Defective expression of apoptosis-related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation.

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis-related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)-based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIMEL , FAS, FASL, A1, BCL2, BCLXL , CFLIPL and CIAP2 genes were up-regulated after AHSCT. With the exception of BIK, BIMEL and A1, all genes reached levels similar to controls at day + 720 post-transplantation. Furthermore, in these patients, we observed increased CD8+ Fas+ T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIPL and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day + 720 post-AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLXL and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl-2 expression before transplantation. At 1 year post-AHSCT, expression of Bak, Bim, Bcl-2, Bcl-xL and cFlip-L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis-related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re-establishment of immune tolerance during the first 2 years post-transplantation.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic ß cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-x(L) and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-x(L) genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-x(L) and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Genetic polymorphisms in the chemokine and chemokine receptors: impact on clinical course and therapy of the human immunodeficiency virus type 1 infection (HIV-1).

The natural history and pathogenic processes of infection by the human immunodeficiency virus type 1 (HIV-1) are complex, variable, and dependent upon a multitude of viral and host factors and their interactions. The CCR5-Delta32 allele remains the most important genetic factor known to be associated with host resistance to the HIV-1 infection. However, other mutations in the CCR5, CCR2, CX(3)CR1, CXCL12 (SDF1), and CCL5 (RANTES) genes have been identified and associated with host resistance and/or susceptibility to HIV-1 infection and disease progression. Some studies have also suggested that chemokine receptor gene polymorphisms may affect response to potent antiretroviral therapy. This article reviews the polymorphisms already described in the mutant chemokine receptors or ligands and their impact on the host susceptibility to HIV-1 infection and on the clinical course of the disease, as well as the development of new anti-HIV therapies that takes into account these potential targets in the host. These genetic polymorphisms could be used as genetic markers to detect individuals at higher risk of developing either a faster disease progression or therapeutic failure. Once these individuals are identified, therapeutic strategies based on either different, more aggressive drugs or combinations of drugs can be used, either alone or in combination with shorter intervals for therapeutic monitoring. Pharmacogenetics is very likely to underlie future therapies for HIV-1 infection, and current patients with multi-resistance to the existing antiretroviral agents could also benefit from this approach. These developments also underscore the importance of continuing the investigation of new therapies targeted to the host in order to inhibit the HIV-1 entry into the host cells.

Six cases of leprosy associated with allogeneic hematopoietic SCT.

We report here the first six cases of leprosy associated with HLA-identical allogeneic SCT in different phases and with different findings and outcomes. Skin and peripheral nerves may be sites of leprosy associated with SCT, stressing the importance of differential diagnosis between leprosy and GVHD or drug reactions. Clinical manifestations of leprosy before or after transplantation did not influence the outcome of SCT in our cases.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant.

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting beta-cell regeneration.

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic beta-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of beta-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce beta-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature beta-cells. There is controversial evidence of the potential of these cell sources to regenerate beta-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic beta-cells and promoting beta-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.

Cherubism, gingival fibromatosis, epilepsy, and mental deficiency (Ramon syndrome) with juvenile rheumatoid arthritis.

This is a report on four persons in one family with a condition similar to that described by Ramon et al [Oral Surg 24:436-48, 1967] in two sibs born to a consanguineous couple. Our patients also had mental deficiency, epilepsy, cherubism due to fibrous dysplasia of the maxillae, gingival fibromatosis, hypertrichosis, and stunted growth. This appears to be an autosomal recessive trait in both families. Our patients are the second set reported with this syndrome; they also have juvenile rheumatoid arthritis, which was not described in the family reported by Ramon et al [Oral Surg 24:436-48, 1967]. We conclude that the Ramon syndrome should also include juvenile rheumatoid arthritis.

Hematopoietic stem cell transplantation for autoimmune diseases in developing countries: current status and future prospectives.

In this paper we present preliminary results of hematopoietic stem cell transplantation for autoimmune diseases in Brazil and China. Chinese experience transplanting lupus is significant and the Brazilian experience with several autoimmune diseases is growing. We discuss peculiar conditions in developing countries which could affect the results, and future prospectives for the organization of phase III randomized trials in those countries.

In vitro comparison of two methods of T cell depletion associated with different rates of graft failure after allogeneic marrow transplantation.

Clinical trials in another center have shown a substantially lower risk of graft failure associated with T cell depletion by treatment of donor marrow with the use of an antibody against CD6 compared to depletion with a mixture of eight antibodies previously used for clinical trials in our center. In order to evaluate mechanisms possibly responsible for this difference, we compared lymphoid cell surface phenotypes and in vitro functions in marrow cells treated by complement-mediated lysis with anti-T12 (CD6) or with the eight antibody mixture. Treatment with the eight antibody mixture produced more than three log depletion of precursors for IL-2-producing cells (pIL-2) and approximately one log depletion of precursors for NK cells. On the other hand, treatment with anti-T12 produced approximately one log depletion of pIL-2 and had no effect on NK precursors. Additional studies were carried out with treated marrow cells cultured in medium containing recombinant IL-2. Compared to cells treated with the eight antibody mixture, the marrow cells that remained after anti-T12 treatment had more cytotoxic activity against K562, Daudi and an EBV-transformed human B cell line during the first 6 days of culture, but marrows treated by the two methods showed similar cytotoxic activity after 10 days of culture. Cultures from marrow treated with anti-T12 contained more CD3+ and CD6+ cells than cultures from marrow treated with the eight antibody mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

CD8+/DR+/CD25--T-lymphocytes associated with marrow graft failure.

Phenotypic and functional characteristics of peripheral blood mononuclear cells (PBMC) were studied in eight patients with poor graft function following HLA-identical T cell-depleted marrow transplantation. Similar patients with good graft function and normal individuals were used as controls. Freshly isolated PBMC from patients with failing grafts contained more CD3+ and CD8+ cells than PBMC from well engrafted patients. The CD8+ cells appeared activated insofar as they expressed DR antigens, but they did not express the low affinity IL-2 receptor recognized by Tac antibody (CD25) and they did not have increased cytolytic activities. After culture with phytohemagglutinin (PHA) and IL-2, PBMC from patients with poor graft function contained fewer CD2+ and CD4+ cells than cultured PBMC from patients with good graft function. Cultured cells from patients with poor graft function acquired lymphokine activated killer (LAK) activity against NK-sensitive and NK-insensitive targets, but still did not express CD25. Host-mediated anti-donor cytotoxic activity could be demonstrated in one patient only after presensitization with donor cells and culture with IL-2 and PHA. The abnormalities in T cell activation observed in patients with poor graft function did not correlate with the donor or host origin of lymphoid cells. These data indicate that some cases of graft failure may be associated with defective T cell maturation. These abnormalities may simply represent a consequence of marrow failure or they may actually contribute to failure by not providing critical hematopoietic accessory functions.

Beneficial effect of intravenous lidocaine in cutaneous chronic graft-versus-host disease secondary to donor lymphocyte infusion.

We report two cases of refractory chronic graft-versus-host disease after donor lymphocyte infusions in which the skin lesions improved dramatically with the use of intravenous pulses of lidocaine. This form of therapy has been used successfully for the cutaneous involvement of scleroderma and may have vasodilator and anti-inflammatory effects.

Influence of menstrual cycle on NK activity.

Natural killer (NK) cells are CD3- CD56+ and/or CD16+ cytotoxic lymphocytes that mediate first-line defense against various types of target cells without prior immunization. To assess the effect of the menstrual cycle and gender on NK activity we evaluated 30 healthy women (mean age 28.1 years, range 21-39) in follicular and luteal phases, 29 postmenopausal women (mean age 58.8 years, range 42-72) and 48 healthy men (mean age 31.6 years, range 21-40). In a flow cytometric test of NK activity, peripheral blood mononuclear effector cells were mixed with K562 targets cells labeled with DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) at effector:target cell ratios of 40, 20, 10 and 5:1. Dead cells were stained with propidium iodide and results were expressed as lytic units per 10(7) cells. In addition, progesterone levels were determined in the luteal phase of the menstrual cycle of healthy women by a chemiluminescence assay. Our results showed that (1) NK cytotoxicity was higher in the follicular than in the luteal phase of the menstrual cycle (P < 0.0001); (2) postmenopausal women and men showed NK activity similar to women in the follicular phase but higher than women in the luteal phase of the menstrual cycle (P < 0.05); and (3) there was no correlation between NK activity and levels of progesterone. The data suggest that progesterone does not influence NK activity directly and that other factors may explain the reduction of NK activity in the luteal phase of the menstrual cycle.

HLA class I and class II profiles of patients presenting with Sydenham's chorea.

Sydenham's chorea (SC) may occur in rheumatic fever (RF) patients without arthritis and carditis. In this study we typed HLA antigens and alleles in patients presenting with the distinct major clinical manifestations of RF, i.e., chorea, carditis, or arthritis, in population and family studies. We evaluated 91 patients with RF for HLA-A, HLA-B, and HLA-DR antigens; of these, 33 had pure chorea, 26 pure carditis, 16 pure arthritis, and 16 carditis plus arthritis. We also typed 24 SC patients and their unaffected siblings for HLA-DRB1 and HLA-DQB1 alleles using molecular methods. HLA-B49 and HLA-DR1 antigens were overrepresented in the total group of patients with RF and in all the subgroups studied, excluding the SC subgroup in which the frequency of HLA-DR1 antigen was not increased. The frequencies of the HLA-DRB1 and HLA-DQB1 alleles in patients with pure chorea were not significantly different from those observed in controls. Similarly, the frequencies of HLA class II alleles in SC patients did not differ significantly from those observed in unaffected siblings. These findings show that immunogenetic susceptibility to RF varies according to the major clinical manifestation presented by the patient.

Experimental nephropathy induced by human polyclonal light chains.

We describe the nephrotoxic effects of a preparation of light chains (LC) obtained by reduction and alkylation of human IgG. The acute renal lesions in rats are dependent upon the dose and route of LC administration. The kidney alterations were not observed in animals treated with comparable amounts of human albumin or the F(ab')2 fragment of human IgG. Intravenous bolus injection of 120 mg LC into 80-100 g hydropenic rats induced extensive cast formation in distal and collecting renal tubules, which were similar to the human "myeloma kidney". In contrast, the slow infusion of the same amount of LC over 1 h produced degenerative changes in the proximal tubular cells without extensive cast formation. The morphological alterations of the kidney were investigated by classical histological methods, by immunofluorescence and in thin sections stained with toluidine blue. Urinary excretion and kidney content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase were increased only in the group of animals infused with LC. These findings may be relevant to the pathogenesis of human nephropathy occurring in multiple myeloma and in some autoimmune diseases.

Expansion of a subset of TCR gamma/delta human lymphocytes from various lymphoid organs cultured with recombinant IL-2.

1. TCR1 cells are a minor component of CD3+ lymphocytes which bear the gamma/delta T-cell receptor. There are limited data concerning the activation of TCR1 cells or TCR1 cell subsets in human lymphoid organs. We analyzed a subset of TCR1 cells (delta TCS1+) in peripheral blood (PBL), spleen (SPL), lymph nodes (LN), bone marrow (BM), and thymus (THY) after activation with IL-2. Lymphoid cells from these organs were cultured with 1500 U/ml IL-2 for 14 days and analyzed at periodic intervals for delta TCS1+ cells. 2. We found increased numbers of delta TCS1+ cells in 6- and 14-day cultures from SPL (20.8 +/- 11.8% positive cells after 14 days of culture), LN, BM and THY but not in peripheral blood (1.8 +/- 0.9%). These delta TCS1+ cells coexpressed CD2, CD3, CD8 and CD56, but were negative for TCR alpha/beta and CD4. We also detected an expansion of TCR1+ cells in IL-2-stimulated PBL employing the pan-gamma/delta marker TCR delta 1; however, in contrast to solid organs, these TCR1+ cells were delta TCS1 negative. 3. Sorting experiments demonstrated directly that delta TCS1 cells from spleen cultures mediate high cytotoxic activity against K562 cell targets (39.4% median specific cytotoxicity) and low activity against Daudi (9.6%), COLO (2.7%) and an antibody-sensitized human B-cell line (17%). 4. These results show expansion and cytotoxic activation by IL-2 of a subset of human TCR1 cells in solid lymphoid organs.

T-cell subsets in patients with aplastic anemia.

Abnormalities of lymphocyte subpopulations have been described in patients with aplastic anemia. In the present report we extend these studies by measuring T cell subsets identified by the presence of Fc receptors for IgM (T mu-lymphocyte) and IgG (T gamma-lymphocyte) in 22 patients and in 48 normal controls. The absolute number of T mu and T gamma lymphocytes was normal in the majority of cases. The percentages of T mu cells was increased in 4 cases and decreased in 2; T gamma cells were increased in 6 patients. The levels of serum immunoglobulins did not correlate with the T mu/T gamma ratio. The pathogenesis of aplastic anemia is discussed in terms of these immunological abnormalities.

Adrenal response to adrenocorticotropin hormone and HLA typing of subjects with different degrees of 21-hydroxylase deficiency.

1. To evaluate different degrees of 21-hydroxylase (21-OH) deficiency we studied the 17-hydroxyprogesterone (17-OHP) and cortisol response to the adrenocorticotropin hormone (ACTH) stimulation test. In a study of 13 families we characterized the relatives of patients with classical 21-OH deficiency using HLA antigen typing and the ACTH test. The subjects were divided into five groups: 12 patients with the classical form, 11 patients with the nonclassical form, 38 heterozygotes, 6 normal homozygotes and 33 controls. 2. The 17-hydroxyprogesterone response to ACTH (mean +/- SD) varied as follows according to the degree of 21-OH deficiency: 25442 +/- 15718 ng/dl for the classical group, 4198 +/- 1637 ng/dl for the nonclassical group, 348 +/- 267 ng/dl for the heterozygotes, 127 +/- 81 ng/dl for normal homozygotes, and 164 +/- 120 ng/dl for the controls. Basal plasma cortisol did not differ among the five groups. The cortisol response to ACTH was not different among controls (30 +/- 8 micrograms/dl), normal homozygotes (28 +/- 7 micrograms/dl) and heterozygotes (26.5 +/- 7 micrograms/dl). The cortisol response was decreased in the patient groups and was lower in the classical (14 +/- 10 micrograms/dl) than in the nonclassical group (20 +/- 4 micrograms/dl). 3. In most families (11/13), HLA typing was informative in identifying the 21-OH deficiency containing haplotype, which correlated with the hormonal profile. In two families there was no correlation between the HLA genotype and the clinical expression of 21-OH activity for two HLA identical pairs of siblings.

Cell-mediated immunity in Brazilian hemophilia patients.

The purpose of this study was to correlate the immunological features of healthy hemophiliac A patients treated with commercial cryoprecipitates with those who received factor VIII concentrates associated or not with cryoprecipitates. The absolute number of total lymphocytes, T3, T4 and B lymphocytes did not differ for either group of patients or the controls. The number of T8 lymphocytes was higher in the group treated only with cryoprecipitates than for the controls. The T4/T8 lymphocyte ratios for both groups of patients were significantly lower than in the controls. This was due to a decrease in the percentage of T4 and increase of T8 lymphocytes. K cell activity was lower in both groups of patients than the controls. These results indicate that both cryoprecipitates and commercial factor VIII concentrate replacements had similar effects on the development of lymphocyte abnormalities.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation.

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Frequency of HLA antigens in a Brazilian type I diabetic population.

1. Sixty-five Brazilian, patients with type I, insulin-dependent diabetes mellitus (IDDM) and 100 unaffected individuals were typed for HLA-A, -B, -C and DR antigens. 2. A significantly higher frequency of HLA-A2 (48% of the patients versus 21% of the controls), B15 (20% of the controls), DR3 (57% of the patients versus 28% of the controls) and DR4 (54% of the patients versus 23% of the controls) was found for IDDM patients compared to the controls. 3. In contrast, DR2 (11% of the patients versus 31% of the controls) and DR7 (3% of the patients versus 21% of the controls) were lower in diabetics, but the difference was not significant. 4. The data reported here, when compared with those of other studies, emphasize the ethnic variability in HLA-IDDM associations.