Alejandro Ruiz-Argüelles

Immunopathogenesis of vitiligo

Vitiligo is a common depigmenting disorder which may have devastating psychological and social consequences and is characterized by the presence of circumscribed white macules in the skin due to the destruction of melanocytes in the epidermis. Various hypotheses have been proposed to explain the pathomechanisms involved in this disease, and studies have shown the participation of autoimmune processes in the pathogenesis of vitiligo. Cellular and humoral immunities have been implicated in the development of vitiligo and their role continues to be investigated. Peripheral blood and skin biopsies of patients with vitiligo show that T-cells, mononuclear cells, various pro-inflammatory cytokines, and auto-antibodies can damage melanocytes. Further research is required to determine whether autoimmunity is the main mechanism of vitiligo or only a consequence.

General concepts about cell sorting techniques

Research involving cell analysis frequently requires isolation of certain cell types or subcellular components either as a final objective or as a preparative tool for further assays. At present, there are a high number of cell sorting methods that are suitable for being used in the clinical laboratory. These methods can be divided into two major groups: (1) bulk sorters and (2) single-cell-based sorters. This latter group mainly refers to fluorescence-activated cell sorting (FACS) by flow cytometry (FCM). In both cases, separation of cell subsets is based on their classification according to one or more cell characteristics. In bulk sorters, cell classification and sorting are usually achieved in a single step; by contrast, in FACS techniques, these two steps are independent sequential processes. In addition, bulk sorters generally use a single-cell characteristic to isolate cell subsets and have a higher throughput rate, as compared with FACS by FCM, where several parameters can be used simultaneously to classify cells for their further isolation. As a consequence of the mechanisms underlying these two cell sorting methods, the balance between cell purity and cell recovery on the sorted fraction are generally different, the single-cell-based methods usually providing both a higher purity and recovery. Thus, in practice, bulk separation methods are frequently used either as a preparative step for FCM-based cell sorting or for the enrichment of the sample in specific cell subsets, when a higher throughput rate is required; in contrast, FACS by FCM is selected for the isolation of cell subsets when a high purity and, especially, recovery of a specific subpopulation of cells present in a sample are needed.

Antibody Penetration Into Living Cells: Pathogenic, Preventive and Immuno-Therapeutic Implications

The dogmatic and at one time prevalent concept that intact antibody molecules were not able to enter into viable cells has now been modified due to the availability of a large amount of experimental findings and clinical observations that indicate otherwise. Penetration of mature autoantibodies into living cells might participate in the pathogenesis of diverse autoimmune diseases, through inducing apoptosis of healthy tissues and cells, but also by contributing to the breakdown of self-tolerance through presentation of self-antigens to the immune system. Conversely, the penetration of naturally occurring autoantibodies into immature lymphoid cells might have a physiological role in the edition of the immune repertoire in healthy individuals. Increasing interest is being paid to the potential immunotherapeutic role of penetrating antibodies as tools to deliver drugs, isotopes or genes into cells.

Deficiency of red cell bound CD55 and CD59 in patients with systemic lupus erythematosus

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. Autoimmune hemolytic anemia (AIHA) has been associated with antiphospholipid antibodies (APLA). The aim of this study was to evaluate the presence of APLA and its possible correlation with diminished CD55 and CD59 in red blood cells from patients with primary AIHA or secondary to systemic lupus erythematosus (SLE). Flow cytometric analyses were performed on CD55 and CD59 stained erythrocytes from 24 patients (primary AIHA, n=8; AIHA plus SLE, n=11; and SLE without AIHA, n=5) and 20 healthy controls. Antibodies to several phospholipids were detected in the sera by ELISA. Most patients with AIHA plus SLE and few with primary AIHA showed deficiency of either or both CD55 and CD59 expression and was not associated to the presence of APLA, while SLE patients exhibited a normal expression of these molecules. Although our findings showed CD55 and CD59 deficiency in primary or secondary AIHA, it appears that this defect plays a facilitator rather than a triggering role for the hemolytic process. Additionally, a role of anti-phospholipid antibodies as causative of this acquired defect is questionable.

Non-cryopreserved peripheral blood stem cells autotransplants for hematological malignancies can be performed entirely on an outpatient basis

We have prospectively performed peripheral blood stem cell autotransplants in six patients with hematological malignancies on an entirely outpatient basis. Patients were conditioned with high‐dose melphalan and received a median of 4.2 × 108/kg non‐cryopreserved, non‐purged mononuclear cells, containing a median of 3.9 × 106/kg CD34 + cells. The median time to achieve > 500 granulocytes/μl was 21 days, with a range of 16 to 40, whereas the median time to achieve > 20,000 platelets/μl was 38 days, with a range of 21 to 48. Only three patients were transfused with platelets whereas packed red blood cells were transfused in two. All patients survived 60 days after the autograft and three are alive at 450, 690, and 1,950 days after the autotransplant. One patient was given an allogeneic bone marrow transplant when relapsing after the autotransplant. One patient had to be admitted to the hospital on day +10 because of fever. A median of 6,500.00 USD per patient was calculated as the total cost of each outpatient autotransplant. Since outpatient autologous transplants with non‐frozen PBSC are feasible, restrictions to PBSC autotransplant programs may be overcome and costs may be diminished. Am. J. Hematol. 58:161–164, 1998.

Report on the second Latin American consensus conference for flow cytometric immunophenotyping of hematological malignancies

On May 3 and 4, 2005, the Second Latin American Consensus Conference for the Immunophenotyping of Hematological Malignancies took place in Queretaro, México, with representatives from 10 countries of the region and two external consultants. This document summarizes the major conclusions for which consensus were achieved. Major differences regarding the recommendations from the first conference, which took place 9 years ago, concern the medical indications and the antibody panels for immunophenotyping. The aim of disseminating these guidelines to the international community is based on the potential interest for other countries with similar socioeconomic conditions.

Results of an Autologous Noncryopreserved, Unmanipulated Peripheral Blood Hematopoietic Stem Cell Transplant Program: A Single-Institution, 10-Year Experience

Background: Methods to simplify the stem cell transplantation procedures are needed mainly in developing countries. We have previously shown that unprocessed leukapheresis material is useful to restore hematopoiesis after high-dose chemotherapy. Methods: Over a 10-year period in a private practice setting, we prospectively performed autotransplants using noncryopreserved and unmanipulated peripheral blood stem cells mobilized from the bone marrow to the peripheral blood by means of filgrastim and using a single-day conditioning regimen with high dose (200 mg/m2) melphalan. Results: Forty-six individuals were given 50 autografts. The median age of the patients was 33 years (range 8–69). Twenty-two patients with acute leukemia (13 with myeloblastic and 9 with lymphoblastic leukemia), 4 with chronic myelogenous leukemia, 6 with multiple myeloma, 7 with Hodgkin’s disease, 3 with non-Hodgkin’s lymphoma and 4 with metastatic breast carcinoma were included. The median time to achieve >0.5 × 109/l granulocytes was 14 days (range 0–86), whereas the median time to achieve >20 × 109/l platelets was 25 days (range 0–102). The 3,300-day posttransplant survival was 63%, the median posttransplant overall survival was over 3,300 days, the 3,300-day disease-free survival was 50% and the transplant-related mortality was 2%. The procedure was performed entirely on an outpatient basis in the case of 48 autografts (96%). The approximate cost of each graft was 7,500 USD. Conclusion: This simplified method to autograft patients, avoiding in-hospital stays, purging procedures and cryopreservation of the cells, is feasible and results in a substantial decrease in the cost of the autologous hematopoietic stem cell transplantation methods.

Report on the first Latin American consensus conference for flow cytometric immunophenotyping of leukemia

On October 16, 1996, the first Latin American Consensus Conference for the Immunophenotyping of Leukemia took place in Puebla, Mexico, with representatives from 10 countries of the region and two external consultants. This document summarizes the major conclusions for which scientific consensus was achieved. The purpose of disseminating these guidelines to the international community is based on the potential interest for other countries with similar social conditions and economical restrictions.

Non-Cryopreserved Unmanipulated Hematopoietic Peripheral Blood Stem Cell Autotransplant Program: Long-Term Results

BACKGROUND Methods to simplify bone marrow transplantation procedures are needed mainly in developing countries. METHODS Between May 1993 and February 1999 in a private-practice setting, we performed 29 autotransplants in 28 patients using non-cryopreserved and unmanipulated peripheral blood stem cells mobilized from the bone marrow to the peripheral blood by means of hematopoietic growth factors. The autografting procedure was performed entirely on an outpatient basis in 19 cases (65%). The median age of the patients was 30 years, with a range of 9-67. There were 15 patients with acute leukemia (9 with acute myelogenous leukemia), 3 with chronic myelogenous leukemia, 2 with multiple myeloma, 3 with Hodgkins disease, 2 with non-Hodgkins lymphoma, and 4 with metastatic breast carcinoma. RESULTS The median time to achieve > 0.5 x 10(9)/L granulocytes was 14 days (range 7-42), whereas the median time to achieve > 20 x 10(9)/L platelets was 20 days (range 5-49). The 64-month post-transplant survival was 38%, whereas the median post-transplant survival was 18 months. The transplant-related mortality was 3.4%. The approximate cost of this simplified procedure was 10.8% for in-hospital procedures and for outpatient autografts, substantially lower than figures reported from the U.S. for autotransplants. CONCLUSIONS This simplified method for autografting patients, avoiding in-hospital stays, purging procedures and cryopreservation of the cells is feasible and results in a substantial decrease of the cost of autologous hematopoietic stem cell transplantation methods.

Donor cell leukemia: A critical review

Approximately 40 cases of DCL have been reported in the literature; cases have been reported after allografts from bone marrow, peripheral blood and cord blood. The study of these cases may provide new insights into the mechanisms of leukemogenesis. Some data suggest that the prevalence of this complication has been under-estimated. Most cases of DCL have occurred following transplantation for leukemia, but there have also been cases reported after transplantation for non-malignant conditions. Various mechanisms have been proposed to explain how DCL arise and are briefly discussed. Additional studies are needed to define with more detail both the true prevalence of this complication and its precise pathogenetic mechanism.