Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A2 from Bothrops jararacussu.

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA(2) edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K(I)) for Lac01 and Lac02 were approximately 740 µM, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 µM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA(2) in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA(2).

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

High remission rate in T-cell prolymphocytic leukemia with CAMPATH-1H.

T-cell prolymphocytic leukemia (T-PLL) is a chemotherapy-resistant malignancy with a median survival of 7.5 months. Preliminary results indicated a high remission induction rate with the human CD52 antibody, CAMPATH-1H. This study reports results in 39 patients with T-PLL treated with CAMPATH-1H between March 1993 and May 2000. All but 2 patients had received prior therapy with a variety of agents, including 30 with pentostatin; none achieved complete remission (CR). CAMPATH-1H (30 mg) was administered intravenously 3 times weekly until maximal response. The overall response rate was 76% with 60% CR and 16% partial remission (PR). These responses were durable with a median disease-free interval of 7 months (range, 4-45 months). Survival was significantly prolonged in patients achieving CR compared to PR or no response (NR), including one patient who survived 54 months. Nine patients remain alive up to 29 months after completing therapy. Seven patients received high-dose therapy with autologous stem cell support, 3 of whom remain alive in CR 5, 7, and 15 months after autograft. Stem cell harvests in these patients were uncontaminated with T-PLL cells as demonstrated by dual-color flow cytometry and polymerase chain reaction. Four patients had allogeneic stem cell transplants, 3 from siblings and 1 from a matched unrelated donor. Two had nonmyeloablative conditioning. Three are alive in CR up to 24 months after allograft. The conclusion is that CAMPATH-1H is an effective therapy in T-PLL, producing remissions in more than two thirds of patients. The use of stem cell transplantation to consolidate responses merits further study.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

The PML-RAR alpha transcript in long-term follow-up of acute promyelocytic leukemia patients.

BACKGROUND AND OBJECTIVES: Detection of PML-RAR alpha transcripts by RT-PCR is now established as a rapid and sensitive method for diagnosis of acute promyelocytic leukemia (APL). Although the majority of patients in long-term clinical remission are negative by consecutive reverse transcription polymerase chain reaction (RT-PCR) assays, negative tests are still observed in patients who ultimately relapse. Conversion from negative to positive PCR has been observed after consolidation and found to be a much stronger predictor of relapse. This study reports on 47 APL patients to determine the correlation between minimal residual disease (MRD) status and clinical outcome in our cohort of patients. DESIGN AND METHODS: The presence of PML-RAR alpha t transcripts was investigated in 47 APL patients (37 adults and 10 children) using a semi-nested reverse transcriptase-polymerase chain reaction to evaluate the prognostic value of RT-PCR tests. RESULTS: All patients achieved complete clinical remission (CCR) following induction treatment with all-trans retinoic acid (ATRA) and chemotherapy (CHT) or ATRA alone. Patients were followed up between 2 and 117.6 months (median: 37 months). Relapses occurred in 11 patients (9 adults and 2 children) between 11.4 and 19 months after diagnosis (median: 15.1 months) while 36 patients (28 adults and 8 children) remained in CCR. Seventy-five percent of patients carried the PML-RAR alpha long isoform (bcr 1/2) which also predominated among the relapsed cases (9 of 11) but did not associate with any adverse outcome (p= 0.37). For the purpose of this analysis, minimal residual disease tests were clustered into four time-intervals: 0-2 months, 3-5 months, 6-9 months and 10-24 months. INTERPRETATION AND CONCLUSIONS: Children showed persisting disease for longer than adults during the first 2 months of treatment. At 2 months, 10 (50%) of 20 patients who remained in CCR and 4 (80%) of 5 patients who subsequently relapsed were positive. Patients who remained in CCR had repeatedly negative results beyond 5.5 months from diagnosis. A positive MRD test preceded relapse in 3 of 4 tested patients. The ability of a negative test to predict CCR (predictive negative value, PNV) was greater after 6 months (>83%), while the ability of a positive test to predict relapse (predictive positive value, PPV) was most valuable only beyond 10 months (100%). This study (i) highlights the prognostic value of RT-PCR monitoring after treatment of APL patients but only from the end of treatment, (ii) shows an association between conversion to a positive test and relapse and (iii) suggests that PCR assessments should be carried out at 3-month intervals to provide a more accurate prediction of hematologic relapses but only after the end of treatment.

BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL).

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods ('live gate') T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of 'empty spaces' (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in 'empty spaces' and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the 'empty spaces' can be easily identified in follow-up BM samples during and after treatment.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL.

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.

The nuclear topography of ABL, BCR, PML, and RARalpha genes: evidence for gene proximity in specific phases of the cell cycle and stages of hematopoietic differentiation.

The mechanisms whereby chromosomal translocations are consistently associated with specific tumor types are largely unknown. A generally accepted hypothesis is that the physical proximity of the involved chromosomal regions may be one important factor in the genesis of these phenomena. Accordingly, a likely possibility is that such a proximity may occur in a cell-lineage and cell-differentiation stage-specific manner. In this work, we have addressed this issue using as models the ABL and BCR genes of t(9;22) and the PML and RARalpha genes of t(15;17). By using in situ hybridization and confocal microscopy, we have measured the distances between these two pairs of genes in three-dimensionally preserved hematopoietic cells belonging to different cell lineages, at various stages of differentiation, and at various stages of the cell cycle, with the following results. (1) Intergenic distances vary periodically during the cell cycle and a significant association of ABL with BCR and of PML with RARalpha is seen at the transition between S and G2, which persists during G2 and prophase (such a behavior is not observed for distances between ABL or PML and the beta-globin genes, used as a control). (2) The proportion of cells in which PML and RARalpha or ABL and BCR are closely associated is higher in hematopoietic precursors than in B-lymphoid cells (whereas the distances between ABL or PML and the beta-globin genes are not affected by cell type). (3) When intergenic distances in unstimulated bone marrow CD34(+) cells were compared with those in CD34(+) cells treated with interleukin-3 (IL-3), a trend towards a higher proximity of the ABL and BCR genes in the former and of the PML and RARalpha genes in the latter is observed. (4) Analysis of B-lymphoid cells during mitosis shows that intergenic distances at metaphase are strongly influenced by physical constraints imposed by the chromosomal location of the gene, by the size of the respective chromosome, and by the geometry of the metaphase plate. These findings suggest that intrinsic spatial dynamics, established early in hematopoiesis and perpetuated differentially in distinct cell lineages, may facilitate the collision of individual genes and thus reciprocal recombination between them at subsequent stages of hematopoietic differentiation.

[The use of peripheral blood progenitor cells as an autologous hematopoietic support in high-dose chemotherapy. II. The experience of the Hematological Intensive Care Unit of the IPOFG. Franciso Gentil Portuguese Institute of Oncology]. / Utilização de células progenitoras do sangue periférico como suporte hematopoiético autólogo de quimioterapias de alta dose. II. Experiência da Unidade de Cuidados Hematológicos Intensivos do IPOFG.

We report the results of PBPC collection by large-volume leukaphereses and the hematologic recovery after high-dose chemotherapy supported by autologous PBPC reinfusion in a series of cancer patients treated at the Hematological Intensive Care Unit (UCHI) (Portuguese Institute of Oncology, Lisbon). Large volume leukaphereses were used to increase the efficacy of the PBPC collection. This modification of the standard apheresis technique allowed the harvesting, in only one session, of enough progenitors to proceed to transplantation in nearly 2/3 of patients and without significant toxicity. From December 1993 until September 1997, 95 autologous PBSC transplants were performed at the UCHI; 45% were performed in solid tumor patients and 55% in patients with hematologic malignancies. Hematologic recovery was similar to that published in the literature and related to the number of CD34+ cells infused. Patients supported with bone marrow in addition to PBPC showed delayed hematopoietic recovery, probably because the bone marrow harvest was only performed when an insufficient number of PBPC had been collected (2 x 10(6) CD34+ cells/Kg). The speed of hematological recovery differed per diagnosis, being higher in multiple myeloma and solid tumor patients and lower in Hodgkin's disease patients.

[The use of peripheral blood progenitor cells as an autologous hematopoietic support in high-dose chemotherapy. I. The rationale and results]. / Utilização de células progenitoras do sangue periférico como suporte hematopoiético autólogo de quimioterapias de alta dose. I. Racional e resultados.

We review the rationale for PBPC transplantation and the results reported in the literature. In order to prolong complete remissions and increase cure rates, high-dose chemotherapy is frequently used in the treatment of selected neoplasias. Hematological toxicity can be overcome by the infusion of autologous hemopoietic progenitors. Recently, peripheral blood is being used as the preferred source for hemopoietic progenitors, since it allows faster hematopoietic recoveries when compared to progenitors harvested from bone marrow. An adequate graft is defined by its content in clonogenic progenitors (mainly CFU-GM) and CD34 positive cells; these two parameters need to be accurately determined by specific laboratory methods. PBPC grafts are harvested using cell separators during leukaphereses; to increase efficiency, hemopoietic progenitors are first mobilized into the circulation with growth factors and or chemotherapy. PBSC transplantation may have procedure-associated toxicity related to the mobilization, harvest or reinfusion of the graft.

Expression of adhesion molecules in chronic B-cell lymphoproliferative disorders.

BACKGROUND AND OBJECTIVE: Abnormalities in the expression of cell adhesion molecules (CAM) are thought to influence the patterns of intranodal growth and hematogeneous spread of malignant cells in chronic lymphoproliferative disorders (LPD). Therefore, the characterization of CAM phenotypic profiles of the neoplastic clones in LPD may help to identify distinct subtypes with prognostic implications. In this work we sought to investigate whether the expression of CAM by circulating malignant cells in patients with B-cell LPD differed from that of normal peripheral blood B-lymphocytes (PBL) and whether the observed phenotypic patterns could be correlated to other biological and clinical parameters of known clinical relevance. DESIGN AND METHODS: Peripheral blood mononuclear cells were obtained from 148 patients with B-cell chronic lymphocytic leukemia (B-CLL), 52 with B-cell non-Hodgkin lymphomas (NHL) and 10 with hairy cell leukemia (HCL). The expression of CAM was analyzed by flow cytometry using monoclonal antibodies against CD49d, CD29, CD11a, CD11b, CD11c, CD18, CD62L, CD54 and CD44. RESULTS: All CAM were detected in normal peripheral blood B-lymphocytes, except CD11c and CD54, which were present in only a minority of cells. Fluorescence mean channel values (FMC) showed that all molecules, with the exception of CD44, were expressed with dim intensity. Emerging patterns of CAM expression, as assessed by FMC values, were observed in different LPD: thus, B-CLL is characterized by a very low expression of CD49d/CD29 and beta 2 integrins. In this disorder, CD49d/CD29, CD11a, and CD54 increase with tumor burden; NHL show high expression of CD29 and CD54; strong expression of all molecules (except CD11a) was found in HCL, particularly CD11c (FMC values 60 times higher than normal). CD62L was faintly expressed in all diagnostic groups, whereas CD11c showed consistently high FMC values. INTERPRETATION AND CONCLUSIONS: The data shows that the phenotypic characterization of LPD can be further refined by the analysis of their patterns of CAM expression which may help to identify distinct subsets within each nosological group.

Large-volume leukaphereses may be more efficient than standard-volume leukaphereses for collection of peripheral blood progenitor cells.

To overcome the need for multiple leukaphereses to collect enough PBPC for autologous transplantation, large-volume leukaphereses (LVL) are used to process multiple blood volumes per session. We compared the efficiency of CD34+ cell collection by LVL (n = 63; median blood volumes processed 11.1) with that of standard-volume leukaphereses (SVL) (n = 38; median blood volumes processed 1.9). To achieve this in patients with different peripheral blood concentrations of CD34+ cells, we analyzed the ratio of CD34+ cells collected per unit of blood volume processed, divided by the number of CD34+ cells in total blood volume at the beginning of apheresis. For LVL, 30% (9%-323%) of circulating CD34+ cells were collected per blood volume compared with 42% (7%-144%) for SVL (p = 0.02). However, in LVL patients, peripheral blood CD34+ cells/L decreased a median of 54% during LVL (similar data for SVL not available). The number of CD34+ cells collected per blood volume processed after 4 and 8 blood volumes and at the end of LVL were 0.32 (0.01-2.05), 0.24 (0.01-1.68), and 0.22 (0.01-2.40) x 10(6) CD34+ cells/kg, respectively (p = 0.0007), despite the 54% decrease in peripheral blood CD34+ cells/L throughout LVL. A median 66% decrease in the platelet count was also observed during LVL. Thus, LVL may be more efficient than SVL for PBPC collection, allowing, in most patients, the collection in one LVL of sufficient PBPC to support autologous transplantation.

HTLV Tax gene expression in patients with lymphoproliferative disorders.

AIMS: To study the expression of the human T lymphotropic virus (HTLV) Tax gene in peripheral blood mononuclear cells. METHODS: Blood was collected from 72 patients with lymphoproliferative disorders. Serum from all patients was assayed for antibodies directed against HTLV-I structural proteins by ELISA and western blotting. RNA was purified from fresh blood cells and amplified by reverse transcription polymerase chain reaction (RT-PCR). After Southern blotting, the PCR products were hybridised with a 32P end-labelled probe specific for the Tax gene. RESULTS: All samples were seronegative. A specific band for the Tax gene was found in five samples. Each of the patients positive for Tax gene expression had a different type of lymphoproliferative disorder. CONCLUSIONS: Infection by HTLV-I cannot be assessed solely by immunological assays, particularly when only disrupted virions are used. Sensitive molecular biology assays are essential for detecting viral gene expression in fresh blood cells.

Natural killer cell numbers and activity in mobilized peripheral blood stem cell grafts: conditions for in vitro expansion.

Peripheral blood stem cells (PBSC) are increasingly being used as an alternative to autologous bone marrow (BM) for hematologic rescue after high-dose chemoradiotherapy in the treatment of hematologic and nonhematologic malignancies. Mobilization procedures such as chemotherapy and/or hematopoietic growth factor administration are employed to allow for the graft enrichment in hematopoietic stem cells and progenitors and to accelerate trilineage recovery after transplant. The influence of these mobilization procedures on the lymphoid populations in the graft and on immunologic recovery after transplant remains to be determined. We studied six consecutive patients undergoing PBSC high-volume collections after cyclophosphamide (Cyc) and granulocyte colony-stimulating factor (G-CSF) administration and observed that NK cell numbers (phenotypically defined as CD3-CD56+ by flow cytometry) and activity (evaluated by a 51Cr release assay) fully recovered after 4-5 weeks; high numbers of functionally active NK cells (42.1-212.1 x 10(6)/kg b.w.) were present in the grafts, and their percentage and cytotoxic activity rose from the beginning to the end of the harvesting procedure in most cases. CD3-CD56+ and CD34+ cell numbers peaked at the same time point during harvesting, which differed from one patient to another. T (CD3+) cells were always present during harvest, and CD4 and CD8 numbers showed interdonor variability. When we cultured leukapheresed PBSC in the presence of interleukin-2 (IL-2) (10-1000 U/mL) for 6-8 days, we were able to expand the NK population three- to 5.4-fold; 100 U/mL appears to be the best concentration to generate high numbers of cytotoxic NK cells. Pilot studies also suggest that this short exposure to IL-2 does not affect the CD34+ cells. We conclude that PBSC grafts mobilized by combined Cyc and G-CSF and harvested through high-volume leukapheresis contain high numbers of cytotoxic NK cells that can be expanded in vitro by exposure to IL-2. In the setting of PBSC transplant, ex vivo immunomodulation aimed at increasing the NK cell numbers and activity is feasible and may prove to be useful in inducing a graft-vs.-tumor effect, thereby decreasing the relapse rate after transplant.