BackgroundIn human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed.MethodsThe serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism).ResultsThe levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma.ConclusionsThis study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.

Background: KELnull (K₀) persons can produce clinically significant anti-KEL5 antibody after transfusion and/or pregnancy, requiring K₀ blood transfusion when indicated. 37 K₀ alleles have been reported in studies over different populations, but none in Amerindian-Caucasian descendants from South America. The aim of this study was to identify the molecular basis of K₀ phenotype in Brazilians. Methods: We investigated three K₀ samples from different Brazilian blood banks (Recife, Manaus, and Vila Velha) in women with anti-KEL5. KEL antigen typing was performed by serologic techniques, and the K₀ status was confirmed by flow cytometry. PCR-RFLP and DNA sequencing of the KEL coding and exon-intron regions were also performed. Results: RBCs of the 3 patients were phenotyped as KEL:-1,-2,-3,-4,-7. The 3 patients had the same KEL*02/02 genotype and were negative for KEL*02.03 and KEL*02.06 alleles. The Recife K₀ patient was homozygous for IVS16 + 1g>a mutation(KEL*02N.31 allele). The flow cytometry with anti-KEL1, anti-KEL2, anti-KEL3, anti-KEL4, and anti-CD238 confirmed the K₀ phenotype. In addition, we found the c.1042C>T mutation (KEL*02N.04 allele) in both the Manaus K₀ and the Vila Velha K₀ patients. Conclusion: This report represents the first study of K₀ molecular basis performed in Amerindian-Caucasian descendants from South America.

Maternal RhD alloimmunization is an inflammatory response against protein antigens in fetal red blood cells (RBC). However, not all women become alloimmunized when exposed to RhD+ fetal RBC. Thus, this study aimed to evaluate levels of inflammatory chemokines in RhD− pregnant women with erythrocyte alloimmunization. CXCL8, CXCL9, CCL5, and CXCL10 levels were determined from cell culture supernatants by flow cytometry in 46 (30 non-alloimmunized RhD− and 16 previously alloimmunized RhD−) pregnant women. CXCL8 levels were significantly higher (P < 0.004), and CXCL9 (P < 0.008) and CXCL10 (P < 0.003) levels were significantly lower in alloimmunized pregnant women. No significant difference in CCL5 levels was detected between the groups. Fetal RHD genotyping was performed in the alloimmunized RhD− group by real-time PCR. Anti-D alloantibody was detected in 10 mothers and anti-D and -C in six mothers. Twelve fetuses were RHD positive and four were RHD negative. Further studies of serum chemokines and placenta tissue could provide a better understanding of the cells involved in the pathogenesis of maternal erythrocyte alloimmunization.

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1111 Kell is the most important blood group system after ABO and Rh because all frequently occurring Kell-specific antibodies must be considered clinically significant. The KEL antigens are carried by the human red cell (RBC) membrane Kell glycoprotein (CD238), a proteolytic enzyme encoded by the 19-exon KEL gene on the long arm of chromosome 7 (7q33). Kell null (K0) is a very rare phenotype characterized by the absence of the Kell protein and all KEL antigens on RBC surface caused by different molecular defects on KEL gene. K0 persons, however, may produce anti-KEL5 (anti-Ku) antibody that is associated to severe hemolytic transfusion reaction and perinatal anemia. Although the K0 phenotype has been known since 1957, the first reports of the K0 molecular basis occurred only in 2001. At present, 23 KELnull alleles are recognized to abolish the KEL antigens expression, most of them due to stop codon and alternative splicing caused by single nucleotide mutation. In this study, we report a novel KELnull allele detected in a Brazilian woman with the K0 phenotype. We investigated a 59-year-old Caucasian-Amerindian descent woman (proband) from Northeast Brazil who showed an antibody that reacted against high-prevalence RBC antigens during a pre-transfusion evaluation for orthopedic surgery. We also were able to evaluated three relatives (proband′s sister and two nieces) and normal RBC controls. We performed standard immunohematological methods and PCR-RFLP KEL genotyping as screening tests. To identify the molecular defect, we used sequencing technique covering all 19 coding and exons-introns regions of the KEL gene. To confirm the K0 phenotype and exclude KELel (Kmod) phenotype we employed flow cytometry, a higher sensitive technique, using soroclone anti-KEL1(anti-K), anti-KEL2(anti-k), anti-KEL3(anti-Kpa), anti-KEL4(anti-Kpb) and anti-CD238 antibodies. Proband′s frozen RBCs were washed 3x in 3ml PBS and 2×106/50μl/tube were incubated for 30min at room temperature with monoclonal antibodies (anti-K, -k, -Kpa, -Kpb – 50μl; anti-CD238 1:200,10μl). FITC conjugated polyclonal rabbit anti-human Ig and goat-anti-mouse Ig were used as second antibodies. Data acquisition and analysis in 20,000 events at RBC region were performed using FACscalibur flow cytometer and CellQuest program. Previously known (A) KEL:-1-,2,-3,4 (K-,k+,Kpa-,Kpb+) and (B) KEL:1,2,-3,4 (K+,k+,Kpa-,Kpb+) RBCs samples were used as positive controls. Fresh and thawed RBCs stained with PE conjugated anti-human glycophorin were also tested. RBCs of the proband were phenotyped as KEL:-1,-2,-3,-4,-6,-7 (K-,k-,Kpa-,Kpb-,Jsa-,Jsb-), and the RBC alloantibody was identified as anti-KEL5 (anti-Ku). RBCs of proband's relatives were phenotyped as wild type KEL:-1,2,-3,4,-6,7 (K-,k+,Kpa-,Kpb+,Jsa-,Jsb+). The proband and her relatives had the same KEL*2/KEL*2, KEL*4/KEL*4, KEL*7/KEL*7 wild type genotype determined by PCR-RFLP. The G>A mutation on intron 16, position +1 downstream of exon 16 (IVS16+1G>A mutation) was identified by KEL sequencing technique at homozygous state on the proband and at heterozygous state on her sister. The IVS16+1G>A mutation introduce an alternative splicing that may skip exon 16 and create a premature stop codon (TGA) on exon 17. No mutation was detected in the two proband's nieces. Proband's RBCs did not react against all antibodies by flow cytometry: MFI (mean fluorescent intensity) of CD238=3.7, KEL1(K)=7.6, KEL2(k)=7.6, KEL3(Kpa)=8.5 and KEL4(Kpb)=8.0 against positive results in control A RBCs [MFI of CD238=77, KEL1(K)=8, KEL2(k)=189.4, KEL3(Kpa)=9.3 and KEL4(Kpb)=114.4] and control B RBCs [MFI of CD238=77, KEL1(K)=76, KEL2(k)=50, KEL3(Kpa)=7.8 and KEL4(Kpb)=49.6]. Results of the RBC phenotyping by flow cytometry of the proband's sister were CD238=156, KEL2(k)=51 and KEL4(Kpb)=48. There were no significant differences between results on fresh and frozen samples. The MFI of KEL2 from proband's sister (KEL*2/KEL*2null) RBCs was similar to that observed in control B (KEL*2/KEL*1 heterozygous), 51 and 50, respectively, and lower than the control A (KEL*2/KEL*2 homozygous), 51 and 189.4, respectively, reflecting the single gene dose in the proband's sister and control B, and double gene dose in control A. In conclusion, we report a novel KELnull allele, IVS16+1G>A, detected for the first time in a Brazilian woman with Kell null phenotype. This study was supported by CNPq and FAPESP. Disclosures: No relevant conflicts of interest to declare.