Combined evaluation of P-glycoprotein expression and activity in childhood acute leukaemia contributes to the identification of patients at risk for relaps

Swerts K. (1,2), De Moerloose B. (1), Dhooge C. (1), Laureys G. (1), Steyaert S. (2), Benoit Y. (1) and Philippé J. (2).
Department of Pediatrics (1), Ghent University Hospital, Ghent, Belgium Department of Clinical Chemistry, Microbiology and Immunology (2), Ghent University Hospital, Ghent, Belgium.

The multidrug resistance protein, P-glycoprotein (P-gp), plays an important role in chemotherapy failure. Studies evaluating the clinical significance of P-gp in childhood acute leukaemia, are hampered by a lack in standardisation of detection techniques and methodologies, leading to divergent data. International workshops have recommended single - cell assays, complemented by PCR or functional tests. The aim of this study was the comparison of two single - cell assays and one functional test in childhood leukaemia. Samples of 103 children (59 de novo ALL, 24 relapse ALL and 20 ANLL) were studied immunocytochemically and/or flow cytometrically using two monoclonal antibodies. P-gp activity was determined by means of rhodamine 123 and the P-gp inhibitor verapamil. The statistical difference in P-gp positivity between ALL en ANLL was noticed for the functional test. The inter - assay agreements between immunocytochemistry and the flow cytometric tests were poor; between both flow cytometric assays only fair. The probability of experiencing a relapse was highest for the ALL patients with a positive functional test result at initial diagnosis. Fifty-seven percent of ALL patients scoring positive for both immunocytochemistry and functional flow cytometry relapsed, while none of those scoring negative for both assays did. Form this study, it is clear that the combined use of immunocytochemistry and a functional flow cytometric test in ALL accurately determines P-gp and identifies patients at higher risk for relapse.

Quantification of the Leukocyte Common Antigen (CD45), FSC and SSC in mature B-cell malignancies.

Hendrickx A and Bossuyt X
Department of Laboratory Medicine, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium.

Aims: To investigate whether the CD45 antigen level, FSC and SSC differ between normal and mature neoplastic B-cells. Material and methods: Bone marrow and peripheral blood from 34 patients with mature B-cell disorders and peripheral blood from 15 healthy adults were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with peridinyl chlorofylline (PerCP)-conjugated CD45 monoclonal antibodies (Becton Dickinson Immunocytometry Systems, CA). Standard microbeads (SpheroTM Califlow kit, Spherotech, IL) with different fluorescence intensity were used to calibrate the instrument for fluorescence intensity (relative fluorescence intensity (RFI)). Results: When compared to the CD45 expression on normal lymphocytes, the CD45 expression on neoplastic B-cells was significantly lower in patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and was significantly higher in patients with hairy cell leukemia (HCL). In patients for which, based on specific markers, the malignant population could be separated from the normal population, the mean (SD) CD45 RFI on normal cells and on malignant cells was respectively, 77629,5 (9298) and 26557 (9060.5) for CLL (n=6), 69887 (3673) and 30288 (6294) for MCL (n=4), 71367 (3984) and 84991 (10155) for HCL (n=7). In CLL patients (n=17) for which the malignant population could not be separated from the normal population, there was an inverse correlation between the CD45 expression on the total lymphocyte population and the percentage of malignant cells in this population. In healthy adults, the CD45 expression in lymphocytes (n=15) was 83232 +/-18898 RFI. When compared to normal cells, both FSC and SSC were significantly higher for hairy cells and significantly lower for CLL cells. For MCL, no difference was found. Conclusions: Quantitative determination of CD45 together with the SSC and FSC may provide additional useful diagnostic information to characterize mature B-cell malignancies.

Biased T cell receptor variable gene usage in activated T lymphocytes from cerebrospinal fluid in multiple sclerosis patients.

Van der Aa A, Medaer R, Raus J and Stinissen P

In vivo activated CD4+ T cells in the cerebrospinal fluid (CSF) of Multiple Sclerosis (MS) patients may play a role in the disease process. We analyzed the phenotypic characteristics and the T cell receptor (TCR) expression of CSF T cells from 6 MS patients and 3 control patients with non-inflammatory neurological diseases (OND). Fresh CSF cells were plated in a single microwell in medium containing autologous serum, IL-2 and irradiated autologous feeder cells. The cells were restimulated 7 days later, and after a total culturing period of 16 to 22 days, the cells were analyzed for their phenotype by flow-cytometry. The TCR BV (beta variable) gene expression was studied by semi-quantitative PCR-ELISA. CDR3 spectratyping and direct sequencing were used to provide information about the clonality of preferentially used BV genes. The majority of the expanded CSF cells were CD3+ TCRab+ T cells. TCR profile analysis showed a rather restricted BV gene expression pattern in the expanded T cells from all subjects, but the overrepresented BV genes (>15% of the total BV repertoire) differed between the patients. CDR3 spectratyping and direct sequencing were then used to analyse the clonal heterogeneity of overrepresented BV families. This analysis revealed that the overrepresented BV genes were mainly of mono- or oligoclonal origin. Interestingly, mono- and oligoclonal T cell populations were observed among CSF T cells from MS patients but also among CSF T cells from control patients. In conclusion, the restricted TCR expression profiles indicate that only a limited number of clonal origins are represented among the in vivo activated T cells in the CSF of MS patients and control OND subjects.

Evaluation of monensin and brefeldin A for flow cytometric determination of IL-1-b, IL-6, TNF-a in monocytes.

Schuerwegh AJ, Stevens WJ, Bridts CH, De Clerck LS.
Department of Immunology, Allergology and Rheumatology. University of Antwerp, UIA, Antwerp, Belgium.

Background
Recently, flow cytometry became a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate blocking of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the blocking capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (IL-1b, IL-6, TNF-a) within peripheral blood monocytes.
Methods
A two colour flow cytometric technique was used to measure intracellular spontaneous and LPS-stimulated IL-1b, IL-6, TNF-a production in monocytes (CD14+) of whole blood cultures of 5 rheumatoid arthritis patients and 5 sex and age matched controls.
Results
The viability of monensin treated monocytes was slightly lower than brefeldin A treated monocytes. The percentage of IL-6 and TNF-a producing monocytes after 8 hours of culture without stimulation revealed lower values for monensin blocked than for brefeldin A blocked monocytes in controls and patients whereas the percentages for stimulated cells did not differ. The spontaneous production of IL-1b, IL-6, TNF-a after 8 hours of culture was higher in brefeldin A treated than in monensin treated monocytes of RA patients. For the LPS-stimulated production of IL-1b, IL-6 and TNF-a, there was a increased level of cytokine production in brefeldin A blocked monocytes in both controls and RA patients.
Conclusions
In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1b, IL-6, TNF-a) in whole blood cultures, brefeldin A is a more potent, effective and less toxic inhibitor of cytokine secretion than monensin.

Resistance to HIV infection in HIV-Exposed Uninfected Female Sex Workers in Abidjan is not associated with decreased CCR5 expression or increased b-chemokine expression.

Jennes W.(1), Sawadogo S.(2), Koblavi-Dème S.(2), Vuylsteke B.(1,2), Maurice C.(2), Roels TH.(2,3), Chorba T.(2,3), Nkengasong JN.(2,3), Kestens L.(1)
1. Institute of Tropical Medicine, Department of Microbiology, Antwerp, Belgium, 2. Project Retro-CI, Abidjan, Côte d'Ivoire, 3. CDC, Division HIV/AIDS prevention, Atlanta, USA.

Introduction: Cellular resistance to infection with HIV may be determined by low HIV-1 coreceptor expression and high intracellular b-chemokine expression. The objective was to compare the expression of HIV-1 coreceptors and b-chemokines in female Blood Donors (BDs), HIV-Exposed Uninfected (EU) and HIV+ Female Sex Workers (FSWs) in Abidjan.
Methods: From November 1999 until May 2000 blood samples were analysed from 26 EU FSWs, 23 HIV+ FSWs and 50 BDs. FSWs were enrolled consecutively at a confidential clinic. The cell surface expression of HIV-1 coreceptors CCR5 and CXCR4 was determined using whole blood and measured by flow cytometry on CD4+/CD45RO+ memory and on CD4+/CD45RO- naïve lymphocytes. Constitutively expressed RANTES (without stimulation) and activation-induced expression of MIP-1a and MIP-1b were measured intracellularly in CD4+ and CD8+ T cells by flow cytometry.
Results: The expression of CCR5 did not differ between EU FSWs and BDs. The expression of CXCR4 was significantly lower in EU FSWs as compared to BDs on both CD4+ memory cells and on CD4+ naïve cells. The same decrease in CXCR4 expression was found in HIV+ FSWs. The intracellular expression of the b-chemokines RANTES, MIP-1a and MIP-1b was similar in EU FSWs and BDs in both CD4+ as CD8+ T cells. In contrast, HIV+ FSWs had significantly increased levels of MIP-1a and MIP-1b in CD4+ and CD8+ T cells and of RANTES in CD8+ T cells.
Conclusions: Protection to HIV infection in EU FSWs in Abidjan is neither related to decreased expression of HIV-1 coreceptor CCR5 nor to increased expression of b-chemokines. EU FSWs display a statistically significant decrease in the expression of CXCR4 for which the physiological significance remains unclear.

Evaluation of Immunostaining Methods for Platelet Counting

Jurdan M., Gérard L., Goffaux M., Cornet Y., Cauchie P., Chatelain C., Chatelain B.
Laboratory of Hematology, Catholic University of Louvain, Mont-Godinne, 5530 Yvoir, Belgium

The automated platelet count is sometimes arduous. Interferences can be observed mainly with samples from thrombocytopenic patients. Therefore we have tested 4 immunostaining methods (manual and automated) including flow cytometry and immunolabelling on blood films using a monoclonal antibody against GP IIIa(CD61) combined or not with a monoclonal antibody against GP IIb/IIIa(CD41). Flow cytometry was performed on Becton Dickinson FacsCalibur (FC) as a manual method and on Abbott Cell-Dyn 4000(CD4000) as an automated one. Counting on immunostained blood films was performed with a manual method(ICCM) and automatically on Olympus Provis Microscope(ICCP). Coulter Gen'S has served as reference after checking the correlation with phase contrast microscopy(n = 22, r = 0.973). A total of 62 samples have been studied including normal and abnormal platelet counts. 6 samples were eliminated from the correlations as an interference was detected with Coulter Gen'S and controlled microscopically(giant platelets (2), platelet aggregates(2), schistocytes(3)). None of the tested methods produces results statistically different from Coulter Gen'S(p>0.05) : Yet, immunostaining on blood films allows a direct and objective control of interferences.

CD11b expression on myeloid cells isolated from bovine bone marrow

Van Merris V, Meyer E, Heyneman R and Burvenich C
Ghent University, Faculty of Veterinary Medicine, Department of Physiology and Biochemistry.

Introduction - Inflammation of the mammary gland (mastitis) causes considerable losses in the dairy industry. For the effective defense against invading udder pathogens, circulating polymorphonuclear leukocytes (PMN) need to migrate into the udder. The increased susceptibility to mastitis observed in the early postpartum period is related to impaired PMN functions. During this period, high numbers of immature PMN are found in circulation. These immature neutrophils have a decreased CD11b (CR3-1) receptor expression. The CD11b molecule is an essential part of the CD11b/CD18 (Mac-1) receptor, which plays a key role in PMN adhesion to endothelium, phagocytosis and respiratory burst.
Materials & Methods - In order to study the expression of CD11b during bovine myeloid differentiation, we developed a three-layer discontinuous gradient centrifugation to fractionate the heterogeneous cell population into three maturational stages. Isolated cells consecutively were incubated with an anti-bovine CD11b mAb and an FITC-labeled secondary antibody. Flow cytometric analysis in combination with fluorescence microscopy were used to evaluate myeloid CD11b expression.
Results - Early (myeloblasts and promyelocytes) and late (myelocytes and metamyelocytes) myeloid cells were selectively retrieved from both interfaces. Bands and segmented cells (mature PMN) were present in the high-density pellet. The number of fluorescence-positive cells, expressing CD11b, increased with maturational stage, being maximal in the mature myeloid population. Microscopic data have shown that the increase in CD11b-positivity is probably related to the maturation-dependent expression of CD11b.
Conclusion - The optimized Percoll separation method allows the enrichment and partial purification of bovine myeloid precursors according to their maturity. These preliminary results provide a basis for further investigation of the relevance of CD11b expression on bovine progenitor cells in relation to mastitis.

This study was supported by the Flemish Institute for the Encouragement of Research in the Industry (IWT-grant SB/991161) and the Fund for Scientific Research (FSR-grant 3G008699).

Study of Ca²⁺ fluxes in IFN-gamma -exposed T-cells of patients with Multiple Sclerosis and Control Subjects.

Buntinx M, Ameloot M, Steels P, Medaer R, Geusens P, Raus J and Stinissen P
Biomedisch Onderzoeksinstituut DWI, Limburgs Universitair Centrum, B-3590 Diepenbeek, Belgium

Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. T-lymphocytes are important mediators in the pathogenesis of MS. Their activity is modulated by a complex network of cytokines, in which interferon-gamma (IFN-gamma) is considered essential. We investigated the presence of an IFN-gamma-activated Ca²⁺-influx in T-cells of MS patients and control subjects. The study group (n=63) consisted of patients with stable and active RR-MS and CP-MS. Stable and active rheumatoid arthritis (RA) patients, flu patients and normal subjects (NS) were studied as controls. T-cells were isolated from PBMC by removing B cells and monocytes using anti-CD19 and anti-CD14 monoclonal antibodies and dynabeads. The depletion was confirmed by flow cytometry. T-cells loaded with the Ca²⁺-indicator FURA-2 AM were consecutively incubated in Hank's Balanced Salt Solution (2 mM Ca²⁺), HBSS supplemented with 3 mM EGTA, HBSS + 1 pg/ml IFN-gamma +3 mM EGTA and finally in HBSS + 1 pg/ml IFN-gamma. We used fluorescence imaging microscopy to measure the intracellular [Ca²⁺]i. No [Ca²⁺] increase was observed in Ca²⁺-free medium. As T-cells were incubated in Ca²⁺-containing medium, we observed a Ca²⁺-influx peak within 3 min, which dropped to a specific [Ca²⁺]i-level depending on the patient's diagnosis. Peak values observed within three minutes after Ca²⁺-reintroduction were higher in patients than in NS. The peak size is probably controlled by CRAC-channels. Experiments with and without IFN-gamma stimulation show that IFN-gamma induces a sustained higher [Ca²⁺]i in RA and MS patients but not in NS and flu patients. We defined D-influx for each individual cell as the difference between the [Ca²⁺]i 6-10min after Ca²⁺-reintroduction and the initial [Ca²⁺]i. ANOVA for nested unbalanced three-factor design was applied on the D-influx dataset. The Ca²⁺-influx in T-cells of MS and RA patients was significantly higher than in NS. The majority of stable MS patients showed lower Ca²⁺-influxes compared to active MS patients. We showed that T-lymphocytes from (active) MS and RA patients are more susceptible to IFN-gamma-induced activation. Our data argue for an increased activation status of T-cells in autoimmune disorders like MS and RA.

Homing and differentiation potentials of mature (CD45-) and immature (CD45+) malignant plasma cells in a murine model of multiple myeloma

Asosingh K.(1), De Raeve H.(2), Croucher P.(3), Goes E.(4), Van Riet I.(1), Van Camp B.(1), Vanderkerken K.(1).
1.Department of Hematology and Immunology, 4.Department of Radiology, Free University Brussels, Brussels, Belgium. 2.Department of Pathology, University Institute Antwerp, Antwerp, Belgium. 3.Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Hill Sheffield, UK.

Introduction: Multiple myeloma (MM), a plasma cell malignancy, is predominantly localized in the bone marrow (BM). These tumoral cells have a heterogeneous expression of CD45. It is however unclear which subset is responsible for the homing and outgrowth of the MM cells in the BM. In this work we investigated the in vivo homing, proliferation and differentiation of both CD45+ and CD45- cells in two murine MM models. Materials and Methods: 5T2MM and 5T33MM in vivo lines of murine MM were used. CD45 and IGF-I receptor expression was analyzed by FACS. Proliferative capacity was measured by in vivo bromodeoxyuridine incorporation. 5TMM cells were sorted into CD45+ and CD45- fractions by MACS. Initial homing was analyzed in vivo by tracing of radioactively labeled cells. MM cells were detected by FACS and histology. Osteolytic lesions were analyzed by radiography. Results: Both CD45+ and CD45- 5TMM cells were able to migrate to the BM, although the homing of the latter subset was lower which was related to a low IGF-I receptor expression. Recipients of both fractions developed MM as evidenced by the presence of serum paraprotein, osteolytic lesions and BM infiltration by MM cells. The tumor load in the recipients of CD45- cells was higher than the CD45+ cells which could be explained by a lower proliferation rate of the latter population. While the separated cells before injection had a homogenous expression of CD45, cells isolated from the BM of these terminally diseased mice had a heterogeneous expression pattern, indicating an in vivo differentiation of CD45- to CD45+ cells and vice versa. Conclusion: We here conclude that both CD45+ and CD45- 5TMM subpopulations contain clonogenic MM cells with BM homing and proliferative capacity.

Identification of lymphocytes, macrophages, neutrophils and apoptotic cells in bovine milk using a simple, rapid flow cytometric method

Dosogne H., Vangroenweghe F., Massart-Leën A.-M., Burvenich C.
Ghent University, Faculty of Veterinary Medicine, Department of Physiology and Biochemistry

Introduction: The number of cells in milk (somatic cell count, SCC) is widely used as a parameter for bovine udder health because an inflammatory reaction of the udder (mastitis) is typically associated with an increased SCC. Although the importance of blood PMN in mastitis is generally accepted, the immunological significance of the resident cells in milk is poorly understood. Different identification methods for the cells in milk have been used, but there is need for an accurate and reproducible standard technique. The purpose of this study was to develop a simple, rapid flow cytometric standard procedure in which the major cell types in milk can be identified. Material and methods: The live cell permeable DNA staining dye SYTO®13 (Molecular Probes) was used to label milk cells. The method was validated with flow cytometric cell sorting, light and confocal laser scanning microscopy. The method was optimized for milk samples with a low SCC. Results: Five different populations in the side scatter/green fluorescence dot plot following SYTO®13 staining were identified using flow cytometry. Following cell sorting, these populations were characterized as large and small lymphocytes (L), polymorphonuclear neutrophils (PMN), macrophages (MF) and apoptotic cells. That apoptotic cells contribute to the SCC was demonstrated in a 3-days experiment where it was observed that, although the SCC remained very constant, this particular population significantly increased. These findings indicate that in the currently used method for determination of SCC both living and apoptotic cells are quantified. The optimal temperature for sample preservation and processing was 20_C and the optimal dilution for milk samples was 37.5 % with PBS. Conclusions: A rapid, simple, accurate and reproducible standard procedure was developed in order to identify MF, PMN, L and apoptotic cells in milk based on DNA staining with the live-cell permeable dye SYTO®13.

This study was supported by the Belgian Ministry of Small Enterprises and Agriculture (S/5871) and by the Belgian Fund for Scientific Research (grant nr. 31504200). The assistance of Prof. J. Plum with cell sorting is greatly appreciated.

Cytokinin analogues : new CDK inhibitors with anti-proliferative effect and induction of apoptosis.

Vermeulen K.(1), Strnad M.(2), Van Onckelen H.A.(3), Lenjou M.(1), Nijs G.(1), Van Bockstaele D.R.(1), Berneman Z.N.(1).
1.Laboratory of Experimental Hematology, Antwerp University Hospital (UZA/UIA), Antwerp, Belgium. 2.Institute of Experimental Botany, Departement of Plant Biotechnology, Olomouc, Czech Republic. 3.Departement of Biology, Antwerp University (UIA), Antwerp, Belgium.

Introduction The development of olomoucine, a C2-, C6-, N9-trisubstituted purine with homology to plant cytokinins and one of the first synthetic cyclin dependent kinase (CDK) inhibitors, has stimulated the search for more chemical CDK inhibitors. Material and Methods and Results Fifteen novel cytokinin analogues were screened for their inhibitory effect on CDK1/CDK2, and these 15 new and 8 known compounds were also screened for effect on the growth of leukemic cell lines. Ten new compounds exhibit stronger inhibitory capacity on CDK1 and CDK2 than olomoucine. Proliferation of the KG1 myeloid leukemia cell line was strongly inhibited by 14 out of 23 tested cytokinin analogues. Proliferation of the Molt3 T-lymphoblastic leukemia cell line was strongly inhibited by 15 cytokinin analogues. The most effective products were purines with an isopropyl group at N9 and a benzylamino- or hydroxybenzylamino-substituent at C6. Substitution of the 6-benzylamino by an extra hydroxyl increased inhibitory activity on CDK and on cell proliferation. Not only previously described C2-substituents but also new ones (amino-, cyano-, thiol-containing or heterocyclic C2 substituents) were associated with cytokinin analogue activity. Effect on cell proliferation was not clearly correlated with effect on CDK. Incubation of cell lines with cytokinin analogues resulted in apoptosis as indicated by morphological changes (after Hoechst 33342 / ethidium bromide staining) and DNA fragmentation (TUNEL technique). Induction of apoptosis, investigated using the Fas receptor blocking antibody ZB4, occurs by a Fas-independent mechanism, while a time-dependent drop in mitochondrial potential (detected with the ??-sensitive probe JC-1) demonstrated mitochondrial involvement. Inhibition of cytokinin analogue-induced apoptosis by the pan-caspase inhibitor Z-VAD.FMK indicated that apoptosis was mediated by caspases. Also, downstream activation of caspase-3 was detected by flow cytometry using an antibody directed against active caspase-3. Conclusion The role of CDK inhibition in induction of apoptosis is now under investigation. The development of now more than twenty compounds with a similar structure and with CDK and cell proliferation activity strongly suggests that we are dealing with a new class of cytostatic agents and we propose to designate them as cytokinin analogues.

Real-time PCR for the measurement of porcine cytokine mRNA.

Herman Ph*, Vaerman JL**, Gianello P $, Latinne D *
IMEX*, SANG**, CHEX$ Units, UCL, 30 clos chapelle-aux-champs, 1200 Brussels

Introduction : The quantification of cytokine mRNA could be of interest in various immunological situations such as inflammatory process, infection or organ transplantation follow-up. Cytokine measurement assays at the level of the protein (ELISA, ELISPOT, Flow Cytometry) are largely described and used in mouse and human models. However, monoclonal antibodies directed against porcine cytokine are not yet available. In order to study "Th1/Th2" cytokine gene expression we have developed a real-time RT-PCR assay to quantify porcine cytokine mRNA. Starting from minute amount of cDNA, this assay allows the measurement of housekeeping (HPRT) and cytokine (Interleukin (IL)2, Interferon-gamma (IFNg), IL4, IL10, IL12p40 and Transforming growth factor-beta1 (TGF-b1)) gene expression.
Materiel and methods : Primers and fluorescent probes were designed by using primer express software (PE biosystems). Primer pairs were chosen to avoid genomic DNA amplification. Total RNA was extracted (Trizol, Gibco Brl) from 107 cultured cells, peripheral blood MNC or liver fine needle biopsies. One µg of RNA was reverse transcribed (M-MLV, Gibco). An equivalent amount of 25 to 50 ng of total RNA was used per PCR. Purified (Concert, Gibco) RT-PCR amplicons for each gene of interest was defined as standard (std). Serial dilutions of known number of copies from std were submitted to PCR for calibration curves construction. HPRT and Cytokine mRNA was quantified using either SybrGreen I reagent (PE) or 5'-exonuclease assay (5'FAM - 3'TAMRA labelled oligonucleotide probes).
Results and Conclusion : We describe here (1) The use of purified RT-PCR products as external standards, (2) the analysis of housekeeping and 6 cytokine gene expression in a single 96-well plate, with a dynamic range of 105 to 101 copies (3) the validation of a real-time RT-PCR assay for porcine cytokine gene expression measurement.

Identification, viability and apoptosis of bovine milk cells: influence of temperature

Vangroenweghe F, Dosogne H, Van Oostveldt K, Burvenich C
Ghent University, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry and Biometrics, Salisburylaan 133, 9820 Merelbeke

Introduction - Mastitis is an important health issue in animal husbandry. The control of mastitis is mainly monitored through counting of cells in milk (somatic cell count, SCC). However, more and more the function of these cells is taken into account. The purpose of this study was to determine the influence of milk sample collection and processing temperature on the characterisation and function of isolated bovine milk cells. Material and methods - Six high yielding Holstein cows (milk production: 28.6 &plusmin; 1.0 l/d), free of udder infection, in mid-lactation stage were used. On collection, each milk sample was divided into three equal parts. After collection, the milk samples were divided in 3 parts and were transported and processed at 4, 20 and 37 C. After dilution of milk (3/5; v/v) with PBS, milk cells were isolated by centrifugation (1000 g, 15 min) at each specific temperature. The identification of bovine milk cells was carried out by flow cytometry using SYTO®13 (Molecular Probes) and the viability of milk PMN was determined using propidium iodide (PI) exclusion. Apoptosis of bovine PMN was determined using Annexin-V-FITC and PI. Results - The optimal temperature for identification of milk cells was 20 C. The viability of milk PMN was sigificantly higher at 20 C compared to 4 C (P < 0.05). No significant differences in viability were found between 4 C and 37 C and between 20 C and 37 C. The degree of apoptosis of milk PMN was highest at 37 C compared to 4 C and 20 C (P < 0.01). No significant differences in the degree of apoptosis were found between 4 C and 20 C. The percentage of living PMN was also significantly higher at 20 C compared to 4 (P < 0.05) and 37 C (P < 0.01). In cows with a high SCC (> 150,000 cells/ml), a significant higher proportion of living PMN cells was found compared to cows with a low SCC (< 75,000 cells/ml). Conclusions - Standardisation of milk sampling procedures for the determination of milk cell functions is more important than for counting. The most optimal temperature for the evaluation of the milk cell characteristics was found to be 20 C. At this temperature, viability was high and apoptosis was low. Additionally, the amount of debris was minimal, resulting in a better identification of the cell types.

This study was supported by the Belgian Ministry for Agriculture and Small Enterprises (grant S/5871) and the National Fund for Scientific Research (FWO, grant 31504200).

Upregulation of VLA-5 during cell cycle transit prevents effective migration of CD34+ cells through fibronectin-coated filters.

Giet O, Huygen S, Beguin Y And Gothot K.
Dpts. Of Clinical and laboratory Hematology, University of Liège, Belgium.

Several reports have provided evidence that engraftment of transplanted hematopoietic stem/progenitor cells is decreased during cell cycle transit. This prompted us to study cell cycle-associated changes in adhesion and migration of primitive progenitor cells to extracellular matrix proteins after 2-day ex-vivo culture in SCF, FL and TPO. In previous studies, we demonstrated that VLA5 expression of CD34+ cells was modulated by cell cycle transit and mediated increased adhesion to fibroneclin after ex vivo culture. In the next step, we examined whether the increase in VLA5 mediated adhesion to Fn had any impact on transmigration. Migration was assayed by placing cells onto 5 µm pore filters in the upper chamber of a Transwell and measuring % cells present in the lower chamber after 3 hours. Spontaneous migration towards control medium through bare filters was observed in 5% of the cells. An additional 15% of input cells migrated when the filter was coated with Fn. When conditloned medium (CM) from the stromal-derived factor-1 (SDF-1) producing cell line MS-5 was placed in the lower chamber, the percentage of migrating (Mg) cells rose to 42%. Migration was dependent on the presence of chemokine receptor CXCR4, as 48% of Mg cells were CXCR4+ versus 20% of non migrating (NMg) cells (n=4; P<.05). When the Transwell filter was coated with BSA, the percentage of CD34+ cells in S/G2+M was similar in Mg and In NMg fractions towards MS-5 CM. In contrast, when the Transwell filter was coated with Fn, the Mg fraction was relatively depleted in cycling cells (33% of Mg cells in S/G2+M versus 41% of NMg cells in SIG2+M1 n=5; P<.05). VLA-5 expression was measured in Mg and NMg cells, and also in cells firmly attached to the filter alter the 3-hour migration assay. Interestingly, VLA-5 expression was minimal in NMg cells, intermediate in Mg cells, and maximal in cells adhering to the Transwell filter. Collectively, these results suggest that SDF-1 displays a cell cycle selectivity when cells have to cross a Fn-coated barrier during transmigration. In this case, moderate expression of VLA-5 facilitates transmigration while maximal VLA-5 expression in cyding cells confers a full adhesive state and prevents or delays effective transmigration.

Monitoring CMV-specific CD8+ T lymphocytes using tetramers: identification of patients at risk for CMV disease after T- cell depleted stem cell transplantation.

Gratama JW, van Esser J, Tournay C, Bolhuis R, Cornelissen J.
Departments of Clinical and Tumor Immunology, and Hematology, University Hospital, Rotterdam, the Netherlands; and Immunotech S.A., Marseille, France.

Recovery of cytomegalovirus (CMV-speciflc) T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. We used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV peptide (NLV) derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 24 HLA-A2+ patients, and to monitor the recovery of these T cells during the first 12 months post SCT. None of the 9 CMV-seronegative patients became infected with CMV, while 13 of 15 CMV-seropositlve patients developed CMV antigenemia post SCT. CMV seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with gancyclovir (GCV) than those of grafts from CMV seropositive donors (3 [1 to 6] vs. I [0 to 3] courses, respectively; P = 0.02). The numbers of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.62; P =0.01). None of 9 CMV seronegative patients mounted a CMV-speclfic immune response as measured by monitoring A2-NLV-specific CD8+ T cells post SCT. All 11 CMV seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells post SCT (P <0.001). Thus, enumeration of HLA-restrlcted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells post SCT is a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.

Analysis of HLA Class I expression in acute and chronic leukemia by serology, flow cytometry and molecular biology.

Maes P, Mulder A, Deneys V, Worsham M, Ferrone S, Claes F and Demanet C.
HLA-lab AZ-VUB, Brussels (B); Univ Med Center, Leiden (NL); UCL, Brussels (B); H Ford Hospital, DT, (USA); Roswell Park Cancer Inst, Buffalo, NY (USA).

Loss or down-regulation of HLA class I is well-documented in solid tumors and is considered to represent a means of escaping immunosurveillance. We studied HLA class I expression on acute (ALL-B, AML) and chronic (CLL-B) leukemia cells by CDC-serology (n=36), flow cytometry (n=44) and molecular biology. All samples were taken at diagnosis. The level of HLA expression on leukemic cells was compared to the remaining patients normal cells (CDC) or to third party normal counterparts (flow). A complete absence of one or two alleles was detected by CDC in 14% of the leukemic cells and confirmed by the use of allele specific mAb's. These selected cases were further sequenced for locus A and/or B, but no mutations were detected compared to the patients normal cells. This suggests a problem at the transcriptional level of HLA class I or other factors in antigen presentation. Flow cytometry showed a mean decrease of total HLA class I of 30% in CLL-B and 60% in ALL-B. In contrast, AML leukemic cells showed an upregulated class I expression. Our conclusions are that HLA class I aberrations in leukemia are more the rule than exception. Further more, our data suggest other mechanisms of altered expression in different types of leukemia.