Lack of association between serum 25-hydroxyvitamin D levels and cervical human papillomavirus infection in systemic lupus erythematosus.

Our objective was to evaluate whether vitamin D deficiency is associated with cervical human papilloma virus (HPV) infection in women with SLE. This is a cross-sectional study of 67 women with SLE. A structured questionnaire was administered to ascertain the possible risk factors associated with cervical HPV infection. A gynaecological evaluation and cervical cytology screening were made. HPV detection and genotyping was made by PCR and linear array assay. Serum 25 hydroxyvitamin D levels were quantified by chemiluminescence immunoassay. Mean age and disease duration were 44.8 ± 10.6 and 42.5 ± 11.8 years, respectively. Demographic characteristics were similar in patients with and without deficiency (<20 ng/ml and ≥20 ng/ml). There were 28.4% of women with cervical HPV infection and 68.4% had high-risk HPV infections. Patients with 25 hydroxyvitamin D levels <20 ng/ml had a higher prevalence of cervical HPV infection than those with levels ≥20 ng/ml (30.7% vs. 25.8%; p = 0.72). We found no significant difference when high-risk HPV infection was evaluated (36.8% vs. 31.5%; p = 0.73). In conclusion, women with SLE have a high prevalence of vitamin D deficiency and cervical HPV infection. However, we found no association between vitamin D deficiency and cervical HPV.

Pharmacogenetic selection of volunteers increases stringency of bioequivalence studies; the case of clopidogrel.

Clinical response to clopidogrel varies widely due to under-dosing, drug interactions and intrinsic interindividual differences resulting from genetic polymorphisms. Cytochrome P450-2C19 is the principal enzyme involved in the activation of the prodrug and loss-of-function alleles have been described. Upon expiration of the pharmaceutical patent of clopidogrel, generic manufacturers have started to subject interchangeable formulations to bioequivalence studies. The purpose of the current investigation was to study the effect of selection of volunteers homozygous for the CYP2C19*1 haplotype on the bioavailability of clopidogrel. A regular 2×2 bioequivalence study between two formulations of clopidogrel was performed in volunteers selected and unselected for relevant CYP2C19 haplotypes for the Mexican population. It was found that selection of volunteers homozygous for the CYP2C19*1 haplotype, increased the stringency of bioequivalence statistics and resulted in bioinequivalence of a generic clopidogrel compound that otherwise proved equivalent when tested in an open unselected population. Augmentation of bioequivalence strictness is expected to result from pharmacogenetic selection of volunteers.

Genetic selection of volunteers and concomitant dose adjustment leads to comparable hydralazine/valproate exposure.

WHAT IS KNOWN AND OBJECTIVE: Hydralazine is an inhibitor of DNA methyltransferases, whereas valproate interferes with histone deacetylation. In combination, they show a marked synergism in reducing tumour growth as well as development of metastasis and inducing cell differentiation. Hydralazine is metabolized by the highly polymorphic N-acetyltransferase 2. The current pilot study was performed to analyse the pharmacokinetic parameters of a single dose of hydralazine in 24 h (one tablet with 83 mg for slow acetylators and one tablet with 182 mg for fast acetylators) and three fixed doses of valproate (one tablet of controlled liberation with 700 mg every 8 h) in healthy genetically selected volunteers. Selection was performed based on their NAT2 activity as deduced from their genotype. METHODS: An open label non-randomized single arm study was conducted in two groups of six healthy volunteers of both genders aged 20-45 years with a body mass index 22·2-26·9 which were classified as fast or slow acetylators after genotyping 3 SNPs that cover 99·9% of the NAT2 variants in the Mexican population. Blood samples were collected predose and serially post-dose in an interval of 48 h. Hydralazine and valproate concentrations were determined by ultra-high performance liquid chromatography (UPLC) coupled to tandem mass spectroscopy (MS/MS). RESULTS AND DISCUSSION: The AUC0-48 h and Cmax of hydralazine were almost identical (1410 ± 560 vs. 1446 ± 509 ng h/mL and 93·4 ± 16·7 vs. 112·5 ± 42·1 ng/mL) in both groups with NAT2 genotype-adjusted doses, whereas the multidose parameters of valproate were not significantly affected neither by the selection of the NAT2 genotype (AUC0-48 h 2064 ± 455 vs. 1896 ± 185 µg h/mL; Cmax 96·4 ± 21·1 vs. 88·8 ± 7·2 µg/mL, for the fast and slow acetylators, respectively) nor the co-administration of 83 or 182 mg of hydralazine. WHAT IS NEW AND CONCLUSION: Comparable hydralazine exposures (differences in AUC0-inf of only 7%) were observed in this study with genetic selection of volunteers and concomitant dose adjustment. However, the conclusions have yet to be confirmed with a full-powered 2 × 2 crossover study.

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.

Diminished expression of complement regulatory proteins (CD55 and CD59) in lymphocytes from systemic lupus erythematosus patients with lymphopenia.

CD55 and CD59 are glycophosphatidylinositol-anchored proteins with complement inhibitory properties. Lymphopenia in systemic lupus erythematosus (SLE) has been associated with autoantibodies targeting nuclear antigens. The aim of this study was to evaluate the surface density of CD55 and CD59 in T and B lymphocytes from patients with SLE and lymphopenia and its possible correlation with the presence of common SLE autoantibodies. Flow cytometric analyses were performed on CD55 and CD59 stained CD3+ and CD19+ cells from 40 SLE patients, 30 with lymphopenia and 10 without it, and 25 healthy controls. Autoantibodies were detected in the sera by enzyme linked immunosorbent assay. The mean fluorescence intensity of CD55 and CD59 in T and B cells was significantly diminished in SLE patients with lymphopenia when compared with healthy subjects. Interestingly, the opposite was found in T and B cells from non-lymphopenic SLE patients. Although there was no correlation between CD55 and CD59 surface density and the presence of any specificity of the autoantibodies tested, higher titres of anti-dsDNA, anti-SM and anti-ribosomal p antibodies were significantly associated with lymphopenia. The deficiency of CD55 and CD59 expression may play a role in the pathophysiology of lymphopenia, most likely by increasing the susceptibility of cells to complement mediated cytolysis.

Features of the engraftment of allogeneic hematopoietic stem cells using reduced-intensity conditioning regimens.

The features of the engraftment in 26 patients allografted using reduced-intensity conditioning regimen (8 with chronic myelogenous leukemia, 6 with acute myelogenous leukemia, 9 with acute lymphoblastic leukemia, 1 with hybrid acute leukemia, 1 with myelodysplasia and 1 with thalassemia major) were analyzed. Patients received a median of 10 x 10(8)/Kg mononuclear cells (range 1.6 to 22.9), and a median of 4.2 x 10(6)/Kg CD34 cells (range 0.3 to 14). There was a linear correlation between the number of infused mononuclear cells (MNC) and that of CD34 cells (r = 0.78, p = 0.002). Three patients (11%) failed to engraft; in those who engrafted, the median time to achieve > 500 granulocytes was 11 days (range 10 to 22), and the median time to achieve > 10,000 platelets was 12 days (range 10 to 41). The three patients who failed to engraft received less than 5 x 10(8)/Kg MNC (1.6, 4.6 and 4.9) and less than 0.5 x 10(6)/Kg CD34; however, five of eight patients who received less than 5 x 10(8)/Kg MNC still engrafted successfully. On the other hand, all the patients who received less than 0.5 x 10(6)/Kg CD34 cells failed to engraft. Within the group of patients who engrafted, it was found that those who received more than 7 x 10(6)/Kg CD34+ cells tended to earlier recover > 20 x 10(9)/L platelets (p = 0.02), and > 0.5 x 10(9)/L neutrophils (p = 0.06) before day 15, than those who received less than 7 x 10(6)/Kg CD34+ cells. No such association could be established between the number of MNC and the time for recovery. In these patients allografted using reduced-intensity conditioning regimens, the target doses of hematopoietic cell used were similar to those described for conventional allografts: The number of CD34 infused cells was significantly related to the possibility of failure to engraft and to the recovery rate of the hemopoiesis.

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.

Penetration of autoantibodies into living cells, 2000.

The formerly prevalent concept that intact autoantibodies could not penetrate into viable cells has been defeated by a large amount of experimental findings and clinical observations that indicate otherwise. The penetration of autoantibodies into living cells seems to participate in the pathogenesis of diverse autoimmune diseases, but it may also play a physiological role in healthy individuals. Although the fine mechanisms of the phenomenon remain to be elucidated, the potential use of penetrating autoantibodies as vectors to deliver molecules into cells, with diverse therapeutic purposes, has gained growing interest during the last few years.

Results of an outpatient-based stem cell allotransplant program using nonmyeloablative conditioning regimens.

Using nonmyeloablative, immunosuppressive, fludarabine (FLU)-based conditioning regimens, we have performed allogeneic peripheral blood stem cell transplants in 26 patients (8 with chronic myelogenous leukemia, 6 with acute myelogenous leukemia, 10 with acute lymphoblastic leukemia, 1 with myelodysplasia, and 1 with thalassemia major). Conditioning consisted of FLU/busulphan/cyclophosphamide/cyclosporin-A (CyA)/methotrexate, or FLU/melphalan/CyA/methotrexate. The median granulocyte recovery time to 0.5 x 10(9)/l was 11 days, whereas the median platelet recovery time to 20 x 10(9)/l was 12 days. Twelve patients did not need red blood cell transfusions, and 8 did not need platelet transfusions. In 21 individuals (81%), the procedure could be completed fully on an outpatient basis. Follow-up times range between 30 and 600 days: one patient failed to engraft and recovered endogenous hemopoiesis; six out of 26 patients developed acute graft-versus-host disease (GVHD) whereas 7/22 developed chronic GVHD. Twelve patients (46%) have died, nine of them with a relapsing disease and three with GVHD; median post-transplant survival (SV) was 300 days, whereas the 12-month SV was 42%. The 100-day mortality was 3.8% and the transplant-related mortality was 11.5%. This procedure is substantially less costly than its counterpart, using in-hospital myeloablative conditioning regimens, and it may represent another approach in the management of patients requiring an allogeneic stem cell transplant.

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.

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.

Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders. Part II: Increased P-glycoprotein activity in lymphocytes from systemic lupus erythematosus patients might affect steroid requirements for disease control.

BACKGROUND: Over-expression of the membrane glycoprotein called P-glycoprotein has been widely observed in a variety of both normal and neoplastic cells. P-glycoprotein is a pump molecule that transports hydrophobic drugs (including steroids) and toxins outside the cells, thus inhibiting their therapeutic or toxic effects. The gene encoding P-glycoprotein is named multidrug resistance-1 (MDR-1). OBJECTIVE: To evaluate the functional activity of P-glycoprotein in lymphocytes and monocytes from patients with systemic lupus erythematosus. METHODS: 30 systemic lupus erythematosus patients and 20 healthy controls were studied. Peripheral blood mononuclear cells isolated by gradient centrifugation were incubated in the presence of daunorubicin (a fluorescent drug extruded by P-glycoprotein) at 37 degrees C or 4 degrees C for 30 min. P-glycoprotein activity was then analyzed using flow cytometry. Results were expressed as the percentage of lymphocytes or monocytes with high P-glycoprotein activity (i.e., low fluorescence). RESULTS: Mean fluorescence values for lymphocytes and monocytes were comparable between patients and healthy controls. However, because our method allowed to measure P-glycoprotein function at the single-cell level, we were able to show that the mean percentage of lymphocytes with high P-glycoprotein activity was increased in the patients (11.51% +/- 14.3%) as compared to the healthy controls (0.71% +/- 0.57%) (P < 0.05). Moreover, P-glycoprotein activity was lower in the patients in clinical remission than in those with active disease. CONCLUSIONS: Our results suggest that P-glycoprotein function might affect glucocorticoid requirements in systemic lupus erythematosus.

Hematopoietic stem cell allografts using a non-myeloablative conditioning regimen can be safely performed on an outpatient basis: report of four cases.

Using a non-myeloablative, immunosuppressive, fludarabine-based conditioning regimen, we performed allogeneic peripheral blood stem cell transplants totally on an outpatient basis in four patients (two with chronic myelogenous leukemia, one with acute myelogenous leukemia and one with thalassemia major). The median granulocyte recovery time to 0.5 x 109/l was 10 days and the lowest absolute neutrophil count was 0.064 x 109/l; only one patient developed thrombocytopenia below 20 x 109/l. No patient required red blood cell transfusions and one was given a single prophylactic platelet transfusion. All patients are alive at 210-390 (median 285) days and have definite evidence of chimerism; one developed biopsy-proven GVHD on day 50, with a limited cutaneous rash. The procedure is less costly than its counterpart using myeloablative conditioning regimens and may represent another approach in the management of patients requiring an allogeneic stem cell transplant. Bone Marrow Transplantation (2000) 25, 131-133.

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.

Primary thrombophilia in Mexico: a prospective study.

A group of 102 Mexican Mestizo patients with appropriate clinical features suggestive of primary thrombophilia was prospectively studied. Thirty-nine percent of them had activated protein C resistance, but only four patients displayed the factor V Leiden mutation. Five percent of the individuals were found to be protein C deficient, whereas 2% had protein S deficiency. No cases of abnormalities in antithrombin III, plasminogen, tissue-type plasminogen activator or plasminogen activator inhibitor were found. The low prevalence of the activated protein C resistance genotype, probably stemming from the genetic admixture of the Mexican Mestizo group is noteworthy.

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.

Molecular follow-up of patients with promyelocytic leukaemia treated with all-trans retinoic acid.

Six consecutive patients with promyelocytic leukaemia displaying the PML/RAR-alpha fusion messenger ribonucleic acid (mRNA)--the molecular marker of the disease--were prospectively treated with all-trans retinoic acid: Haematological complete remission was achieved in all of them. However, in three of them the PML/RAR-alpha mRNA persisted. Subsequent treatment with ablative chemotherapy rendered the molecular remission in all cases. Two of the patients that did not achieve the molecular remission with ATRA were autografted with peripheral blood stem cells after clearing the PML/RAR-alpha fusion mRNA. One patient with tretinoin-induced molecular remission relapsed two years after diagnosis and died; the remaining five survive in complete haematological and molecular remission for periods ranging from 405 to 1695 days. Additional studies are needed to clarify the significance of the molecular follow-up of patients with PML.