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Biomedical subjects

R Haas

Publications and source records attributed to R Haas.

At least 199 records · Page 11Linked to original sources
PubMed1996

Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain.

The N-terminal sequence of a protein, originally described as an adhesin of Helicobacter pylori, was used in an oligonucleotide-based screening procedure of an H. pylori plasmid library in Escherichia coli. Five independent plasmid clones were isolated, all mapping to the same chromosomal region and encoding the H. pylori catalase. The gene, designated katA, comprises 1,518 nucleotides and encodes a putative protein of 505 amino acids with a predicted Mr of 58,599. A second open reading frame, orf2, encoding a putative 32,715-Da protein of unknown function, follows katA. The transcriptional start site of katA mRNA was determined, but no typical consensus promoter sequence was present. A potential binding site for the Fur protein is located upstream of katA. When introduced into the catalase-deficient E. coli double-mutant UM255, the cloned gene readily complemented E. coli for catalase activity. H. pylori KatA is highly homologous to catalases in both prokaryotes and eukaryotes, with the highest homology being shown to Bordetella pertussis (64.9%), Bacteroides fragilis (59.8%), and Haemophilus influenzae (57.9%) catalases. Transposon insertion mutants were generated in three independent H. pylori strains by TnMax5-mediated transposon shuttle mutagenesis. In contrast to the wild-type strains, no significant catalase-specific enzymatic activity could be detected in the mutant strains, consistent with the fact that no additional katA-homologous gene copies were found in the H. pylori chromosome. No significant difference between wild-type and mutant strains for binding to epithelial cells was apparent, suggesting that KatA is not involved in H. pylori adhesion. The cloning and genetic characterization of katA are essential steps for further investigation of the role of catalase in the defense of H. pylori against oxygen-dependent killing mechanisms by polymorphonuclear granulocytes, a process not well understood for this chronically persisting pathogen.

Amino Acid Sequence↗
PubMed1996

An epidemiologic study of early biologic effects of benzene in Chinese workers.

Benzene is a recognized hematotoxin and leukemogen, but its mechanisms of action in humans are still uncertain. To provide insight into these processes, we carried out a cross-sectional study of 44 healthy workers currently exposed to benzene (median 8-hr time-weighted average; 31 ppm), and unexposed controls in Shanghai, China. Here we provide an overview of the study results on peripheral blood cells levels and somatic cell mutation frequency measured by the glycophorin A (GPA) gene loss assay and report on peripheral cytokine levels. All peripheral blood cells levels (i.e., total white blood cells, absolute lymphocyte count, platelets, red blood cells, and hemoglobin) were decreased among exposed workers compared to controls, with the exception of the red blood cell mean corpuscular volume, which was higher among exposed subjects. In contrast, peripheral cytokine levels (interleukin-3, interleukin-6, erythropoietin, granulocyte colony-stimulating factor, tissue necrosis factor-alpha) in a subset of the most highly exposed workers (n = 11) were similar to values in controls (n = 11), suggesting that benzene does not affect these growth factor levels in peripheral blood. The GPA assay measures stem cell or precursor erythroid cell mutations expressed in peripheral red blood cells of MN heterozygous subjects, identifying NN variants, which result from loss of the GPA M allele and duplication of the N allele, and N phi variants, which arise from gene inactivation. The NN (but not N phi) GPA variant cell frequency was elevated in the exposed workers compared with controls (mean +/- SD, 13.9 +/- 8.4 mutants per million cells versus 7.4 +/- 5.2 per million cells, (respectively; p = 0.0002), suggesting that benzene produces gene-duplicating but not gene-inactivating mutations at the GPA locus in bone marrow cells of exposed humans. These findings, combined with ongoing analyses of benzene macromolecular adducts and chromosomal aberrations, will provide an opportunity to comprehensively evaluate a wide range of early biologic effects associated with benzene exposure in humans.

Adult↗
PubMed1996

High-dose therapy with peripheral blood progenitor cell transplantation in low-grade non-Hodgkin's lymphoma.

It was the objective of our study to evaluate the efficacy of a sequential high-dose therapy with peripheral blood progenitor cell (PBPC) support in patients with low-grade non-Hodgkin's lymphoma (NHL). Since July 1991, 48 patients (23 male/25 female) with a median age of 43 years (range 26-55) were included in the study. At the time of entry, 28 patients were in first and seven in second or higher remission. Twelve patients had relapse of disease and one patient had tumor progression. PBPC were collected during granulocyte colony-stimulating factor (G-CSF)-enhanced leukocyte recovery following treatment with high-dose cytarabine and mitoxantrone (HAM). A median of two leukaphereses (range 2-7) resulted in 6.9 x 10(6) CD34+ cells/kg (median, range 2.1 x 10(6)-38.8 x 10(6)). A comparison was made between the harvests obtained from patients in first remission and those from patients in second remission, in relapse or progressive disease. Patients mobilized in first remission tended to have a greater collection efficiency for CD34+ cells comprising a significantly greater proportion of more primitive CD34+/Thy-1+ progenitor cells. Conversely, leukapheresis (LP) products collected during first remission contained a significantly smaller proportion of CD34+/CD45RA+ cells and CD34+/c-kit+ cells, subsets which reflect a more differentiated progenitor cell stage. Following high-dose therapy and PBPC autografting, the median time to reach platelets > or = 20 x 10(9)/l and neutrophils > or = 0.5 x 10(9)/l and 12 and 13 days, respectively. Two patients died of treatment-related toxic organ failure. Thirty-nine patients are alive in remission after a median follow-up time of 15 months (range 1-31), while seven patients relapsed between 5 and 29 months post-transplantation. Except for one patient autografted in first remission, the patients with relapse had a history of previous relapse or progressive disease. Since the probability of disease-free survival appears to be related to the disease status at the time of autografting, PBPC-supported high-dose therapy including total body irradiation should be investigated further for patients with low-grade NHL while they are in first remission.

Adult↗
PubMed1996

Mobilization of peripheral blood progenitor cells with high-dose cyclophosphamide (4 or 7 g/m2) and granulocyte colony-stimulating factor in patients with multiple myeloma.

High-dose cyclophosphamide (HD-CY) has been shown to decrease the tumor mass in multiple myeloma (MM) patients and to be effective in the mobilization of PBPC. By administering hematopoietic growth factor the quantity of progenitor cells in the peripheral blood increased and the hematological toxicity of CY could be reduced. Thirty-two patients with stage II and stage III MM were treated to mobilize and harvest a sufficient amount of PBPC for autologous transplantation. Sixteen patients received 4 g/m2 CY and 16 patients 7 g/m2 CY in divided doses of 1 g/m2 every 2 h. Both patient groups were comparable for disease stages as well as previous therapies. Twenty-four hours after chemotherapy 300 micrograms GCSF were administered subcutaneously once daily until the last day of leukapheresis. Administration of 7 g/m2 HD-CY resulted in statistically significantly higher peak values for CD34+ progenitor cells (47.86/microliters vs 18.75/microliters, P = 0.0198) in the peripheral blood. PBPC autografts containing > 2.5 x 10(6) CD34+ cells/kg BW could be obtained at the first attempt from 14 of 16 patients treated with 7 g/m2 CY as compared to 10 of 16 patients treated with 4 g/m2 CY (P = 0.11). The analysis of potentially malignant CD19+ B cells showed a highly significant lower mean CD19+ cell content/kg BW per leukapheresis in the 7 g/m2 compared to the 4 g/m2 CY group (0.75 vs 1.81 x 10(6), P = 0.001). WHO grade IV treatment-related non-hematologic toxicity was not observed. We prefer the 7 g/m2 CY dosage followed by cytokine administration for the mobilization of PBPC in advanced state MM patients pretreated with alkylating agents.

Adult↗
PubMed1996

Myeloablative therapy with blood stem cell transplantation is effective in mantle cell lymphoma.

Long-term disease-free survival following conventional cytotoxic therapy is extremely rare in patients with advanced-stage mantle cell lymphoma (MCL). High-dose conditioning therapy consisting of hyperfractionated total body irradiation (TBI, 14.4 Gy) and cyclophosphamide (200 mg/kg) was therefore offered to 13 patients (four females/nine males) with advanced-stage MCL. The patients were relatively young with a median age of 49 years (range 30-60). High-dose cytarabine and mitoxantrone with granulocyte colony-stimulating factor (G-CSF) support were given for second-line therapy and mobilization of peripheral blood stem cells (PBSC). During cytokine-stimulated marrow recovery, a median of two leukaphereses (range 1-4) were performed. Using direct immunofluorescence analysis including two-color staining, the proportion of CD19+ B cells and CD34+/CD19+ B lymphoid progenitor cells was found to be extremely low with quantities below detection limit in approximately 50% of the autografts. At the time of autografting, nine patients (pts) were in first partial (five pts) or complete (four pts) remission, while four patients had achieved a second complete remission. Following myeloablative therapy a median number of 7.5 x 10(6) CD34+ cells/kg were autografted. The median time for neutrophil (> or = 0.5 x 10(9)/l) and platelet recovery (> or = 20 x 10(9)/l) was 13 and 10 days, respectively. Hematological recovery was delayed in a patient who received 5.8 x 10(6) positively selected CD34+ cells/kg. There was one toxic death 17 days post-transplantation because of overwhelming interstitial pneumonia. Two patients with a history of previous treatment failure relapsed 10 and 11 months post-transplantation, respectively, at sites of previous disease. Ten patients are disease-free with a median follow-up time of 18 months (range 10-47). The results presented here suggest that PBSC-supported high-dose therapy including TBI may provide long-term disease-free survival for patients with advanced-stage mantle cell lymphoma.

Adult↗
PubMed1995

An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions.

A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.

Base Sequence↗
PubMed1995

Genetic organization of a small cryptic plasmid of Helicobacter pylori.

A 2.9-kb cryptic plasmid of Helicobacter pylori (Hp), pHel1, was isolated and the complete nucleotide (nt) sequence was determined. An open reading frame (ORF1) was identified encoding a putative polypeptide of 63,709 Da, the existence and correct size of which was confirmed by T7 promoter expression analysis. The ORF1 sequence showed strong amino-acid sequence identity to a recently identified putative ORF1 protein of a cryptic Hp plasmid, pHPM180, and significant homologies to putative Rep proteins of Campylobacter coli (RepB) and Pediococcus halophilus (RepA), and was therefore designated RepA. A functional role of RepA in replication of pHel1 was demonstrated by the fact that only pHel1 plasmid derivatives with an intact repA gene were able to autonomously replicate in Hp. Upstream of repA, a 22-bp sequence was recognized which was tandemly repeated four and a half times, a feature typical for many replication origins (ori) and commonly termed a DNA iteron. Analysis of the repA upstream region by primer extension identified a transcription start point for the repA mRNA, but did not correspond to known consensus promoter sequences. Southern hybridizations using pHel1 as a probe under stringent conditions revealed that homologous sequences to pHel1 were present in nearly all plasmid-carrying Hp strains, but not in a plasmid-carrying Helicobacter felis strain, suggesting that this type of replicon is predominantly found in the Hp species.

Amino Acid Sequence↗
PubMed1995

Cloning of the Helicobacter pylori recA gene and functional characterization of its product.

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned a recA homolog from Helicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to the Campylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus sigma 70 promoter sequence was found upstream of recA. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream of recA. Compared to the wild-type strains, isogenic H. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinant H. pylori RecA protein produced in Escherichia coli strain GC6 (recA-) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein in H. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of the H. pylori mutant using the cloned recA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA in H. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced in E. coli, giving rise to a smaller but inactive protein.

Amino Acid Sequence↗
PubMed1995

Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support.

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non-Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.

Adolescent↗
PubMed1995

Peripheral blood progenitor cell (PBPC) counts during steady-state hematopoiesis allow to estimate the yield of mobilized PBPC after filgrastim (R-metHuG-CSF)-supported cytotoxic chemotherapy.

Peripheral blood progenitor cells (PBPC) can be mobilized using cytotoxic chemotherapy and cytokines. There is a substantial variability in the yield of hematopoietic progenitor cells between patients. We were looking for predictive parameters indicating a patient's response to a given mobilization regimen. Multiparameter flow-cytometry analysis and clonogenic assays were used to examine the hematopoietic progenitor cells in bone marrow (BM) and peripheral blood (PB) before filgrastim (R-metHuG-CSF; Amgen, Thousand Oaks, CA)-supported chemotherapy and in PB and leukapheresis products (LPs) in the recovery phase. Fifteen patients (four with high-grade non-Hodgkin's lymphoma [NHL], two with low-grade NHL, two with Hodgkin's disease, two with multiple myeloma, three with breast cancer, one with ovarian cancer, and one with germ cell tumor) were included in this study. The comparison of immunofluorescence plots showed a homogenous population of strongly CD34+ cells in steady-state and mobilized PB whereas in steady-state BM, the CD34+ cells ranged from strongly positive with continuous transition to the CD34- population. Consistent with the similarity in CD34 antigen expression, a correlation analysis showed steady-state PB CD34+ cells (r = .81, P < .001) and colony-forming cells (CFCs; r = .69, P < .01) to be a measure of a patient's mobilizable CD34+ cell pool. Individual estimates of progenitor cell yields could be calculated. With a probability of 95%, eg, 0.4 steady-state PB CD34+ cells x 10(6)/L allowed to collect in six LPs 2.5 x 10(6) CD34+ cells/kg, the reported threshold-dose of progenitor cells required for rapid and sustained engraftment after high-dose therapy. For the total steady-state BM CD34+ cell population, a weak correlation (r = .57, P < .05) with the mobilized CD34+ cells only became apparent when an outlier was removed from the analysis. Neither the CD34+ immunologic subgroups defined by the coexpression of the myeloid lineage-associated antigens CD33 or CD45-RA or the phenotypically primitive CD34+/HLA-DR- subset nor the BM CFC count had a predictive value for the mobilization outcome. This may be caused by the additional presence of maturing progenitor cells in BM, which express lower levels of the CD34 antigen and do not circulate. Our results permit us to recognize patients who are at risk to collect low numbers of progenitor cells and those who are likely to achieve sufficient or high progenitor cell yields even before mobilization chemotherapy is administered.

Adult↗
PubMed1995

Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin A locus in exposed humans.

Occupational exposure to benzene is known to cause leukemia, but the mechanism remains unclear. Unlike most other carcinogens, benzene and its metabolites are weakly or nonmutagenic in most simple gene mutation assays. Benzene and its metabolites do, however, produce chromosomal damage in a variety of systems. Here, we have used the glycophorin A (GPA) gene loss mutation assay to evaluate the nature of DNA damage produced by benzene in 24 workers heavily exposed to benzene and 23 matched control individuals in Shanghai, China. The GPA assay identifies stem cell or precursor erythroid cell mutations expressed in peripheral erythrocytes of MN-heterozygous subjects, distinguishing the NN and N phi mutant variants. A significant increase in the NN GPA variant cell frequency (Vf) was found in benzene-exposed workers as compared with unexposed control individuals (mean +/- SEM, 13.9 +/- 1.7 per million cells vs. 7.4 +/- 1.1 per million cells in control individuals; P = 0.0002). In contrast, no significant difference existed between the two groups for the N phi Vf (9.1 +/- 0.9 vs. 8.8 +/- 1.8 per million cells; P = 0.21). Further, lifetime cumulative occupational exposure to benzene was associated with the NN Vf (P = 0.005) but not with the N phi Vf (P = 0.31), suggesting that NN mutations occur in longer-lived bone marrow stem cells. NN variants result from loss of the GPA M allele and duplication of the N allele, presumably through recombination mechanisms, whereas NO variants arise from gene inactivation, presumably due to point mutations and deletions. Thus, these results suggest that benzene produces gene-duplicating mutations but does not produce gene-inactivating mutations at the GPA locus in bone marrow cells of humans exposed to high benzene levels. This finding is consistent with data on the genetic toxicology of benzene and its metabolites and adds further weight to the hypothesis that chromosome damage and mitotic recombination are important in benzene-induced leukemia.

Adolescent↗
PubMed1995

Blood-derived autografts collected during granulocyte colony-stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells.

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR- cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony-forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.

Adult↗
PubMed1995

Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase.

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [14C]HCHO and NaCNBH3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mixture of constant composition (approximately 11% H2O-89% CH3CN), so that a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.

Acetonitriles↗
PubMed1995

Large-bore central venous catheters for the collection of peripheral blood stem cells.

In order to establish a peripheral blood stem cell graft, repeated apheresis are necessary in the majority of patients. Each apheresis requires withdrawal and reinfusion of blood with high flow rates. To guarantee these flow rates, large-bore catheters are needed for central venous access. Subcutaneously tunneled silicone catheters (Hickman) caused venous thrombosis in 10-40% of the patients. We therefore used polyurethane large-bore catheters only for the time of peripheral blood stem cells (PBSC) collection. Via a Seldinger guidewire following delineation of the right (160 patients) or left (23 patients) internal jugular vein by ultrasound, 183 apheresis catheters have been inserted when the white blood cell count was > 1.0 x 10(9)/L and a measurable population of CD34+ cells was detected by fluorescence-activated cell sorter analysis. The median flow rate was 70 ml/min (range 50-80 ml/min). We observed the following complications: puncture of the carotid artery in 2%, pneumothorax in 0.5%, local infection in 3%, and catheter-related septicemia in only 2% of the patients. At the time of the removal of the catheters, we detected thrombosis of the internal jugular vein in 5% of the patients by ultrasound. The collection of PBSC with short-term, large-bore catheters is effective and is associated with a low incidence of infection and thrombosis.

Acinetobacter Infections↗
PubMed1995

High-dose therapy with peripheral blood progenitor cell support in patients with non-Hodgkin's lymphoma.

Between September 1991 and April 1995, high-dose therapy with peripheral blood progenitor cell (PBPC) support was administered to 105 patients with non-Hodgkin's lymphoma (NHL). Thirty-three patients had high-grade NHL, while 72 patients had different forms of low- or intermediate-grade NHL. Except for three patients who received G-CSF during steady-state hematopoiesis, PBPCs were collected following cytokine-supported cytotoxic chemotherapy. This included G-CSF or the sequential administration of interleukin 3 (IL-3) and GM-CSF. Assessing bone marrow (BM) samples before the start of chemotherapy and leukapheresis (LP) products collected during cytokine-enhanced marrow recovery, a 2.3-fold greater mean concentration of CD34- cells was found in peripheral blood (p < 0.005). The blood-derived progenitor cells were enriched with a particular subset of more primitive progenitors, as the mean proportion of CD34+/Thy-1+ cells in LP products was three-fold greater in comparison to premobilization BM samples, respectively (p < 0.001). In contrast, the mean proportion of CD34+/CD19+ and CD19+ cells in LP products was 8.8- and 80-fold smaller compared to BM samples, respectively (p < 0.001). Following high-dose conditioning therapy including TBI in 74 patients, reinfusion of PBPC resulted in rapid and sustained engraftment in the majority of patients, while in seven patients an unsubstituted platelet count of greater than 20 x 10(9)/l was reached between 31 and 51 days. Five patients died of treatment-related complications between 13 and 188 days following transplantation. The probability of long-term disease-free survival at 30 months in patients autografted while they were in first remission was 70% in high-grade and 83% in low-grade NHL, respectively. The data may provide the rationale for the use of PBPC-supported high-dose regimens as first-line treatment for patients at high risk of treatment failure.

Adult↗
PubMed1995

Sequential high-dose treatment with peripheral blood progenitor cell transplantation in patients with multiple myeloma.

In June 1992, we started a dose-escalated cytotoxic therapy with peripheral blood progenitor cell (PBPC) transplantation in patients with chemosensitive multiple myeloma (MM). At the time of best response to conventional treatment, 70 patients received high-dose cyclophosphamide (HD-CY) or, in case of pre-existing heart disease, dose-escalated ifosfamide/mitoxantrone followed by filgrastim (R-metHuG-CSF, 300 micrograms/day). PBPC collection was commenced when CD34+ cells were detectable using direct immunofluorescence analysis. Fifty-four out of 70 patients were successfully harvested (> or = 2.5 x 10(6) CD34+ cells/kg body weight [BW]) after the first cycle of HD chemotherapy. Conditioning therapy consisted of 140 mg/m2 melphalan plus TBI (14.4 Gy hyper-fractionated) or 200 mg/m2 melphalan in patients not eligible for TBI because of previous radiotherapy. To date, 56 patients have been transplanted. Autografts contained a median of 3.4 x 10(6) CD34+ cells/kg BW. Following reinfusion of PBPC, rapid engraftment was achieved in 54 out of 56 patients with a median of 14 days (range 9-23) to reach 0.5 x 10(9)/l neutrophils and 10 days (range 5-22) for an unsubstituted platelet count of > 20 x 10(9)/l. One patient died of transplantation-related complications. Sequential HD treatment improved the remission status (European Bone Marrow Transplantation criteria) in 19 out of 46 patients (9 patients too early). Of note, in 11 patients the immunofixation became negative and a polyclonal immunoglobulin reconstitution was achieved. Our protocol provides an effective treatment strategy for patients with advanced MM combined with low treatment-related toxicity.

Adult↗