Vß T cell receptor (TCR) genes in circulating cells of patients with systemic lupus erythematosus and their healthy relatives / Genes del receptor variable beta de células T en células circulantes de pacientes con lupus eritematoso generalizado y sus familiares sanos.

Objective: We investigated the proportion of Vß T cell receptor (TCR) gene expression in peripheral CD3+ lymphocytes in familial and non-familial systemic lupus erythematosus (SLE) patients. Method: The Vß TCR repertoire was studied in 14 families in which several members had SLE. The Vß TCR usage in SLE patients (n = 27) was compared with that in healthy members of these multiplex families (n = 47), in 37 sporadic SLE patients who had no relatives with SLE, and in 15 healthy unrelated controls. Vß TCR repertoire expression was studied by multiparameter flow cytometry with the use of an array of 24 different Vß TCR gene family-specific monoclonal antibodies. Results: We found the same Vß TCR expression profile in the comparisons between sporadic SLE and familial SLE cases and healthy relatives, which included increased expression of Vß 5.2, Vß 11 and Vß 16, and lower expression of Vß 3, Vß 4, Vß 7.1 and Vß 17. Interestingly, solely Vß 17 was differentially expressed among sporadic and familial SLE. Also, increased expression of Vß 9 was the hallmark among familial SLE (casesand h ealthy relatives) in comparison to controls. Conclusion: These results highlight the notion that the final profile of the Vß TCR repertoire seen in familial and non-familial SLE seems to arise from the interaction of genetic, environmental, and immunoregulatory factors. Furthermore, it may contribute to the immunologic abnormalities affecting relatives of SLE patients.

Objetivo: Se investigó la proporción de la expresión génica del receptor variable beta de células T (Vß TCR) en linfocitos periféricos CD3+ en pacientes con lupus eritematoso generalizado (LEG) familiar y no familiar. Método: El repertorio de Vß TCR se estudió en 14 familias que presentaban más de un miembro con LEG. El uso de Vß TCR en pacientes con LEG (n = 27) se comparó con el de los miembros sanos de estas familias (n = 47), con 37 pacientes con LEG esporádico y con 15 controles sanos. La expresión del repertorio de Vß TCR se estudió por citometría de flujo multiparamétrica utilizando un arreglo de 24 diferentes anticuerpos monoclonales específicos de genes familiares para Vß TCR. Resultados: Se encontró el mismo perfil de expresión en las comparaciones entre los casos de LEG esporádico y familiar, así como en los consanguíneos sanos de las familias multicasos, que incluía una expresión incrementada de Vß 5.2, Vß 11 y Vß 16, y una menor expresión de Vß 3, Vß4, Vß 7.1 y Vß 7. De manera interesante, solo Vß 17 se expresó de modo diferente entre casos familiares y esporádicos de LEG. Igualmente, la expresión incrementada de Vß 9 fue el distintivo entre los casos de LEG familiar (casos y consanguíneos sanos) y los controles sanos. Conclusiones: Estos resultados refuerzan la noción de que el perfil final del repertorio Vß TCR observado en LEG familiar y no familiar parece surgir de la interacción de factores genéticos, ambientales e inmunorreguladores, además de que pueden explicar las alteraciones inmunitarias que se observan en los consanguíneos sanos de pacientes con LEG.

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

BACKGROUND: The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. METHODS: MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. RESULTS: The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. CONCLUSIONS: MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society.

Clinical relevance of P-glycoprotein activity on peripheral blood mononuclear cells and polymorphonuclear neutrophils to methotrexate in systemic lupus erythematosus patients.

To elucidate the relationship between P-glycoprotein activity on peripheral blood leukocytes of systemic lupus erythematosus (SLE) patients with lupus arthritis and the clinical response to methotrexate. An observational study was made in patients with SLE according to ACR criteria 1997 who had arthralgia and arthritis and received methotrexate for ≥3 months. Methotrexate responders and non-responders were compared according to the Clinical Disease Activity Index. Mononuclear cells and polymorphonuclear neutrophils were isolated from SLE patients and P-glycoprotein expression was measured using the relative fluorescence index and percentage of positive cells. The chi-square test was used to compare P-glycoprotein activity between responders and non-responders. Thirty-two patients with a mean age of 45.4 ± 10.7 years were included: 34.4% had a response to methotrexate and 65.6% did not. Mean relative fluorescence units of both mononuclear cells and polymorphonuclear neutrophils were significantly lower in patients with a good response (7.0 ± 4.3 vs. 9.6 ± 3.8; p = 0.041 and 4.2 ± 3.5 vs. 7.6 ± 4.0; p = 0.004). The prevalence of low fluorescence levels (<6 relative fluorescence units), signifying higher P-glycoprotein activity of both mononuclear cells and polymorphonuclear neutrophils, was higher in methotrexate responders than in non-responders (27.3 vs. 4.8%; p = 0.10 and 81.8 vs. 23.8%; p = 0.003, respectively). In SLE patients with joint involvement treated with methotrexate, P-glycoprotein activity was higher in responders to methotrexate than in non-responders. Further studies are required to determine the mechanisms behind this finding and whether P-glycoprotein activity mediates alterations in methotrexate efficacy.

Serum Antibodies to Melanocytes in Patients With Vitiligo Are Predictors of Disease Progression.

The aim of this study was to investigate whether the amount of serum antibodies to melanocyte antigens could predict clinical activity or disease progression in patients with vitiligo. A solid-phase enzyme immunoassay was developed to semiquantitate serum antibodies to a human melanocyte extract and was used in 127 patients, 93 of whom showed clinical progression of the disease, while the remaining 34 were quiescent. Results showed different values for clinical sensitivity and specificity depending on the cutoff level for decision, but the overall performance of the test was adequate and supported statistical significance to predict clinical activity/progression or quietness of the disease process. The test might prove useful in deciding the indication and aggressiveness of immunosuppressive therapy in patients with vitiligo. Previous findings suggest that melanocyte-specific antibodies might play a pathogenetic role in the depletion of melanocytes, which characterizes this disorder, and that this depletion might be due to apoptosis following antibody internalization.

Potential use of the Macrobrachium rosenbergii lectin for diagnosis of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia is the most common form of cancer in children. Lectins are proteins or glycoproteins from plants or animals that recognize oligossacharides on the cell surface and have been used to characterize the structural changes of oligosaccharides in leukemias. In this study, we used the lectin from the freshwater prawn Macrobrachium (M. rosenbergii), specific for acetyl groups in sialylated glycans, because increased sialylation of glycoproteins and glycolipids has been identified in lymphoblastic leukemias. We compared the specificity of the M. rosenbergii lectin for lymphoblastic leukemias with the specificities of the lectins from Triticum vulgaris, Solanum tuberosum, Arachis hipogaea, and Phytolacca americana. By morphologic and phenotype characterization with a panel of monoclonal antibodies, we identified four types of leukemias from 106 leukemia patients: 11 cases of T-cell acute lymphoblastic leukemia, 61 cases of B-cell acute lymphoblastic leukemia, 24 cases of acute myeloblastic leukemia, and 10 cases of acute biphenotypic leukemia. As determined by cytofluorometric assays, nine of the eleven cases with T-cell acute lymphoblastic leukemia (8 +/- 3 years old) were specifically identified with the lectin from M. rosenbergii. In contrast, only six cases of B-cell leukemia, one case of myeloblastic leukemia, and 2 cases of biphenotypic leukemia were identified with this M. rosenbergii lectin. The other lectins tested showed no capacity to differentiate, in a significant manner, any of the four types of leukemias tested. Thus, the lectin from M. rosenbergii could be considered a useful tool for the diagnosis and study of T-cell acute lymphoblastic leukemia.

Apoptosis of melanocytes in vitiligo results from antibody penetration.

Vitiligo is a rather common disease characterized by depigmentation of skin and mucosae due to the loss of melanocytes, most likely as a result of autoimmune phenomena. In this study we demonstrated apoptotic markers in residual melanocytes in skin biopsies of patients with vitiligo, as well as the presence of serum antibodies to melanocyte-specific antigens in the vast majority of patients. Moreover, we were able to prove that serum IgG antibodies from vitiligo patients, but not from healthy controls, were capable to penetrate into cultured melanocytes in vitro, and trigger them to engage in apoptosis. Our results are consonant with the theory that melanocyte-specific antibodies are responsible for the deletion of melanocytes through antibody penetration and apoptosis.

Minimal residual disease testing in acute leukemia by flow cytometry immunophenotyping: prognostic significance.

Two main techniques are being used for the detection of minimal residual disease (MRD) in acute leukemia (AL): immunophenotypic analysis and polymerase chain reaction (PCR). In this paper, we analyze the results of assessing MRD by means of flow cytometry (FC) in a group of 93 patients with AL who were prospectively studied and treated in a single institution over a 9-year period. The presence or absence of MRD was established at a cut-off level of 2%, as judged by FC; a single result above this level was considered to define the positivity. The patients were grouped in 4 subsets: (1) acute lymphoblastic leukemia (ALL) patients with MRD (n = 36); (2) acute myeloblastic leukemia (AML) patients with MRD (n = 13); (3) ALL patients without MRD (n = 31); and (4) AML patients without MRD (n = 13). The relapse rates in these groups were 17%, 8%, 0%, and 0%, respectively, whereas the overall 7-year survival was 65%, 69%, 83%, and 98%, respectively. Our results support the usefulness of serially assessing MRD in patients with AL by means of FC; because this method is applicable to all cases of AL, despite being less sensitive than a molecular biology study; it is a good option to follow-up patients and to decide therapeutic and timely interventions.

A single-institution, 20-year prospective experience with an affordable Fc-receptor blockade method to treat patients with chronic, refractory autoimmune thrombocytopenic purpura.

In a 20-year period in a single institution, 34 patients with chronic, refractory autoimmune thrombocytopenic purpura were prospectively treated with ex vivo anti-D opsonized autologous red blood cells. All patients had received previous treatment with steroids and/or immunosuppressive agents, and 11 had been splenectomized. Twenty one patients had an increase in the platelet count; in five cases, the increase was more than 50 x 10(9)/L platelets and in 16 the increase was more than 100 x 10(9)/L platelets. Early responses were observed in 20 patients and late responses in seven, whereas seven patients (20%) did not respond at all. Nine of the 20 individuals who achieved an ER had a subsequent drop in the platelet count; however, only three had a drop below 50 x 10(9)/L. When last censored, of the 34 patients, 24 (70%) had a platelet count above 50 x 10(9)/L. The 84-month thrombocytopenia-free (over 50 x 10(9)/L platelets) status of the whole group is 70%, whereas the 84-month complete remission (over 100 x 10(9)/L platelets) status of the whole group is 50%. It is concluded that the use of ex vivo anti-D opsonized red blood cells may represent another, substantially cheaper treatment of patients with chronic, refractory, autoimmune thrombocytopenic purpura.

Instrument- and protocol-dependent variation in the enumeration of CD34+ cells by flow cytometry.

BACKGROUND: The information regarding the minimum number of CD34+ cells that are necessary to reconstitute hematopoiesis in patients undergoing peripheral blood progenitor cell transplantation is quite controversial. Some of the differences in these figures might be due to the selection of antibodies, staining protocols, and acquisition strategies for the flow cytometric enumeration of these cells. STUDY DESIGN AND METHODS: Twenty-seven human umbilical cord blood samples and 33 leukapheresis products were consecutively collected for this study. Cells were stained following two different protocols, both using monoclonal antibodies to CD45 and CD34, and analyzed by the same operator in two different flow cytometers to enumerate the percentage of CD34+ mononuclear cells. RESULTS: Relevant differences in the proportion of cells were encountered, and the correlation between the results yielded by both instruments and protocols, although statistically valid, was suboptimal. CONCLUSIONS: Both interinstrument and interprotocol variation can provide additional explanation for the redundantly reported discrepancies concerning the numbers of CD34 cells that suffice to secure hemopoietic grafting. These results point to the need for new and different standardization approaches in this clinically relevant field.

Parvovirus B19 infection in medical students during a hospital outbreak.

From March to May 2002, a parvovirus B19 (B19) outbreak was identified at a general hospital that serves as a teaching facility for the Universidad Autónoma de San Luis Potosí, Mexico. Medical students attending the hospital presented with symptoms suggestive of B19 infection. Previous studies have suggested that apparent hospital-related B19 outbreaks may be a reflection of B19 infection in the community. A study was undertaken to assess whether exposure to the hospital was a risk factor for B19 infection and to determine to what extent medical students were infected during this outbreak. The incidence of B19 infection in medical students attending the teaching hospital during the outbreak (n=211) was determined and compared to students not attending the hospital (n=96). To assess if a community-wide outbreak had occurred, 80 blood donors were also evaluated for the presence of B19 antibodies. Acute B19 infection was identified in 40 of 119 (33.6%) susceptible students attending the hospital and in 20 of 47 (42.6%) susceptible students not attending the hospital. The frequency of acute infection among susceptible blood donors was lower (9.5%) than in students, but higher than the rate expected during non-epidemic periods. Most infections (68.3%) were asymptomatic. Symptoms reported by infected subjects were not specific for B19 infection. Only 11.7% of subjects with acute infection fulfilled the clinical surveillance definition used to detect cases during the outbreak. In conclusion, hospital exposure was not associated to increased risk of B19 infection among medical students. Medical students may be at increased risk for acquiring and transmitting B19 infection during outbreaks.

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 x 10(9)/l granulocytes was 14 days (range 0-86), whereas the median time to achieve >20 x 10(9)/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.

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.

Assessment of residual disease in acute leukemia by means of polymerase chain reaction / Evaluación de enfermedad residual en leucemia aguda por medio de reacción en cadena de la polimerasa

Empleando reacción en cadena de la polimerasa (PCR) se buscaron marcadores moleculares específicos en 75 pacientes consecutivos con leucemia aguda en una sola institución a lo largo de un periodo de cinco años. En leucemia aguda linfoblástica se buscaron BCR/ABL t (9:22), TEL-AML1, t (12:21) y rearreglos clonotípicos de los genes de inmunoglobulinas, en tanto que en leucemia aguda mieloblástica se buscaron PML-RARa t (15:17), AML1-ETO t (8:21) y CBFb-MYH11 (inv16). Se identificaron marcadores moleculares en 15 de 75 casos: cuatro LAL (tres rearreglos de Ig y uno BCR/ABL) y 11 LAM (9 PML/RARa y 2 AML1/ETO). A lo largo de seguimientos de 1 a 60 meses después de haber hecho el diagnóstico, en siete pacientes se eliminó la enfermedad residual evaluada por PCR (ER-PCR) y en ocho persistió. Para pacientes sin o con ER-PCR, la supervivencia (SV) a 30 meses fue de 86 por ciento y 14 por ciento, y la mediana de SV >60 y dos meses, respectivamente (p < 0.01). Seis de los ocho pacientes con ER-PCR fallecieron y en dos se pudo lograr una nueva remisión molecular. Dos de las tres recaídas moleculares pudieron detectarse antes de la recaída hematológica florida. Se concluye que la PCR es útil para vigilar la persistencia de ER en leucemia aguda y en ocasiones, para decidir los tratamientos antileucémicos de rescate, de manera oportuna.

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 Fenruary 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 growht factors. The autografting procedure was performed entirely on an autpatient basis in 19 cases (65 percent). THe median age of the patients was 30 years, with a range of-967. There were 15 patients with acute leukemia (9 with acute myelogenous leukemia), 3 with chronic myelogenous leukemia, 2 with multiple myeloma, 3 with Hodgkin's disease, 2 with non-Hodgkin's lymphoma, and 4 with metastatic breast carcinoma. Results. The median time to achieve >0.5 X 10 a to nine/L granulocytes was 14 days /range 7-42), whereas the median time to achieve >20 X 10 a to nine/L platelets was 20 days (range 5-49). The 64-month post-transplant survival was 38 percent, whereas the median post-transplant survival was 18 months. The transplant-related mortality was 3.4 percent. The approximate cost of this simplified procedure was 10.8 percent for in-hospital procedures and for outpatient autografts, substantially lower than figures reported from the U.S. for autotransplants. Conclusions. This simplified method for autografiting 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

Deficiencia de proteínas de superficie ancladas por glucosilfosfatidilinositol en células de pacientes mestizos mexicanos con hipoplasia medular / Clycosilphosphatidylinositol anchored cell surface proteins deficiency in mexican mestizo patients with aplastic anemia

Se estudiaron de manera prospectiva 10 sujetos con diagnóstico histológico de hipoplasia medular para identificar por medio de citometría de flujo las características de las moléculas de superficie CD55 y CD59, ancladas a la superficie celular a través de glucosilfosfatidilinositol (GPI). En cinco se identificó una distribución anormal de estas moléculas; las pruebas de hemólisis ácida, por insulina y sacarosa, así como la investigación de hemosiderinuria fueron anormales en dos de los cinco pacientes. Cinco de ellos fueron tratados con globulina antitimocito y ciclosporina-A y tres se encuentran en remisión completa, en tanto que cinco enfermos fueron tratados con andrógenos y ninguno logró la remisión. De los pacientes en remisión completa, uno tuvo trastornos en las moléculas ancladas por GPI ocurren con frecuencia en pacientes con hipoplasia medular, que la identificación de estas alteraciones es más sensible que las pruebas tradicionales para investigar hemoglobinuria paroxística nocturna (HPN), que las formas aplásticas de HPN son frecuentes en nuestro país y que el tratamiento con inmunosupresión intensa puede ser efectivos en algunas formas hipoplásicas de la HPN. La identificación citofluorográfica de las alteraciones en las moléculas ancladas por GPI debieran reemplazar a las pruebas ®tradicionales¼ para identificar a la HPN

Hb Lepore Washington-Boston in two Mexican mestizo families

Se describen dos familias mestizas mexicanas con Hb lepore Washington-Boston: una familia es de Córdova en el estado de Veracruz, ubicado en la costa del este de México en la cual el caso índice es un varón asintomático de 44 años de edad con ancestros italianos; la otra es una nativa de Durango, en el noreste del país, en que el caso es una mujer embarazada de 32 años de edad con ancestros franceses. En ambos casos la Hb leporese identificó por electroforesis alcalina y se caracterizó por cromatografía líquida de alta resolución y PCR con oligonucleótidos específicos flanqueando el marco de la deleción. El haplotipo en ambas familias fue +----++, que es el haplotipo ß más comúnmente reportado con esta mutación. Estos son los primeros casos de esta entidad de México

Tetraploidia restringida a blastos bifenotípicos CD2/CD19: demostración por citometría multiparamétrica / Tetraploidy restricted biphenotypic (CD2/CD19)blast cells:demostration by multiparameter flow cytometry

Se describe el caso de un paciente masculino de 17 años de edad con leucemia aguda linfoblástica (LAL), en el que la inmunotipificación de las celulas de la médula ósea reveló el caracter bifenotípico de las células neoplásicas, al co-expresar un antígeno pan-T (CD2) con uno pan-B (CD19). La tinción simultánea del antígeno CD2 con ficoeritrina, del CD19 con isotiocianato de fluoresceína y del ácido desoxirribonucleico (ADN) con ioduro de propedio, permitió demostrar que todas las células con el fenotipo CD2+/CD19+, y solo ellas, mostraban cantidades anormales de ADN indistinguibles de la tetraploidia.Este caso podría representar el primero descrito en la literatura, en que se ha empleado el análisis simultáneo de dos antígenos de membrana y ADN en una sola preparación

Tetraploidia restringida a blastos bifenotípicos CD2/CD19: demostración por citometría multiparamétrica / Tetraploidy restricted biphenotypic (CD2/CD19)blast cells:demostration by multiparameter flow cytometry

Se describe el caso de un paciente masculino de 17 años de edad con leucemia aguda linfoblástica (LAL), en el que la inmunotipificación de las celulas de la médula ósea reveló el caracter bifenotípico de las células neoplásicas, al co-expresar un antígeno pan-T (CD2) con uno pan-B (CD19). La tinción simultánea del antígeno CD2 con ficoeritrina, del CD19 con isotiocianato de fluoresceína y del ácido desoxirribonucleico (ADN) con ioduro de propedio, permitió demostrar que todas las células con el fenotipo CD2+/CD19+, y solo ellas, mostraban cantidades anormales de ADN indistinguibles de la tetraploidia.Este caso podría representar el primero descrito en la literatura, en que se ha empleado el análisis simultáneo de dos antígenos de membrana y ADN en una sola preparación