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.

Effects of prophylactic red blood cell (RBC) transfusion with extended antigen matching on alloimmunization in patients with Sickle Cell Disease (SCD).

BACKGROUND: RBC alloimmunization remains a significant problem for many patients with SCD. To reduce alloimmunization some strategies have been implemented to provide limited or extended antigen matched RBC transfusions to patients with SCD who need chronic transfusion support. The aim of this study was to evaluate the effects of prophylactic RBC transfusion with extended antigen matching on alloimmunization in patients with SCD. METHODS: This is a 20-year retrospective study of patients with SCD transfused with RBCS that were prospectively matched for D, C, c, E, e, K, Fya/Fyb, Jka/Jkb and S antigens. Our study included 95 patients, and none had antibodies documented before their first transfusion. Patients and donors were phenotyped and molecular typing was performed in all patients who had recent transfusions or a positive direct antiglobulin test to predict their antigen profile. Unexpected antibodies to the Rh system, meaning anti-Rh antibodies in patients whose serologic phenotype was Rh positive, were investigated by molecular genotyping for RH variant alleles. RESULTS: During this study-period, 12 (12.6%) were alloimmunized and 83 (87.4%) were not. Among the 12 patients who alloimmunized, 7 (58.3%) developed antibodies to Rh antigens and 5 (41.7%) produced antibodies to low prevalence antigens. All patients who developed Rh antibodies had RH variant alleles. Autoantibodies were found in 16 (16.8%) transfused patients. CONCLUSION: SCD patients benefit from receiving prophylactic RBC transfusions with extended antigen matching, as demonstrated by the reduction on the rates of alloimmunization and the lack of antibodies to K, FY, JK and S antigens, however, this strategy does not avoid alloimmunization to Rh and low-prevalence antigens.

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.

Recombinant erythropoietin as alternative to red cell transfusion in sickle cell disease.

Disturbances in the physiological regulation of erythropoietin (EPO) in patients with sickle cell disease (SCD) may contribute to worsening anaemia and increased transfusion requirements, but the use of recombinant EPO in this group of patients is controversial. The objective of this study was to evaluate the use of this drug in adult patients with SCD and its effects on haemoglobin levels and transfusion requirements. We conducted a retrospective analysis at the University of Campinas, with nineteen adults with a diagnosis of SCD (HbSS and HbS/ß+ thalassaemia), who had received at least 1 year of EPO therapy between 2007 and 2014. Haemoglobin concentrations and trends of variation in transfused RBC volumes were compared before and after EPO administration. We observed that seven patients had a good response to treatment (Hb increment higher than 1·5 g/dl) and nine had a partial response (0·5-1·5 g/dl increment) and there was a significant decrease in the need for transfusion amongst those who usually required regular transfusions. There were no increases in the rates of vaso-occlusive crisis or venous thromboembolism in comparison to the year before the onset of the therapy. Erythropoietin therapy led to a marked increase in haemoglobin concentration with a concomitant decrease in the demand for transfusion. Considering all complications related to allogeneic transfusion, we believe that EPO therapy represents an important therapeutic tool in sickle cell anaemia.

Deferasirox associated with liver failure and death in a sickle cell anemia patient homozygous for the -1774delG polymorphism in the Abcc2 gene.

This manuscript describes the case of a patient with sickle cell anemia who died of fulminant hepatitis after therapy with the iron chelator Deferasirox. The patient was homozygous for the -1774delG polymorphism in the Abcc2 gene, which raises the concern about the use of hepatotoxic drugs in this specific context.

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.

Cytokine polymorphisms in sickle cell disease and the relationship with cytokine expression.

Sickle cell disease is a chronic inflammatory condition characterized by elevated levels of inflammatory cytokines, which may be regulated by genetic polymorphisms and could be associated with diverse disease presentations and alloimmunization. The aim of this study was to evaluate Treg and Th17 cell frequencies, cytokine gene polymorphisms, and their association with cytokine expression profile in patients with sickle cell disease. For that purpose, we evaluated the IL intron 3 variable number tandem repeat (VNTR, genotypes 1.1, 1.2, 2.2, and 2.3), IL4-T590C>T, IL6-174G>C, TNFα-308G>A, IL10-819T>C, IL10-592A>C, and IL10-1082A>G polymorphisms and their correlation with TGFß, IL4, IL6, and IL10 gene expression in sickle cell patients. We observed a significant decrease in Treg frequency together with a substantial increase in Th17 response in patients with sickle cell disease compared with healthy controls (p < 0.001 and p = 0.014, respectively). There was also a higher prevalence of the IL4-590T/T genotype in patients with sickle cell disease than in Afro-Brazilian descendent controls (p < 0.001) and higher expression of IL4 in patients with the 1.1 genotype of IL4 intron 3 VNTR (p = 0.06). Significantly greater gene expression of TGFß, IL6, and IL10 was observed in sickle cell patients when compared with controls (p = 0.01, 0.03, and <0.001, respectively). Moreover, higher levels of interleukin-6 and -10 were observed in the group of alloimmunized patients. These new data bring insights into the deregulation in the immune system affecting sickle cell patients and must be further investigated in larger cohorts to better characterize individual variations in immune responses and new markers for disease morbidity.

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.

Molecular matching for Rh and K reduces red blood cell alloimmunisation in patients with myelodysplastic syndrome.

BACKGROUND: Matching for Rh and K antigens has been used in an attempt to reduce antibody formation in patients receiving chronic transfusions but an extended phenotype matching including Fy(a) and Jk(a) antigens has also been recommended. The aim of this study was to identify an efficient transfusion protocol of genotype matching for patients with myelodysplastic syndrome (MDS) or chronic myelomonocytic leukaemia. We also examined a possible association of HLA class II alleles with red blood cell (RBC) alloimmunisation. MATERIALS AND METHODS: We evaluated 43 patients with MDS undergoing transfusion therapy with and without antibody formation. We investigated antigen-matched RBC units for ABO, D, C, c, E, e, K, Fy(a), Fy(b), Jk(a), Jk(b), S, s, Do(a), Do(b) and Di(a) on the patients' samples and on the donor units serologically matched for them based on their ABO, Rh and K phenotypes and presence of antibodies. We also determined the frequencies of HLA-DRB1 alleles in the alloimmunised and non-alloimmunised patients. RESULTS: Seventeen of the 43 patients had discrepancies or mismatches for multiple antigens between their genotype-predicted profile and the antigen profile of the units of blood serologically matched for them. We verified that 36.8% of patients had more than one RBC alloantibody and 10.5% of patients had autoantibodies. Although we were able to find a better match for the patients in our extended genotyped/phenotyped units, we verified that matching for Rh and K would be sufficient for most of the patients. We also observed an over-representation of the HLA-DRB1*13 allele in the non-alloimmunised group of patients with MDS. DISCUSSION: In our population molecular matching for C, c, E, e, K was able to reduce RBC alloimmunisation in MDS patients. An association of HLA-DRB1*13 and protection from RBC alloimmunisation should be confirmed.

Variant RH alleles and Rh immunisation in patients with sickle cell disease.

BACKGROUND: Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. MATERIALS AND METHODS: RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. RESULTS: We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. DISCUSSION: In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.