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Biomedical subjects

M Moos

Publications and source records attributed to M Moos.

At least 19 recordsLinked to original sources
PubMed2002

Stable mixed chimerism after T cell-depleted allogeneic hematopoietic stem cell transplantation using conditioning with low-dose total body irradiation and fludarabine.

Although reduced intensity conditioning (RIC) before allografting is associated with low treatment-related morbidity and mortality, graft-versus-host disease (GVHD) remains a significant complication of hematopoietic stem cell transplantation (HSCT). T cell depletion (TCD) has been successfully used in conventional allotransplantation to reduce the incidence of GVHD, but was associated with an increased rate of engraftment failure. In a small cohort of six patients at high risk of developing GVHD we have determined whether sustained engraftment could be achieved using reduced intensity conditioning and T cell depletion in combination. All patients engrafted and 5/6 developed high levels (i.e. > or =95%) of donor chimerism, even though mismatched related or matched unrelated donors were used. Only one patient developed acute GVHD, as he received donor lymphocyte infusions (DLI) for relapse. In summary, TCD might be a useful prophylactic tool in RIC allogeneic HSCT. Although TCD after RIC might be associated with high relapse rate, as 5/6 patients are not in remission, this combined strategy might be appropriate for patients with less aggressive malignant or non-malignant diseases in which high transplant-related morbidity and mortality is not acceptable.

Adult↗
PubMed2002

Wetland management to reduce Baltic Sea eutrophication.

Seven regions with coastal eutrophication problems in the Baltic Sea, including the Kattegat, constitute the BERNET project (Baltic Eutrophication Regional Network). To counteract eutrophication and associated severe biological conditions the countries around this large brackish water body must all cooperate. The regions are characterized by large differences in land use, e.g. agricultural intensity, and losses of retention capacity in the catchments due to wetland reclamation. Initially it has been necessary to identify nutrient sources--especially nitrogen--and technical, economical and even administrative obstacles to initiate eutrophication management measures. Nitrogen retention in different types of wetlands in the Baltic Sea Region has been analysed. The wetlands generally have a positive effect on reduced nitrogen transport to aquatic environments and it is generally accepted that measures leading to decreased losses of nutrients to the aquatic environment must be combined with measures leading to increased retention of nutrients in catchments. Data analysed in the BERNET project show that the potential for such a measure is large. Therefore, conservation and restoration initiatives for wetlands is an essential part of the work in the BERNET project. Wetlands have been drained or totally eliminated due to intensive agriculture in some regions while large scale rehabilitation of wetlands occurs in regions with less intensive agriculture. Based on land use data from the seven regions, the working group for wetland management within the BERNET project has identified the possible use of wetlands as building blocks as a contribution to the management of the Baltic Sea eutrophication. Several recommendations are presented on the wise use of existing and constructed wetlands for water quality management in relation to non-point nutrient pollution.

Agriculture↗
PubMed2002

Regulatory philosophy for comparability protocols.

Continuing improvements in analytical biochemistry and biophysics make it reasonable to evaluate their use in obviating the need to use clinical studies for confirmation of product comparability following a change in manufacturing. It is essential to consider these analytical techniques in the context of specific manufacturing processes with well-defined parameters and specific products that have been characterized fully. Existing analytical technologies continue to be limited in dynamic range, so that small amounts of potentially harmful impurities may not be detected. In most cases, biochemical or biophysical techniques are neither sensitive nor robust enough to substitute entirely for some sort of biological potency assay, though there are important exceptions. Prudent design of comparability protocols will thus reflect the international consensus set down by ICH that sets of specifications on which such protocols are based comprise "one part of a total strategy designed to ensure product quality and consistency. Other parts of this strategy include thorough product characterization during development,...adherence to Good Manufacturing Practices, a validated manufacturing process, raw materials testing, in-process testing," and so forth.

Chemistry Techniques, Analytical↗
PubMed2001

Anti-CD20 antibody as consolidation therapy in a patient with primary plasma cell leukemia after high-dose therapy and autologous stem cell transplantation.

In multiple myeloma (MM), circulating malignant B cells are proposed as the proliferative compartment of the disease. In view of the close relationship between multiple myeloma and primary plasma cell leukemia (PCL), an anti-CD20 antibody treatment might also be considered as consolidation for patients with PCL. A 55-year-old patient diagnosed with PCL achieved complete remission after autologous transplantation. A total of four weekly courses of rituximab (375 mg/m(2)) were administered. Prior to antibody therapy, CD20+ cells comprised 22.6% of the mononuclear cells in peripheral blood (PB) assessed by flow cytometry and were enriched by magnetic activated cell sorting (MACS). In the enriched CD20+ fraction, 0.093% clonotypic cells were detected using a quantitative polymerase chain reaction (PCR) assay based on limiting dilutions. The proportion of clonotypic cells was 0.034% in PB and 0.032% in bone marrow (BM). Rituximab depleted CD20+ cells completely in PB and BM. Tumor load in PB and BM at day 40 and in PB at day 70 did not change in comparison to prior to therapy (0.037% in PB, 0.026% in BM). At day 90, the tumor load increased to 0.066% in PB. At day 120, the patient relapsed with 0.65% CD38++/CD138+/CD20- plasma cells and furthermore no CD20+ B cells in PB. The expansion of plasma cells was accompanied by an increase in the tumor load in both compartments (PB: 0.65%, BM: 1.8%). The accumulation of plasma cells during disease progression without the reappearance of CD20+ cells did not sustain the role of circulating clonotypic B cells as proliferative compartment in our patient. However, it cannot be excluded that rituximab was not able to eradicate malignant B cells, which subsequently contributed to relapse.

Antibodies, Monoclonal↗
PubMed2001

Variant toxin B and a functional toxin A produced by Clostridium difficile C34.

A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first 'S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a 'D-type' enterotoxin. Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect.

Animals↗
PubMed2001

Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation.

The clinical relevance of the assessment of minimal residual disease (MRD) in patients with multiple myeloma (MM) to predict disease recurrence has not been proven. In the present study, we retrospectively analyzed the tumor load in peripheral blood (PB) and bone marrow (BM) samples of 13 patients with MM both in remission after high-dose therapy (HDT) with autologous PBSC transplantation (PBSCT) and at the time of progressive disease (PD). For six patients, subsequent samples obtained in remission could be included in the study. Tumor cells were assessed by means of quantitative PCR with allele-specific oligonucleotides (ASO-qPCR) based on the method of limiting dilutions. PD was documented with ASO-qPCR in BM samples (median concentration of tumor cells in remission vs at PD: 0.18% vs 4.6%) representing a significant increase by a median factor of 8.7. In PB, the median tumor load was 799 cells/ml in remission and 23 400 cells/ml at PD. With a median factor of 45, the increase was much more pronounced. Comparing the results of the molecular monitoring in PB with those of the determination of the monoclonal protein, routinely applied as parameter for the course of the disease, revealed a superiority of the molecular monitoring because of the significantly higher increase in the tumor load. Analyzing the subsequent remission samples showed an increase of the malignant cells in four out of six PB samples and in all four BM samples available, indicating the potential of ASO-qPCR for an early PD recognition.

Adult↗
PubMed2000

Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma.

The aim of this investigation was to examine the possible clinical significance of the kinetics of bone marrow (BM) tumor load during the course of sequential high-dose therapy (HDT) as assessed by quantitative PCR in patients with multiple myeloma. In 20 patients with multiple myeloma (MM) treated with two consecutive cycles of HDT followed by autologous peripheral blood stem cell transplantation (PBSCT), clonotypic cells in the peripheral blood (PB) and BM were quantitated by PCR using allele-specific oligonucleotides (ASO) prior to the first, immediately prior to the second, and after the second HDT. The median proportion of clonotypic cells in the BM was 1.27% before the first HDT (range, 0.03-70%), 0.17% after the first (range, 0.001-22%), and 0.05% after the second HDT (range, 0.00009-1.44%). The median number of circulating clonotypic cells was 65/ml (range, 0.9-10842) prior to HDT, 2.7/ml (range, 0-315) after the first, and 3.5/ml PB (range, 0.7-97) after the second HDT. While the median BM tumor load decreased during the first (P = 0.03) and second (P = 0.044) HDT cycles, only the first cycle resulted in a reduction of clonotypic cells in the PB (P = 0.00078 and P= 1.0, respectively). In seven patients, the BM tumor load did not decrease below the initial level after one or two cycles of HDT. All of these patients developed progressive disease (median, 19 months post first cycle; range, 10-21). Of the remaining 13 patients, only four relapsed (18, 19, 21 and 22 months after the first cycle of HDT), while nine remain in response (median followup, 29 months; range, 18-41) (log-rank test P = 0.0009). Our results indicate that the kinetics of the BM tumor load is a predictive parameter in patients with MM and identifies those patients who could benefit from further therapy including new treatment modalities.

Adult↗
PubMed2000

In vivo depletion of B cells using a combination of high-dose cytosine arabinoside/mitoxantrone and rituximab for autografting in patients with non-Hodgkin's lymphoma.

We performed a pilot study including rituximab (Mabthera; IDEC-C2B8, Hoffmann-La Roche) with a sequential high-dose therapy protocol in 15 patients with follicular and three patients with mantle cell lymphoma and studied the potential of the chemoimmunotherapy to induce depletion of malignant B cells in vivo. Our treatment protocol included induction with three cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy, followed by peripheral blood stem cell (PBSC) mobilization using high-dose cytosine arabinoside (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3) (HAM), preceeded by rituximab (375 mg/m2). The proportion of CD19+ B cells in blood and bone marrow decreased from 1.2 +/- 0.4% to 0.13 +/- 0. 1% (P = 0.01) and from 2.7 +/- 0.8% to 0.8 +/- 0.5% (P = 0.03) respectively. The number of t(14;18)-positive cells in blood and bone marrow progressively decreased with treatment, as assessed by the quantitative real-time PCR assay in four patients. Conversion to PCR-negativity was achieved in the peripheral blood (PB) of seven informative patients. Leucaphereses were performed during the granulocyte colony-stimulating factor (G-CSF)-supported leucocyte recovery phase. In 17 of 18 patients, a median of 15.1 x 106 CD34+ cells/kg body weight (BW) could be harvested by a single procedure for enrichment by an immunomagnetic method. Leucapheresis products contained 51.3 +/- 28.8 x 104 CD19+ B cells/kg BW (mean) and were t(14;18) PCR negative in all seven informative patients. These data compare favourably with results obtained in patients treated with the same regimen without rituximab. The high-dose therapy (n = 12 patients), including total body irradiation (14.4 Gy) and cyclophosphamide (200 mg/kg BW), was also preceeded by rituximab. Recovery of neutrophils to > 0.5 x 109/l and of platelets to > 20 x 109/l required a median of 13.5 and 11.5 d (range 11-24 and 9-24 d) respectively. In conclusion, the addition of the CD20 antibody to chemotherapy ensured tumour depletion in vivo and allowed the collection of PBSCs devoid of tumour cells and with conserved engraftment capability.

Adult↗
PubMed2000

A chimeric ribozyme in clostridium difficile combines features of group I introns and insertion elements.

CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element. We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic and non-toxinogenic C. difficile strains, indicating the mobility of CdlSt1. In strain C34, we characterize 10 CdlSt1 variants; all are highly homologous to CdlSt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdlSt1 and are precisely spliced from their primary transcripts. CdlSt1-like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.

Amino Acid Sequence↗
PubMed1999

Immunolocalization of the neural cell adhesion molecule L1 in epithelia of rodents.

The expression of the neural cell adhesion molecule L1 was analyzed in several non-neural tissues of the mouse using immunohistochemical and immunochemical techniques. In the adult mouse, L1 immunoreactivity was detectable in the basal and intermediate layers of epidermal and lingual epithelia, in the outer sheath of hair roots and in the single-layered endodermal epithelia of lung, small intestine, and colon. Epithelia of salivary glands also showed L1 immunoreactivity, while endothelial cells of blood vessels did not express detectable levels of L1. The epithelia of the kidney showed expression only in the collecting tubule system. In single-layered kidney epithelia and stratified epithelia, L1 expression was confined to lateral cell contacts and basal infoldings of the epithelial cells but was absent from apical and basal cell surface membranes. Also, in cultured keratinocytes L1 was confined to cell-cell contacts. During development of the epidermis, L1 immunoreactivity was first detectable at the onset of keratinization around embryonic day 16. At this age LI was detectable in the kidney on branching tubules of the ureter. Western blot analysis showed that L1 immunoreactivity in epidermis and kidney appeared as two bands of 190-210 and 210-230 kDa. Northern blot analysis of mRNA from the L1-immunopositive HEL-30 keratinocyte cell line revealed a single band with the expected size of 6 kb. The presence of L1 in epithelia indicates that this molecule may be involved in interactions between epithelial cells and thereby may affect differentiation and maintenance of epithelial tissues.

Animals↗
PubMed1999

Bone marrow derived dendritic cells from patients with multiple myeloma cultured with three distinct protocols do not bear Kaposi's sarcoma associated herpesvirus DNA.

BACKGROUND: An association between Kaposi's sarcoma associated herpesvirus (KSHV) and the pathogenesis of multiple myeloma (MM) was postulated recently. The dendritic cells of patients with MM were proposed to be infected with the virus. PATIENTS AND METHODS: Bone marrow mononuclear cells (MNC) of 23 patients, 22 with MM and one with MGUS, were cultured according to three distinct protocols for the generation of dendritic cells. One was essentially the stromal cell culture protocol described by Rettig et al. (Science 1997; 276: 1851-4), while the two other protocols comprised growth factors. Cultured cells were characterised by FACS analysis and assessed for the presence of KSHV DNA with a highly sensitive and specific nested PCR assay detecting the KS 330233 sequence of the virus genome followed by hybridisation with a KSHV specific oligonucleotide. RESULTS: FACS analysis of the cells with the specific markers CD1a, CD86 and HLA-DR, characteristic for dendritic cells, revealed differences in the expression pattern depending on the protocol used. The proportion of CD1a+ cells was very low in the stromal cell cultures (median 0.4%), while a higher percentage of CD14+ cells could be observed (median 37.8%). Growth factor containing cultures revealed a distinctly higher median percentage of CD1a+ cells of 32.5%. The proportion of CD86+ cells varied between 10.4% and 78.5% and HLA-DR+ cells between 26% and 94.4%. Examination of those cells with PCR did not reveal positivity for KSHV in any of the 34 samples assessed. Amplification of seven samples revealed PCR products of approximately the size of the KS 330(233), which, however, could not be confirmed as KSHV specific after hybridisation. CONCLUSION: We have no evidence that bone marrow derived dendritic cells from patients with MM are infected with KSHV.

Adult↗
PubMed1999

Variability of polymerase chain reaction detection of the bcl-2-IgH translocation in an international multicentre study.

BACKGROUND: The capacity of the polymerase chain reaction (PCR) to detect very low numbers of cells bearing a t(14;18) translocation has led to its application in assessment of the results of treatment for follicular lymphoma, and suggestions that therapy might be guided by molecular studies. To test the reliability of PCR a collaborative study was undertaken to compare results from different laboratories in Europe and North America. METHODS: Twenty laboratories with records of publication in molecular diagnostics were sent blood from normal donors with varying numbers of t(14;18)-bearing cells added from a cell line with a translocation in the major breakpoint region (MBR) of the bcl-2 gene. Samples contained 1000, 100, 10, 1 or 0 cells per ml of whole blood and were sent blinded in duplicate. PCR methodology varied widely, with the total number of amplification cycles between 30 and 70, and 13 different primers used for the MBR region. Twelve laboratories used nested PCR and eight single round amplification. RESULTS: The sensitivity of nested and single round PCR was similar at 100 cells/ml but below this the nested method proved significantly more sensitive. The false positive rate was 28%, with 11 samples from 9 laboratories reported as positive when no t(14;18) cells were added. PCR product size and sequence analysis showed that false positives were due to contamination from cell-line DNA rather than background translocations in the donors. There was no significant difference in false positive rates between nested and single round techniques. CONCLUSION: The polymerase chain reaction to detect bcl-2-IgH rearrangements is presently carried out with widely disparate results. Further effort is required to bring forward a standard PCR protocol which can be re-tested in different laboratories to improve accuracy and reproducibility. The application of quantitative techniques such as real-time PCR may resolve many of the problems presently encountered.

Adult↗
PubMed1999

Analysis of circulating tumor cells in patients with multiple myeloma during the course of high-dose therapy with peripheral blood stem cell transplantation.

In multiple myeloma (MM) circulating CD19+ cells have been considered as myeloma precursors. As these cells are also possibly a reservoir of treatment resistant disease evaluation of the CD19+ cells during the course of high-dose therapy has to be a major concern. We determined the number of tumor cells in the CD19+ as well as CD19- fractions of PB of eight patients with disease sensitive to VA[I]D chemotherapy, of 10 patients who achieved partial or complete remission post-high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) and of a further seven patients with disease progression post-transplantation. CD19+ cell fractions were obtained by preparative sequential magnetic and fluorescence activated cell sorting with a median purity of 97.1%. In addition, PB samples of seven patients post-transplantation were sorted for CD20+ cells (median purity, 98.7%). The number of tumor cells in the CD19+, the CD19- and the CD20+ fractions were determined using a quantitative CDR3 PCR assay. The number of CD19+ tumor cells in patients in remission post-HDT was similar to those of the patients post-VA[I]D (median, 1.05 vs 0.92 CD19+ tumor cells/ml PB, P = 0.72) providing evidence for the persistence of this tumor cell fraction during the course of HDT. This was in contrast to the CD19- compartment, in which the number of tumor cells was significantly reduced in those patients in remission post-transplantation (median, 53 vs 0 CD19- tumor cells/ml PB; P = 0.006). In patients with progressive disease the number of tumor cells in both cell fractions was significantly higher (CD19+: median, 1.05 vs 21 tumor cells/ml PB, P = 0.05; CD19-: 0 vs 63 tumor cells/ml PB, P = 0.008). While the absolute number of CD19+ cells was reduced in the group of patients after VA[I]D treatment, a polyclonal CD19+ reconstitution had occurred in patients responding to HDT. The tumor cell content in the CD19+ fractions could be confirmed by the results obtained analyzing the CD20+ cell fractions. In conclusion, these results indicate that disease progression after PBSCT in MM is accompanied by an expansion of tumor cells in both the CD19+ and CD19- fractions. Similar numbers of CD19+ clonotypic cells post-HDT suggest that these cells persist and thus, contribute to disease dissemination and relapse.

Adult↗
PubMed1999

Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content.

The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non-Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy-1 and Leu-8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR-negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high-dose therapy, a mean number of 4.7x 10(6) CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back-up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.

Antigens, CD34↗
PubMed1999

Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation.

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.

Adult↗