Massive hemolysis following allogeneic peripheral blood stem cell transplantation with minor ABO incompatibility

We observed massive hemolysis after allogeneic peripheral blood stem cell transplantation with minor ABO incompatibility (the donor was group O and the recipient group A). The patient was a 21-year-old man diagnosed as having Philadelphia chromosome-positive chronic myelogenous leukemia. On day 7 post-transplant, there was abrupt onset of massive hemolysis necessitating supportive treatment with transfusions. During the allogeneic peripheral blood stem cell transplantation the patient had received 2 x 10(8) CD3 positive cells/kg and 7.8 x 10(7) CD19 positive cells/kg donor lymphocytes with stem cells. The present case shows that in allogeneic peripheral blood stem cell transplantation with ABO incompatibility, severe hemolysis occurs early after transplantation presumably due to the transfusion of a large number of lymphocytes.     

Monitoring of CD59 expression in paroxysmal nocturnal hemoglobinuria treated with danazol.

We describe a 52-year-old man with paroxysmal nocturnal hemoglobinuria (PNH) and a moderate transfusion requirement. Prior to and during sequential therapy with androgen (metenolone), glucocorticoid, and danazol, we evaluated CD59-negative expression (PNH clone) in red blood cells, neutrophils, lymphocyte subsets, and bone marrow (BM) CD34(+) cells. Although androgen and glucocorticoid were not effective for recovery of blood cell counts, the hemoglobin and platelet levels increased immediately after the therapy with danazol and the patient became transfusion independent. However, neither the serum level of LDH nor the percentage of PNH clone in each cell lineage, including BM CD34(+) cells, decreased. The number of nucleated cells in BM increased drastically after the start of danazol. These findings suggest that the efficacy of danazol was not only due to the impediment of hemolysis but also due to stimulation of PNH clone proliferation in BM.     

Wiskott-Aldrich syndrome in a female

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema, and various degrees of immune deficiency. Carriers of mutated WASP have nonrandom X chromosome inactivation in their blood cells and are disease-free. We report data on a 14-month-old girl with a history of WAS in her family who presented with thrombocytopenia, small platelets, and immunologic dysfunction. Sequencing of the WASP gene showed that the patient was heterozygous for the splice site mutation previously found in one of her relatives with WAS. Sequencing of all WASP exons revealed no other mutation. Levels of WASP in blood mononuclear cells were 60% of normal. Flow cytometry after intracellular staining of peripheral blood mononuclear cells with WASP monoclonal antibody revealed both WASP(bright) and WASP(dim) populations. X chromosome inactivation in the patient's blood cells was found to be random, demonstrating that both maternal and paternal active X chromosomes are present. These findings indicate that the female patient has a defect in the mechanisms that lead in disease-free WAS carriers to preferential survival/proliferation of cells bearing the active wild-type X chromosome. Whereas the patient's lymphocytes are skewed toward WASP(bright) cells, about 65% of her monocytes and the majority of her B cells (CD19(+)) are WASP(dim). Her naive T cells (CD3(+)CD45RA(+)) include WASP(bright) and WASP(dim) populations, but her memory T cells (CD3(+)CD45RA(-)) are all WASP(bright). After activation in vitro of T cells, all cells exhibited CD3(+)CD45RA(-) phenotype and most were WASP(bright) with active paternal (wild-type) X chromosome, suggesting selection against the mutated WASP allele during terminal T-cell maturation/differentiation.     

Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles

Circulating CD34(+) cells are used in reparative medicine as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are confined to bone marrow (BM) niches until they differentiate into B cells and that they do not circulate in blood. The prevailing convention is that BCP transit a CD34(+)CD19(-)10(+) early-B-->CD34(+)CD19(+)CD10(+) B-cell progenitor (pro-B)-->CD34(-)CD19(+)CD10(+) B-cell precursor (pre-B) differentiation pathway within BM. However, populations of CD34(+)CD10(+) and CD34(+)CD19(+) cells circulate in adult peripheral blood and neonatal umbilical cord blood (CB) that are operationally taken as BCPs on the basis of their phenotypes, although they have not been submitted to a systematic characterization of their gene expression profiles. Here, conventional CD34(+)CD19(+)CD10(+) and novel CD34(+)CD19(+)CD10(-) BCP populations are characterized in CB by single-cell sorting and multiplex analyses of gene expression patterns. Circulating BCP are Pax-5(+) cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-J(H), CD79, VpreB, recombination activating gene (RAG), and terminal deoxynucleotidyl transferase (TdT) expression. Contrary to the expectation, circulating CD34(+)CD19(-)CD10(+) cells are essentially devoid of Pax-5(+) BCP. Interestingly, the novel CD34(+)CD19(+)CD10(-) BCP appears to be the normal counterpart of circulating preleukemic BCPs that undergo chromosomal translocations in utero months or years before their promotion into infant acute lymphoblastic B-cell leukemia after secondary postnatal mutations. The results underscore the power of single-cell analyses to characterize the gene expression profiles in a minor population of rare cells, which has broad implications in biomedicine.     

Blood levels of immune cells predict survival in myeloma patients: results of an Eastern Cooperative Oncology Group phase 3 trial for newly diagnosed multiple myeloma patients.

Previously, it was reported that patients with multiple myeloma (MM) who have higher baseline levels of blood CD4(+) or CD19(+) cells have longer survival. This article extends the analysis of immune cell levels and survival in a large cohort (N = 504) of patients with MM entered on Eastern Cooperative Oncology Group (ECOG) phase 3 trial (9486). Newly diagnosed patients with MM received 2 cycles of vincristine, bischloroethylnitrosourea, melphalan, cytoxan, prednisone (VBMCP) and were treated on one of 3 randomized arms: VBMCP with either interferon or high-dose cyclophosphamide, or VBMCP alone. Blood immune cell levels were studied at trial entry (baseline), after 2 cycles of chemotherapy, after 2 years of therapy, and at relapse. Baseline CD3(+), CD4(+), CD8(+), CD19(+), and CD4(+) subset cell levels were all positively associated with survival (P =.0087 to P <.0001). A multivariate analysis incorporating CD4(+) and CD19(+) cell levels defined 3 separate groups of patients with MM to survival outcome. Higher CD19(+) blood levels were positively associated with MM-patient survival at entry to the study, at year 2, and at relapse (P <.0001 at all 3 timepoints). Patients with MM had evidence of immune cell reconstitution after 2 years of therapy, but the rate and extent of recovery was greater for CD8(+), which was greater than CD4(+), which was greater than CD19(+). This latter data affirms the positive relationship between the quantitative status of the blood immune system in MM and survival. In addition, the importance of the CD19(+) blood cells to survival is evident throughout the course of MM. Therapeutic efforts to maintain an intact immune system may be crucial in maximizing chemotherapeutic and/or immunotherapy efforts in this disease.     

Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response

Maintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19(+)/CD27(high)/intracellular immunoglobulin G(high) (IgG(high))/HLA-DR(high)/CD38(high)/CD20(-)/CD95(+) tetanus toxin-specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19(+)/CD27(high)/intracellular IgG(high)/HLA-DR(low)/CD38(+)/CD20(-)/CD95(+) cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.     

Autologous peripheral blood stem cells harvesting in Kaohsiung Medical University Hospital

We report our clinical experience in autologous peripheral blood stem cells harvesting. A total of 40 patients with 112 apheresis procedures were analyzed, 88 with Cobe system and 24 with MCS3P system. Our results revealed that there was no significant difference in efficiency of CD34(+) cell harvesting between both apheresis systems, but the Cobe system had more nucleated cells collected and less red cell contaminated in the final PBSCs collections. The percentage of CD34(+) cells collected decreased significantly following the first day's harvesting (p = 0.026). There was a good correlation between the percentage of CD34(+) cells in PBSCs and colony forming units-granulocyte macrophage (CFU-GM) or burst forming unit-erythrocyte (BFU-E)(r = 0.909, p < 0.0001; r = 0.788, p < 0.0001, respectively). However, it was negatively correlated with the patient's age. The CD34(+) cells collected in patients with acute leukemias were also higher than those patients with other solid tumors. Ten cases (13%) with 15 apheresis procedures experienced side effects like numbness, nausea, fever, or headache etc. The Cobe system seemed to have higher frequency of side effects than that of MCS3P system (16% vs 4%). From our results, we concluded that both COBE and MCS3P system have similar efficiency and all patients could tolerate the apheresis procedures in peripheral blood stem cells harvesting. The CD34(+) cell can be used as a good parameter to estimate the amount of stem cells collected. The patient's age and disease pattern were significant factors influencing the CD34(+) stem cells collection in autologous peripheral blood stem cells harvesting.     

Prolonged ex vivo culture of cord blood CD34(+) cells facilitates myeloid and megakaryocytic engraftment in the non-obese diabetic severe combined immunodeficient mouse model

A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.     

IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.     

Allogeneic graft CD34(+) cell dose correlates with dendritic cell dose and clinical outcome, but not with dendritic cell reconstitution after transplant

OBJECTIVE: This study examined whether the CD34(+) cell dose in allografts correlates with the dose of myeloid dendritic cells (mDC) and plasmacytoid DC (pDC), and with DC reconstitution and clinical outcome after a myeloablative HLA-matched transplant. PATIENTS AND METHODS: Fifty-three patients were included in this study: 37 who had undergone a granulocyte colony-stimulating factor mobilized peripheral blood stem cells (PBSC) transplant from related donors and 16 who had undergone a marrow transplant from unrelated donors. The number of CD34(+) cells, lin(-)HLA-DR(+)CD11c(+) mDC, lin(-)HLA-DR(+)CD123(+) pDC, CD14(+) monocytes, and CD3(+)CD4(+), CD3(+)CD8(+), CD56(+), and CD19(+) lymphocytes was compared in the graft, as well as in the peripheral blood after transplant, in patients receiving more than versus less than or equal to the median number of CD34(+) cells in PBSC (5.78 x 10(6)/kg) or in marrow (2.8 x 10(6)/kg). RESULTS: A higher CD34(+) cell dose was associated with larger numbers of mDC in PBSC (p=0.01) and pDC in marrow grafts (p=0.004). However, neither mDC nor pDC recovery after transplant correlated with the number of CD34(+) cells infused. Finally, higher doses of CD34(+) cells appeared to negatively affect (p=0.02) the overall survival in PBSC transplantation and were associated with a trend for higher acute graft-vs-host disease in PBSC and lower acute graft-vs-host disease in marrow transplant. CONCLUSIONS: CD34(+) cell dose correlates with the dose of different DC subsets in PBSC and marrow grafts, but it does not affect DC reconstitution after transplant. Higher doses of CD34(+) cells in PBSC, but not in marrow, seem to adversely affect survival after transplant.     

High-dose therapy in patients with Hodgkin's disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells.

Register data suggest that patients with Hodgkin's disease (HD) given high-dose therapy (HDT) with peripheral blood progenitor cells (PBPC) have a less favourable prognosis as compared to those given bone marrow as stem cell support. Since this can be due to infusion of tumour cells contaminating the PBPC grafts, we initiated a feasibility study in which PBPC grafts from HD patients were purged by CD34(+) cell enrichment. Controversy exists about whether the use of CD34(+) enriched stem cells leads to a delayed haematological and immune reconstitution. We compared these parameters, including risk of infections and clinical outcome after HDT, in patients with HD given either selected CD34(+) cells or unmanipulated PBPC as stem cell support. From October 1994 to May 2000, 40 HD patients with primary refractory disease or relapse were treated with HDT and supported with either selected CD34(+) cells (n = 21) or unmanipulated PBPC (n = 19) as stem cell support. All patients had chemosensitive disease at the time of transplantation. A median of 5.8 (range 2.7-20.0) vs 4.5 (range 2.3-17.6) x 10(6) CD34(+) cells per kilo were reinfused in the CD34(+) group and PBPC group, respectively. No difference was observed between the two groups with regard to time to haematological engraftment, reconstitution of B cells, CD56(+) cells and T cells at 3 and 12 months and infectious episodes after HDT. Two (5%) treatment-related deaths, one in each group, were observed. The overall survival at 4 years was 86% for the CD34(+)group and 74% for the PBPC group with a median follow-up of 37 months (range 1-61) and 46 months (range 4-82), respectively (P = 0.9). The results of this study demonstrate that the use of CD34(+) cells is safe and has no adverse effects either with respect to haematological, immune reconstitution or to infections after HDT.     

CD81 expression for discrimination between sustained virologic response and relapse in patients with chronic hepatitis C

Abstract

Background and aim. The hepatitis C virus (HCV) receptor CD81 is overexpressed on peripheral blood mononuclear cells (PBMC) in patients chronically infected with HCV compared with healthy controls, and expression declines during antiviral therapy. The aim of this study was to prospectively investigate CD81 expression on PBMC for early discrimination between sustained virologic response (SVR) and relapse (REL) to pegylated interferon alfa-2b and ribavirin treatment. Methods. Sixty-one patients with chronic HCV infection (genotype, GT, 1 and low baseline viremia <600,000 IU/ml, n = 30; GT 2 or 3, n = 31) were investigated. CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was measured at baseline, therapy week (TW) 4 and 12 during antiviral therapy by fluorescence-activated cell sorting (FACS) analysis. Results. Baseline levels of CD81 on CD4(+), CD8(+), and CD56(+) cells were similar between patients who achieved a SVR (n = 42) and those who relapsed (n = 19). On CD19(+) cells, baseline CD81 expression was higher in patients with SVR than in patients with virologic relapse (REL) (p < 0.006). A cutoff value of 720 relative fluorescence units (RFU) discriminated correctly between SVR and REL with a sensitivity and specificity of 73.7% and 66.7%, respectively. SVR patients showed a significant decline of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells (p < 0.01 for all) while in REL patients a significant decline of CD81 expression was observed on CD8(+) and CD56(+) cells, only (p = 0.050 and p = 0.038, respectively). Conclusions. The current study confirms significant down-regulation of CD81 expression on different lymphocyte subpopulations during pegylated interferon alfa-based antiviral therapy in patients with chronic hepatitis C. Baseline CD81 expression on CD19(+) cells was found to discriminate between SVR and REL.     

Functional analyses of cord blood natural killer cells and T cells: a distinctive interleukin-18 response

OBJECTIVE: To search for the functional property of cord blood (CB) cells, the effects of interleukin-18 (IL-18) on interferon-gamma (IFN-gamma) production of T cells or natural killer (NK) cells were compared between CB and adult peripheral blood (PB). MATERIALS AND METHODS: T cells, CD45RA(+) T cells, and NK cells were purified from CB and adult PB mononuclear cells using magnetic beads or a cell sorter. After stimulation with or without IL-18 in the presence of IL-12 for 48 hours (NK cells) or 72 hours (T cells or CD45RA(+) T cells), IFN-gamma concentration was measured in each subset. Although IL-18 induced significant IFN-gamma production from both CB and adult PB T cells in the presence of IL-12, the IFN-gamma levels from CB T cells were lower than those from adult PB T cells. However, CD45RA(+) T cells from CB and from adult PB produced similar levels of IFN-gamma after stimulation with IL-18 + IL-12. On the other hand, CB NK cells exhibited higher IFN-gamma production and CD69 expression than adult PB NK cells after stimulation with IL-18 + IL-12. Cytolytic activity of CB NK cells increased to a level comparable to that of adult PB NK cells after the same IL-18/IL-12 stimulation. CONCLUSIONS: These results suggest that a low response of CB T cells to IL-18 is due to a higher proportion of naive (CD45RA(+)) T cells in CB, which may be one of the factors responsible for the neonatal immaturity of the immune system as well as the low incidence of graft-vs-host disease in patients receiving CB stem cell transplantation. On the other hand, a high response of CB NK cells to IL-18 may contribute to the host defense during the neonatal period and antitumor effects in CB stem cell transplantation.     

CD4+ T-helper cells stimulated in response to placental ischemia mediate hypertension during pregnancy

We have shown that hypertension in response to chronic placental ischemia is associated with elevated inflammatory cytokines and CD4(+) T cells. However, it is unknown whether these cells play an important role in mediating hypertension in response to placental ischemia. Therefore, we hypothesize that reduced uterine perfusion pressure (RUPP)-induced CD4(+) T cells increase blood pressure during pregnancy. To answer this question, CD4(+) T cells were isolated from spleens at day 19 of gestation from control normal pregnant (NP) and pregnant RUPP rats, cultured, and adjusted to 10(6) cells per 100 μL of saline for intraperitoneal injection into NP rats at day 13 of gestation. On day 18, in the experimental groups of rats, arterial catheters were inserted, and on day 19 mean arterial pressure was analyzed. Inflammatory cytokines and antiangiogenic factor soluble fms-like tyrosine kinase 1 were determined via ELISA. Mean arterial pressure increased from 104±2 mm Hg in NP rats to 124±2 mm Hg in RUPP rats (P<0.001) and to 118±1 mm Hg in rats receiving RUPP CD4(+) T cells (P<0.001). Circulating tumor necrosis factor-α and soluble fms-like tyrosine kinase 1 were elevated in recipients of RUPP CD4(+) T cells to levels similar to control RUPP rats. In contrast, virgin rats injected with NP or RUPP CD4(+) T cells exhibited no blood pressure changes compared with control virgin rats. Importantly, mean arterial pressure did not change in recipients of NP CD4(+) T cells (109±3 mm Hg). These data support the hypothesis that RUPP-induced CD4(+) T cells play an important role in the pathophysiology of hypertension in response to placental ischemia.     

Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell-depleted bone marrow, we measured the appearance of CD4(+)CD45RA(+)RO(-) naive T cells (thymic rebound), restoration of the diversity of the T-cell-receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4(+)CD45RA(+)RO(-) thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4(+) T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4(+)CD45RA(+)RO(-) T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4(+)CD45RA(+)RO(-) T cells less than 30 microL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4(+)CD45RA(+)RO(-) naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.     

Increased activated human T cell lymphotropic virus type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load

To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I (HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.     

Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors

Immature and predendritic cells (pre-DCs) of human blood are the most readily accessible human DC sources available for study ex vivo. Murine homologues of human blood DCs have not been described. We report the isolation and characterization of 2 populations of precursor DCs in mouse blood. Mouse blood cells with the surface phenotype CD11c(lo)CD11b(-)CD45RA(hi) closely resemble human plasmacytoid cells (or pre-DC2) by morphology and function. On stimulation with oligonucleotides containing CpG motifs (CpG), these cells make large amounts of type 1 interferons and rapidly develop into DCs that bear CD8, though they may be distinct from the CD8(+) DCs in the unstimulated mouse. A second population of cells with the surface phenotype CD11c(+)CD11b(+)CD45RA(-) closely resembles the immediate precursors of pre-DC1, rapidly transforming into CD8(-) DCs after tumor necrosis factor-alpha (TNF-alpha) stimulation. These findings indicate the close relationship between human and mouse DCs, provided cells are obtained directly from equivalent source materials.     

A prospective study of positive/negative ex vivo B-cell depletion in patients with chronic lymphocytic leukemia

OBJECTIVE: Autologous peripheral blood stem cell (PBSC) transplantation is increasingly being used in patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have prospectively investigated the feasibility and efficacy of ex vivo double purging of PBSC grafts in an open, nonrandomized, single-center phase I/II clinical study. MATERIALS AND METHODS: Twenty consecutive patients with poor-risk CLL underwent uniform stem cell mobilization with chemotherapy and granulocyte colony-stimulating factor (G-CSF). Double B-cell depletion of the harvested PBSC products was performed using immunomagnetic CD34(+) cell selection (Isolex300i Nexell, Irvine, CA) followed by a negative step with anti-CD19/20/23/37-labeled immunomagnetic beads. The purified PBSC were reinfused after myeloablative treatment with TBI/CY. RESULTS: A total of 25 separation runs was accomplished using collection products containing 3.4% (1.1-8.1) CD34(+) cells and 1.2% (0.1-42) CD19(+)CD5(+) CLL cells. After double selection, 33% (15-67) CD34(+) cells were recovered with a purity of 98.8% (89.1-99. 8). CLL cells were undetectable by high-resolution flow cytometry in 15 of 25 final products; median purging efficacy was 5 (4.1-6) log. The CD34(+) content of the 20 final grafts was 4.6 (2.2-6.5) x 10(6)/kg. Rapid and durable engraftment developed in all cases. With a median follow-up of 20 (6-29) months, 17 patients live in complete clinical remission, two have recurrent disease, and one patient died due to pulmonary embolism five months after transplant. Persistence of the leukemic clone on the molecular level was demonstrated by dot blotting with clone-specific CDR3 probes in an additional five patients. Serious or unexpected infectious complications did not occur. CONCLUSIONS: Positive/negative purging with the Isolex system allows preparation of highly purified CD34(+) fractions and up to six log of tumor cell depletion in patients with B-CLL and can be safety reinfused after myeloablative therapy without affecting hematopoietic engraftment.     

No immunological benefit of selenium in consecutive patients with autoimmune thyroiditis.

BACKGROUND: Recently it has been demonstrated that after selenium (Se) supplementation in autoimmune thyroiditis (AIT) patients, there was a significant decrease of thyroid peroxidase (TPO) autoantibody (TPOAb) levels. The aim of our study was to evaluate the immunological benefit of Se administration in unselected AIT patients and thus address the question whether Se administration should generally be recommended for AIT patients. METHODS: Thirty-six consecutive AIT patients (aged 19-85 years) were included in the present study. In addition to their levothyroxine (LT(4)) treatment, 18 patients received 200 microg (2.53 micromol) sodium selenite per day orally for the time span of 3 months, whereas 18 patients received placebo. All patients had measurement of thyroid hormones, thyrotropin (TSH), autoantibodies (thyroglobulin antibodies [TgAb] and TPOAb), Se levels, and intracellular cytokine detection in CD4(+) and CD8(+) T cells of peripheral blood mononuclear cells (PBMC) by flow cytometry before and after Se or placebo administration. RESULTS: No significant difference in the TPOAb levels was found after Se administration (524 +/- 452 vs. 505 +/- 464 IU/mL; p > 0.05). Furthermore, we found no significant differences in the CD4(+) or CD8(+) cytokine pattern (IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-alpha, TNF-beta) in patients before and after Se administration, in patients before and after placebo administration and between Se group and placebo group before and after Se vs. placebo administration. CONCLUSION: We demonstrate that Se administration in our AIT patient's cohort does not induce significant immunological changes, either in terms of cytokine production patterns of peripheral T lymphocytes or of TPOAb levels. Our data suggest that AIT patients with moderate disease activity (in terms of TPOAb and cytokine production patterns) may not (equally) benefit as patients with high disease activity.     

Simian immunodeficiency virus infection of CD4+CD8+ T cells in a macaque with an unusually high peripheral CD4+CD8+ T lymphocyte count

We assessed the possible role in vivo CD4(+) CD8(+) T cells as a viral reservoir for simian immunodeficiency virus (SIV), in a macaque with 50% CD4(+) CD8(+) T cells in peripheral blood. During primary infection (day 14) of this rhesus macaque with the pathogenic SIVmac251 strain, proviruses were detected at similar frequencies in CD4(+) CD8(+) T cells (1/10) and CD4(+) T cells (1/10) and at a lower frequency in CD8(+) T cells (1/800). On day 235, no viral DNA was detected in CD8(+) cells, despite the persistent high viral load, indicating that CD8(+) cells do not constitute a reservoir during the chronic phase of SIV infection. Infection induced early lymphopenia of CD4(+), CD4(+) CD8(+), and CD8(+) cells; only the CD8(+) cell population returned to initial levels and expanded further. We found that CD4(+) CD8(+) T cells expressed the costimulatory CD28 molecule less and were more prone to die in vitro after phytohemagglutinin/interleukin 2 stimulation than were CD4(+) T cells. Taken together, massive death of CD4(+) CD8(+) T cells during acute stages of SIV infection may explain why CD8(+) T cells did not represent a major reservoir for SIV at the onset of infection.