screening for renal carcinoma associated mutations in the von Hippel-Lindau tumor suppressor gene by temperature gradient gel electrophoresis.

Renal cell carcinoma is the most common neoplastic disease of the adult kidney and occurs in its sporadic and hereditary form. Approximately 57% of all renal carcinomas of the clear cell type analyzed revealed a mutation in the von Hippel-Lindau disease (VHL) gene. In the present work, temperature gradient gel electrophoresis (TGGE) is presented as a rapid and precise polymerase chain reaction (PCR)-employing methodology for the detection of mutations in the VHL gene. The theoretical efficiency of TGGE to detect mutations in every base pair of the gene was calculated. According to computer analysis, at least 92% of all known mutations in the VHL gene are detectable. This calculated figure appears to be in agreement with the experimental results. Primary difficulties in analyzing exon 1 of the VHL gene were overcome by the employment of psoralen-cross-linked PCR fragments. In addition, TGGE analysis was used in screening for possible mutations in thirteen renal carcinoma samples. With this protocol TGGE is successfully added to the array of methods for the screening of VHL mutations.

Aged↗
PubMed1997

Large volume leukapheresis maximizes the progenitor cell yield for allogeneic peripheral blood progenitor donation.

We have investigated the efficiency and safety of large volume leukapheresis (LVL) for the collection of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) from healthy donors. In six apheresis sessions in four healthy individuals on a COBE-BCT Spectra cell separator (median processed volume 3.5 X total blood volume, TBV, range 3.3-4.4 X TBV), harvested cells were collected sequentially into three single bags. The collection bags were changed after processing 33%, 66%, and 100% of the prospective apheresis volume, allowing analysis of PBPCs collected at different periods during one harvest. Mononuclear cells (MNCs), CD34+ cells, CD34+ subsets, and lymphocyte subsets were determined in each bag. Substantially more PBPCs were harvested than were in the circulation before G-CSF administration preceding LVL (median 171%, range 69-267%), reflecting progenitor release during the procedure. In donors 1 and 3, the CD34+ cell yields decreased in the third bag to 53% and 42% of that collected in the first bag, whereas the progenitor cell yields in donors 2 and 4 were stable or rose during the procedure, achieving in the third bag 157% and 105% of the number of CD34+ cells collected in the first bag. Minor changes were found in the subsets of CD34+ cells, lymphocytes, and monocytes collected at different periods during a single harvest. LVL was well tolerated. Reversible thombocytopenia developed in all cases. No late effects attributable to LVL or G-CSF were found in the 4 donors and 16 other healthy individuals who have undergone LVL in our institution. We conclude that LVL is safe and maximizes PBPC yields for allogeneic transplantation.

Adult↗
PubMed1997

Idarubicin, melphalan and cyclophosphamide: an intensified high-dose regimen for the treatment of myeloma patients.

Following conventional chemotherapy, eight myeloma patients presenting with advanced tumor stages were treated with an intensified high-dose regimen and autologous peripheral blood stem cell transplantation. High-dose chemotherapy consisted of idarubicin 20 mg/m2 on days -13, -12 and -11, melphalan 100 mg/m2 on days -5 and -4 and cyclophosphamide 60 mg/kg (plus mesna 60 mg/kg) on days -3 and -2 (IMC). Seven patients achieved a complete remission or a very good partial remission (reduction of M-component > or =90%). There were no toxic deaths. Severe mucositis and fever of unknown origin were seen in all patients. Reversible supraventricular tachycardias without clinical signs of cardiac failure occurred in five patients. One patient developed a persistent deterioration of cardiac function. We surmise that high-dose chemotherapy with IMC is very effective and well tolerated in myeloma patients.

Adult↗
PubMed1996

Quantitative assessment of the human TCRBV repertoire by competitive PCR.

A novel quantitative protocol was developed for the measurement of the relative expression levels of the human TCRBV genes based on reverse transcription (RT) and subsequent competitive polymerase chain reaction (cPCR). Competitor DNA templates for the analysis of 24 different TCRBV families were generated by a simple and rapid one-step PCR procedure with a special PCR primer, which introduces a deletion in the constant region gene segment. A defined amount of TCRBV family-specific competitor DNA and the reverse transcribed cDNA of interest were amplified in the same tube with the same primer pair in a competitive way. The resulting fragments were separated on agarose gels and the densitometrical values were evaluated directly without the requirement for additional hybridization steps. For all of the 24 different TCRBV family-specific cPCRs equal amplification efficiencies were demonstrated by titration experiments for wild-type and competitor templates. For TCRBV repertoire studies, a short form of the cPCR assay was performed, requiring only one cPCR for quantitation of each TCRBV family. The exact initial amount of wild-type template in each cPCR was interpolated from TCRBV family-specific reference calibration curves. The RT-cPCR assay was applied for the quantitative assessment of the TCRBV repertoire of CD4+ and CD8+ T cell subsets in unstimulated PBMC and compared to flow cytometric analyses with a panel of monoclonal antibodies specific for TCRBV determinants. The RT-cPCR experiments revealed a differential expression of several BV families in either the CD4+ or the CD8+ fraction. The TCRBV family-specific cPCR assay presented here combines the simplicity and speed of conventional TCRBV family-specific PCR with the quantitative features of competitive PCR. TCRBV family-specific RT-cPCR has a broad application for all kinds of quantitative T cell repertoire studies and could be easily adapted for the usage with different BV-specific primer sets.

Antibodies, Monoclonal↗
PubMed1996

Genetic influence on the shaping of the human T-cell receptor repertoire: quantitative assessment by competitive polymerase chain reaction.

It has been difficult to define the different factors which contribute to the shaping of the human T-cell receptor (TCR) repertoire. In this study, the influence of the polymorphic human leucocyte antigen (HLA) genes and non-HLA genes on the phenotype of the TCRBV segment repertoire was assessed in a population of HLA class I-matched individuals including three pairs of siblings. The gene expression levels of 24 TCRBV families were evaluated in the CD4+ and CD8+ T-cell subsets of unstimulated peripheral blood mononuclear cells (PBMC) by reverse transcription (RT) and a newly developed competitive polymerase chain reaction (cPCR) assay. Titration experiments demonstrated that the RT-cPCR assay was suitable for an accurate quantification of the relative TCRBV segment expression levels. The T-cell repertoires of HLA-identical siblings were found to be more similar than the repertoires of unrelated individuals. On the other hand, there was no difference in the degree of similarity between the TCRBV repertoires of CD4+ T-cells of HLA class II identical or non-identical unrelated individuals. Furthermore, although most of these individuals had identical HLA class I genes and non-identical HLA class II genes, the TCRBV repertoires of the CD4+ T cells exhibited significantly lower variabilities than the repertoires of the CD8+ T cells. The results of the RT-cPCR assay were supported by flow cytometric analysis of the CD4+ and CD8+ T-cell subsets of the same eight individuals employing 10 different TCRBV segment-specific monoclonal antibodies. These observations argue for a predominant role of non-HLA or non-polymorphic HLA determinants for the shaping of the TCRBV repertoire.

Adult↗
PubMed1996

Hematopoietic transplant potential of unrelated cord blood: critical issues.

To date, hematopoietic stem and progenitor cells from human umbilical cord blood (CB) have been employed in approximately 90 allogeneic (56 sibling and 34 unrelated) matched and mismatched transplantations worldwide with easy and successful restoration of hematopoiesis. Requests for stem cell preparations from CB will continue to increase. Thus, as a pilot study, the examination and standardization of unrelated cord blood-derived stem cell preparations and banking as well as their biologic characterization were initiated. Up to October 1995, a total of 574 samples [mean volume 79 +/- 26 ml, total nucleated cells (NC) 8.5 +/- 5 x 10(8), BFU-E 9.5 +/- 8.6 x 10(5), CFU-GM 5.7 +/- 6.3 x 10(5), CFU-GEMM 1.6 +/- 1.9 x 10(5)] from cord-derived or placental-derived residual blood have been defined by hematologic, immunologic, and microbiologic criteria. These CB samples were collected from the umbilical cord vein immediately after vaginal full-term delivery (n = 450) or cesarean section (n = 124) and stored frozen in liquid nitrogen. Seven percent of all samples collected could not be considered for potential transplants because of volumes < 40 ml. Only 5.0 ml of a CB sample is required for routine laboratory testing, consisting of HLA class I typing, HLA class II typing by sequence-specific oligonucleotide probes (PCR-SSOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony-forming and LTC-IC assays, and CD34+ status. To assess the potential problem of contaminating maternal cells, a PCR was performed on 7 representative samples. During the initial 6 months of the unrelated CB collection program, a median bacterial contamination rate of 18% (20% skin flora species, 80% perineal flora species) was encountered, which has since been reduced to < 1% through practical experience. With regard to viral infections, maternal sera was tested for HBsAg (0.6% positive), anti-HCV (0%), anti-HAV (IgG 18%, IgM 0%), anti-HIV-1-2 (0%), anti-EBV (IgG 98%, IgM 0%), anti-HTLVI-II (0%), anti-CMV (IgG 43%, IgM 0.4%), toxoplasmosis (46%) and syphilis (0%). In addition, all cord blood samples were tested by PCR for CMV infection. With regard to its clinical relevance, it is important that only 0.3% of all the samples were positive for CMV by this sensitive method. This may represent a critical advantage of CB grafts over bone marrow (BM) since, in contrast, > 40% of the unrelated BM donors have been identified to be positive for CMV. An additional advantage of CB is that since 20% of CB samples were collected from ethnic minorities, it appears possible to balance common HLA types and uncommon HLA types represented in this group. In summary, with the extensive practical experience of the obstetric collection team as well as the stem cell-processing laboratory, it appears feasible to obtain a 90% yield of unrelated CB-derived stem cell preparations for banking, which clearly should meet the medical and regulatory qualification criteria required for clinical transplantation. To test the feasibility of hematopoietic transplant potential of unrelated CB for adult patients, ex vivo expansion of CD34+-enriched stem/progenitor cell populations isolated from fresh or frozen CB was attempted in the presence of rh-IL-3, rh-IL-6, rh-EPO, rh-GM-CSF, and rh-SCF with or without fit 3. At varying time points (days 0, 2, 4, 7, 14, 21), the contents of these cultures were analyzed for the numbers of cells, CFC (BFU-E, CFU-GM, CFU-GEMM), and LTC-IC. In this setting, the increase of cells was 200-fold, that of CFC 70-fold, and most importantly that of LTC-IC was 4.5-fold after 7 days in culture in the presence of flt3. In conclusion, LTC-IC derived from CB can be maintained and considerably expanded ex vivo from highly enriched CD34 + CB cell populations from fresh or frozen CB samples.

ABO Blood-Group System↗
PubMed1996

Qualitative assessment of mixed chimerism after allogeneic bone marrow transplantation with regard to leukemic relapse.

Serial peripheral blood specimen from eight adult patients after sex-mismatched bone marrow transplantation (BMT) for Chronic Myeloid Leukemia (CML) (N = 3). Ewing sarcoma (N = 1), Acute Myeloid Leukemia (AML) in second remission (N = 1), Acute Lymphoid Leukemia (ALL) (N = 1), of multiple myeloma (N = 2) were analyzed by the simultaneous immunophenotypic (moAbs/ APAAP-staining) and genotypic analysis (for X and Y chromosomes) of interphase cells to characterize mixed chimerism, residual host cells, and leukemic relapse. Although a stable donor chimerism for T cells, myelomonocytic cells, and granulocytes was developed in seven of the eight patients at Days +21 to +28 post BMT, 0.5 to 1% host cells of different lineages remained continuously in five of the eight patients post BMT (> day 100). In two patients, one with common ALL and the other with multiple myeloma and long-term stable mixed chimerism, a tumor cell relapse was detected first in a sample at Day +176 and confirmed at Day +294. These malignant cells were genotypically of host origin and presented phenotypes identical to those at diagnosis. In the three patients with CML, residual host cells were identified as CD13 (Patient 3) of CD13/CD34 (Patient 4) positive and in one case as CD4/CD8 positive (Patient 7). Since no exclusive antigenic marker is available for this discrimination in these CML patients, normal host hematopoiesis can interfere with the identification of residual disease. Therefore, the identification of the bcr-abl transcripts by a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) was included in this analysis. Patient 3 was bcr-abl positive at [Days +21, +28, +35, and +311, but negative at Days +121 and +400; Patient 4 was bcr-abl positive at only Day +166 post BMT. These results are interpreted as signaling a continuing risk of relapse. In Patient 7, the bcr-abl RT-PCR was negative at Days +142, +166, and +237. Thus, the combination of the simultaneous immunophenotypic and genotypic analysis and the bcr-abl detection by RT-PCR clearly improves the discrimination between malignant cells and normal residual host cells.

Acute Disease↗
PubMed1995

Enhancement of neutrophil function by in vivo filgrastim treatment for prophylaxis of sepsis in surgical intensive care patients.

PURPOSE: To determine the kinetics of leukocyte counts and of oxygen radical production of neutrophils from postoperative/posttraumatic patients with or without infusion of filgrastim (recombinant human granulocyte colony-stimulating factor, rhG-CSF) as prophylaxis against sepsis. METHODS: Twenty postoperative/posttraumatic patients with a Therapeutic Intervention Scoring System (TISS) score greater than 30 were included in this study. In the 10 patients of the study group, filgrastim (1 microgram/kg/d) was infused continuously within the first 3 days and tapered to 0.5 microgram/kg/d on the following 4 days or until discharge from the surgical intensive care unit. Ten patients without administration of filgrastim served as controls. Oxygen radical production of isolated neutrophils of these patients was tested by N-formyl-methionyl-leucyl-phenylalanine (FMLP)- and zymosan-induced chemiluminescence from serial blood samples, taken until the 16th postoperative day. RESULTS: Compared with the first postoperative day, in vitro FMLP-induced neutrophil chemiluminescence was significantly increased during the following 4 postoperative days in the patients with filgrastim infusion; however, only during the first 2 postoperative days in the control group. The increase in the FMLP-induced neutrophil chemiluminescence was significantly greater (P < .05) in the study group than in the control group on the third and on the fourth postoperative day. Tapering of filgrastim by 0.5 microgram/kg/d in the study group resulted in a reduction of FMLP-induced neutrophil oxygen radical production within 48 hours. In contrast, zymosan-induced neutrophil chemiluminescence was not measurably affected in both groups. Leukocyte count of the study group significantly (P < .05) exceeded the leukocyte count of the control group from the third up to the 10th postoperative day. None of the patients treated with filgrastim developed sepsis; however, three patients within the control group did. CONCLUSIONS: Prolonged enhancement of neutrophil count and function induced by rhG-CSF may be useful in the prophylaxis of sepsis in posttraumatic/postoperative patients at high risk of sepsis.

Adolescent↗