The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Contemporary analysis of the influence of acute kidney injury after reduced intensity conditioning haematopoietic cell transplantation on long-term survival.

We evaluated retrospectively the incidence of acute kidney injury (AKI), defined by risk, injury, failure, loss and end-stage kidney disease (RIFLE) and its influence on long-term survival, in 82 patients aged 18-60 years who underwent a reduced intensity conditioning (RIC) haematopoietic cell transplantation (HCT). Patients (53.6%) developed AKI after HCT: 25% were on risk, 45.5% on injury and 29.5% on failure. In all, 64 patients survived after 100 days of post transplant and were available for long-term survival analysis. At follow-up, 43.7% of patients died. A 5-year overall survival of AKI patients was 41.6% as compared with 67.1% for those who did not develop AKI (P=0.028), and decreased according to AKI severity (risk, 55.6%; injury plus failure, 33.3%; P=0.045). After adjusting for age, history of cardiovascular disease, high-risk disease and chronic GVHD, AKI predicted 5-year overall mortality (AKI: adjusted hazards ratio (AHR), 2.36, 95% CI: 1.03-5.37; P=0.041). Moreover, moderate and severe AKI (injury plus failure) was also associated with an increased 5-year overall mortality (injury plus failure: AHR, 1.64, 95% CI: 1.06-2.54; P=0.024). According to RIFLE, 53.6% of patients had AKI after RIC HCT. Such patients have poor long-term survival, particularly in moderate or severe AKI.

Allogeneic stem cell transplantation in patients with myelodysplastic syndrome: outcome analysis according to the International Prognostic Scoring System.

We determined the outcome of patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML) after allogeneic stem cell transplantation according to their international prognostic scoring system (IPSS) risk categories at diagnosis. A total of 11 females and 7 males, with a median age of 45 years, were transplanted. With a median follow-up of 60 months, the 6-year actuarial event-free survival (EFS) for Less Advanced (Low and Intermediate-1 risk IPSS) and Advanced (Intermediate-2 and High risk IPSS) MDS was 71.4% and 43.6%, respectively (p=0.002). We did not observe a difference in EFS depending on cytogenetics at diagnosis (good risk 53.8% Vs intermediate and high risk 53.3%, p=ns), neither on the type of conditioning regimen used (myeloablative 50% Vs reduced intensity 52.2%, p=ns). Our results support that IPSS score at diagnosis may be used to predict EFS in patients with MDS undergoing allogeneic SCT.

Haploidentical stem cell transplantation with purified CD34 cells after a chemotherapy-alone conditioning regimen.

We investigated whether a novel chemotherapy-alone conditioning regimen would permit durable engraftment of standard doses of CD34+ purified stem cell grafts from full-haplotype mismatched related donors. We also examined the role of infusing limited doses of donor leukocytes for prevention of leukemia relapse. Our conditioning regimen consisted of thiotepa, fludarabine, rabbit antithymocyte globulin, melphalan, cyclosporin, and prednisolone. Since October 1998, 14 patients with high-risk leukemia were treated; 13 donor-patient pairs shared 3 of 6 HLA antigens, and 1 pair shared 5 of 6 HLA antigens. A median of 5.4 x 10(6) CD34+ cells per kilogram, 1.62 x 10(4) CD3+ cells per kilogram, and 9.32 x 10(4) CD19+ cells per kilogram were infused. T-cell depletion was the only graft-versus-host disease (GVHD) prophylaxis. All patients had prompt engraftment, and no late graft rejections were observed. All surviving patients received at least 1 infusion of donor whole blood containing 5, 7, 10, 25, or 50 x 10(3) CD3+ cells per kilogram between days 25 and 95 after transplantation, after which 8 developed acute GVHD (3 grade I, 2 grade II, 2 grade III, and 1 grade IV) and 2 developed a bronchiolitis obliterans-like syndrome. After attaining complete remission, 5 patients relapsed and died with active leukemia. The estimated relapse-related mortality at 4 years is 38.1%. As of June 15, 2003, 6 of 14 patients have survived a median of 43.5 months after transplantation with 100% donor cells. All 6 surviving patients developed acute GVHD and had a natural killer cell mismatch with their donors in the direction of graft versus host. The estimated overall survival and event-free survival for the 14 patients at 4 years is 41.7% +/- 13.5%.

[Donor leukocyte infusion after allogeneic stem cell transplantation]. / Infusão de leucócitos de dador após a transplantação alogénica de células estaminais.

Adoptive cellular immunotherapy with donor leukocytes of patients submitted to allogenic stem cell transplantation has had significant success in the past few years, especially in the treatment of primary disease relapse and in the prevention and treatment of some post-transplant infectious complications. Most patients treated with donor leukocytes had a relapse of chronic myelogenous leukemia, which was successfully re-induced into remission. The most significant toxicities of this treatment are the development of graft versus host disease and marrow aplasia. Three strategies were developed to limit the former: the infusion of graded doses of donor leukocytes, the depletion of CD8+ cells and the transfer of donor leukocytes transvected with a timidine kinase gene, which renders these cells sensitive to gancyclovir. The post-transplant infectious complications treated successfully with donor leukocytes were Epstein-Barr virus-induced lymphoproliferative disorders and cytomegalovirus infection. The former, arising most frequently in recipients of unrelated and/or mismatched T-cell depleted grafts, were treated with donor unseparated leukocytes or Epstein-Barr virus-specific T-cells. Cytomegalovirus infection in the early post-transplant period was largely prevented by the infusion of virus-specific T-cell clones, which restored donor-specific immunity to cytomegalovirus in the recipient.

Characteristics of human EBV-specific cytotoxic T lymphocytes utilized for adoptive immunotherapy of EBV-induced lymphoproliferations in xenografted SCID mice.

Human Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) prolong the survival of mice with severe combined immune deficiency bearing the autologous, but not HLA-mismatched, human EBV-induced lymphoproliferative disorders (EBV-LPDs). In the present study, we demonstrate that the HLA-restricted activity displayed by EBV-CTLs both in vitro and in vivo correlates with their in vivo homing pattern, and further characterize these effectors. EBV-CTLs were CD3+, CD16/56-, TCR alpha/beta+, predominantly CD8+ and CD4-, and had a high expression of T-cell activation antigens. EBV-CTLs were positive for CD11a/CD18, CD54, CD58, CD44, CD49d, CD28, and CD45RO, and negative for CD45RA, CD11b, CD11c. After 26 days in culture, EBV-CTLs displayed strong cytotoxicity against the autologous EBV-transformed B-cell line (EBV-LCL), which was inhibited by the addition of anti-CD3 MoAb and mostly HLA class I-restricted. Unirradiated and irradiated EBV-CTLs in the absence of IL-2 failed to proliferate after more than 2 days in culture with the autologous EBV-LCLs, while unirradiated EBV-CTLs with IL-2 formed large colonies and had a high thymidine incorporation both on days 5 and 8. The cytotoxicity of irradiated EBV-CTLs against the autologous EBV-LCLs was conserved. It remains to be determined whether irradiated EBV-CTLs are capable of homing to EBV-LPDs in vivo and to mediate a therapeutic response comparable to that observed with unirradiated EBV-CTLs.

Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice.

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3-activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV-specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.

Administration of rabbit anti-asialo GM1 antiserum facilitates the development of human Epstein-Barr virus-induced lymphoproliferations in xenografted C.B-17 scid/scid mice.

Mice with severe combined immune deficiency (C.B-17 scid/scid [SCID mice]) lack functional B and T lymphocytes and are permissive for the growth of human xenografts. However, the development of functional NK cells is not affected by the scid mutation. Mouse NK cells express the surface glycolipid asialo GM1 and are implicated in the rejection of heterotransplanted cells. In the present study, we demonstrate that SCID mice treated with rabbit anti-asialo GM anti-serum (alpha-asialo GM1), for in vivo depletion of endogenous NK cell function, develop lethal Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (EBV-LPD) at lower doses od inoculated EBV-transformed lymphoblastoid B cell lines (EBV-LCL) than untreated animals. Furthermore, at any given dose of EBV-LCL inoculated, EBV-LPD developed earlier and induced lethality sooner in alpha-asialo GM1-treated animals. We also demonstrate that SCID mice treated with alpha-asialo GM1 have reduction in the number of asialo GM1-expressing splenocytes. Moreover, splenic cell suspensions derived from alpha-asialo GM1-treated SCID mice show lower cytotoxicity against the mouse NK-sensitive cell line YAC-1 and human EBV-LCL than splenocytes obtained from untreated SCID mice.

Selective treatment of SCID mice bearing methotrexate-transport-resistant human acute lymphoblastic leukemia tumors with trimetrexate and leucovorin protection.

Impaired transport of methotrexate (MTX) is a common resistance mechanism of tumor cells to this drug. Trimetrexate (TMTX), a second-generation folate antagonist, is still active against MTX-transport-resistant cells because it enters cells by passive diffusion and does not use the reduced folate transport system for cell entry. Therefore, although leucovorin (LV) protects MTX-sensitive cells from TMTX toxicity, MTX-transport defective cells are poorly rescued by LV. Severe combined immunodeficiency mice bearing MTX-transport-resistant CCRF-CEM acute lymphoblastic leukemia tumors were treated with TMTX alone or with the combination of TMTX and LV, with tumor regressions in both groups (P < .001) and without significant toxicity. These results indicate that TMTX with LV protection may be a useful therapeutic regimen for patients with MTX-transport-defective acute lymphoblastic leukemia. Furthermore, resistance to TMTX plus LV may result in reversion to MTX sensitivity.

[Treatment of acute promyelocytic leukemia with trans-retinoic acid. Experience of the Santa Maria Hospital, Medical School of Lisbon]. / Terapêutica da Leucemia promielocítica aguda com ácido Trans-Retinóico. Experiência do Hospital de Santa Maria, Faculdade de Medicina de Lisboa.

Acute promyelocytic leukemia (APL) is a rare subtype of acute myelogenous leukemia that is usually associated with a fatal hemorrhagic diathesis. All trans-retinoic acid (ATRA) is an active metabolite of vitamin A that differentiates the malignant cell clone, corrects the coagulopathy, and induces complete remission in the vast majority of patients with APL. Between June 1992 and September 1993, 8 patients with APL (4 previously untreated, 3 in first relapse and 1 in second relapse) received ATRA. Complete remission was achieved in 7 patients; in 5 with ATRA alone and in 2 with ATRA followed by cytotoxic chemotherapy due to the development of asymptomatic hyperleukocytosis. The earliest signs of response were the correction of the coagulopathy and an increase in the white blood cell count. Sequential morphological and immunophenotypical analyses of the bone marrow revealed differentiation of the malignant cell clone, in the absence of bone marrow hypoplasia. 4 of 5 patients treated only with ATRA until complete remission had late leukopenia. The most frequent adverse effects were dryness of skin and mucosae, hypertrigliceridemia and hypercholesterolemia, and a moderate increase in liver transaminases. An increase in the white blood cell count was common, and in two cases exceeded 35.0 x 10(9)/l. One of these patients developed multiple thrombosis of the extremities after cytotoxic chemotherapy. We frequently observed an increase in lactic dehydrogenase levels that was concomitant with the peak in the white blood cell count. The only patient on whom complete remission was not achieved was 60 years old, had chronic obstructive pulmonary disease, and died in the third week of therapy with a pulmonary distress syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)