Self-reported changes in the expanded disability status scale score in patients with multiple sclerosis after autologous stem cell transplants: real-world data from a single center.

In order to reset the immune system to baseline function, autologous hematopoietic stem cell transplantation (HSCT) has been performed in patients with multiple sclerosis (MS). After June 2015, 617 new consecutive patients with MS were autografted in our center with non-frozen peripheral blood stem cells. The autografts were performed on an out-patient basis, after conditioning with cyclophosphamide and rituximab. The aim of the study was the assessment of both safety and efficacy of the method. The study's primary co-end-points were recovery of granulocyte and platelet counts and transplant-related mortality. Secondary end-points were overall survival and clinical response (improvement or stabilization of the self-reported expanded disability status scale score). The protocol was registered in ClinicalTrials.gov identifier NCT02674217.0. We included 401 females and 216 males, with a median age of 46 years. A total of 259 patients had relapsing-remitting MS (RRMS), 228 had secondary progressive (SPMS) and 130 had primary progressive (PPMS) multiple sclerosis. All procedures were initially performed on an out-patient basis and only 32 individuals (5%) required hospitalization. One to three aphereses (median 1) were required to harvest at least 1 × 106 /kg viable CD34+ cells. The total number of viable CD34+ infused cells ranged between 1 and 37·83 × 106 /kg (median 5·68). Patients recovered more than 0·5 × 109 /l absolute granulocytes by day 8 (median, range = 2-14), and platelet values were above 20 × 109 /l by day 4 (median, range = 0-11). Eleven individuals required red blood cells and six needed platelet transfusions. To date, there have been no deaths attributable to the transplant, yielding a 30-month overall survival of 100%. Patients have been followed for 3-42 months (median = 12). The overall response rate (decrease or stabilization of the self-reported EDSS score) at 12 months was 78% for all patients (83% in RRMS, 78% in PPMS and 73% in SPMS), while the disability progression-free survival was 82% for all patients (86% in RRMS, 78·5% in SPMS and 78% in SPMS). Changes in the self-reported EDSS score in parallel with neurological improvement were observed in people with all types of MS after HSCT, employing the 'Mexican method'.

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.

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.

Immunophenotypic characterization of lymphoid cell infiltrates in vitiligo.

The pathogenesis of vitiligo is still controversial. The purpose of this study was to gain insight into the nature of lymphoid cells infiltrating depigmented areas of skin in vitiligo. Immunochemical procedures were carried out in biopsies from 20 patients with active lesions to search for cells expressing CD1a, CD2, CD3, CD4, CD5, CD8, CD20, CD25, CD30, CD56, CD68 and CD79a. Results indicate that early lesions are infiltrated mainly by dendritic cells, whereas older lesions display significantly lower proportions of these cells and increased percentages of mature T cells. This finding might suggest that the autoimmune reactivity towards melanocyte antigens might be T cell-dependent and antigen-driven. It is possible that a non-immune offence of melanocytes is responsible for the exposure of intracellular antigens, while autoreactivity might be a secondary, self-perpetuating mechanism.

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.

Spontaneous reversal of acquired autoimmune dysfibrinogenemia probably due to an antiidiotypic antibody directed to an interspecies cross-reactive idiotype expressed on antifibrinogen antibodies.

A young man with a long history of abnormal bleeding was seen in January 1985. Coagulation tests showed dysfibrinogenemia and an antifibrinogen autoantibody was demonstrable in his serum. This antibody, when purified, was capable of inhibiting the polymerization of normal fibrin monomers, apparently through binding to the alpha fibrinogen chain. 6 mo later the patient was asymptomatic, coagulation tests were normal, and the antifibrinogen autoantibody was barely detectable. At this time, affinity-purified autologous and rabbit antifibrinogen antibodies were capable of absorbing an IgG kappa antibody from the patient's serum, which reacted indistinctly with both autologous and xenogeneic antifibrinogen antibodies in enzyme immunoassays. It has been concluded that the patient's dysfibrinogenemia was the result of an antifibrinogen autoantibody, and that later on an anti-idiotype antibody, which binds an interspecies cross-reactive idiotype expressed on anti-human fibrinogen antibodies, inhibited the production of the antifibrinogen autoantibody which led to the remission of the disorder.

Decreased circulating thymus-derived cells with receptors for the Fc portion of immunoglobulin G in systemic lupus erythematosus.

Thymus-derived cells with receptors for the Fc portion of immunoglobulin G (Fcgamma+ T cells) have recently been found to have a suppressor function, a function that is decreased in systemic lupus erythematosus (SLE). Fcgamma+ T cells were found significantly diminished in 21 untreated SLE patients, particularly in the 7 patients who had active disease. Most Fcgamma+ T cells were separated with a subpopulation of T cells with low affinity for sheep erythrocytes. Decrease of this subpopulation was dependent on the decrease in Fcgamma+ T cells. Non-T cells with Fcgamma receptors were also diminished in SLE patients, but their decrease did not correlate with disease activity. The decrease in suppressor-cell function in SLE may be a result of loss, rather than of dysfunction, of the suppressor Fcgamma+ T cells.

Autologous rosette-forming T cells as the responding cells in human autologous mixed-lymphocyte reaction.

Autologous rosette-forming cells (Tar cells) have surface and functional characteristics of post-thymic precursors and among these characteristics there are some that have been identified in the responsive cell of the autologous mixed-lymphocyte reaction (AMLR). We therefore did AMLR with circulating mononuclear cells from normal subjects using as responding cells either total T cells, T cells depleted of Tar cells, or purified Tar cells. The response of Tar cells in AMLR was significantly greater than that of total T cells and these responded significantly more than Tar-depleted T cells. Conversely, Tar cells responded less than total T cells or T cells depleted of Tar cells in allogeneic mixed-lymphocyte reactions. Increasing numbers of Tar cells gave significantly greater AMLR responses both alone and when added to diminishing proportions of Tar-depleted T cells to keep the number of T cells constant in the system. Tar cells are the responding cells in AMLR but not in allogeneic mixed-lymphocyte reactions.

P-glycoprotein in autoimmune rheumatic diseases.

P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases.

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.

Laser nephelometry quantitation of fibrinogen-related antigens.

A laser nephelometric procedure to quantitate fibrinogen related antigens (FRA), that joined the best characteristics of the immunological available techniques, is proposed. It is sensitive, rapid, simple, reproducible, antiserum saving and accurate since it correlates very well with the tanned red cell hemagglutination inhibition immunoassay (TRCHII) at the most commonly found clinical levels (r = 0.98, p less than 0.0005), but with the advantage of a more objective quantitation and thus, reliable for any concentration of FRA. Prepared samples of different known levels of fibrinogen, assayed with the laser method and the TRCHII, showed a higher correlation with the laser procedure (r = 0.97, p less than 0.0005) than the obtained with the TRCHII (r = 0.85, p less than 0.001). The technic is useful in emergency situations, routine work and research.

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.

Increased detection of human immunodeficiency virus antigen carriers by simultaneous assay of plasma and extracts from resting and phytohaemagglutinin-stimulated peripheral blood mononuclear cells.

Antigenaemia due to human immunodeficiency virus (HIV) is thought to be significant either before the appearance of a specific antibody response, or after its decline during terminal stages. In order to increase the rate of detection of HIV antigen carriers, regardless of the stage or despite the presence of specific serum antibodies, we assayed, simultaneously, plasma samples and extracts from resting and phytohaemagglutinin-stimulated mononuclear cells from 25 infected, anti-body-positive individuals and 10 healthy, antibody-negative female volunteer blood donors. We detected the presence of HIV antigen in at least one of the three types of specimens obtained from all the 25 infected subjects but in none of the 10 healthy blood donors. This approach might prove most useful for the study of patients with controversial or equivocal antibody test results.

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.

Tuberculosis-associated fatal hemophagocytic syndrome in a patient with lymphoma treated with fludarabine.

A patient with a stage IV high-grade non-Hodgkin's lymphoma who developed a fatal hemophagocytic syndrome is presented: When the patient had achieved complete remission and receiving fludarabine and chlorambucil/prednisone, she developed miliary tuberculosis, the CD4+ T-cell count then being 50/microL; the hemophagocytic syndrome ensuing at this point was fatal. Speculations about the predisposing factors that could have led to this complication are discussed focusing on the severe cellular immunosuppression which developed probably related to the use of fludafabine: it could be useful in the future to use anti-tuberculous prophylaxis in selected patients treated with this purine nucleoside analog.

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.

Antiphospholipid syndrome in patients with cyanotic congenital heart disease.

Patients with cyanotic congenital heart disease exhibit an increased incidence of thrombotic episodes and are frequently thrombocytopenic. We studied the sera of 15 patients with this type of heart malformation, searching for anticardiolipin antibodies. 3/15 had positive results. The three of them were adult females; two had thrombotic episodes and a false positive VDRL. Thus, cyanotic congenital heart disease may be another disease entity associated with the antiphospholipid syndrome.

Phenotypic heterogeneity in the expression of a 30 kDa cysteine proteinase in axenic cultures of Entamoeba histolytica.

A polyclonal antibody raised against a purified approximately 30 kDa cysteine proteinase derived from extracts of axenically grown trophozoites of E. histolytica strain HM-1:IMSS was used to stain 72 h cultures of the same amebas by indirect immunofluorescence. Fluorescence was limited to the outer membrane of the parasite and was either uniformly distributed or more condensed on a segment, at times on a single point of the membrane. In relation to the intensity of fluorescence staining, three distinct amebic populations were present: negative, weakly stained and intensely stained. The relative numbers of these three groups remained quite constant for at least one year under the same culture conditions. Flow cytometry was used to quantitate simultaneous variations in amebic size and intensity of fluorescence at various times after different treatments. Amebic size was registered in three levels: small (< 7 microns), medium (7-20 microns), and large (> 20 microns). Staining intensity was measured in arbitrary units. Exposure to 100% fresh hamster serum, phagocytosis of erythrocytes, exposure to cysteine proteinase inhibitors E-64 and cystatin, and to calmodulin antagonist W-7, resulted in various modifications of the phenotype of amebas in very short time periods. We conclude that the expression of the membrane approximately 30 kDa cysteine proteinase in axenic amebic cultures is phenotypically heterogeneous, and that such heterogeneity is modulated and not constitutive.

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.

Activated protein C resistance phenotype and genotype in patients with primary antiphospholipid syndrome.

We studied both inherited and acquired activated protein C (APC) resistance in a group of 22 patients with primary antiphospholipid syndrome (APS). The APC resistance genotype was assessed using a PCR-based analysis for the factor V R506Q (Leiden) mutation. One patient with primary APS was found to be heterozygous for the factor V Leiden mutation. He and other family members were affected by severe thrombophilia and had a familial form of primary APS. The APC resistance phenotype was assessed by measuring the prolongation of the activated partial thromboplastin clotting time in response to APC. It was found in five out of six patients with APS, in one of them transiently. We have found that the APC resistance phenotype is more frequent than the genotype in primary APS. It would seem that patients with thrombophilia should be investigated for APC resistance even if found to have antiphospholipid antibodies and/or lupus anticoagulants.