Impact of HLA-G +3142C>G on the development of antibodies to blood group systems other than the Rh and Kell among sensitized patients with sickle cell disease.

BACKGROUND: Patients' inflammatory history is an important factor underlying red blood cell (RBC) alloimmunization, which is a frequent transfusion complication among individuals with sickle cell disease (SCD). HLA-G has been associated with different inflammatory and auto - immune diseases. Our goal was to verify whether the HLA-G + 3142 C>G and 14-bp Ins/Del variations are associated with RBC antibody development among SCD patients. METHODS: This was a single-center case-control study. SCD patients were randomly selected for the study and divided into two groups: 'Alloimmunized' and 'Nonalloimmunized' depending on the presence of irregular antibodies. The 'Alloimmunized'group was further divided into two subgroups according to the presence of only antibodies against the Rh and Kell blood group systems or the existence of antibodies to antigens of the other blood group systems. RESULTS: A total of 213 patients were included in the study (110 alloimmunized and 103 non-alloimmunized). The 'Alloimmunized' and 'Non-alloimmunized' groups did not differ statistically regarding the HLA-G + 14 bp Ins/Del ( p = 0.494) and + 3142 C>G ( p = 0.334). Individuals who had only antibodies against the Rh and Kell antigens had a frequency of HLA-G + 3142GG genotype almost twice as high compared to the groupwith antibodies against less immunogenic antigens ( p = 0.043). CONCLUSIONS: The genotype frequency of HLA-G + 3142 C>G differs among alloimmunized SCD patients, depending on the presence of antibodies against low immunogenic RBC antigens. This highlights a possible role played by the HLA-G molecule in the RBC alloimmunization process.

Variant genotypes associated with reduced expression of RhCE antigens among Brazilian blood donors.

BACKGROUND: The genetic diversity of the RHCE gene locus has been explored in diverse populations of different racial backgrounds. Data referring to the diversity of RHCE encoding weakened expression of C, c, E, and e in multiethnic populations is still incomplete. METHODS: Samples from Brazilian blood donors presenting reduced expression of C, c, E, or e on gel method were selected for the study. All exons and flanking introns of RHCE were genotyped though direct Sanger sequencing for the included donors. RESULTS: Sixty-six donors were included: 23 with weak C, 22 with weak c, 6 with weak E, 14 with weak e, and 1 with weak c and E. Among the samples with weak C, the following altered RH*C were encountered: RHCE*CeMA (n = 3), RHCE*Ce941C (n = 1), and RHCE*CeVA (n = 1). RHD*D-CE(4-7)-D was detected in six cases, RHCE*CE was presumably present in five cases, and seven cases were unexplained. Two altered alleles underlay the weak c phenotype: RHCE*ceJAL (n = 20) and RHCE*ce340T (n = 2), and two altered RHCE justified weak e: RHCE*ceMO (n = 6) and RHCE*ceJAL (n = 8). Three variant RHCE were associated with weak E: RHCE*cEJU (n = 4), RHCE*cE382C (n = 1), and RHCE*cEIV (n = 1). The RHCE*cE905A justified one case of weak c and E. CONCLUSION: We describe the distribution of RHCE variants found in association with weak expression of C, c, E, and e in blood donors of multiethnic origin, which differs in comparison to that previously reported for people of African or Caucasian descent.

Weak D types 38 and 11: determination of frequencies in a Brazilian population and validation of an easy molecular assay for detection.

CONCLUSIONS: Recent evidence shows that, among Brazilians, the distribution of weak D types significantly differs from that represented in people of European descent, with a high percentage of weak D types 38 and 11. Our goal was to determine the population frequencies of weak D types 38 and 11 in a Brazilian population and to validate a molecular approach to identify these two variants. Blood donors were sequentially enrolled in the study in a 5-year period. Donors with serologic weak D phenotype had the RHD coding region sequenced. The frequencies of weak D type 38 and weak D type 11 (CDe-associated) were calculated. Two allele-specific-polymerase chain reaction (AS-PCR) assays were designed to detect RHD*weak D type 38 and RHD*weak partial 11 and were validated with samples positive and negative for these two variants, respectively. A total of 618,542 donors were enrolled, of which 265 presented with a serologic weak D phenotype. When considering all donors evaluated, the frequencies of weak D types 38 and 11 were 0.013 and 0.002 percent, respectively. In the subgroup of donors with a serologic weak D phenotype, the frequencies of weak D types 38 and 11 were 30.2 and 4.9 percent, respectively. The two proposed AS-PCR assays for detection of RHD*weak D type 38 and RHD*weak partial 11 showed 100 percent accuracy. The frequencies of weak D types 38 and 11 among Brazilians are high compared to that previously described for other populations. The AS-PCR assays to detect RHD*weak D type 38 and RHD*weak partial 11 represent potentially helpful tools for investigating Brazilian individuals with these weak D phenotypes.

Topical application of lectin Artin M improves wound healing in defects created in the palatal mucosa: an in vivo study in dogs.

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6 mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21 days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.

SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

Prevalence and laboratorial determinants of the clinical relevance of antibodies of undetermined specificity.

BACKGROUND AND OBJECTIVES: Antibodies of unknown specificity (AUS) are frequently identified in the pre-transfusion testing. These antibodies can be insignificant or potentially cause post-transfusion haemolysis. Information about the prevalence of clinically relevant AUS is still lacking. Our aim was to predict the potential clinical relevance of AUS using the monocyte monolayer assay (MMA) and to identify the clinical and laboratorial determinants of AUS' significance. MATERIALS AND METHODS: Antibodies of unknown specificity identified at a single institution from 2015-2017 were evaluated through MMA. A monocyte index (MI) of more than 5% was predictive of potential post-transfusion haemolysis. RESULTS: Thirty-two patients with AUS were included in the study. Of the studied AUS, 37·5% (12/32) presented with a monocyte index (MI) more than 5%. In the group of significant AUS, 41·7% of the patients presented with sickle cell disease (SCD) and the AUS were associated with Rh antibodies in 75% of the cases. In the group of insignificant AUS, only 10% of the patients had SCD and the association with Rh antibodies was detected in 20% of the cases. The presence of Rh antibodies was independently associated with the AUS clinical relevance (P = 0·012). CONCLUSION: More than one-third of the AUS are potentially clinically relevant, and the association with Rh antibodies is predictive of AUS relevance. Services must honour AUS in the pre-transfusion process in order to ensure transfusion safety.

Defining the clinical relevance of red blood cell autoantibodies by Monocyte Monolayer Assay.

BACKGROUND: The Monocyte Monolayer Assay (MMA) is an in vitro simulation of red blood cell (RBC) alloantibody behavior. It has been classically applied to predict the risks of post-transfusion hemolytic reactions when transfusing incompatible RBC units. Quantifying erythrophagocytosis by MMA may be an interesting option for situations where there is doubt whether a RBC autoantibody is mediating significant hemolysis. Here, we present three situations involving RBC autoantibodies in which the MMA was decisive for clarifying the diagnosis and choosing the best clinical treatment. CASE REPORT: Case 1. Pregnant patient with severely anemic fetus exhibited warm autoantibody without signs of hemolysis. MMA revealed 30% of monocyte index (MI) highlighting that fetal hemolysis was caused by maternal autoantibody. Prednisone was prescribed with fetal clinical improvement. Cases 2 and 3. Two patients with the diagnosis of mixed auto-immune hemolytic anemia and poor response to corticosteroids were evaluated using MMA. The resulting MI was less than 10% in both cases, suggesting that the cold-agglutinin rather than the warm auto-IgG was responsible for overt hemolysis. Treatment with rituximab was begun, with good clinical response. CONCLUSION: MMA can be used to evaluate the ability of RBC autoantibodies to mediate overt hemolysis. It can be especially useful to determine the role played by cold and warm auto-antibodies in mixed auto-immune hemolytic disease, helping to define the best treatment option.

Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center.

INTRODUCTION: The Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. OBJECTIVE: In this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. METHODS: Samples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. MAIN RESULTS: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. CONCLUSION: Our study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Association between FY*02N.01 and the severity of COVID-19: initial observations.

Introduction: The pro-inflammatory immune response underlies severe cases of COVID-19. Antigens of the Duffy blood group systems are receptors for pro-inflammation chemokines. The ACKR1 c.-67T>C gene variation silences the expression of Duffy antigens on erythrocytes and individuals presenting this variant in homozygosity have impaired inflammatory response control. Our aim was to evaluate the association between the ACKR1 c.-67T>C and the severity of COVID-19. Methods: This was a retrospective single-center case-control study, enrolling 164 participants who were divided into four groups: 1) Death: COVID-19 patients who died during hospitalization; 2) Hospital Discharge: COVID-19 patients who were discharged for home after hospitalizations; 3) Convalescent Plasma Donors: COVID-19 patients who were not hospitalized, and; 4) Controls: patients with diagnosis other than COVID-19. Patients were genotyped for the ACKR1 c.-67T>C (FY*02 N.01 allele) and the frequency of individuals presenting the altered allele was compared between the groups. Results: The groups significantly differed in terms of the percentage of patients presenting at least one FY*02N.01 allele: 36.8% (Death group), 37% (Hospital Discharge group), 16.1% (Convalescent Plasma group) and 16.2% (Control group) (p = 0.027). The self-declared race (p < 0.001) and the occurrence of in hospital death (p = 0.058) were independently associated with the presence of the FY*02N.01 allele. Hypertension (p < 0.001), age (p < 0.001) and the presence of at least one FY*02N.01 allele (p = 0.009) were independently associated with the need for hospitalization. Conclusion: There is a suggestive association between the presence of the FY*02N.01 and the severity of COVID-19. This may be a mechanism underlying the worse prognosis for Afro-descendants infected with SARS-CoV-2.

Transfusion of ABO non-identical platelets increases the severity of trauma patients at ICU admission.

BACKGROUND: Transfusion of ABO-compatible non-identical platelets (PTLs), fresh plasma (FP) and red blood cells (RBCs) has been associated with increased morbidity and mortality of recipients. Trauma victims are frequently exposed to ABO non-identical products, given the need for emergency transfusions. Our goal was to evaluate the impact of the transfusion of ABO non-identical blood products on the severity and all-cause 30-day mortality of trauma patients. METHODS: This was a retrospective single-center cohort, which included trauma patients who received emergency transfusions in the first 24 h of hospitalization. Patients were divided in two groups according to the use of <3 or ≥3 ABO non-identical blood products. The patient severity, measured by the Acute Physiology and Chronic Health Evaluation (APACHEII) score at ICU admission, and the 30-day mortality were compared between groups. RESULTS: Two hundred and sixteen trauma patients were enrolled. Of these, 21.3% received ≥3 ABO non-identical blood products (RBCs, PLTs and FP or cryoprecipitate). The transfusion of ≥3 ABO non-identical blood products in the first 24 h of hospitalization was independently associated with a higher APACHEII score at ICU admission (OR = 3.28 and CI95% = 1.48-7.16). Transfusion of at least one unit of ABO non-identical PTLs was also associated with severity (OR = 10.89 and CI95% = 3.38-38.49). Transfusion of ABO non-identical blood products was not associated with a higher 30-day mortality in the studied cohort. CONCLUSION: The transfusion of ABO non-identical blood products and, especially, of ABO non-identical PLTs may be associated with the greater severity of trauma patients at ICU admission. The transfusion of ABO non-identical blood products in the trauma setting is not without risks.

Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

ABSTRACT Introduction: The Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. Objective: In this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. Methods: Samples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. Conclusion: Our study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Association between FY* 02N. 01 and the severity of COVID-19: initial observations

ABSTRACT Introduction The pro-inflammatory immune response underlies severe cases of COVID-19. Antigens of the Duffy blood group systems are receptors for pro-inflammation chemokines. The ACKR1 c.-67T>C gene variation silences the expression of Duffy antigens on erythrocytes and individuals presenting this variant in homozygosity have impaired inflammatory response control. Our aim was to evaluate the association between the ACKR1 c.-67T>C and the severity of COVID-19. Methods This was a retrospective single-center case-control study, enrolling 164 participants who were divided into four groups: 1) Death: COVID-19 patients who died during hospitalization; 2) Hospital Discharge: COVID-19 patients who were discharged for home after hospitalizations; 3) Convalescent Plasma Donors: COVID-19 patients who were not hospitalized, and; 4) Controls: patients with diagnosis other than COVID-19. Patients were genotyped for the ACKR1 c.-67T>C (FY*02 N.01 allele) and the frequency of individuals presenting the altered allele was compared between the groups. Results The groups significantly differed in terms of the percentage of patients presenting at least one FY*02N.01 allele: 36.8% (Death group), 37% (Hospital Discharge group), 16.1% (Convalescent Plasma group) and 16.2% (Control group) (p= 0.027). The self-declared race (p < 0.001) and the occurrence of in hospital death (p= 0.058) were independently associated with the presence of the FY*02N.01 allele. Hypertension (p < 0.001), age (p < 0.001) and the presence of at least one FY*02N.01 allele (p= 0.009) were independently associated with the need for hospitalization. Conclusion There is a suggestive association between the presence of the FY*02N.01 and the severity of COVID-19. This may be a mechanism underlying the worse prognosis for Afro-descendants infected with SARS-CoV-2.

Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1ß) release; otherwise, transforming growth factor-beta (TGF-ß) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.

Transfusion of ABO non-identical platelets increases the severity of trauma patients at ICU admission

ABSTRACT Background: Transfusion of ABO-compatible non-identical platelets (PTLs), fresh plasma (FP) and red blood cells (RBCs) has been associated with increased morbidity and mortality of recipients. Trauma victims are frequently exposed to ABO non-identical products, given the need for emergency transfusions. Our goal was to evaluate the impact of the transfusion of ABO non-identical blood products on the severity and all-cause 30-day mortality of trauma patients. Methods: This was a retrospective single-center cohort, which included trauma patients who received emergency transfusions in the first 24 h of hospitalization. Patients were divided in two groups according to the use of <3 or ≥3 ABO non-identical blood products. The patient severity, measured by the Acute Physiology and Chronic Health Evaluation (APACHEII) score at ICU admission, and the 30-day mortality were compared between groups. Results: Two hundred and sixteen trauma patients were enrolled. Of these, 21.3% received ≥3 ABO non-identical blood products (RBCs, PLTs and FP or cryoprecipitate). The transfusion of ≥3 ABO non-identical blood products in the first 24 h of hospitalization was independently associated with a higher APACHEII score at ICU admission (OR = 3.28 and CI95% = 1.48-7.16). Transfusion of at least one unit of ABO non-identical PTLs was also associated with severity (OR = 10.89 and CI95% = 3.38-38.49). Transfusion of ABO non-identical blood products was not associated with a higher 30-day mortality in the studied cohort. Conclusion: The transfusion of ABO non-identical blood products and, especially, of ABO non-identical PLTs may be associated with the greater severity of trauma patients at ICU admission. The transfusion of ABO non-identical blood products in the trauma setting is not without risks.

Neutrophil activation induced by ArtinM: release of inflammatory mediators and enhancement of effector functions.

The D-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin, is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by d-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB(4) and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections.