Prevalence of scleroderma in Spain: an approach for estimating rare disease prevalence using a disease model.

PURPOSE: To estimate scleroderma prevalence in Spain. METHODS: As no data were available for Spain we used reported scleroderma incidence (Silman's study on UK and Alamanos' on Greece), scleroderma cause-specific mortality, obtained through the National Institute of Statistics (codes included in M34 of the International Classification of Diseases, 10th revision) in Spain and remission data. Estimates were applied to the Spanish population and to all-cause mortality for 2004, broken down by age group and gender. Estimators were assumed to follow a Poisson distribution. DisMod-II software was used for this purpose. RESULTS: Estimated prevalence was 0.23 and 0.71 cases per 10,000 people for men and women, respectively, based on UK incidence, and 0.28 and 2.58 cases per 10,000 people, respectively, based on Greek incidence. Estimated age at disease onset ranged from 50 to 58 years among men and from 52 to 55 among women, according to UK and Greek data, respectively. Mean duration of the disease was estimated to be in the range of 17-20 years for men and 19-20 for women. CONCLUSIONS: DisMod-II is useful for obtaining, modelling and confirming variability ranges of prevalence found in literature. It also provides information for orphan drug designation and for supporting public health decisions regarding rare diseases.

Severe hematopoietic alterations in vitro, in bone marrow transplant recipients who develop graft-versus-host disease.

Graft-versus-host disease (GVHD) is currently one of the major obstacles for successful allogeneic bone marrow transplantation (BMT). GVHD results from a complex set of interactions between donor T cells and a variety of target tissues from the host. To gain a better understanding of the biology of the human hematopoietic system in GVHD patients, in the present study we have determined the progenitor cell content in bone marrow (BM) samples from BMT recipients, with and without GVHD, and followed their growth kinetics in Dexter-type long-term marrow cultures (LTMC). We have also assessed some aspects regarding the composition of the hematopoietic microenvironment developed in vitro. As compared to normal subjects, BMT recipients showed decreased numbers of myeloid, erythroid, and multipotent progenitor cells. Interestingly, progenitor levels were significantly lower in GVHD patients (7% of the levels in normal marrow) than in those without GVHD (44% of the levels in normal marrow). When marrow cells from BMT recipients were cultured in LTMC, hematopoiesis was sustained at lower levels and for shorter periods of time, as compared to cultures from normal subjects. The hematopoietic deficiencies observed in this in vitro system were also more pronounced in GVHD patients. In terms of the microenvironment elements, reduced numbers of fibroblastic progenitors and adherent stromal cells were observed in BMT recipients, as compared to normal subjects, who showed 7 colony-forming unit fibroblast (CFU-F)/10(5) marrow cells and 320,000 adherent cells in LTMC. Again, GVHD patients showed more severe deficiencies (0.16 CFU-F/10(5) marrow cells and 34,000 adherent cells in LTMC) than patients without GVHD (2 CFU-F/10(5) marrow cells and 122,000 adherent cells in LTMC). Our results demonstrate that the hematopoietic system of BMT recipients is impaired, both in terms of its in vitro composition and function, and that these deficiencies are clearly more pronounced in patients with GVHD than in those without GVHD. Finally, although the evidence is still preliminary, our results also indicate that the severity of the hematopoietic alterations may be greater in acute GVHD than in chronic GVHD.

Tumor necrosis factor-alpha levels in long-term marrow cultures from patients with aplastic anemia: modulation by granulocyte-macrophage colony-stimulating factor.

We have previously shown that the levels of hematopoietic progenitors in long-term marrow cultures (LTMC) from patients with aplastic anemia (AA) are drastically reduced, as compared to normal LTMC. We have also reported that when LTMC from AA patients are supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) there is an increase in colony-forming cell (CFC) levels. However, such a stimulation is only transient and it is followed by an inhibition in CFC growth. Based on these observations, in the present study we have tested the hypothesis that the levels of tumor necrosis factor-alpha (TNF-alpha), an inhibitor of hematopoiesis, are increased in AA LTMC and that such levels are further increased after rhGM-CSF has been added to the cultures for several weeks. Accordingly, we have determined the levels of TNF-alpha in the supernatant of LTMC established from normal (n = 8) and AA (n = 6) bone marrow and in AA LTMC supplemented with rhGM-CSF (n = 6). At the time of culture initiation, TNF-alpha levels were below detection in all the samples analyzed. After 5 weeks of culture, TNF-alpha levels in normal LTMC were very low, with a median of 7.3 pg/mL. In contrast, AA LTMC contained higher levels of TNF-alpha (median of 49.6 pg/mL). In keeping with our hypothesis, addition of rhGM-CSF to AA LTMC resulted in a significant further increase of TNF-alpha levels (median of 135.4 pg/mL). Our results demonstrate an inverse correlation between reduced hematopoiesis in AA LTMC and increased levels of TNF-alpha in this culture system. Based on the results presented here, together with previous reports indicating that TNF-alpha is a potent inducer of apoptosis in hematopoietic progenitor cells, it seems reasonable to suggest that TNF-alpha is implicated in the pathophysiology of AA.

Deficient proliferation of myeloid, erythroid, and multipotent progenitor cells in long-term marrow cultures from patients with aplastic anemia treated with immunosuppressive therapy.

By using Dexter-type long-term marrow cultures (D-LTMC), it has been shown previously that hematopoietic progenitor cells (HPC) from patients with aplastic anemia (AA) have a deficient proliferation in vitro. The studies reported to date, however, have focused exclusively on granulomonocytic progenitors and no information exists on erythroid or multipotent progenitor cells. On the other hand, in such studies, the input progenitor cell numbers were significantly below normal levels, thus suggesting that the rapid disappearance of myeloid progenitor cells from AA D-LTMC could also be due, at least in part, to their reduced number at culture onset. In the present study, we have followed the kinetics of myeloid, erythroid, and multipotent progenitors, from 24 AA patients subjected to immunosuppressive therapy (including patients that achieved complete, partial, or no remission at all), throughout a seven-week culture period. For analysis, we grouped all the patients based on their initial content of all three types of progenitors. Thus, we were able to evaluate separately the kinetics of these cells in D-LTMC from patients with normal and subnormal levels of progenitor cells. At the time of marrow sampling, most patients showed decreased levels of HPC; in fact, only 21%, 8%, and 16% of them showed normal levels of myeloid, erythroid, and multipotent progenitors, respectively. When cultured in D-LTMC, HPC from all AA patients analyzed showed a relatively fast disappearance from the cultures. Indeed, myeloid progenitors could be detected for only six weeks, whereas erythroid and multipotent progenitors disappeared from the cultures after two and one weeks of culture, respectively. In contrast, in normal marrow D-LTMC, myeloid, erythroid, and multipotent progenitors were detected for at least seven, five, and three weeks, respectively. Such a deficient proliferation was observed even in cultures of AA patients that contained normal levels of HPC at culture onset. Interestingly, no correlation was found between HPC proliferation in D-LTMC and response to treatment. Thus, the results of this study indicate the presence of a functional in vitro deficiency in the hematopoietic system of patients with AA, including those that achieved partial or complete remission after immunosuppressive treatment. Furthermore, this work suggests that such a proliferation deficiency is more pronounced in erythroid and multipotent progenitors than in their myeloid counterparts.

Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells.

In the present study, we have established Dexter-type long-term cultures (D-LTC) from human umbilical cord blood (UCB) and followed the kinetics of different hematopoietic progenitor cells (HPCs)--including multipotent (colony forming unit [CFU]-Mixture), erythroid (CFU-erythroid, BFU-E), and myeloid (CFU-granulocyte, CFU-macrophage, CFU-granulocyte/marcophage) progenitors as well as of morphologically recognizable erythroid, myeloid and lymphoid cells--during a nine-week culture period. D-LTC were also established from adult bone marrow (BM) as controls. On day 0, both UCB and BM showed similar total numbers of HPCs (about 310/10(5) cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU-Mixture, CFU-granulocyte/macrophage and BFU-E). A poor adherent cell layer, consisting almost exclusively of macrophages, was developed in UCB D-LTC and this correlated with a continuous decline in HPC numbers throughout the culture period. In contrast, adherent cell numbers in BM D-LTC, including fibroblasts and macrophages, were two- to fourfold higher than in UCB cultures, and the numbers of HPCs were also significantly higher, reaching plateau levels between weeks 6 and 9. In both types of cultures, erythroid and multipotent progenitors declined relatively fast, reaching undetectable levels after five weeks of culture. Myeloid progenitors, on the other hand, were sustained longer (always at higher levels in BM cultures) and were still detected by week 9. Among myeloid progenitors, a shift towards the predominance of macrophage HPCs was observed, both in UCB and BM D-LTC, and this correlated with an increase in the proportion of mature monocytes and macrophages. Taken together, our results indicate that myeloid progenitor cell growth is deficient in UCB D-LTC and suggest that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures regardless of the number and types of adherent cells present; this suggests that the stroma developed in D-LTC is not sufficient for the proliferation of these progenitor cells.

[5 episodes of transient aplastic anemia in 3 patients]. / Cinco episodios de anemia aplástica transitoria en tres enfermos.

We report five episodes of severe aplastic anemia (AA) followed by spontaneous remission in three patients. They were classified as transient aplastic anemia (TAA). Two were females and one male of 32, 56 and 41 years of age, respectively; the man had two recurrences. They had been in contact with insecticides, solvents or drug ingestion. The three had fever, anemia and muco-cutaneous purpura. Supportive measures were used (transfusion of packed red blood cells and platelets, antibiotics, corticosteroids and danazol, the latter two given for ten days in three episodes). They showed spontaneous remission after 16 to 45 days of evolution. The patients did not suffer infection or myeloproliferative disorders which might explain the AA. Transient AA is infrequent and should be considered a variant of AA.

Treatment results of 23 cases of severe aplastic anemia with lymphocytapheresis.

We report the results of 23 patients with aplastic anemia (AA) treated with a program of 14 lymphocytapheresis (LC). Treatments were performed with apheresis machines, models Haemonetics 30-S and Baxter CS3000, using the standard program. This procedure was done because AA in many cases appears as a result of the action of a T cell population that inhibits hematopoiesis. Theoretically, removal of this clonal population would produce hematopoietic recovery. Of the total of 23 patients, 9 were excluded for final evaluation of treatment results because 7 died during or shortly after treatment (0.7-3 months); one patient abandoned treatment after three LC and another died 7 months later because of transformation to acute leukemia. The remaining 14 patients were included in the final evaluation of treatment; seven females and seven males, average age 46.1 years (range 22-69); 13 with severe, and one with moderate AA; 11 with recently diagnosed, and 3 with chronic AA; 12 without previous treatment and two treated before with antilymphocyte globulin + oxymetholone (OXM) + cyclosporine A (CsA) with transient partial remission (PR). Besides lymphocytapheresis, 13 patients received OXM; 4 of them GM-CSF and one low dose CsA. Four patients had complete remission lasting > 59.5 months (range 42-78); eight PR (average duration of > 38.6 months), and two minimal remission (> 37 and 29 months). Platelet, reticulocyte and granulocyte counts increased on average at 48.7, 73.3 and 91.4 days, respectively. In conclusion, 14 (60.8%) of 23 patients with AA showed an improvement related to LC treatment, with a survival probability of 63% from the fourth month, the latter with an added beneficial effect of the other therapies used. Larger numbers of patients have to be treated with LC to determine its real usefulness, mechanism of action and the best conditions for its use.

[Blood erythropoietin levels in healthy subjects studied in the valley of Mexico]. / Niveles séricos de eritropoyetina en sujetos sanos estudiados en el valle de México.

Erythropoietin (EPO) is the hematopoietic growth factor that regulates red cell production. There is a direct relationship between its secretion and tissue hypoxia. Above sea level, oxygen concentration diminishes. This causes an increase of hemoglobin and hematocrit; this effect could be the consequence of higher EPO levels. Currently, evaluation of baseline serum EPO levels is very important in the differential diagnosis of anemia and erythrocytosis. The purpose of the present work was to report the EPO levels on a group of healthy blood donors living in Mexico City, 2,240 m above sea level. Two-hundred twenty blood donors were selected to measure serum EPO; there were 168 males and 52 females. Median EPO levels of the entire population were 7.5 mU/mL (percentile interval, PI, 1-18). Median EPO levels were 7.6 (PI 1-18) and 7.5 (PI 1-16.9) for men and women, respectively. We did not find differences in serum EPO levels among previous reports in other populations and the values determined in this study.

[Treatment of paroxysmal nocturnal hemoglobinuria with antilymphocyte globulin]. / Tratamiento de la hemoglobinuria paroxística nocturna con globulina antilinfocitaria.

OBJECTIVE: To evaluate the effectiveness of antilymphocyte globulin therapy (ALG) in patients with paroxysmal nocturnal hemoglobinuria (PNH). DESIGN: Prospective, non-controlled trial. SETTING: Hematology Service, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, IMSS, Mexico City. PATIENTS: Six patients were included. The median age was 37.5 years and the male/female ratio was 1:1. All the patients had clinical disease consistent with PNH (hemolytic anemia with some degree of transient or persistent pancytopenia) and also erythrocytes with enhanced sensitivity to complement mediated lysis in vitro, as documented by either the Ham test or the sucrose lysis assay. The criterion for severity was the existence of continuous hemolysis in all and transfusion requirements of two or more packed red cells per month in four cases. Prior to ALG therapy, androgens and/or steroids had been given to five patients with no improvement. INTERVENTION: A single batch of ALG was used during the trial (E 0034, Lymphoglobulin Mérieux, Lyon, France). Patients received an infusion of 10 mg/kg per day in a 20 hours lapse during four consecutive days. Also 500 mg/day of methylprednisolone were started simultaneously with the ALG; it was given for seven days and was gradually tapered off and stopped on day 30. MEASUREMENTS: The increases in hemoglobin, granulocytes and/or platelets as well as decreases in red cell transfusion requirements were used to evaluate the results of therapy. RESULTS: Two patients suffered anaphylaxis after the first administration of ALG and were withdrawn from the study. Two of the four remaining patients responded, one response was total and the other minimal. The responses were transient, and no response was seen in the follow-up of 11-14 months. CONCLUSION: ALG therapy for PNH in the doses and time periods used by us had no beneficial effect in patients with a severe form of PNH.

[Advances in the treatment of aplastic anemia]. / Avances en el tratamiento de la anemia aplástica.

The purpose of this paper is to describe the current advances in the pathogenesis, classification and treatment of acquired aplastic anemia (AA). The therapeutical experience obtained at the Servicio de Hematología, Centro Medico Nacional, Siglo XXI is described. Bone marrow transplantation is the first choice therapy for severe AA. This procedure succeeds in obtaining complete remission in nearly 80% of the cases. Nevertheless, few patients are eligible for such therapy, consequently other treatments should be considered. In this context some immunosuppressive therapies such as antilymphocyte globulin had shown to produce favorable responses in 60% of the patients. In addition, androgens and immunosuppressive drugs like methylprednisolone bolus and cyclosporin A do not have a definitive place in severe AA. Finally, it is important to describe the experience with lymphocytapheresis, a new procedure, that decreases the immunological response against the normal hematopoiesis by removing the population of T-lymphocytes inducing complete remission in a few patients.

[Treatment of intracranial hemorrhage in chronic idiopathic thrombocytopenic purpura]. / Tratamiento de la hemorragia intracraneana en la púrpura trombocitopénica idiopática crónica.

Conventional treatment of two patients with chronic idiopathic thrombocytopenic purpura (CITP) and petechial intracranial hemorrhage was ineffectual. However, after a treatment with vincristine, the neurological manifestations disappeared and platelet counts improved; clinical recovery was complete. This success contrast with results informed in similar patients treated with corticosteroids or splenectomy, among whom the mortality is as high as 100%. The favorable reaction may be related to the various mechanisms of the action of vincristine, which usually lead to control of CITP. The use of this drug is proposed in association of conventional therapy of this severe complication, especially in patients in whom splenectomy is contraindicated.