Changes of immunological markers after autologous peripheral blood stem cell transplantation

PURPOSE: Recently high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important treatment for hematological and solid tumors. METHODS: Immunological parameters were examined before and after PBSCT in 9 patients with lung cancer and 13 patients with malignant lymphoma. Findings were compared with those for bone marrow transplantation (BMT). Peripheral blood cells were analyzed for phenotype and the levels of cytokines and soluble factors were measured. RESULTS: After PBSCT, activated T cells (CD3+HLA-DR+ cells, CD8+HLA-DR+ cells) and suppressor/cytotoxic T cells (CD8+CD11b- cells) were significantly higher in the patients with lung cancer than in those with malignant lymphoma. Serum levels of interleukin-4 and soluble interleukin-2 receptor were also significantly higher in the patients with lung cancer than in those with lymphoma. On the other hand, the serum levels of interferon gamma, tumor necrosis factor alpha, interleukin-6, soluble human leukocyte antigen class 1, and soluble thrombomodulin were significantly increased after bone marrow transplantation. The transfused peripheral stem cells of lung cancer and lymphoma patients had a similar number of granulocyte/macrophage-colony-forming units, but lung cancer patients had significantly more CD34-positive cells. CONCLUSION: By reinfusing large numbers of autologous immune cells, PBSCT may accelerate immune reconstitution, with T cells being likely to have a marked therapeutic potential. The changes after PBSCT were greater in patients with lung cancer than in lymphoma patients. These blood cells are potent mediators of anticancer activity and could play an important role in the elimination of autologous malignant cells.     

CD30 positive large T-cell primary cutaneous lymphoma

The primary cutaneous CD30 positive large cell lymphoma is a rare tumor, confined to the skin. The characteristic clinical picture is a large, often exulcerating sometimes spontan regressing tumor or nodule. Dense infiltration of large, anaplastic or non-anaplastic T or non T, non B cell of the dermis is characteristic. Generalization, lymph node or internal manifestation is rare, the prognosis is favourable. A 25-year-old male patient is presented, in whom generalised skin symptoms-itching, reddish-brownish papules with central necrosis developed. Two years later general symptoms-fever, fatigue, lymph node and spleen enlargement, increased in white blood cell count with prominent eosinophilia, increase in CD4 number occurred. The histology and immunohistology of the skin and peripheral lymph node showed large, anaplastic, CD30 positive T cell infiltration. CHOP, then BACOP treatment resulted in regression of the skin and the internal symptoms.     

中剂量依托泊苷联合粒细胞集落刺激因子动员恶性淋巴瘤的外周血造血干/祖细胞

目的 观察中剂量依托泊苷(VP16)和粒细胞集落刺激因子(G-CSF)在恶性淋巴瘤患者动员采集自体外周血造血干/祖细胞的有效性和安全性.方法 31例恶性淋巴瘤患者(非霍奇金淋巴瘤30例,霍奇金淋巴瘤1例),VP16 1.2 g/m2分3 d静脉滴注,外周血白细胞降至最低点时给予G-CSF每天5μg/kg,分2次,皮下注射,直至采集结束.结果 VP16应用后12 d(10～15 d)开始采集外周血造血干/祖细胞,获得单个核细胞(MNC)7.8×108/kg[(5.2～11.3)×108/kg],CD+34细胞7.2×106/kg[(5.3～13.1)×106/kg] 18例患者采集1次,13例采集2次.所有患者移植后均恢复造血,外周血粒细胞＞0.5×109/L的中位时间为12 d(9～18 d),血小板＞20×109/L的中位时间为14d(10～21 d).患者无严重不良反应结论中剂量VP16和G-CSF动员恶性淋巴瘤患者外周血干/祖细胞有效、安全,可获得满意的动员采集效果.     

难治性淋巴瘤患者外周血T淋巴细胞亚群与自然杀伤细胞活性的检测及其临床意义

目的 探讨外周血T淋巴细胞亚群及自然杀伤(NK)细胞活性水平与难治性淋巴瘤的相关性.方法 采用流式细胞术(FCM)检测60例初治淋巴瘤患者化疗前外周血T淋巴细胞亚群水平与NK细胞的活性,化疗后随访分为难治组30例、有效组30例,以20名健康者为健康对照组.结果 淋巴瘤患者组化疗前外周血CD+4、CD+4/CD+8、NK细胞数比健康对照组低(30.17±8.63与46.52±1.39,t=12.218,P＜0.05;0.86±0.45与1.64±0.05,t=11.225,P＜0.05;12.39±7.08与19.29±0.84,t=6.365,P＜0.05),CD+3、CD+8细胞数比健康对照组高(76.14±10.71与70.48±1.44,t=-3.439,P＜0.05;40.28±14.03与28.35±0.73,t=-5.625,P＜0.05).难治组化疗前外周血CD+4、CD+4/CD+8、NK细胞数比有效组低(27.70±7.81与33.13±8.82,t=2.163,P=0.036;0.67±0.27与1.10±0.52,t=3.272,P=0.003;9.87±6.60与15.40±6.58,t=2.771,P=0.008),而CD+3、CD+8细胞数比有效组高(79.67±8.18与71.91±12.00,t=-2.540,P=0.015;44.70±13.99与34.98±12.41,t=-2.416,P=0.020).结论 淋巴瘤初治患者化疗前外周血T淋巴细胞亚群水平及NK细胞活性的检测,对判断、预测容易转归为难治的患者可能有一定的参考价值.     

Therapy of highly malignant non-Hodgkin lymphoma with high-dose chemotherapy and stem cell transplantation

Patients with intermediate or high-grade non-Hodgkin's lymphoma are rarely cured of their disease after the failure of conventional therapy. High-dose chemotherapy followed by transplantation of either autologous marrow (ABMT) or peripheral stem cells (PBSCT) offers such patients a new possibility of cure. To patients younger than 60 years and without contraindications high-dose chemotherapy should be offered in case of relapse and bad prognosis. The PARMA-study demonstrated, that high-dose chemotherapy with subsequent autologous stem cell transplants is superior to conventional chemotherapy for relapsing patients. However high-dose chemotherapy in first complete or partial remission followed by ABMT and PBSCT as post-remission therapy for adult NHL has yielded only similar results randomized to those achieved with conventional chemotherapy in several prospectively studies. This approach should be limited to patients with bad prognostic factors (high-intermediate or high-risk groups according to the International Prognostic Factor Project). The success of ABMT and PBSCT in treating these diseases mandates exploration of the best high-dose chemotherapy, the use of autologous or allogenic bone marrow resp. peripheral stem cell for haematopoietic reconstitution and technical aspects such as purging of tumor cells, the short- and long-term transplant-related mortality continues to be a major concern.     

Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily

The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.     

Development of a high-resolution pinhole SPECT system using dual-head gamma camera for small animal studies]

Cyclosporin A (CsA) can induce graft-versus-host disease (GVHD) following autologous bone marrow transplantation (ABMT) and autologous peripheral blood stem cell transplantation (APBSCT) in adults. We investigated whether GVHD can be induced following ABMT and APBSCT in childhood, and which cells are involved in the pathogenesis of this syndrome. We conducted a prospective study of 20 children and adolescents with hematological malignancies receiving CsA after ABMT and APBSCT. Skin biopsies were obtained on day 21 after transplantation or in the event of a rash. Immunophenotypic analysis of peripheral blood lymphocytes was performed on days 14, 21, 28 and 60 after transplantation. Clinical GVHD of the skin, confirmed by histological criteria, occurred in five patients. Five patients had no clinical GVHD but had acute GVHD alterations on routine skin biopsy. In all 10 patients with a positive skin biopsy for GVHD, CD4+ lymphocytes were the predominant cells in the epidermis. Immunophenotypic analysis of peripheral blood lymphocytes revealed a significantly increased CD4/CD8 ratio in patients with a positive skin biopsy (P < 0.01). Our findings indicate that it is possible to induce acute GVHD following ABMT and APBSCT in childhood. In addition, CD4+ lymphocytes play an important role in the pathogenesis of CsA-induced GVHD.     

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.     

Recipient polyclonal B cell activation and immunoglobulin production induced by priming with a retroviral superantigen

The superantigen vSAG-7 (or MIs 1a) is a membrane glycoprotein encoded by the endogenous retrovirus mouse mammary tumor virus 7 (MMTV-7) and is highly stimulatory for V beta 6/CD4+ T cells. Priming of adult MMTV-7-negative mice with vSAG-7-expressing cells initially results in the activation of the peripheral V beta 6/CD4+ T cell compartment and is followed by T cell tolerance to the superantigen. During the course of tolerance induction the number of recipient B lymphocytes increases in the lymph nodes, but not the spleen, of vSAG-7-primed recipients. These B cells also express increased levels of class II MHC and present passively acquired superantigen. In this study we asked if these effects on the host B cell compartment are followed by the production of immunoglobulin. Priming of MMTV-7-negative BALB/c or CB.17 mice with vSAG-7-expressing cells from DBA/2 mice induced increases of both IgM and IgG2a in the serum. Use of Igh congenic CB.17 (IgMb) mice as recipients of the vSAG-7-presenting cells from DBA/2 (IgMa) donors indicated that the IgM and IgG produced were entirely of host origin. Priming with vSAG-7 also amplified (four- to fivefold) the antibody-producing cell response induced to a suboptimal dose of sheep RBC. Priming with purified B cells from vSAG-7 donors resulted in recipient V beta 6/CD4+ T cell activation and increased numbers of recipient B cells in the lymph nodes, but did not induce immunoglobulin production. In contrast, priming with purified CD8+ T cells resulted in increased quantities of serum IgM but not vSAG-7-reactive T cell activation or increased numbers of recipient B cells in the lymph nodes. These results indicate that the T cell activation and increased B cell number with upregulated class II MHC expression initially observed following vSAG-7 priming of adult MMTV-7-negative recipients are not linked to the induction of the recipient-derived immunoglobulin production.     

An aggressive case of Burkitt's lymphoma with t(8;14) and c-myc rearrangement transformed from CD5+ B-cell lymphoma

We experienced a case of Burkitt's lymphoma showing an unusual surface phenotype, CD5 expression, at an early stage of the disease. Initially, this patient showed massive abdominal para-aortic lymph node swelling which rapidly developed into leukemic change. Based on the clinical course and cytogenetic features of lymphoblasts in the bone marrow, which showed t(8;14) and c-myc gene rearrangement, the patient was diagnosed with Burkitt's lymphoma. Combination chemotherapy induced short-term remission, but central nervous system (CNS) involvement developed, followed by a regrowth of lymphoma cells in the bone marrow. The bone marrow at the end stage showed monotonous expansion of large cells with conspicuous vacuolation in the basophilic cytoplasm. The initial lymphoma cells showed pan-B markers and were CD5 positive but weakly CD10 positive; however, the lymphoma cells obtained from the bone marrow at the terminal stage did not express CD5. The chromosomal t(8;14) was seen, and identical rearrangement of immunoglobulin heavy chain joining gene and c-myc gene were detected by Southern blot analysis in the bone marrow lymphoblasts throughout the clinical course. This case is evidence that remarkable transformation of CD5-positive lymphoblasts to CD5-negative lymphoblasts occurred in an identical clone of Burkitt's lymphoma.     

Thought control strategies in acute stress disorder

Relapse after transplant for malignant lymphomas remains the main cause of treatment failure. Most conditioning regimens contain total body irradiation (TBI). We investigated the toxicity and efficacy of an intensified chemotherapy conditioning regimen without TBI in patients with relapsed or high-risk malignant lymphoma who had received prior radiation therapy and were therefore not eligible for TBI. Twenty patients with a median age of 38 (18-56) and relapsed or high-risk malignant non-Hodgkin's lymphoma (NHL, n = 16) or Hodgkin's disease (HD, n = 4) underwent high-dose chemotherapy consisting of busulfan (16 mg/kg), cyclophosphamide (120 mg/kg) and etoposide 30 mg/kg (n = 8) or 45 mg/kg (n = 12) followed by peripheral stem cell support (n = 14), autologous bone marrow (n = 3), allogeneic (n = 2) or syngeneic (n = 1) transplantation. All but two had chemosensitive disease before high-dose chemotherapy. The main toxicity -- according to the Bearman score -- was mucositis II in 18 (90%) patients; five patients (25%) suffered a grade I hepatic toxicity. GI toxicity I occurred in three (15%) and renal toxicity I in two patients (10%). Sixty percent of the patients developed transient dermatitis with erythema and three of them (15%) had skin desquamation; one patient experienced asymptomatic pancreatitis. Toxicity was slightly higher in patients treated with 45 mg/kg etoposide. One patient (5%) died of treatment-related venoocclusive disease. After a median follow-up of 50 months (24-84) the disease-free and overall survival were 50% and 55%. One of the nine relapsing patients developed secondary AML 18 months after transplant. High-dose busulfan, cyclophosphamide and etoposide is an effective regimen resulting in long-term disease-free survival in 50% of patients with relapsed malignant lymphoma and prior radiation therapy. The toxicity is moderate with a low treatment-related mortality (5%).     

The value of sequential CD 34 measurement for carrying out stem cell pheresis

The recent significant improvement in disease-free survival in patients with certain haematological malignancies is due to high-dose chemotherapy and subsequent autologous bone marrow and/or stem cell transplantation. The proliferation and egression of stem cells into the peripheral blood must first be stimulated by defined chemotherapy and/or by administration of cytokines. However, the increase of circulating stem cells in peripheral blood is limited to only a few days. By immunologically analysing white blood cells for the expression of the surface antigen CD 34 it is possible to calculate the numbers of haematopoietic progenitor cells. Thus, besides monitoring haematopoietic recovery, the estimation of CD34+ cells in the peripheral blood can be used to indicate the optimal time point for stem cell collection. Two to four stem cell pheresis (one per day) may then yield sufficient stem cells to enable the safe and rapid reconstitution of haematopoiesis following supralethal chemotherapy.     

Prognosis of the skin UV-incidence increase in Poland

We studied the morphologic and immunohistochemical features of 10 peripheral T-cell lymphomas of a cytotoxic phenotype (CD3+/CD4-/CD8+), encountered among 98 peripheral T-cell lymphomas (PTCLs). Nine tumors were positive for both cytotoxic molecules, namely perforin (Pf) and granzyme B (GrB), and strong positivity was seen in the majority of the malignant cells. We also studied the expression of these molecules in 92 other cases of T-cell and natural killer (NK) cell neoplasms; 18 anaplastic large cell lymphomas (ALCLs); 63 CD4+ PTCLs; 10 CD56+ nasal lymphomas; and 1 NK-cell leukemia. Most of the CD4+ PTCLs (62 of 63) were negative for GrB, but all of the nasal lymphomas and the NK cell leukemia were positive for both Pf and GrB. Variable expression was seen among the 18 ALCLs. Within the 10 CD8+ PTCLs, 4 involved the skin, 3 of which were diagnosed as primary cutaneous lymphomas. Five patients died within 1 year of diagnosis. According to the Revised European-American Classification of Lymphoid Neoplasms, seven cases were categorized as "PTCL, unspecified," and three as "angioimmunoblastic T-cell lymphoma," "adult T-cell lymphoma/leukemia," or "small cell lymphoma," respectively. Three cases had characteristic morphologic features consisting of large lymphomatous cells with massive necrosis and nuclear fragmentation. Epstein-Barr virus mRNA was detected by in situ hybridization in three cases. Although the degree of apoptosis varied, apoptotic cells were detected in all cases by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end labeling. We conclude that CD8+ PTCLs are relatively rare, often involve extranodal sites, have an aggressive clinical course, and are often associated with Epstein-Barr virus. Compared with ALCLs, which have recently been considered as neoplasms of cytotoxic T-cells, we think that CD8+ PTCLs are more lineage-specific neoplasms of mature, cytotoxic, T lymphocytes.     

2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1

The objective of this study was to identify factors associated with poor mobilization of peripheral blood progenitor cells (PBPCs) or delayed platelet engraftment after high-dose therapy and autologous stem cell transplantation in patients with lymphoma. Fifty-eight patients with Hodgkin's disease or non-Hodgkin's lymphoma underwent PBPC transplantation as the "best available therapy" at Memorial Sloan-Kettering Cancer Center (New York, NY) between 1993 and 1995. PBPCs were mobilized with either granulocyte colony-stimulating factor (G-CSF) alone (n = 19) or G-CSF following combination chemotherapy (n = 39). Forty-eight of these patients underwent a PBPC transplant, receiving a conditioning regimen containing cyclophosphamide, etoposide, and either total body irradiation, total lymphoid irradiation, or carmustine. A median number of 4.6 x 10(6) CD34+ cells/kg were obtained with a median of three leukapheresis procedures. Mobilization of PBPCs using chemotherapy plus G-CSF was superior to G-CSF alone (6.7 x 10(6) versus 1.5 x 10(6) CD34+ cells/kg; P = 0.0002). Poorer mobilization of progenitor cells was observed in patients who had previously received stem cell-toxic chemotherapy, including (a) nitrogen mustard, procarbazine, melphalan, carmustine or > 7.5 g of cytarabine chemotherapy premobilization (2.0 x 10(6) versus 6.0 x 10(6) CD34+ cells/kg; P = 0.005), or (b) > or = 11 cycles of any previous chemotherapy (2.6 x 10(6) versus 6.7 x 10(6) CD34+ cells/kg; P = 0.02). Platelet recovery to > 20,000/microliter was delayed in patients who received < 2.0 x 10(6) CD34+ cells (median, 13 versus 22 days; P = 0.06). Patients who received > or = 11 cycles of chemotherapy prior to PBPC mobilization tended to have delayed platelet recovery to > 20,000/microliter and to require more platelet transfusions than less extensively pretreated patients (median, 13.5 versus 23.5 days; P = 0.15; median number of platelet transfusion episodes, 13 versus 9; P = 0.17). These data suggest that current strategies to mobilize PBPCs may be suboptimal in patients who have received either stem cell-toxic chemotherapy or > or = 11 cycles of chemotherapy prior to PBPC mobilization. Alternative approaches, such as ex vivo expansion or the use of other growth factors in addition to G-CSF, may improve mobilization of progenitor cells for PBPC transplantation.     

Femur head necrosis and bone marrow edema syndrome in pregnancy

Patients with angioimmunoblastic lymphadenopathy (AILD)-type T-cell lymphoma may develop hypergammaglobulinemia. Among four cases of AILD-type T-cell lymphoma that we have studied, we detected a correlation between the number of plasma cells in tissue and the extent of interleukin-6 (IL-6) expression in lymphoma cells. We did not detect IL-6 in three patients who had no hypergammaglobulinemia and whose tissues showed only minimal plasma cell infiltration. In the fourth patient we observed an abundant IL-6 production by lymphoma cells, which accounted for a B-cell plasmacytic tissue response and for hypergammaglobulinemia. The pathogenic significance of IL-6 was substantiated by a concomitant decrease in the serum IL-6 level, measurable tumor mass, and immunoglobulin levels, as well as by a decline in the proportion of plasmacytoid cells in peripheral blood promptly on administration of chemotherapy. Plasmacytoid B cells could be maintained in culture in the presence of IL-6, but viability was lost on co-incubation with anti-IL-6. Interleukin-1 and tumor necrosis factor were not produced by T lymphoma cells and were incapable of sustaining plasmacytoid B-cell viability in vitro. Small amounts of IL-4 were noted in T lymphoma cells. Thus, in this case of AILD-type T-cell lymphoma, tumor cells with a T-cell phenotype produced IL-6 in large quantities, explaining the accompanying B-cell and plasmacytic histologic changes and humoral disease manifestations, including marked hypergammaglobulinemia. Although not all cases of AILD-type T-cell lymphoma have an accompanying plasma cell proliferation and hypergammaglobulinemia, and although the cytokine network in these patients may be more complex than has been recognized, this case with IL-6 expression serves to illustrate the utility of cytokine assays in the analysis of the histopathologic and clinical heterogeneities of peripheral T-cell lymphomas.     

Does eradication of Helicobacter pylori impair healing of nonsteroidal anti-inflammatory drug associated bleeding peptic ulcers? A prospective randomized study

High-dose therapy with peripheral blood stem cell (PBSC) support is a frequently used treatment option in younger patients with poor prognosis histologically indolent (low-grade) non-Hodgkin's lymphoma (NHL), usually at the time of second or subsequent response to conventional-dose therapy. We have undertaken PBSC collection in 57 patients with histologically indolent NHL mobilized with either cyclophosphamide 1.5 g/m2 or the ESHAP regimen, followed by daily G-CSF. Progenitor cell yields were determined by quantification of CD34+ cells and GM-CFC. Twelve patients (21%) failed to achieve the minimum progenitor cell requirements of 1 x 10(6)/kg CD34+ cells or 1 x 10(5)/kg GM-CFC in their pooled harvests and 40 patients (70%) failed to achieve the optimal harvest thresholds of 3.5 x 10(6)/kg CD34+ cells or 3.5 x 10(5)/kg GM-CFC. This high failure rate is significantly higher than that in patients with histologically aggressive NHL or Hodgkin's disease. A multivariate analysis was performed to identify factors contributing to the low stem cell yields in this group. This identified the time interval from the last chemotherapy to the priming chemotherapy as the most important predictive factor. With respect to CD34 and GM-CFC numbers, on the single harvest on the day the white cell count first exceeded 5 x 10(9)/l the P values were 0.0078 and 0.0065, respectively, and for the progenitor cell values on the pooled harvests the P values were 0.004 for CD34+ cells and 0.015 for GM-CFC. Progenitor cell yields may therefore be improved in patients with low grade lymphoma by harvesting at diagnosis if no marrow disease is present, or by delaying mobilization for 6 months post-chemotherapy in patients in first or subsequent remission.     

Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38- cells

In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45(+) cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 10(7) unfractionated cells, approximately 10(5) purified CD34+ cells, or 5 x 10(3) purified CD34+CD38- cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 x 10(4)/mouse) failed to engraft, whereas CD34- cells (10(7)/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38- cells produced in vivo showed a second-order relation to CD34+ graft size, and mice engrafted with purified CD34+CD38- grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38- cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38- cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38- cells in vivo when cotransplanted with purified CD34+CD38- grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.     

Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+ Thy1+ Lin- peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin- progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of >/=7.2 x 10(5) cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 x 10(9)/L, 16 days) and platelet recovery (platelets > 50 x 10(9)/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.