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'.

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.

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.

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.

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.

Cytokine production and expression of leucocyte-differentiation antigens by human mononuclear cells in response to mycobacterium tuberculosis antigens.

The aim of this work was to characterize a leucocyte-differentiation antigen or chemokine receptor that allows the identification of type 1 (T helper 1 (Th1), Tc1) and type 2 (Th2, Tc2) lymphocytes in short-term-cultured human peripheral blood mononuclear cells. In addition, we assessed the type of response induced by mycobacterial antigens in tuberculosis patients and healthy contacts. Cells were stimulated with an unfractionated culture filtrate or 30 kDa antigen from Mycobacterium tuberculosis. Then, CD4 and CD8 cell labelling was combined with CD30, CD27, CD28, CD45RA or CD45R0 staining, detection of intracellular interferon-gamma (IFN-gamma) or interleukin-4 (IL-4) and analysis by three-colour flow cytometry. In separate experiments, the expression of different chemokine receptors (CCR1, CCR3, CCR5, CXCR3 and CXCR4) was also studied. We found that none of the cell-surface molecules studied was preferentially expressed by Th1 or Th2 cells. Thus, our results indicate that these lymphocyte subsets cannot be identified in short-term-cultured mononuclear cells on the basis of preferential expression of the cell markers studied, and that it is necessary to look for additional molecules that allow the discrimination of Th1 and Th2 cells.

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.

Novel facts about an old marker: the LE cell.

For more than fifty years, lupus erythematosus (LE) cells were believed to result from in vitro opsonization of bare nuclei by serum antinuclear antibodies and their ultimate phagocytosis by neutrophils. Twenty years ago, we described that certain antinuclear antibodies could enter into viable cells, and later on, it was proved that penetration of anti-DNA antibodies into cells results in protracted active cell death. Recent findings indicate that the material engulfed by LE cells are apoptotic blebs as residuals of cells dying after penetration of anti-DNA antibodies. These observations not only change the interpretation of the presence of the LE cell phenomenon, but also stress the potential pathophysiological role of antibodies to intracellular antigens in autoimmune diseases.

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.

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.

'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.