Insufficient ex vivo expansion of Valpha24(+) natural killer T cells in malignant lymphoma patients related to the suppressed expression of CD1d molecules on CD14(+) cells.

BACKGROUND: Valpha24(+) natural killer T (NKT) cell is a human counterpart of mice Valpha14(+) NKT cell that has a regulatory role for innate and acquired potential antitumor activity. The efficient expansion of NKT cells is an obstacle to the clinical application of Valpha24(+) NKT cells for immunotherapy. METHODS: We used mononuclear cells (MNC) obtained from the peripheral blood (PB) of normal healthy donor (HD) and malignant lymphoma (ML) patients before and after granulocyte colony-stimulating factor (G-CSF) treatment. MNC were cultured for 12 days with alpha-galactosylceramide (100 ng/mL) and interleukin-2 (IL-2; 100 U/mL). RESULTS: The fold expansion of Valpha24(+) NKT cells was higher in HD than in ML patients (208 versus 0.00), despite comparable numbers of Valpha24(+) NKT cells before culture. G-CSF administration enhanced the predominance of Valpha24(+) NKT cell fold expansion in HD compared with ML patients (1935 versus 1.95). After treatment with G-CSF, the expression of CD1d molecules was up-regulated in CD14(+) cells from HD but not ML patients. The fold expansion of Valpha24(+) NKT cells and CD1d expression on CD14(+) cells was strongly correlated in both HD and ML patients (r(2)=0.84). However, replacement of a patient's CD14(+) cells with HD cells did not increase the efficacy of Valpha24(+) NKT cell expansion. DISCUSSION: G-CSF-mobilized PB from ML patients has inhibitory characteristics for Valpha24(+) NKT cell expansion as a result of both monocytes and Valpha24(+) NKT cells. Multiple procedures would be needed for the expansion of patients' Valpha24(+) NKT cells.

Functional analysis of cytomegalovirus-specific T lymphocytes compared to tetramer assay in patients undergoing hematopoietic stem cell transplantation.

In order to evaluate whether we could predict reactivation of CMV by monitoring the number of CMV-specific cytotoxic T-lymphocytes (CTL), tetramer analysis was performed in 37 patients who underwent hematopoietic stem cell transplantation (HSCT). The results disclosed that the mean number of CMV-specific CTL at day 30 did not differ among patients who developed CMV antigenemia (22/microl) and those who did not (12/microl). Serial tetramer analysis showed that 21% of the patients had >10/microl CMV-specific CTL at the first detection of CMV antigenemia and 67% of the patients had more than 10/microl CMV-specific CTL at the onset of CMV disease. Intracellular staining upon stimulation by CMV lysates and peptide in patients with CMV colitis revealed that both IFN-gamma producing CD4+ and CD8+ lymphocytes were suppressed at the onset of CMV colitis (1.6 and 8/microl), which increased with recovery of the disease (19 and 47/microl). These data suggest that it is difficult to predict CMV reactivation solely by the number of CMV-specific CTL. We suggest that additional functional analysis by intracellular cytokine assay may be useful for immunomonitoring against CMV.

Allo-SCT using reduced-intensity conditioning against advanced pancreatic cancer: a Japanese survey.

Pancreatic cancer is a frequent cause of cancer-related mortality and has an extremely poor prognosis. To evaluate the efficacy of allogeneic hematopoietic SCT with reduced-intensity conditioning (RICT) against pancreatic cancer, we analyzed the clinical data of 22 patients. After a fludarabine-based conditioning regimen followed by the infusion of PBSCs, all but two achieved engraftment. Complete, partial and minor response was observed in 1, 2 and 2 patients, respectively, with an overall response rate of 23%. Median survival was only 139 days and the major cause of death was tumor progression. Poor performance status before RICT and a lower number of infused CD34-positive cells were associated with shorter survival after RICT. Patients who developed chronic GVHD tended to survive longer than those who did not. These findings support the investigation of a novel treatment strategy to enhance the immunological effect against pancreatic cancer.

Infectious complications in chronic graft-versus-host disease: a retrospective study of 145 recipients of allogeneic hematopoietic stem cell transplantation with reduced- and conventional-intensity conditioning regimens.

To assess infectious complications associated with chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) with reduced- and conventional-intensity conditioning regimens (RIC, n=91; CIC, n=54, respectively), we retrospectively analyzed data from 145 consecutive patients with cGVHD after allogeneic HSCT from a human leukocyte antigen-matched related or unrelated donor. In the present retrospective analysis, 57% (83/145) of patients with cGVHD developed infections, with a mortality rate of 27% (22/83). The incidences of bacteremia (n=28), central venous catheter-related infections (n=11), bacterial pneumonia (n=4), invasive aspergillosis (n=7), and adenoviral hemorrhagic cystitis (n=8) were significantly higher in patients with prednisolone dose >or=1 mg/kg at the time of diagnosis of cGVHD. The present results suggest that infections associated with cGVHD, especially after high-dose prednisolone, are predictive of poor outcome regardless of whether the patient received RIC or CIC.

Population pharmacokinetics of intravenous busulfan in patients undergoing hematopoietic stem cell transplantation.

A population pharmacokinetic analysis was performed in 30 patients who received an intravenous busulfan and cyclophosphamide regimen before hematopoietic stem cell transplantation. Each patient received 0.8 mg/kg as a 2 h infusion every 6 h for 16 doses. A total of 690 concentration measurements were analyzed using the nonlinear mixed effect model (NONMEM) program. A one-compartment model with an additive error model as an intraindividual variability including an interoccasion variability (IOV) in clearance (CL) was sufficient to describe the concentration-time profile of busulfan. Actual body weight (ABW) was found to be the determinant for CL and the volume of distribution (V) according to NONMEM analysis. In this limited study, the age (range 7-53 years old; median, 30 years old) had no significant effect on busulfan pharmacokinetics. For a patient weighting 60 kg, the typical CL and V were estimated to be 8.87 l/h and 33.8 l, respectively. The interindividual variability of CL and V were 13.6 and 6.3%, respectively. The IOV (6.6%) in CL was estimated to be less than the intraindividual variability. These results indicate high interpatient and intrapatient consistency of busulfan pharmacokinetics after intravenous administration, which may eliminate the requirement for pharmacokinetic monitoring.

Peripheral blood stem cell mobilization by granulocyte colony-stimulating factor alone and engraftment kinetics following autologous transplantation in children and adolescents with solid tumor.

In 56 pediatric and adolescent patients (median age 7 years, range 1-21) with various solid tumors, peripheral blood stem cells (PBSC) were mobilized with granulocyte colony-stimulating factor (G-CSF) alone, and the yields of PBSC and engraftment kinetics following autologous peripheral blood stem cell transplantation (PBSCT) were evaluated retrospectively. Granulocyte colony-stimulating factor (10 microg/kg) was injected subcutaneously for mobilization when patients showed no influence of previous chemotherapy, and administration was continued for 5 days. The peaks of CD34+ cells and colony-forming units-granulocyte/macrophage in the blood were observed on days 4 through 6 of G-CSF administration in all patients. Peripheral blood stem cell harvest was commenced on day 5 of G-CSF treatment. Compared to the results in patients mobilized by chemotherapy plus G-CSF (N=18), the progenitor cell yields were lower in patients mobilized with G-CSF alone. However, there were no significant differences in WBC and ANC engraftment compared to the chemotherapy plus G-CSF mobilization group. Platelet recovery following autologous PBSCT was delayed in patients mobilized with G-CSF alone. The median time taken for ANC and platelet counts to reach 0.5 x 10(9) and 20 x 10(9)/l was 12 days (range: 9-28) and 15 days (8-55), respectively, in all patients who received PBSC mobilized by G-CSF alone. In summary, mobilization with G-CSF alone can mobilize sufficient CD34+ cells for successful autografting and sustained hematological reconstitution in pediatric and adolescent patients with solid tumors, and even in heavily pre-treated patients.

Serum thrombopoietin levels in patients receiving high-dose chemotherapy with support of purified peripheral blood CD34+ cells.

In a case control study, serum levels of thrombopoietin (TPO) were determined by a sandwich ELISA in 20 patients (median age, 7 years; range, 2-56 years) with various malignancies who received high-dose chemotherapy and a stem cell rescue operation. The patients received two different transplant modalities: (a) 12 patients received purified autologous peripheral blood CD34+ cells; and (b) 8 patients received cells in the CD34(-) fraction, which still contains many CD34+ cells. No significant differences were observed between the two groups with regard to the duration required to achieve an absolute granulocyte count of >0.5 x 10(9)/liter, the duration of dependence on platelet transfusion, or the number of platelet transfusions. In both groups, the serum TPO levels were inversely correlated with the circulating platelet count. Multivariate analysis demonstrated that significant determinants of the serum TPO level included the circulating platelet count (standardized regression coefficient = -0.5179), transplantation with cells in the CD34(-) fraction (0.2414), solid tumor (0.1420), and the age of the patient (-0.1236; r2 = 0.3021; P < 0.0001). These results suggest that the mode of stem cell support (ie., the presence of accessory cells in the inoculum), age, or the type of preceding chemotherapy affects serum TPO levels after transplantation.

Efficacy of the mobilization of peripheral blood stem cells by granulocyte colony-stimulating factor in pediatric donors.

The advantages/disadvantages of the use of peripheral blood stem cells (PBSCs) for allogeneic transplantation still need to be clarified, particularly in children. We compared the kinetics, efficacy, and safety of PBSC mobilization by granulocyte colony-stimulating factor (G-CSF) and collection by apheresis between healthy pediatric and adult donors. A total of 19 pediatric (median age, 6 years) and 25 adult healthy donors (median age, 37 years) were given 10 micro/kg/day of G-CSF for 5 consecutive days for PBSC mobilization, which were harvested by apheresis on days 5 and/or 6. All of the donors tolerated the whole procedures. Serum trough levels of G-CSF determined by ELISA were significantly lower in the 16 pediatric donors evaluated than in adults (n = 16) on days 3 and 4 (P < 0.05). Although the WBC counts on days 4 and 5 were significantly higher in adults than in children (P = 0.006 and 0.004, respectively), the numbers of circulating CD34+ cells/unit of blood were identical. The number of blood CD34+ cells collected per unit of blood processed was identical in both donor populations. We propose that PBSCs could be effectively mobilized and collected in small children so that they could be donors for adult patients.

Frequent aberration of FHIT gene expression in acute leukemias.

We analyzed the mRNA expression of the FHIT gene by reverse transcription-PCR (RT-PCR) in 54 cases of acute lymphoblastic leukemia (ALL; 11 cases of T-cell ALL [T-ALL] and 43 cases of non-T-ALL) and 40 cases of acute myeloid leukemia (AML). In 46% of the ALL cases and 55% of the AML cases, FHIT expression was absent or markedly decreased. Only abnormal short bands were detected in 30% of the ALL cases and 5% of the AML cases. Eighteen of 19 abnormal transcripts had the same fusion of exons 2-7, and all lacked the starting codon in exon 5. No obvious normal-sized PCR products were detected in cases exhibiting abnormal transcripts. These findings suggest that the expression of functional FHIT protein was lost in the majority of ALL (76%) and AML (60%) cases. Differential quantitative PCR of exons 3-9 of the FHIT gene and RT-PCR of the PTPRG gene, which is centromeric to the FHIT gene, showed the presence of the target sequences. Fluorescence in situ hybridization analysis using probes covering exons 5 and 8 revealed no difference in the signal patterns between leukemia and normal cells, showing one or two signal doublets in more than 90% of nuclei, and indicated that gross segments of the FHIT gene were not homozygously deleted in these cases. A small number of transcripts with an aberrant fusion between exons 2 and 7 were detected by RT-PCR in the bone marrow cells from four healthy individuals. Granulocytes, lymphocytes, and monocytes in the bone marrow cells of a healthy individual contained transcripts with the same fusion. This unique fusion of exons 2 and 7 might be preferentially seen in either neoplastic or normal hematopoietic cells, regardless of their lineage. The finding that FHIT expression was abolished in the majority of leukemia cases might support the hypothesis that the FHIT gene acts as a tumor suppressor, at least in leukemia.

Evaluation of cytomegalovirus-specific cytotoxic T-lymphocytes in patients with the HLA-A*02 or HLA-A*24 phenotype undergoing hematopoietic stem cell transplantation.

Cytomegalovirus-specific cytotoxic T-lymphocytes (CMV-CTL) are essential for the control of CMV reactivation. To monitor the quantity and function of CMV-CTL after hematopoietic stem cell transplantation (HSCT), two CMV epitopes that bind to HLA-A*0201 NLVPMVATV (A*02NLV) and HLA-A*2402 QYDPVAALF (A*24QYD) were evaluated for their immunological potential. Samples from patients with the HLA-A*02 or HLA-A*24 serotype were analyzed by tetramer, intracellular cytokine staining and enzyme-linked immunospot (ELISPOT) assay. There were significantly more A*02NLV-specific CMV-CTL than A*24QYD (23 x 10(6) vs 0.4 x 10(6)/l). The frequency of IFN-gamma-producing cells was also higher upon stimulation with A*02NLV than with A*24QYD (2.5 vs 0.1%/CD8). Furthermore, the magnitude of CMV-CTL expansion was two- to 50-fold when cells were cultured with A*02NLV, while only an insignificant increase was observed in culture with A*24QYD. Although the number of A*24QYD-specific CMV-CTL was very low in most of the HLA-A*24 patients, the incidence of CMV reactivation did not differ between those with HLA-A*02 and HLA-A*24 serotype alone. These results suggest that an epitope other than A*24QYD plays a major role in patients with HLA-A*24. Our study also showed that A*02NLV may be a useful epitope for monitoring CMV-CTL not only in patients with HLA-A*0201 but also in those with the A*0206 genotype.

Optimal initial dose of oral cyclosporine in relation to its toxicities for graft-versus-host disease prophylaxis following reduced-intensity stem cell transplantation in Japanese patients.

Since the introduction of reduced-intensity stem-cell transplantation (RIST), allogeneic stem-cell transplantation has become available for elderly patients. While pharmacokinetics of cyclosporine might differ according to age or other factors, cyclosporine is uniformly started at an oral dose of 6 mg/kg/day. We retrospectively reviewed medical records of 35 patients aged between 32 and 65 (median 52) years who had undergone RIST. Doses of cyclosporine were adjusted to the target blood trough level of 150-250 ng/ml. Cyclosporine dosages were changed in 33 patients (94%). Dose reduction was required in 32 patients because of high blood levels (n=25), renal dysfunction (n=3), hepatic dysfunction (n=2), and hypertension (n=2). Cyclosporine doses were increased in one because of the suboptimal level. The median of the achieved stable doses was 3.1 mg/kg/day (range, 1.0-7.4). Five patients sustained Grade III toxicities according to NCI-CTC version 2.0: renal dysfunction (n=4), hyperbilirubinemia (n=2), and hypertension (n=2). No patients developed grade IV toxicity. There was no statistically significant difference in the frequency and severity of cyclosporine toxicities between patients aged 50 years and above and those below 50 years. The initial oral cyclosporine dose of 6 mg/kg/day was unnecessarily high irrespective of age. The possible overdose of cyclosporine might have aggravated regimen-related toxicities.

A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement.

The prognosis of patients with metastatic retinoblastoma is poor with conventional chemotherapy and radiation. Since retinoblastoma is highly chemosensitive, dose-escalation of chemotherapeutic agents with stem cell support should be promising. We report our experience with high-dose chemotherapy (HDC) and autologous stem cell transplantation (SCT) in patients with metastatic retinoblastoma. Five patients with metastatic retinoblastoma underwent HDC with autologous SCT following conventional chemotherapy and local radiation therapy. Stem cells (bone marrow in four and peripheral blood stem cells in one) were collected after marrow involvement was cleared. Melphalan was a key drug in all patients, and was administered in combination with other agents such as cisplatin, cyclophosphamide, carboplatin or thiotepa. Three patients are currently alive disease-free at 113, 107 and 38 months, respectively, from the time of SCT. They had no central nervous system (CNS) involvement. The two patients who died of disease had CNS involvement. No long-term sequelae of HDC have been noted. Our treatment strategy using HDC appears to be effective for treating metastatic retinoblastoma without CNS involvement.

Reduced-intensity hematopoietic stem-cell transplantation for malignant lymphoma: a retrospective survey of 112 adult patients in Japan.

We conducted a nation-wide survey of 112 adult Japanese patients who underwent reduced-intensity stem cell transplantation (RIST) from 1999 to 2002. Underlying diseases included indolent (n=45), aggressive (n=58) and highly aggressive lymphomas (n=9). Median age of the patients was 49 years. A total of 40 patients (36%) had relapsed diseases after autologous stem cell transplantation and 36 patients (32%) had received radiotherapy. RIST regimens were fludarabine-based (n=95), low-dose total body irradiation-based (n=6) and others (n=11). Cumulative incidences of grade II-IV acute graft-versus-host disease (GVHD) and chronic GVHD were, respectively, 49 and 59%. Cumulative incidences of progression and progression-free mortality were 18 and 25%, respectively. With a median follow-up of 23.9 months, 3-year overall survival rates were 59%. A multivariate analysis identified three significant factors for progression, which are history of radiation (relative risk (RR) 3.45, confidential interval (CI) 1.12-10.0, P=0.03), central nervous system involvement (RR 6.25, CI 2.08-20.0, P=0.001) and development of GVHD (RR 0.28, CI 0.090-0.86, P=0.026). RIST may have decreased the rate of transplant-related mortality, and GVHD may have induced a graft-versus-lymphoma effect. However, whether or not these potential benefits can be directly translated into improved patient survival should be evaluated in further studies.

Value of surveillance blood culture for early diagnosis of occult bacteremia in patients on corticosteroid therapy following allogeneic hematopoietic stem cell transplantation.

Bloodstream infection (BSI) is a significant complication following allogeneic hematopoietic stem cell transplantation (allo-SCT). Corticosteroids mask inflammatory responses, delaying the initiation of antibiotics. We reviewed medical records of 69 allo-SCT patients who had been on >0.5 mg/kg prednisolone to investigate the efficacy of weekly surveillance blood cultures. A total of 36 patients (52%) had positive cultures, 25 definitive BSI and 11 probable BSI. Pathogens in definitive BSI were Staphylococcus epidermidis (n=7), S. aureus (n=4), Entrococcus faecalis (n=3), Pseudomonas aeruginosa (n=5), Acenitobacter lwoffii (n=4), and others (n=10). The median interval from the initiation of corticosteroids to the first positive cultures was 24 days (range, 1-70). At the first positive cultures, 15 patients with definitive BSI were afebrile. Four of them remained afebrile throughout the period of positive surveillance cultures. Patients with afebrile BSI tended to be older (P=0.063), and had in-dwelling central venous catheters less frequently than febrile patients (P<0.0001). Bloodstream pathogens were directly responsible for death in two patients with afebrile BSI. This study demonstrates that cortisosteroid frequently masks inflammatory reactions in allo-SCT recipients given conrticosteroids, and that surveillance blood culture is only diagnostic clue for 'occult' BSI.

Comparison between reduced intensity and conventional myeloablative allogeneic stem-cell transplantation in patients with hematologic malignancies aged between 50 and 59 years.

To evaluate the efficacy of reduced-intensity stem-cell transplantation (RIST), we retrospectively compared outcomes of 207 consecutive Japanese patients aged between 50 and 59 years with hematologic malignancies who received RIST (n=70) and conventional stem-cell transplantation (CST) (n=137). CST recipients received total body irradiation (TBI)-based or busulfan/cyclophosphamide-based regimens. RIST regimens were purine analog-based (n=67), 2 Gy TBI-based (n=2), and others (n=1). Most CST recipients (129/137) received calcineurin inhibitors and methotrexate as graft-versus-host (GVHD) prophylaxis, while 32 RIST recipients received cyclosporin. In all, 23 CST and five RIST recipients died without disease progression within 100 days of transplant. Grade II to IV acute GVHD occurred in 56 CST and 38 RIST recipients. There was no significant difference in overall survival (OS) and progression-free survival between CST and RIST. On multivariate analysis on OS, five variables were significant: preparative regimens (CST vs RIST) (hazard ratio=1.92, 95% confidence interval, 1.25-2.97; P=0.003), performance status (2-4 vs 0-1) (2.50, 1.51-4.16; P<0.001), risk of underlying diseases (1.85, 1.21-2.83; P=0.004), acute GVHD (2.57, 1.72-3.84; P<0.001), and CML (0.38, 0.21-0.69; P=0.002). We should be careful in interpreting results of this small-sized retrospective study; however, reduced regimen-related toxicity might contribute to better survival in RIST. The low relapse rates following RIST suggest a strong antitumor activity through allogeneic immunity.

Prospective monitoring of minimal residual disease during the course of chemotherapy in patients with acute lymphoblastic leukemia, and detection of contaminating tumor cells in peripheral blood stem cells for autotransplantation.

A prospective study for detecting minimal residual disease (MRD) was conducted on children with acute lymphoblastic leukemia (ALL). Thirty-nine patients (38 B-lineage ALL, one T-ALL) with TCR delta rearrangements could be followed for 21 to 44 months (mean 30.9 months) excluding four patients who died. One hundred and ninety four bone marrow (BM) samples and 13 peripheral blood stem cell (PBSC) grafts were available for detection of MRD. Initially 34 cases were treated prospectively according to the CCLSG risk-stratified protocols for ALL (ALL874 or ALL911), and five cases according to the other protocols. Conventional chemotherapy was replaced by autologous PBSC transplantation (PBSCT) in five patients, by allogenic BM transplantation (BMT) in one patient, or suspended in another patient. Twenty-nine of 32 children in whom conventional chemotherapy could be continued without interruption remain in complete remission (CR). In 24 of the 29 patients MRD became undetectable within 12 months of their diagnosis. In five cases, BM samples obtained during maintenance therapy exhibited residual leukemia cells, and yet none of them relapsed (mean follow-up period 28.6 months). Our results thus indicate that intensive maintenance therapy for patients with PCR-positive results during consolidation therapy may prevent subsequent relapse. Nine events of relapse were diagnosed in eight patients (five BM, two isolated central nervous system (CNS), one combined BM and CNS, one isolated skin relapse). An increase or a re-emergence of MRD was detected in BM samples obtained from patients prior to BM relapse, but one patient remained in CR despite reappearance of leukemic cells following a PCR-negative status. Monitoring of MRD failed to predict isolated CNS or skin relapse. PBSCT allows high-dose cytoreduction therapy for patients with refractory neoplasia. In our study, leukemic cells were identified in eight of 13 PBSC grafts harvested from five patients. Three of four children who received PBSC grafts containing leukemic cells relapsed within 6 months after PBSCT. Monitoring of MRD as part of quality control of PBSC grafts may ultimately contribute to improvements in PBSCT procedures.

Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage.

We analyzed homozygous deletions and mutations of the CDKN2(p16(INK4A)/MTS1) gene, using polymerase chain reaction and Southern blot analysis, in 120 children with acute lymphoblastic leukemia (ALL). Homozygous deletion was found in 17 of 89 (19%) precursor B-ALL patients, in 11 of 24 (46%) T-ALL patients, and in 0 of 7 other phenotype ALL patients. After excluding 28 (23%) patients who showed a homozygous deletion of CDKN2, we found that three patients (3%) had mutation at exon 2 of CDKN2 using PCR-SSCP and sequencing strategy. One had a CGA to TGA nonsense mutation (Arg to stop) at codon 72, one had a 1-bp deletion at codon 117, and the third had a 2-bp deletion at codon 70, resulting in frameshifts in the two latter patients. All three of these patients were T phenotype ALL, and the incidence of mutation in the 24 T-ALL patients examined was 13%. In contrast, no mutation was detected in the remaining patients with precursor-B or other type ALL (0/96). Our results suggest that mutational inactivation of the CDKN2 gene may contribute to the leukemogenic growth, especially in some patients with T-ALL.

Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia.

We investigated TEL/AML1 fusion mRNA in 108 children with acute lymphoblastic leukemia (ALL) (86 B-lineage ALL, 15 T-ALL, two mixed lineage ALL, and five other phenotypes) using reverse transcriptase-polymerase chain reaction (RT-PCR). TEL/AML1 transcripts were found in 14 patients (13%) including three relapsed patients, and were unexceptionally limited to B-lineage ALL patients. The incidence of TEL/AML1 transcripts among B-lineage ALL was 16% (14/86). The reciprocal AML1/TEL transcripts were detected in 12 (86%) of the 14 cases expressing a TEL/AML1 transcript. In three cases, the TEL gene was fused to exon 3 of the AML1 gene, and to exon 2 in the remaining cases. To evaluate the amount of TEL/AML1 molecules for the quantification of a minimal residual disease (MRD), a plasmid vector which contained either a long TEL/AML1 PCR product (464 bp) or a short one (425 bp) was used as a competitor. We amplified RNAs obtained from bone marrow (BM) at complete remission or from peripheral blood stem cell (PBSC) harvests in two representative cases. For one PBSC harvest showing a positive result, a competitive PCR was carried out to quantify the amount of MRD. A 1:4 dilution series of competitor vectors was constructed, and each vector was added to a PCR reaction which contain a constant amount of cDNA obtained from the PBSC harvest. An equivalent point was compared to that of corresponding samples at diagnosis. Using this method, MRD in the PBSC harvest was 3.9:10(3). Our results elucidated the incidence, lineage-specificity, and variant forms of TEL/AML1 fusion transcripts in childhood ALL. Since the percentage of other chromosomal translocations in childhood ALL is not more than 5%, TEL/AML1 transcript would be the most feasible clone-specific marker for these patients. In addition, our method could be a powerful tool for quantification of the TEL/AML1 transcript and for the detection of MRD.

T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells.

We present evidence that T-cell-conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell-free supernatants of anti-CD3-activated T cells contained several soluble factors including CD40-ligand (sCD40L), TNF-alpha, and IFN-gamma. In contrast to moderate up-regulation of costimulatory molecules by the addition of individual cytokines or monocyte-conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose-dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF-alpha, and IFN-gamma, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM-treated DC can produce significantly higher levels of IL-12 and are highly effective stimulators in allogenenic and autologous mixed-lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen-specific immune responses against cancer and viruses.

Cell processing protocol for allogeneic peripheral blood stem cells mobilized by granulocyte colony-stimulating factor.

Although there is a growing body of information available regarding restoration of hematopoiesis with peripheral blood stem cell (PBSC) autografts, few studies have explored this procedure using allografts. In this study with healthy donors, we investigated the feasibility of a protocol for mobilizing PBSC using recombinant human granulocyte colony-stimulating factor (G-CSF) and subsequent bulk depletion of T cells from apheresis-harvested cells. Nine informed healthy donors were given G-CSF subcutaneously at two different dosing schedules (5 micrograms/kg/d in five donors and 2 micrograms/kg/d in four) for 5 consecutive days, and serial changes in blood components, including hematopoietic progenitor cells, were monitored. After 5 days of stimulation with G-CSF, PBSCs were collected by apheresis, and yields were compared. The number of white blood cells (WBC) reached a plateau level on either day 2 (5 micrograms) or 3 (2 micrograms), but the numbers of red blood cells and platelets were not affected. Circulating colony-forming unit-granulocyte/macrophage (CFU-GM) levels started to increase 1 or 2 days after the increase in the WBC count. By performing a 3L apheresis, the number of CFU-GM harvested was 4.6 +/- 3.3 x 10(6) (mean +/- standard error of the mean [SEM]) in the 5-micrograms group and 1.8 +/- 0.7 x 10(6) in the 2-micrograms group. Different procedures for depleting T cells, including the use of L-phenylalanine methyl ester (PME) and flasks coated with anti-CD5/CD8 monoclonal antibodies or neuraminidase-treated sheep red blood cells (SRBC), were also tested on the harvested cells. We found that cell lysis with PME before selective removal of T cells was very effective in reducing the number of cells that required further processing and was suitable for routine use. However, our current procedure resulted in unsatisfactory depletion of T cells (99.5% removal) while retaining hematopoietic progenitor cells (7.5% recovery). Further research is required in this area.