Vascular Endothelial Growth Factor as Potential Biomarker for COVID-19 Severity.

INTRODUCTION: COVID-19 is characterized by immunological responses to viral replication and coherent with endothelitis, microvascular disturbance of lung vasculature and coagulopathy. Vascular Endothelial Growth Factor (VEGF) is a proangiogenic mediator regulating endothelial changes. It is induced by proinflammatory signaling and hypoxia. We sought to determine whether VEGF levels differ between SARS-CoV-2-positive patients of different disease severity and whether VEGF might be useful in risk stratification. METHODS: After retrospective screening of all SARS-CoV-2-positive patients treated in Unfallkrankenhaus Berlin in 2020, we included those with documented VEGF measurement. We extracted laboratory values and clinical parameters. An exploratory data analysis was performed to detect possible relations between VEGF level and clinical disease features. RESULTS: We included 167 SARS-CoV-2-positive patients of which 139 suffered from COVID-19. Seventy-one of the COVID-19 patients had to be treated in the intensive care unit (ICU), those patients exhibited higher VEGF levels than those being admitted to normal wards (535 vs 279 pg/L, P < .001). APACHE-2 (Acute Physiology And Chronic Health Evaluation Score) correlated with mortality and patients with high values showed higher VEGF concentrations on admission (456 vs 875 pg/L, p = 0.006). Receiver operating characteristic analytic revealed that the occurrence of organ dysfunctions like acute respiratory distress syndrome (ARDS), shock, or acute kidney injury could be predicted by VEGF. It was significantly higher in patients who later died compared to survivors (637 vs 389 pg/mL, P = 0.041) and predicted mortality with same accuracy as established markers. In our cohort, association of VEGF above 277 pg/L on admission with risk of ARDS could be confirmed in logistic regression adjusting for possible confounding factors (odds ratio 3.1, 95% confidence interval: 1.34-7.7). DISCUSSION: Even though there are several limitations to this retrospective study it revealed that in COVID-19 patients VEGF can contribute to the prediction of necessity of ICU, mortality and the prediction of ARDS, kidney injury or shock. Its use in risk stratification and potential pathogenetic involvement should be further investigated.

Coronavirus Disease 2019-Related Alterations of Total and Anti-Spike IgG Glycosylation in Relation to Age and Anti-Spike IgG Titer.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been affecting the world since January 2020 and has caused millions of deaths. To gain a better insight into molecular changes underlying the COVID-19 disease, we investigated here the N-glycosylation of three immunoglobulin G (IgG) fractions isolated from plasma of 35 severe COVID-19 patients, namely total IgG1, total IgG2, and anti-Spike IgG, by means of MALDI-TOF-MS. All analyses were performed at the glycopeptide level to assure subclass- and site-specific information. For each COVID-19 patient, the analyses included three blood withdrawals at different time-points of hospitalization, which allowed profiling longitudinal alterations in IgG glycosylation. The COVID-19 patients presented altered IgG N-glycosylation profiles in all investigated IgG fractions. The most pronounced COVID-19-related changes were observed in the glycosylation profiles of antigen-specific anti-Spike IgG1. Anti-Spike IgG1 fucosylation and galactosylation showed the strongest variation during the disease course, with the difference in anti-Spike IgG1 fucosylation being significantly correlated with patients' age. Decreases in anti-Spike IgG1 galactosylation and sialylation in the course of the disease were found to be significantly correlated with the difference in anti-Spike IgG plasma concentration. The present findings suggest that patients' age and anti-S IgG abundance might influence IgG N-glycosylation alterations occurring in COVID-19.

Association of Genetic Polymorphisms of Fibrinogen, Factor XIII A-Subunit and α2-Antiplasmin with Fibrinogen Levels in Pregnant Women.

Fibrinogen synthesis is stimulated by proinflammatory triggers and depends on α-, ß- and γ-fibrinogen (FGA, FGB, FGG) genotypes. Constellations of fibrinogen, factor XIII A-subunit (F13A) and α2-antiplasmin (A2AP) genotypes predisposing for dense fibrin gels with high antifibrinolytic capacity (e.g., FGB rs1800790 A-allele carriage in F13A 34Val/Val or A2AP 6Arg/Arg wildtypes) are related with reduced inflammation. As both relationships are likely to influence each other, we tested whether the association of fibrinogen genotypes with fibrinogen levels is influenced by F13A and A2AP genotypes in a population under proinflammatory stress. In total, 639 women were followed during pregnancy (2218 observations). The relationship between fibrinogen genotypes and levels was statistically assessed in univariate and multivariate analyses without and with stratification for F13A Val34Leu and A2AP Arg6Trp. Strong associations with fibrinogen levels could be found for FGB rs1800790G > A, FGA rs2070016T > C and FGG rs1049636T > C. For FGB rs1800790G > A and FGA rs2070016T > C, this relationship significantly depended on F13A Val34Leu and A2AP Arg6Trp genotypes. Specifically, in F13A 34Val/Val wildtypes, carriage of FGB rs1800790A was related to significantly lower fibrinogen levels compared with FGB rs1800790GG wildtypes (p < 0.01). For A2AP 6Arg/Arg wildtypes, a comparable relationship could be found (p < 0.04). As these genotype constellations related to lower fibrinogen levels have previously been shown to be associated with reduced inflammatory activity, these findings suggest that the influence of fibrinogen, F13A and A2AP genotypes on inflammation could affect the control of fibrinogen levels and vice versa.

Sequence-specific primers for MNS blood group genotyping.

BACKGROUND: Various techniques of genotyping the MNSs blood group have been described, but none of them enables the complete detection of all MNS antigens. MATERIALS AND METHODS: Blood samples were obtained from blood donors. Primers were created using the published DNA sequences for glycophorins A and B. Genotyping was performed using polymerase chain reaction sequence-specific primers (PCR-SSP). RESULTS: A total of seven primers were found to specifically amplify the most common MNS antigens. The use of these primers has enabled us to correctly genotype all blood samples tested so far (n=116). DISCUSSION: Specifically created primers enable genotyping of the MNS antigens in a single PCR-SSP run. The method is reliable, easy to perform, and can be used in routine practice.

High variability of peptidylarginine deiminase 4 (PADI4) in a healthy white population: characterization of six new variants of PADI4 exons 2-4 by a novel haplotype-specific sequencing-based approach.

Seven single nucleotide polymorphisms (SNPs) of the peptidylarginine deiminase 4 (PADI4) gene have recently been reported to be strongly associated with rheumatoid arthritis in Japanese individuals. These SNPs are located in or close to exons 2-4 of PADI4 and are organized in at least four different haplotypes. However, a detailed sequencing-based characterization of the PADI4 gene in other populations is still lacking. We therefore analyzed exons 2-4 of the PADI4 gene in 102 healthy white Germans individuals by DNA sequencing and characterized new variants and haplotypes by a novel haplotype-specific sequencing-based approach. The haplotypes 2/3 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*T, padi4_95*C, padi4_96*C), and haplotype 4 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*C, padi4_95*G, padi4_96*T) conferring susceptibility to rheumatoid arthritis were detected at frequencies of 30.9% and 7.8%, respectively. In addition, three novel coding SNPs in exons 2, 3, and 4, and three SNPs in introns 2 and 3 located near the exon-intron boundaries were identified in 11 individuals (10.8%). The so-called nonsusceptibility haplotype 1 (padi4_89*A, padi4_90*C, padi4_92*C, padi4_94*C, padi4_104*C, padi4_95*G, padi4_96*T) occurred at a frequency of 58.3%. Additionally, we identified a closely related novel haplotype, haplotype 1B (2.9%), that differs from haplotype 1 only by padi4_92*G/padi4_96*C. This haplotype was not described in the Japanese population. Our results indicate that the PADI4 gene exhibits a remarkable variability and a rather complex haplotypic organization. Further studies on disease association of PADI4 should be performed by haplotype-specific sequencing-based approaches to identify the exact genotype of the PADI4 fragment of interest.

Polymorphism of the alpha4-subunit of VLA-4 integrin and bone marrow transplantation.

Integrin alpha4beta1 is an important homing molecule on stem cells. Two genetic variants of this integrin are known, alpha4-mas and alpha4-tex. We assessed the potential influence of this polymorphism in 37 patients undergoing allogeneic bone marrow transplantation. None of the constellations of variants influenced the outcome, as determined by the recovery of leukocytes or platelets, hospitalization time, and the development of graft-versus-host disease.

Screening for multiple hereditary hypercoagulability factors using the amplification refractory mutation system.

Many hereditary factors have been implicated in the development of arterial and/or venous thromboembolic diseases. A number of these risk factors can be identified by the amplification refractory mutation system (ARMS). However, the underlying technical conditions for performing ARMS are highly variable, and depend on which risk factors are being analyzed. We have now developed a novel ARMS-based system to simultaneously screen for multiple hypercoagulability factors under identical PCR conditions. This can greatly simplify the process of screening for hereditary hypercoagulability.

Acute intravascular hemolysis after transfusion of a chimeric RBC unit.

BACKGROUND: Natural blood cell chimerism rarely occurs in humans. The case of a patient who developed transfusion reaction due to the transfusion of chimeric RBCs is reported. CASE REPORT: A 61-year-old male patient with blood group O received two units of packed and O-grouped RBCs after elective kidney surgery. Immediately after blood transfusion, the patient developed a hemolytic transfusion attack. The serologic re-examination revealed only a mixed-field pattern of agglutination of RBCs in one of the two transfused units. The donor of this unit was an apparently healthy 24-year-old male with a twin sister. Both of them showed an identical mixture of roughly 95 percent group O and 5 percent group B RBCs by gel agglutination technology and flow cytometry. The results were also confirmed by ABO blood group genotyping. CONCLUSIONS: This is the first report of a hemolytic transfusion reaction related to the transfusion of chimeric RBCs.

The DAU allele cluster of the RHD gene.

Variant D occurs frequently in Africans. However, considerably less RHD alleles have been described in this population compared with Europeans. We characterized 5 new RHD alleles, dubbed DAU-0 to DAU-4, that shared a T379M substitution and occurred in a cDe haplotype. DAU-1 to DAU-4 were detected in Africans with partial D phenotypes. They harbored one and 2 additional missense mutations, respectively, dispersed throughout the RhD protein. An anti-D immunization was found in DAU-3. DAU-0 carrying T379M only was detected by screening European blood donors and expressed a normal D phenotype. Within the phylogeny of the RHD alleles, DAU formed an independent allele cluster, separate from the DIVa, weak D type 4, and Eurasian D clusters. The characterization of the RH phylogeny provided a framework for future studies on RH alleles. The identification of the DAU alleles increased the number of known partial D alleles in Africans considerably. DAU alleles may be a major cause of antigen D variability and anti-D immunization in patients of African descent.