Treatment of vitiligo with a chimeric monoclonal antibody to CD20: a pilot study.

Five patients with active disseminated vitiligo were given 1g of a chimeric (murine/human) monoclonal antibody to CD20 in a single intravenous infusion and followed-up for 6 months. Three of the patients showed an overt clinical and histological improvement of the disease, one presented slight improvement and the remaining patient showed no changes. Improvement was neither associated with changes in laboratory parameters nor to a specific human leucocyte antigen D-related (HLA-DR) phenotype. We believe that these preliminary results are encouraging, and further clinical trials should be undertaken. An important aim should be the finding of a marker with a good response to this therapeutic approach.

Decreased dopaminergic tone and increased basal bioactive prolactin in men with human immunodeficiency virus infection.

OBJECTIVE: The aims of the study were: (1) to assess dopaminergic tone in a group of HIV infected men and the bioactivity and the molecular species of their circulating PRL in comparison with healthy men and (2) to search for a correlation between serum PRL and CD4+ T lymphocytes and viral load. DESIGN: In a cross-sectional study the effect of acute dopaminergic blockade with intravenous metoclopramide on serum PRL (both immunoreactive and biologically active), TSH and PRL circulating molecular isoforms was evaluated. PATIENTS: Twenty untreated HIV infected men category C2 or C3, mean (SD) age 26.9 (6.3) years, were compared to 14 clinically healthy HIV-negative men, age 25.4 (2.3) years. MEASUREMENTS: Under fasting conditions and following metoclopramide administration duplicate measurements of serum immunoreactive PRL, bioactive PRL (PRL dependent Nb2 lymphoma cell assay) and immunoreactive TSH were performed. The molecular species of circulating PRL were determined by immunoblot analysis, CD4+ T lymphocytes by flow cytometry and the viral load using a nucleic acid sequence-based amplification assay. RESULTS: In HIV infected men fasting bioactive (but not immunoreactive) PRL was higher (P = 0.03), but the stimulated PRL (both immunoreactive and bioactive) was lower than in healthy men throughout the test (P < or = 0.01). Fasting serum TSH was similar in HIV-infected and healthy men while its response to metoclopramide was absent in the former but not in the latter (P = 0.049). A 23.5-kD PRL was the predominant circulating isoform both in patients and healthy men. Considering HIV-infected and healthy men, CD4+ T lymphocytes correlated negatively with fasting bioactive PRL (P = 0.008) and positively with the area under the PRL (both immunoreactive and bioactive) curves (P < 0.001). The viral load was negatively correlated with the area under the curve of the bioactive/immunoreactive ratio (P = 0.008). CONCLUSIONS: The raised fasting bioactive PRL, the diminished response of both immunoreactive and bioactive PRL and the absent TSH response to metoclopramide in HIV infected men, suggest the existence of a decreased, but not absent dopaminergic tone. A monomeric form of PRL was the predominant circulating species, as in healthy men, and this hormone seems to be associated both with CD4+ T lymphocytes and the viral load.

Serum prolactin is associated with apoptosis in men with human immunodeficiency virus infection.

We examined the in vivo and in vitro production of prolactin (PRL) in 20 untreated HIV-infected men compared to 14 uninfected men and its association with the cell cycle and apoptosis. Compared to uninfected men, the HIV-infected men had: (i) higher fasting serum bioactive (BIO) PRL; (ii) lower serum immunoreactive (RIA) and BIO-PRL responses to intravenous metoclopramide; (iii) greater BIO-RIA PRL ratio both fasting and during intravenous metoclopramide; (iv) lower percentage of non-stimulated PBMC in the G0/G1 phase, but a higher percentage in the S phase, of the cell cycle with normal response to Concanavalin-A; and (v) higher in vitro production of BIO-PRL by non-stimulated PBMC, which was blocked after Concanavalin-A. Fasting serum BIO-PRL positively correlated with the percent of non-stimulated PBMC in S + G2/M phases. The percentage of apoptotic PBMC negatively correlated with CD4+ T lymphocytes and with the area under the serum RIA-PRL curve, but positively correlated with the area under the curve for the BIO/RIA ratio. These results suggest that in these HIV-infected men: (i) a diminished dopaminergic tone may exist, as an adaptive mechanism attempting to survive; and (ii) BIO-PRL may participate as a cofactor in the stimulation of T-cell proliferation.

Immunophenotypic analysis of peripheral blood lymphocytes.

This unit on basic phenotyping describes two basic and two alternate protocols for the immunophenotypic identification and classification of human peripheral blood lymphocytes. The presented protocols comply with consensus recommendations from professional organizations that regulate the clinical use of such assays. The authors discuss whole blood assay systems as well as enriched systems from several perspectives, including absolute number determination.

'LE cells' result from phagocytosis of apoptotic bodies induced by antinuclear antibodies.

Lupus erythematosus (LE) cells are believed to represent phagocytosis by granulocytes of cell nuclei whose DNA has been 'depolymerized' and opsonized by serum factors, most likely antinuclear antibodies and C3b. Since it is known that certain antinuclear antibodies are capable of inducing apoptosis after intracellular penetration; and that the resulting apoptotic bodies can be ingested by non-professional phagocytes, we decided to investigate the possibility that LE cells could result from the phagocytosis of apoptotic bodies induced by antinuclear antibodies. We demonstrate herein, through different methodological approaches, that the ingested material within LE cells corresponds to apoptotic bodies, and that the LE cell phenomenon can be reproduced, in the absence of other serum factors, by penetrating murine monoclonal anti DNA antibodies.

Assessment of residual disease in acute leukemia by means of polymerase chain reaction.

Along a 5-year period in a single institution, specific molecular markers were prospectively looked for in consecutive patients with acute leukemia, by means of polymerase chain reaction (PCR): In patients with acute lymphoblastic leukemia (ALL), the BCR/ABL and TEL-AML1 fusion transcripts as well as clonotypic immunoglobulin gene rearrangements were investigated, whereas in patients with acute myelogenous leukemia (AML) the PML-RAR alpha, AML1-ETO and CBF beta-MYH11 fusion proteins were assessed. Specific molecular markers were identified in 15/75 patients: Four with ALL (three with clonotypic IgG rearrangements and one with BCR/ABL) and 11 with AML (nine with the PML/RAR alpha fusion protein--M3 AML-, and two with the AML1/ETO fusion protein--M2 AML-). During follow-up periods ranging from 1 to 60 months, seven patients cleared the residual disease assessed by PCR (RD-PCR), whereas eight patients had either persistence of RD-PCR or a molecular relapse. For patients without or with RD-PCR, the 30-month survival (SV) was 86% and 14%, respectively, whereas median SV was > 60 and two months, also respectively (p < 0.01). Six of eight patients with detectable RD-PCR died, all of them within three months after the detection of the RD-PCR, whereas two of the patients that relapsed were rescued with treatment and entered a second molecular remission. Two of the three molecular relapses were detected without an overt morphological relapse. It is concluded that PCR is a valuable method for assessing residual disease and that early diagnosis of relapses may lead into effective salvage treatment in some instances.

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 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(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.

Chronic lymphocytic leukemia is infrequent in Mexican mestizos.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults living in Western countries, and accounts for approximately 30% of adult leukemias. In a 15-year period in a single institution, we identified 19 patients with CLL in a group of 211 adults with leukemia (9% of adult leukemias). Of these 19 CLL patients, 8 had a Caucasian phenotype, 4 were born outside the country, and only 11 were Mexican mestizos. On the other hand, in a multicenter experience involving 1968 Mexican adults with leukemia, CLL represented 6.6% of the cases, a figure significantly lower than that reported in Caucasians (P < 0.01). CLL is the least frequent type of leukemia in Mexican mestizos, and this low prevalence may stem from the genetic origin of this racial group. The data also suggest a genetic predisposition of Caucasians to suffer from this disease.

[Glycosilphosphatidylinositol-anchored cell surface protein deficiency in Mexican mestizo patients with aplastic anemia]. / Deficiencia de proteínas de superficie ancladas por glucosilfosfatidilinositol en células de pacientes mestizos mexicanos con hipoplasia medular.

The peripheral blood cells of ten patients with biopsy-proven aplastic anemia were studied by means of flow-cytometry in order to assess the expression of two phosphatidylinositol-anchored surface proteins: CD55/DAF (decay accelerating factor) and CD59/MIRL (membrane inhibitor of reactive lysis). An abnormal expression was found in five of these ten patients, whereas the "traditional" tests for paroxysmal nocturnal hemoglobinuria (PNH) were positive only on two of these five individuals. Five of the aplastic patients were treated with anti-thymocyte globulin and cyclosporin-A and three entered a complete remission; of the latter, one had CD55/CD59 deficiencies whereas two did not. Along the study period one patient with a hemolytic pattern of PNH was identified. It is concluded that CD55 and/or CD59 abnormalities are frequent in Mexican mestizo patients with aplastic anemia, that the aplastic presentation of PNH is more frequent in Mexico than the hemolytic presentation, that the flow-cytometric identification of CPI-anchored proteins is more sensitive than the "traditional" PNH tests, and that some patients with PNH-aplasia may respond to intensive immunosuppressive treatment. The flow-cytometric identification of GPI-anchored cell surface proteins should replace the "traditional" tests in the identification of patients with PNH.

Penetration of anti-DNA antibodies into immature live cells.

Several data suggest that immature lymphoid cells are more prone to penetration and therefore are affected more by antibodies than their mature counterparts. In this study, we examined the penetration of monoclonal anti-DNA antibodies in several models of immature cells. Results confirm that most anti-DNA antibodies penetrate larger proportions of immature cells than normal adult cells. It was also proven that anti-DNA antibodies induce larger fractions of immature cells to undergo apoptosis than mature cells; however, there is not a numerical association between penetration and apoptosis. Additionally, the penetration and induction of apoptosis of several anti-DNA monoclonal antibodies into U937 and NIH-3T3 cells followed a rather heterogeneous pattern. When mature and immature cells were stimulated polyclonally, it was shown that polyreactive antibodies might act as an accessory signal to induce apoptosis in immature cells. This process could contribute to the edition of the immune repertoire. We propose that naturally occurring polyreactive antinuclear antibodies, through penetration and deletion of self-reacting cells, could participate, either as a unique or secondary signal, in the mechanism of self tolerance. If these polyreactive antibodies undergo affinity maturation, it is possible they may develop into pathogenic antibodies.

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 x 10(8)/kg non-cryopreserved, non-purged mononuclear cells, containing a median of 3.9 x 10(6)/kg CD34 + cells. The median time to achieve > 500 granulocytes/microl was 21 days, with a range of 16 to 40, whereas the median time to achieve > 20,000 platelets/microl 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.

Hb Lepore Washington-Boston in two Mexican mestizo families.

Two Mexican mestizo families with Hb Lepore Washington-Boston are described. One family is from Cordova, in the State of Veracruz, in the East coast of Mexico: the proband is a 44-year old asymptomatic male with italian ancestors; the other family is from the city of Durango, State of Durango, in the northwestern part of the country: the propositus is a 32-year old pregnant female with French ancestors. In both cases the Hb Lepore was identified by alkaline electrophoresis and characterized by high performance liquid chromatography and PCR with specific probes flanking the deletion frame. The beta-haplotype in both families was +(-)-(-)-(++), the commonest beta-haplotype reported with this mutation. This paper describes the first cases of this entity in Mexico.

Serum antibodies to distinct epitopes of the tissue-type plasminogen activator (t-PA) in patients with systemic lupus erythematosus.

Defective fibrinolysis due to decreased tissue-type plasminogen activator (t-PA) activity is a well-established finding in patients with systemic lupus erythematosus (SLE). The possibility that this decrease in t-PA activity may be related to the presence of autoantibodies directed against t-PA, and the possible role of these autoantibodies in the pathophysiology of fibrinolysis in SLE, were investigated. Serum samples from 115 SLE patients and 63 normal volunteers were analyzed for the presence of such antibodies. The search for antibodies to t-PA was performed by means of several systems, allowing for the identification of epitopes presented in different conformational physical states of t-PA: free or associated to its inhibitor (PAI-1) in plasma, specifically bound to fibrin surface, or passively adsorbed to solid supports. Antibodies in variable amounts were detected by all systems used; however, t-PA activity was not inhibited by the IgG fraction of the positive sera in a fibrin-agar fibrinolysis system. Moreover, the demonstration of serum anti-t-PA antibodies was not associated with clinical or laboratory abnormalities related to vasoocclusive episodes. These results indicate that, as in the case of other autoantibodies, their detection in serum does not imply their direct participation in the pathophysiology of thrombosis in SLE.

Filgrastim-mobilized peripheral-blood stem cells can be stored at 4 degrees and used in autografts to rescue high-dose chemotherapy.

We studied 21 filgrastim (G-CSF)-mobilized peripheral blood stem cells (PBSC) apheresis products obtained from seven patients, and stored at 4 degrees C for periods of up to 96 hr prior to their reinfusion, to rescue high-dose chemotherapy. The apheresis products contained a median of 106 x 10(8)/L mononuclear cells (MNC), 14.6% of them displaying the CD34 antigen; the viability was over 90% in all samples studied at 24, 48, and 72 hr after harvesting. These PBSC were successfully used to rescue high-dose chemotherapy; patients received a median of 4.8 x 10(8)/Kg MNC; the median time to achieve > 500 granulocytes was 14 days (range 11-26) and the median time to achieve > 20,000 platelets was 20 days (range 11-40). Since autologous transplants with nonfrozen PBSC are feasible and less costly than those using frozen PBSC, restrictions to PBSC autotransplant programs may be overcome and costs may be diminished.

Presence of serum antibodies to coagulation protein C in patients with systemic lupus erythematosus is not associated with antigenic or functional protein C deficiencies.

Antibodies directed to immunopurified coagulation protein C (PC) were investigated in serum samples from 108 patients with systemic lupus erythematosus (SLE) and found in 12 of them. However, their presence was not associated with antigenic or functional deficiencies of PC, which were documented in 6 and 17 patients, respectively.

The infusion of anti-RhO-(D) opsonized erythrocytes may be useful in the treatment of patients, splenectomized or not, with chronic, refractory autoimmune thrombocytopenic purpura--a prospective study.

Six patients with chronic refractory autoimmune thrombocytopenic purpura (ATP) were treated with five doses of autologous red blood cells (RBCs) opsonized ex vivo with anti-RhO-(D) IgG. Increases in platelet counts were observed in all cases; complete responses requiring no further treatment--for periods of 8-72 months--were recorded in four patients, three of them splenectomized. The cost of the procedure was significantly lower than other "Fc receptor (FcR) blockade" treatments.

Protein S deficiency associated to anti-protein S antibodies in a patient with mixed connective-tissue disease and its reversal by danazol.

We describe here a female patient with mixed connective tissue disease, secondary antiphospholipid syndrome, pulmonary hypertension and severe acquired, probably autoimmune, antibody-mediated type I coagulation protein S deficiency (total, free and C4bp-bound). No previous instance of anti-PS antibody-associated PS deficiency had been reported. The patient was treated initially with prednisone, but the protein S levels did not rise until danazol was added, and dropped again after its withdrawal.

A prospective trial of the treatment of acute megakaryoblastic leukaemia.

Acute megakaryoblastic leukaemia, the M-7 variant of acute leukaemia according to the French-American-British (FAB) co-operative group, comprises 8.4% of all cases of acute leukaemia in the city of Puebla, Mexico. The malignancy can be identified by means of monoclonal antibodies or electron microscopy. Using two monoclonal antibodies, Hp1-1d that binds the glycoprotein IIb/IIIa complex (CDw 41) and W1-23 that recognizes the factor VIII:von Willebrand fraction, we have found 19 cases of M-7 leukaemia. Fourteen of these were entered in a prospective therapeutic trial, seven were treated with low-dose (LD) Ara-C (10 mg/m2, delivered subcutaneously every 12 h in 21-day courses). The median age was 14 years, four were female and three male. The remaining seven patients were treated with HOP (adriamycin 25 mg/m2 + vincristine 1.4 mg/m2 orally, daily, for the same period. The median age was 20 years, three were females and four males. Patients were followed for periods of 1-24 months. Six of seven patients in each group achieved remission; however, 18-month disease-free survival was 14% for the LD Ara-C group and 42% for the HOP-treated group. All patients in the LD Ara-C group were dead at 24 months; three patients in the HOP group survived at 12, 14 and 18 months. Differences between these two groups are probably not significant due to the small number of patients involved.