Marc De Waele

Correlations between complaints, inflammatory cells and mediator concentrations in nasal secretions after nasal allergen challenge and during natural allergen exposure

A quantitative determination of the inflammatory mediators was performed and correlated with complaints and the measurement of the inflammatory cells in nasal secretions of 18 seasonal allergic rhinitis patients (group 1) outside the pollen season and 40 symptomatic patients (group 2) with seasonal allergic rhinitis during the pollen season. Ten nonallergic subjects (group 3) were also studied as a normal control group. In group 1, 17 (94%) out of 18 patients had an immediate response of nasal symptoms accompanied by a significant increase of histamine, leukotriene C4 (LTC4), and tryptase 5 min after nasal allergen challenge (NAC). One hour later, a simultaneous increase was seen both in the percentage of the eosinophils and in the eosinophil cationic protein (ECP) concentration. The eosinophil count reached a peak 2 h after NAC with a duration of 8 h, while the highest ECP level was reached only after 24 h with no clear-cut plateau. In group 2, a high percentage of eosinophils was observed. Mostly one observed significantly (p < 0.01) higher concentrations of ECP, LTC4 and histamine but not of tryptase than the baseline values of group 1. The authors concluded that during the pollen season allergic rhinitis reflects mainly a chronic state of allergic inflammation of the nasal mucosa involving various inflammatory components induced by one or more episodes of early-phase type allergic reaction. Infiltration of eosinophils and consequently release of the various late-phase inflammatory mediators into the nasal secretions are certainly believed to be the predominant pathophysiologic condition in the patients.

Platelet 3H-paroxetine binding in depressed patients

Studies using 3H-imipramine binding in platelets as a means of exploring the serotonin transport system in depressed patients have produced inconsistent findings. For this reason, we studied 3H-paroxetine binding in platelets, because we believed it might provide a more reliable ligand for studying serotonin transport. Subjects were 23 depressive inpatients and 23 normal controls. No differences in the maximal number of binding sites (Bmax) or in the dissociation constant (Kd) were found between depressives and controls.

Adhesive interactions between tumour cells and bone marrow stromal elements in human multiple myeloma

Abstract:  Long‐term bone marrow cultures (LTBMC) were established from marrow samples obtained from 6 myeloma patients and 5 healthy donors and were examined by in situ immunogold–silver staining. During the culture period, the established stroma in myeloma LTBMC revealed a lower level of confluency compared to the normal LTBMC. In addition, an increasing proportion of macrophages and osteoclasts was observed in the myeloma stroma throughout the culture period. Moreover, plasma cells were detectable by wk 8, mostly organized in small clusters. They strongly expressed VLA‐4 (6/6), H‐CAM (6/6), ICAM‐1 (6/6) and N‐CAM (3/6). In most cases, a weak expression of the other members of β1‐integrins was observed. The expression of β2‐integrins was always absent. Stromal fibroblasts were found to be weakly positive for VLA‐2, VLA‐3 and VLA‐5 and showed strong expression of VCAM‐1, H‐CAM and ICAM‐1. N‐CAM expression could not be detected. By comparing the adhesion molecule profile of the stromal cells in myeloma cultures with normal bone marrow (BM) cultures, no particular defects could be observed. The stroma displayed most of the potential ligands which could interact with adhesion molecules detected on the myeloma cells. Among these ligands we could find fibronectin and VCAM‐1 for VLA‐4, collagen I for VLA‐2 and VLA‐3 and laminin for VLA‐2, 3 and 6. Four myeloma cell lines, i.e. OPM‐1, U266, RPMI 8226 and JJN3, with a representative phenotype, were used to study the adhesive interactions of myeloma cells with the BM microenvironment. All the myeloma cell lines bound strongly to the marrow cell layers and also showed a high binding to purified fibronectin (FN). However, the adhesion of the cell lines to intact stroma could not be significantly inhibited by anti‐FN receptors antibodies. Nor could it be prevented when the latter were combined with anti‐H‐CAM, V‐CAM and ICAM‐1 antibodies, as tested in the JJN3 cell line. This implies that other unknown mechanisms contribute to the myeloma cell binding.

Effect of Topical Applications of Budesonide and Azelastine on Nasal Symptoms, Eosinophil Count and Mediator Release in Atopic Patients after Nasal Allergen Challenge during the Pollen Season

We studied the activity of a topical form of a corticosteroid (budesonide) and an antihistamine (azelastine) in the treatment of seasonal allergic rhinitis by including an assessment of mediator concentrations and the percentage of eosinophils in the nasal secretions before and after the treatment. Nasal allergen challenge (NAC) during the season was performed to mimic an acute attack of allergic rhinitis and to objectively evaluate the effect of the drugs on the early-phase reaction. The study compared in a randomized way (2 parallel groups) the effect of budesonide (Rhinocort Aqua) and azelastine (Allergodil nasal spray) in a group of 14 patients during the pollen season. The study showed that azelastine significantly reduced sneezing, total nasal resistance and increased nasal airflow even when significant increases in histamine, tryptase and leukotriene C4 (LTC4) concentrations in nasal secretions were evidenced immediately after NAC. Budesonide showed a strong (p<0.05) decrease in infiltration and activation of eosinophils, and on tryptase and LTC4 release after NAC. These effects (not for LTC4) lasted at least for 1 week after therapy. Azelastine is a powerful topical antihistamine, while budesonide appears to be a potent long-acting anti-inflammatory agent.

Monitoring nasal allergic inflammation by measuring the concentration of eosinophil cationic protein and eosinophils in nasal secretions

Quantitative measurement of the eosinophil cationic protein (ECP) concentration and the percentage of eosinophils in nasal secretions has greatly improved our understanding of the inflammatory process after natural allergen exposure. ECP and eosinophils were measured in the nasal secretions of 40 symptomatic patients with seasonal allergic rhinitis during the pollen season. Results showed a significant relationship between a high concentration of ECP (median: 410 ng/g, range: 6–2380 ng/g) and a high percentage of eosinophils (median: 13.5%, range: 1–85%). This quantitative study again demonstrated that infiltration by eosinophils and release of ECP play a key role in allergic rhinitis. It also suggests that the combined measurement of the percentage of eosinophils together with the ECP concentration in nasal secretions seems to be a very useful model in monitoring and assessing the condition of chronic nasal inflammation in patients with allergic rhinitis.

Use of Likelihood Ratios Improves Interpretation of Laboratory Testing for Pulmonary Sarcoidosis

Laboratory tests for pulmonary sarcoidosis (percentage lymphocytes and CD4/CD8 ratio in bronchoalveolar lavage fluid and serum angiotensin-converting enzyme activity) lack sensitivity and specificity. In a retrospective study of 153 subjects under suspicion of pulmonary sarcoidosis (36 cases and 117 patients with other diseases [control patients]), we defined likelihood ratios (LRs) for rationally selected result intervals of these tests, which improve clinical interpretation as compared with dichotomous interpretation based on a single cutoff value. By using logistic regression analysis, we further integrated the 3 individual tests into a unified algorithm that could rule out diagnosis in 57 (48.7%) of the 177 control subjects and confirm diagnosis in 12 (33%) of the 36 pulmonary sarcoidosis cases. We conclude that use of LRs improves interpretation of laboratory tests for pulmonary sarcoidosis. In addition, we present a prediction algorithm based on the combination of laboratory tests that helps clinicians confirm or exclude diagnosis in almost half of the study population.

Reference values for new red blood cell and platelet parameters on the Abbott Diagnostics Cell-Dyn Sapphire

1 Department of Haematology UZ Brussel, Brussels , Belgium 2 Department of Medicine , UZ Brussel, Brussels , Belgium Keywords: ageing; normal values; red blood cells; reticulo-cytes; variability. A set of new parameters became available by the recent soft-ware upgrade (version 4) on the Cell-Dyn Sapphire haema-tology cell counter (Abbott Diagnostics, Santa Clara, CA, USA). Some of these parameters are linked with the size of red blood cells, such as the percentage of microcytic (pMIC) and macrocytic (pMAC) red blood cells. The percentage of hypochromic (pHPO) and hyperchromic (pHPR) erythro-cytes and haemoglobin distribution width (HDW) refl ect the haemoglobin (Hb) concentration of the cells. In addition, the mean corpuscular volume (MCVr), mean content of haemo-globin (MCHr) and concentration of haemoglobin (CHCr) of reticulocytes are measured. Finally, the percentage of reticulated platelets (pRP) is determined. Homologues of some of these parameters, determined on other analysers, have been shown to be clinically useful in the assessment of different conditions. In patients undergoing haemodialy-sis the European Best Practice Guideline uses pHPO and MCHr for assessment of anaemia due to iron defi ciency (1) . Although pHPO seems one of the best predictors of iron defi ciency (2) , the US Kidney Disease Outcomes Quality Initiative (KDOQI) only recommends MCHr because pHPO is very sensitive to ageing of the sample (3) , due to a swell-ing of the red blood cells. The impact of ageing seems to be different according to the technology used, as suggested for the percentage of hypochromic red blood cells by Buttarello et al. (4) . A rise of the percentage of reticulated plate-lets (pRP) precedes that of the platelet count after a bone marrow graft (5) . In this study, a complete peripheral blood count, includ-ing reticulocyte parameters, was performed on 151 healthy age of 26.0 years (interquartile range of 22.2 – 38.0). The large majority of the volunteers (144/151) were of Caucasian eth-nicity. Reference values were defi ned as the 2.5th and 97.5th percentiles of the results according to CLSI guidelines (6) . Outliers were removed when the difference with the median value exceeded at least 2.5 times the interquartile range. In addition we determined the reproducibility for these para-meters and studied the effect of ageing of the sample at room temperature on the test results. Reference values for a population of healthy adults are given in Table 1

Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma

Mobilized CD34+ blood cells were immunomagnetically enriched from leukapheresis products in five multiple myeloma (MM) patients. Thawed samples of selected CD34+ cells were cultured for up to 21 d in a liquid and stroma‐free culture system with different combinations of recombinant cytokines. The most successful cell expansion was obtained when a combination of rh‐IL‐1β, rh‐IL‐3, rh‐IL‐6, rh‐SCF, rh‐G‐CSF and rh‐GM‐CSF was used. After 14 d this mixture gave a 120–187‐fold overall increase of total nuclear cells and a 4–8‐fold overall increase of early CFU‐GM numbers. In four patients a very sensitive patient‐specific PCR analysis showed the presence of monoclonal cells in the initial leukapheresis products. After immunomagnetic separation a tumour cell depletion of 2–4 logs was observed, although all samples still contained malignant cells. Cell suspensions that were cultured with the most potent cytokine combination showed tumour contamination in two‐thirds of evaluable cases at the moment of maximal CFU‐GM output. Serial cDNA dilution experiments indicated that the positive PCR results at day 14 reflected the persistence of pre‐culture tumour cells rather than in vitro expansion of tumour cells in two cases. This study demonstrates that ex vivo expansion of myeloid precursor cells from mobilized CD34+ cells in MM patients does not always result in an effective purging of residual tumour cells. On the other hand, our culture conditions do not seem to favour in vitro expansion of malignant cells, despite the use of a cytokine cocktail that includes potential myeloma growth factors.

Aging-associated subpopulations of human CD8+ T-lymphocytes identified by their CD28 and CD57 phenotypes.

BACKGROUND During organismal aging, human T-cells shift towards less functional phenotypes, often called senescent cells. As these cells have not been well characterized, we aimed to relate surface markers of human T-cell senescence with characteristics of in vitro cellular aging and to further characterize these cells. METHODS We identified, by flow cytometry, subpopulations of CD8+ T-cells based on CD57 and CD28 expression, and tested them for some markers of cellular senescence, apoptosis, differentiation and homing. RESULTS Elderly persons presented significantly higher proportions not only of CD28-CD57+, but also of CD28+CD57+ cells. CD28+CD57+ cells had the highest expression of p16, p21, Bcl-2, CD95, CD45RO, CCR5 and PD-1, thereby arguing in favor of a senescent phenotype. CONCLUSION Among CD8+ T-lymphocytes, CD28+CD57+ cells represent a subset with some senescent features that are distinct from the CD28-CD57+ cells.

Different expression of adhesion molecules on myeloid and B-lymphoid CD34+ progenitors in normal bone marrow

Abstract:  The expression of adhesion molecules was studied on B lymphoid and myeloid CD34+ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34+ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B‐lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34+ cells in the 3 subpopulations expressed VLA‐4, VLA‐5, LFA‐3 and H‐CAM. B‐lymphoid progenitors showed a higher density of VLA‐4 and VLA‐5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B‐lymphoid progenitors. The majority of the CD34+ cells also expressed LFA‐1 and L‐selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L‐selectin. We conclude that B lymphoid and early and late myeloid CD34+ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34+ precursors to circulate.