M. De Waele

Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation.

Serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-alpha were frequently measured during the first 30 days after allogeneic bone marrow transplantation (BMT) in 84 consecutive adult patients. Major transplant-related complications (MTCs) occurred in 33% of cases and included veno-occlusive liver disease, idiopathic pneumonia syndrome, severe endothelial leakage syndrome and >grade II acute graft-versus-host disease. Compared with patients having minor complications, those with MTCs developed higher levels at times of maximal clinical signs (all cytokines, P<0.001), between days 0–5 post-BMT (IL-6 and IL-8, P<0.05) and days 6–10 (L-6, P<0.001; IL-8 and TNF, P<0.01) post-BMT. We could not discriminate patterns of cytokine release that were specific for any subtype of MTC. Higher levels of IL-8 during days 0–5 were associated (P=0.044) with early (<40 days) death. Multivariate analysis including patient and transplant characteristics as well as post-BMT levels of C-reactive protein showed that high average levels of one or more of the cytokines within the first 10 days post-BMT were independently associated with MTC (Odds ratio: 2.3 [1.2–4.5], P=0.011). This study shows that systemic release of proinflammatory cytokines contributes to the development of MTC and provides a rationale for pre-emptive anti-inflammatory treatment in selected patients.

VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections.

AIMS--To characterise a new mouse monoclonal antibody, VS38, which recognises an intracytoplasmic antigen of 64 kilodaltons present in normal and neoplastic plasma cells; and to establish its value as a diagnostic reagent for routine pathological practice. METHODS--A range of normal and neoplastic tissue sections, both frozen and routinely fixed, were immunostained, using the microwave method of antigen retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range of human cell lines. The molecular weight of the antigen recognised by the antibody was obtained by western blot analysis. FACS analysis was used to demonstrate the cellular location of the antigen and its presence on tonsil cell suspensions and myeloma cases. RESULTS--VS38 recognised normal and neoplastic plasma cells in all of the tissues, including all routinely fixed plasma cell neoplasms tested. The antibody also weakly stained epithelial elements within the tissue but was absent from haemopoietic cells of other lineages. CONCLUSION--Antibody VS38 is of potential value in identifying myeloma or plasmacytoma in bone marrow or other tissues. It differentiates lymphoplasmacytoid lymphoma from lymphocytic and follicular lymphoma. It also subdivides large cell lymphomas into two groups which may be a more reliable method of separating these tumours than morphology alone.

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

An early increase in serum levels of C-reactive protein is an independent risk factor for the occurrence of major complications and 100-day transplant-related mortality after allogeneic bone marrow transplantation

We monitored levels of C-reactive protein (CRP) in 96 consecutive adult allogeneic BMT patients (age 15–50 years) transplanted in our unit. Major transplant-related complications (MTC) occurred in 32% of cases and included: hepatic veno-occlusive disease, pneumonitis, severe endothelial leakage syndrome and >II acute GVHD. Transplant-related mortality (TRM) before day 100 post-BMT was 13.5%. Variables included in a stepwise logistic regression model were: gender, age, disease category, donor type, T cell depletion, TBI, use of growth factors, bacteremia, mean CRP-levels >50 mg/l between days 0 and 5 (CRP day 0–5) and >100 mg/l between days 6 and 10 (CRP day 6–10) post-BMT. Only high CRP-levels (for MTC and TRM) (P < 0.001) and donor-type (for TRM) (P = 0.02) were independent risk factors. The estimated probability for MTC was 73% (CRP day 6–10 >100 mg/l) vs 17% (CRP day 6–10 <100 mg/l). Using the same cut-off levels, the probabilities for TRM were 36.5% vs 1% in the identical sibling donor situation and 88% vs 12.5% in other donor-type transplants. We conclude that the degree of systemic inflammation, as reflected by CRP-levels, during the first 5–10 days after BMT identifies patients at risk of MTC and TRM. Our data may be useful in selecting patients for clinical trials involving pre-emptive anti-inflammatory treatment.

Monitoring of C-reactive protein after allogeneic bone marrow transplantation identifies patients at risk of severe transplant-related complications and mortality

Patterns of C-reactive protein (CRP) release were derived from frequent CRP measurements in a cohort of 66 consecutive patients receiving allogeneic bone marrow transplants (BMT) in our unit. Based on a retrospective study of clinical events occurring within the first 40 days after BMT, patients with major transplant-related complications (MTC+ group, n = 22) could be separated from those with fever or mild complications only (MTC− group, n = 44). Treatment-related mortality in the MTC+ group was significantly higher: 32 vs 0% (P < 0.001). major complications included veno-occlusive liver disease (vod), severe endothelial leakage syndrome (els), pneumonitis and acute gvhd >II. The severity of complications was reflected by the patterns of CRP release with continuously high levels preceding the maximal signs and symptoms of MTC. Univariate analysis showed that, among other variables (sex, age, disease status at transplant, ±TBI in the conditioning regimen, ± use of myeloid growth factors after BMT, time to reach PN >200/mm3), three factors were significantly associated with MTC: maximal levels of CRP during the post-transplant episode (CRPmax) (296.6 ± 91.8 vs 88.9 ± 55.8 mg/100 ml, P < 0.001), the use of unmanipulated graft (no t depletion) (46.9 vs 12.5%, P < 0.009) and the crp level on the day of bmt (crpo) (42.7 ± 55.4 vs 18.2 ± 19.5, P = 0.045). In multivariate analysis, using a stepwise logistic regression model including the same variables, CRPmax appeared to be the strongest independent variable (P < 0.001) and a reliable (94% accuracy) parameter to assess the risk of mtc. based on this model, crp levels of 200 and 300 mg/100 ml are associated with a risk of 48 and 94% of developing mtc, respectively. we conclude that crp monitoring after bmt identifies patients at risk of severe transplant-related complications and mortality.

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Circulating CD34+ cells in cord blood and mobilized blood have a different profile of adhesion molecules than bone marrow CD34+ cells.

Abstract: The expression of adhesion molecules was studied on CD34+ hematopoietic precursors in cord blood, bone marrow and mobilized blood. The samples were labeled in a double immunofluorescence procedure with a CD34 monoclonal antibody and with antibodies against maturation and differentiation antigens and adhesion molecules. Myeloid precursors formed the majority of the CD34+ cells in all samples. In bone marrow a separate cluster of B‐cell precursors with low forward scatter was present. Nearly all CD34+ cells in normal bone marrow expressed VLA‐4 and VLA‐5, PECAM‐1, LFA‐3 and HCAM. The majority of the CD34+ cells also had LFA –1 and L‐selectin on the surface membrane. A small subset was VLA‐2, VLA‐3, ICAM‐1 or Mac‐1 positive. CD34+ cells expressing the vitronectin receptor or the CD11c antigen were rare. Cord blood and mobilized blood CD34+ cells had a lower expression of VLA‐2, VLA‐3 and VLA‐5 and a higher expression of LFA‐1, ICAM‐1 and L‐selectin than bone marrow CD34+ cells. Except for LFA‐1, this was not due to the presence of more myeloid precursors in these samples. Low β1 integrin expression may lead to less adhesion to the extracellular matrix. High expression of L‐selectin may facilitate interaction with endothelial cells. Therefore, this phenotype may favour mobilization.

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy.

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2)

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin κ light chain RNA sequence as the patient’s original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic κ light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 μm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein–Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.