Production of dendritic cell vaccines using different methods with equivalent results: Implications for emerging centers

Introduction Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients. Method: In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation. Results The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor. Conclusion These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option.

Production of dendritic cell vaccines using different methods with equivalent results: Implications for emerging centers.

INTRODUCTION: Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients. METHOD: In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation. RESULTS: The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor. CONCLUSION: These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option.

The challenges of handling deferasirox in sickle cell disease patients older than 40 years.

OBJECTIVES: Deferasirox is an oral iron chelator with established dose-dependent efficacy for the treatment of iron overload secondary to transfusion. However, there is few data reporting the use of Desferasirox in adult patients with sickle cell disease (SCD) and transfusional iron overload. METHODS: We conducted a prospective, single center, nonrandomized study from January 2014 to March 2015 in Campinas, Brazil. Seven patients (five women, median age 50 y.o.) who were followed up on regular transfusion program were treated with a single daily dose of deferasirox (median dose 20 mg/kg). They were monitored for clinical symptoms, renal function and hepatotoxicity. RESULTS: One patient discontinued the study due to lack of compliance. Two patients reported mild to moderate adverse events (gastrointestinal disturbances). Five patients had the drug discontinued due to worsening of renal function. One patient had the drug discontinued due to severe hepatotoxicity that evolved to death; no patient finished the study. Discussion and conclusions: Deferasirox does not appear to be well tolerated in SCD patients older than 40 years, in which complications of the underlying disease are already fully installed. The choice of the ideal iron chelator for this population should include an evaluation of comorbidities and organic dysfunctions, as well as the need to find pharmacogenetic safety markers in this group of patients.

Echocardiografic abnormalities in patients with sickle cell/β thalassemia do not depend on the β-thalassemia phenotype

ABSTRACT Objectives and methods: We evaluated possible relationships between echocardiographic findings and clinical and laboratory parameters, in a cohort of Brazilian patients diagnosed with sickle cell/β-thalassemia, to better understand the cardiac involvement in this disease. Results: Left atrial (LA) and left ventricular (LV) dilation were found in 19.5 and 11% of patients, respectively; systolic left ventricular dysfunction was present in a single patient. There were no differences in masses and volumes of cardiac chambers comparing Sβ0 with Sβ+ patients, and no relationship between these parameters and specific complications of the disease. However, parameters of altered ventricular geometry were significantly correlated with serum creatinine, hepatic transaminases and bilirubin levels. Moreover, 3 patients presented stroke; they were significantly older [53 (41-56) × 37.5 (18-70), p = 0.048], had higher values of LV posterior wall diastolic thickness [10 (10-11) × 8 (6-14), p = 0.03], LV mass [226 (194-260) × 147 (69-537), p = 0.039] and LA/aortic ratio [1.545 (1.48-1.61) × 1.26 (0.9-1.48), p = 0.032]. Conclusions: Cardiac involvement in this disease does not appear to depend on the thalassemia phenotype. The presence of signs of myocardial remodeling in this group of patients was related to multi-organ impairment and rendered a higher propensity for stroke in older patients, suggesting the need for greater vigilance and control of associated factors.

Echocardiografic abnormalities in patients with sickle cell/ß-thalassemia do not depend on the ß-thalassemia phenotype.

OBJECTIVES AND METHODS: We evaluated possible relationships between echocardiographic findings and clinical and laboratory parameters, in a cohort of Brazilian patients diagnosed with sickle cell/ß-thalassemia, to better understand the cardiac involvement in this disease. RESULTS: Left atrial (LA) and left ventricular (LV) dilation were found in 19.5 and 11% of patients, respectively; systolic left ventricular dysfunction was present in a single patient. There were no differences in masses and volumes of cardiac chambers comparing Sß0 with Sß+ patients, and no relationship between these parameters and specific complications of the disease. However, parameters of altered ventricular geometry were significantly correlated with serum creatinine, hepatic transaminases and bilirubin levels. Moreover, 3 patients presented stroke; they were significantly older [53 (41-56)×37.5 (18-70), p=0.048], had higher values of LV posterior wall diastolic thickness [10 (10-11)×8 (6-14), p=0.03], LV mass [226 (194-260)×147 (69-537), p=0.039] and LA/aortic ratio [1.545 (1.48-1.61)×1.26 (0.9-1.48), p=0.032]. CONCLUSIONS: Cardiac involvement in this disease does not appear to depend on the thalassemia phenotype. The presence of signs of myocardial remodeling in this group of patients was related to multi-organ impairment and rendered a higher propensity for stroke in older patients, suggesting the need for greater vigilance and control of associated factors.

Red blood cell alloimmunization in patients with sickle cell disease: correlation with HLA and cytokine gene polymorphisms.

BACKGROUND: The reason for the difference in susceptibility to red blood cell (RBC) alloimmunization among patients with sickle cell disease (SCD) is not clearly understood and is probably the result of multiple factors. Our hypothesis is that genetic polymorphisms are associated with RBC alloimmunization. STUDY DESIGN AND METHODS: We investigated the possible association of susceptibility to RBC alloimmunization with polymorphisms of HLA and cytokines genes in 161 SCD patients prior exposed to RBC transfusion. Cytokine gene polymorphisms were analyzed by polymerase chain reaction (PCR) and TaqMan assays. HLA Class I genotyping was performed using PCR-specific sequence of oligonucleotides. Polymorphism frequencies were compared using the Fisher's exact test. RESULTS: Our results revealed increased percentage of the A allele and the GA genotype of the TNFA -308G/A cytokine among alloimmunized patients when compared to nonalloimmunized patients (A allele, 16.4% vs. 6.8%, p = 0.004; GA genotype, 32.8% vs. 11.7%, p = 0.0021). In addition, the IL1B -511T allele and the IL1B -511TT and CT genotype frequencies were overrepresented among alloimmunized patients (T allele, 53.0% vs. 37.5%, p = 0.0085; CT + TT genotypes, 81.82% vs. 60.87%, p = 0.0071). In relation to HLA Class I, we found a higher frequency of HLA-DRB1*15 among patients alloimmunized to Rh antigens when compared to nonalloimmunized patients (15.63% vs. 6.98%, p = 0.044). CONCLUSION: Brazilian SCD patients with the TNFA, IL1B, and HLA-DRB1 gene polymorphisms were at increased risk of becoming alloimmunized by RBC transfusions. These findings may contribute to the development of future therapeutic strategies for patients with SCD with higher susceptibility of alloimmunization.

Sickle cell/ß-thalassemia: Comparison of Sß0 and Sß+ Brazilian patients followed at a single institution.

OBJECTIVES: In sickle cell/ß-thalassemia, mutations in the corresponding ß-globin genes are responsible for complex pathological events resulting in diverse clinical complications. The objective of this study was to provide an overview of the clinical and laboratory characteristics of patients with the syndrome, and of the degree of severity of clinical manifestations resulting from the ß-thalassemia mutation. METHODS: A retrospective chart review was performed on 46 patients with sickle cell/ß-thalassemia (31 Sß° and 15 Sß+), evaluating hematological parameters and end organ damage. Statistical analyzes were carried out in order to highlight differences between the two groups according to the nature of the thalassemia mutation. RESULTS: As expected, patients with the Sß0 phenotype had a higher degree of hematological involvement in comparison to Sß+ patients; with lower hemoglobin levels, and signs of more intense chronic hemolysis. However, Sß+ patients were more prone to the occurrence of acute chest syndrome. The impact of the thalassemia mutation upon total body and bone composition was also evident, as Sß0 patients presented lower body mass index (BMI) and bone mineral density. The degree of bone damage correlated to lower BMI and hemoglobin levels, as well as plaquetosis, monocytosis and elevated lactate dehydrogenase, possibly reflecting the effects of hemolysis and inflammation upon bone metabolism and body constitution. CONCLUSIONS: This study identified significant differences among sickle cell/ß-thalassemia patients according to the beta mutation involvement, pointing to an important predictor of disease severity.

The effects of exchange transfusion for prevention of complications during pregnancy of sickle hemoglobin C disease patients.

BACKGROUND: Pregnancy represents a challenge for women with sickle cell disease (SCD), with higher rates of both maternal and fetal complications. The aim of this study was to evaluate the impact of prophylactic transfusion support administered specifically to pregnant women with sickle hemoglobin C disease. MATERIALS AND METHODS: Patients were divided into two groups according to the type of transfusion support received: 10 women received prophylactic erythrocytapheresis or manual exchange transfusion at 28 weeks of gestation, and 14 received transfusions only on demand, due to acute complications, or received no transfusions at all. RESULTS: Our results indicated higher frequencies of SCD-related complications in the group that did not receive prophylactic transfusion support (35.7% vs. only 10% in the erythrocytapheresis group). Furthermore, these complications were more severe in this group and included all cases of acute chest syndrome. A significant difference was observed concerning gestational age at birth (38.7 weeks in the transfusion group vs. 34.4 weeks, p = 0.037), with a higher frequency of preterm births in the nontransfused group (69.23% vs. 30% in the transfusion group). CONCLUSION: We demonstrated a clear reduction of unfavorable outcomes in patients receiving prophylactic transfusions, probably reflecting better maternal and fetal conditions, which corroborated to the more satisfactory indices of vitality, observed in newborns. Considering that erythrocytapheresis or manual exchange transfusions both represent feasible and safe procedures, they could represent important tools for the optimal management of these patients.

An efficient protocol for the generation of monocyte derived dendritic cells using serum-free media for clinical applications in post remission AML patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on "good manufacture practices" (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Obesity and inflammation and the effect on the hematopoietic system.

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

Obesity and inflammation and the effect on the hematopoietic system

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

IL4 and IFNalpha generation of dendritic cells reveals great migratory potential and NFkB and cJun expression in IL4DCs.

Dendritic cells (DCs) recently revealed as a potent tumor vaccine component, are commonly differentiated from monocytes by cultivation with IL-4 and GM-CSF. Despite the different opinions, the use of IFNalpha can promote DCs differentiation and activation. The aim of this study was to compare the functionality and phenotypic characterization of monocyte-derived DC generated by IL-4 (IL4DC) and IFNalpha (IFNalphaDC) modified protocols. To this aim, we investigated the expression of maturation markers, co-stimulatory molecules, relevant miRNA, cytokine and migratory profiles and the functional ability of these cells to stimulate autologous T cells in vitro. We herein investigated the molecular mechanism underlying the parameters previously described, as the relative expression of NF-kB p65, c-fos and c-jun, transcription factors. Our results demonstrated that IL4DC presented a stable phenotype, an increase in migratory capacity and NF-KB activation, in addition to lower levels of miR-146 a and miR-221. We believe that the IL4DC migratory potential and increase in NFkBp65 expression may be involved in higher IL12 expression and migration, suggesting a preferential activation of TH1 immune responses by IL4DC.

Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients.

OBJECTIVE: To evaluate the usefulness of DNA methods to provide a means to precisely genotypically match donor blood units for the antigen-negative type of 35 sickle cell disease patients. METHODS: Red blood cell units were investigated for ABO, D, C, c, E, e, K, Fy(a), Fy(b), Jk(a), Jk(b), S, s, Di(a) and RH variants by performing a molecular array (Human Erythrocyte Antigen BeadChip(TM), BioArray Solutions), polymerase chain reaction followed by restriction fragment length polymorphism analysis and sequencing of patient samples and donor units that had been serologically matched based on the ABO, Rh and K phenotypes and the presence of antibodies. RESULTS: Matches for 21 of 35 sickle cell disease patients presented discrepancies or mismatches for multiple antigens between the genotype profile and the antigen profile of their serologically-matched blood units. The main discrepancies or mismatches occurred in the RH, FY, JK and MNS systems. Eight Rh alloimmunized patients presented RHD and RHCE variants that had not been serologically identified. According to these results better matches were found for the patients with genotyped units and the patients benefited as shown by better in vivo red blood cell survival. CONCLUSION: Molecular matching is superior to serological matching in sickle cell disease patients, decreasing the risk of transfusion reactions, especially delayed transfusion reactions to existing alloantibodies and preventing alloimmunization.

IL10 inversely correlates with the percentage of CD8⁺ cells in MDS patients.

The role of the immune system in myelodysplastic syndrome (MDS) progression has been widely accepted, although mechanisms underlying this immune dysfunction are not clear. CD4(+) and CD8(+) lymphocyte profiles in the peripheral blood of MDS patients were evaluated and correlated with clinical characteristics, the expression of FOXP3 and the anti-inflammatory cytokines IL10, TGFß1 and CTLA4. IL10 expression inversely correlated with the percentage of CD8(+) cells and was higher in high-risk MDS. Our findings provide further evidence for the role of T cell-mediated IL10 production in MDS and strengthen the idea of distinct cytokine profiles in low and high-risk MDS.