Lower access to risk stratification tests and drugs, and worse survival of chronic lymphocytic leukaemia patients treated in public as compared to private hospitals in Brazil: A retrospective analysis of the Brazilian registry of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) has a highly variable clinical course. In addition to biological factors, socioeconomic factors and health system characteristics may influence CLL outcome. Data from the Brazilian Registry of CLL were analyzed to compare clinical and treatment-related characteristics in patients with CLL, from public or private institutions. A total of 3326 patients from 43 centres met the eligibility criteria, of whom 81% were followed up at public hospitals and 19% at private hospitals. The majority were male (57%), with a median age of 65 years. Comparing public and private hospitals, patients in public hospitals were older, had more advanced disease at diagnosis, and more frequently had elevated creatinine levels. All investigated prognostic markers were evaluated more often in private hospitals. First-line treatment was predominantly based on chlorambucil in 41% of the cases and fludarabine in 38%. Anti-CD20 monoclonal antibody was used in only 36% of cases. In public hospitals, significantly fewer patients received fludarabine-based regimens and anti-CD20 monoclonal antibodies. Patients from public hospitals had significantly worse overall survival (71% vs. 90% for private hospitals, p < 0.0001) and treatment-free survival (32% vs. 40%, for private hospitals, p < 0.0001) at seven years. Our data indicate striking differences between patients followed in public and private hospitals in Brazil. A worse clinical condition and lack of accessibility to basic laboratory tests and adequate therapies may explain the worse outcomes of patients treated in public institutions.

Zinc Supplementation Attenuates Cardiac Remodeling After Experimental Myocardial Infarction.

BACKGROUND/AIMS: The objective of our study was to evaluate the effects of zinc supplementation on cardiac remodeling following acute myocardial infarction in rats. METHODS: Animals were subdivided into 4 groups and observed for 3 months: 1) Sham Control; 2) Sham Zinc: Sham animals receiving zinc supplementation; 3) Infarction Control; 4) Infarction Zinc. After the followup period, we studied hypertrophy and ventricular geometry, functional alterations in vivo and in vitro, changes related to collagen, oxidative stress, and inflammation, assessed by echocardiogram, isolated heart study, western blot, flow cytometer, morphometry, and spectrophotometry. RESULTS: Infarction induced a significant worsening of the functional variables. On the other hand, zinc attenuated both systolic and diastolic cardiac dysfunction induced by infarction. Considering the infarct size, there was no difference between the groups. Catalase and superoxide dismutase decreased in infarcted animals, and zinc increased its activity. We found higher expression of collagens I and III in infarcted animals, but there was no effect of zinc supplementation. Likewise, infarcted animals had higher levels of IL-10, but without zinc interference. Nrf-2 values were not different among the groups. Infarction increased the amount of Treg cells in the spleen as well as the amount of total lymphocytes. Zinc increased the amount of CD4+ in infarcted animals, but we did not observe effects in relation to Treg cells. CONCLUSION: zinc attenuates cardiac remodeling after infarction in rats; this effect is associated with modulation of antioxidant enzymes, but without the involvement of collagens I and III, Nrf-2, IL-10, and Treg cells.

pVAXhsp65 Vaccination Primes for High IL-10 Production and Decreases Experimental Encephalomyelitis Severity.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating pathology of the central nervous system (CNS) used as a model to study multiple sclerosis immunopathology. EAE has also been extensively employed to evaluate potentially therapeutic schemes. Considering the presence of an immune response directed to heat shock proteins (hsps) in autoimmune diseases and the immunoregulatory potential of these molecules, we evaluated the effect of a previous immunization with a genetic vaccine containing the mycobacterial hsp65 gene on EAE development. C57BL/6 mice were immunized with 4 pVAXhsp65 doses and 14 days later were submitted to EAE induction by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) emulsified in Complete Freund's Adjuvant. Vaccinated mice presented significant lower clinical scores and lost less body weight. MOG35-55 immunization also determined less inflammation in lumbar spinal cord but did not change CD4+CD25+Foxp3+ T cells frequency in spleen and CNS. Infiltrating cells from the CNS stimulated with rhsp65 produced significantly higher levels of IL-10. These results suggest that the ability of pVAXhsp65 vaccination to control EAE development is associated with IL-10 induction.

Vitamin D Deficiency and Rheumatoid Arthritis.

Vitamin D (VitD) is a hormone primarily synthesized in human skin under the stimulation of ultraviolet radiation. Beyond its endocrine role in bone metabolism, VitD is endowed with remarkable immunomodulatory properties. The effects of VitD on the immune system include the enhancement of microbicidal ability of monocytes/macrophages and the down-modulation of inflammatory cytokines produced by T lymphocytes. VitD deficiency is involved in many health problems, including immune-mediated diseases such as autoimmune disorders. Rheumatoid arthritis (RA) is a chronic inflammatory systemic autoimmune disease that compromises the joints, causing cartilage destruction and bone erosion. RA treatment usually consists of combined therapies that generally suppress the entire immune response leading to increased susceptibility to infections. This review describes the main effects of VitD on innate and adaptive immune system and also VitD status in inflammatory rheumatic diseases such as RA. Despite some controversies, the majority of reports reinforce the idea that lower VitD levels correlate with more severe clinical manifestations in RA and other rheumatic diseases. Therefore, supplementation with VitD to achieve normal serum levels is worthwhile as an aforethought. Original data concerning the potential applicability of 1,25-dihydroxyvitamin D3 (VitD3), the active form of vitamin D, as a tolerogenic adjuvant are also included. In this sense, the effect of VitD3 associated with proteoglycan (PG), which is a specific cartilage antigen, was tested in the course of experimental arthritis. This association significantly lowered clinical scores and local histopathological alterations. Even though local analysis of T cell subsets and cytokine production did not reveal any difference between the experimental groups, VitD3+PG association significantly reduced cytokine production by spleen cells. These results suggest that VitD3 played a role as a tolerogenic adjuvant by down-modulating the course of experimental RA. Considering this tolerogenic effect of VitD3+PG association, further investigations will reveal its plausible use in human RA.

Diagnosis and treatment of chronic lymphocytic leukemia: recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia

ABSTRACT Chronic lymphocytic leukemia is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+, CD5+ and CD23+. The lymphocytes usually infiltrate the bone marrow, peripheral blood, lymph nodes, and spleen. The diagnosis is established by immunophenotyping circulating B-lymphocytes, and prognosis is defined by two staging systems (Rai and Binet) established by physical examination and blood counts, as well as by several biological and genetic markers. In this update, we present the recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia for the diagnosis and treatment of chronic lymphocytic leukemia. The following recommendations are based on an extensive literature review with the aim of contributing to more uniform patient care in Brazil and possibly in other countries with a similar social-economic profile.

Diagnosis and treatment of chronic lymphocytic leukemia: recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+, CD5+ and CD23+. The lymphocytes usually infiltrate the bone marrow, peripheral blood, lymph nodes, and spleen. The diagnosis is established by immunophenotyping circulating B-lymphocytes, and prognosis is defined by two staging systems (Rai and Binet) established by physical examination and blood counts, as well as by several biological and genetic markers. In this update, we present the recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia for the diagnosis and treatment of chronic lymphocytic leukemia. The following recommendations are based on an extensive literature review with the aim of contributing to more uniform patient care in Brazil and possibly in other countries with a similar social-economic profile.

Systemic Administration of Proteoglycan Protects BALB/c Retired Breeder Mice from Experimental Arthritis.

This study was undertaken to evaluate the prophylactic potential of proteoglycan (PG) administration in experimental arthritis. Female BALB/c retired breeder mice received two (2xPG50 and 2xPG100 groups) or three (3xPG50 group) intraperitoneal doses of bovine PG (50 µg or 100 µg) every three days. A week later the animals were submitted to arthritis induction by immunization with three i.p. doses of bovine PG associated with dimethyldioctadecylammonium bromide adjuvant at intervals of 21 days. Disease severity was daily assessed after the third dose by score evaluation. The 3xPG50 group showed significant reduction in prevalence and clinical scores. This protective effect was associated with lower production of IFN-γ and IL-17 and increased production of IL-5 and IL-10 by spleen cells restimulated in vitro with PG. Even though previous PG administration restrained dendritic cells maturation this procedure did not alter the frequency of regulatory Foxp3(+) T cells. Lower TNF-α and IL-6 levels and higher expression of ROR-γ and GATA-3 were detected in the paws of protected animals. A delayed-type hypersensitivity reaction confirmed specific tolerance induction. Taken together, these results indicate that previous PG inoculation determines a specific tolerogenic effect that is able to decrease severity of subsequently induced arthritis.

Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia.

Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia

Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

Treatment with Vitamin D/MOG Association Suppresses Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model to study multiple sclerosis (MS). Considering the tolerogenic effects of active vitamin D, we evaluated the therapeutic effect of myelin oligodendrocyte glycoprotein (MOG) associated with active vitamin D in EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG emulsified with Complete Freund's Adjuvant plus Mycobacterium tuberculosis. Animals also received two intraperitoneal doses of Bordetella pertussis toxin. One day after immunization, mice were treated with 0,1 µg of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) every other day during 15 days (on days 1, 3, 5, 7, 9, 11, 13 and 15). MOG (150 µg) was co-administered on days 3 and 11. The administration of 1,25(OH)2D3 or MOG determined significant reduction in EAE incidence and in clinical scores. When MOG was associated with 1,25(OH)2D3 the animals did not develop EAE. Spleen and central nervous system (CNS) cell cultures from this group produced less IL-6 and IL-17 upon stimulation with MOG in comparison to the EAE control group. In addition, this treatment inhibited dendritic cells maturation in the spleen and reduced inflammatory infiltration in the CNS. The association of MOG with 1,25(OH)2D3 was able to control EAE development.

Aging does not affect the ability of human monocyte-derived dendritic cells to phagocytose Candida albicans.

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells, playing a key role in induction of both innate and adaptive immunity. Immunosenescence refers to age-associated changes in the immune system, which may be associated with susceptibility to infections and their clinical complications. The precise effects of aging on DCs in immunity to infections are not well understood. Among the common pathogenic microorganisms, the fungus Candida albicans is an important pathogen for the development of invasive infections, especially in immunocompromised individuals, as well as during aging. AIMS: To make a comparative in vitro evaluation of the immunomodulatory function of DCs challenged with C. albicans, by phagocytosis of the fungal cells, and determine the involvement of TLR2 and TLR4 receptors. For this purpose, DCs were generated with the use of peripheral blood monocytes from healthy young and aged subjects. RESULTS: The phagocytosis of C. albicans is developed by DCs in TLR2- and TLR4-dependent way. This mechanism is not affected by aging. CONCLUSION: Given the important role of the DCs in responses against the fungus, it is evident that if changes in phagocytosis occurred with aging, impairment in the elderly could develop. However, the evidence that phagocytosis of this fungus by DCs is not impaired with aging, brings us to the question of which are the mechanisms truly associated with the prevalence of certain diseases in the elderly.

Low Counts of Plasmacytoid Dendritic Cells after Engraftment Are Associated with High Early Mortality after Allogeneic Stem Cell Transplantation.

Dendritic cells (DCs) are antigen-presenting cells that drive immune responses and tolerance and are divided in different subsets: myeloid DCs (mDCs: lineage-; HLA-DR+, 11c+), plasmacytoid dendritic cells (pDCs: HLA-DR+, CD123+), and monocyte-derived DCs (moDC: lineage-, 11c+, 16+). After hematopoietic stem cell transplantation (HSCT), low DC counts in the recipients' peripheral blood (PB) have been associated with worse outcomes, but the relevance of DC graft content remains unclear, and there are few data in the setting of unrelated donor HSCT. We evaluated the DC graft content and monitored DC recovery in PB from 111 HSCT recipients (median age, 17 years; range 1 to 74), who received bone marrow (46%), umbilical cord blood (32%), or PB (22%) from unrelated (81%) or related donors (19%). In 86 patients with sustained allogeneic recovery, patients with higher counts of all DC subsets (pDC, mDC, and moDC) 3 weeks after engraftment had lower incidence of nonrelapse mortality (NMR) and acute graft-versus-host disease (aGVHD) and better survival. pDC counts were associated with more striking results: patients with higher pDC counts had much lower incidences of NRM (3% versus 47%, P < .0001), lower incidence of aGVHD (24% versus 67%, P < .0001), and better overall survival (92% versus 45%, P < .0001). In contrast, higher pDC counts in the graft was associated with an increased risk of aGVHD (55% versus 26%, P = .02). Our results indicate that DC counts are closely correlated with HSCT outcomes and warrant further prospective evaluation and possible early therapeutic interventions to ameliorate severe aGVHD and decrease mortality.

First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX.

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

Regulatory T cells in the actinic cheilitis.

BACKGROUND: Actinic cheilitis (AC) is an oral potentially malignant lesion which is the counterpart of actinic keratosis of the skin and has potential to develop into squamous cell carcinoma. Regulatory T cells (Tregs) have a critical role in modulating the antitumor immune responses. The presence of regulatory T cells in potentially malignant lesions has not been described. We chose investigate the involvement of regulatory T cells in potentially malignant lesions. METHODS: The frequency, phenotype, and activity of CD4+CD25+ T cells isolated from blood and lesion of AC patients were analyzed by flow cytometry. Cytokines were quantified by ELISA. Data were compared with samples from healthy subjects. RESULTS: The frequency and suppressor activity of circulating CD4+CD25+ T cells was similar in AC patients and control subjects. However, the frequencies of IL-10-positive Tregs were higher in AC patients, and these cells inhibited interferon-gamma (IFN-γ) and increased interleukin (IL)-10 productions in co-cultures. Furthermore, CD4+CD25+ T cells accumulate in AC lesions. Lesions-derived regulatory T cells suppressed lymphocyte proliferation and pro-inflammatory cytokine production. Moreover, high levels of IL-10 and transforming growth factor-ß (TGF-ß), and low IFN-γ were detected in the potentially malignant lesions. CONCLUSION: Therefore, our data show that Tregs accumulate in AC lesions, and these cells could be suppressing immune responses in a potentially malignant microenvironment.