Sickle cell disease (SCD) results from mutations in the β-globin gene, producing abnormal hemoglobin S (HbS) and leading to complications causing significant morbidity and mortality.1 One of the hallmark consequences of SCD is the occurrence of vaso-occlusive crises (VOCs), which arise from the interplay of factors in the disease's pathophysiology, involving abnormal hemoglobin polymerization, inflammation, endothelial dysfunction, and activation of the immune system, culminating in the painful obstruction of blood vessels by sickled red blood cells, that tend to obstruct blood vessels, leading to reduced blood flow and oxygen supply. This vicious cycle of ischemia followed by reperfusion constitutes the ischemia-reperfusion model.2 The complement system, a complex defense mechanism, is implicated in various diseases through unregulated activation.3 However, diagnostic challenges hinder patient selection for complement inhibition.4 Preliminary data from our group using novel assays indicate complement activation even at a steady state in a limited patient population.5 Limited information exists on additional markers in the complement activation and endothelial dysfunction cycle in SCD. Neutrophil extracellular traps (NETs), indicative of thromboinflammation, are elevated in SCD patients, even during steady state.6 ADAMTS13 (A Disintegrin and Metalloproteinase with Thrombospondin motifs), studied for its role in SCD vasculopathy, shows conflicting results as a potential biomarker.7, 8 Genetic variants and autoantibodies leading to unregulated complement activation are implicated in the pathogenesis of various human diseases.9 Despite the lack of specific biomarkers or targeted treatments for crises, our hypothesis posits the presence of complement activation and thromboinflammation in SCD, particularly during complications, with distinct yet unexplored clinical or genetic features in these patients. Our study's methods regarding patient population, observation period, functional assays, and genetic, bioinformatic, and statistical analysis are demonstrated in supplementary materials. Our study included 81 adult SCD patients who are treated in different Hemoglobinopathies Units across Northern Greece. Their median age was 41 years, and 50 were female (61.7%). As expected in our population, the majority had the S/beta genotype (62), while 19 patients had the S/S genotype. Twenty-three presented SCD complications during the observation period (17 vaso-occlusive crises and six proteinuria/nephropathy) and were studied during this complication. Importantly, none of the patients that presented with renal damage, was on deferasirox, or other iron chelation therapy. The remaining 58 patients were studied at the end of the observation period. The comprehensive characteristics and parameters studied are summarized in Table 1. First, we measured soluble C5b-9 and modified Ham test at a steady state and during the observation period. At steady state, soluble C5b-9 was above normal limits in a significantly higher percentage of patients who then presented a SCD complication compared to those who did not (7/23, 30%, p = 0.028). Similarly, patients who presented a complication had a significantly higher rate of modified Ham test (5/23, 21%, p = 0.001, Figure 1A) at steady state. During the observation period, we detected a significant increase in soluble C5b-9 (p < 0.001). This increase was significantly higher in patients with complications (p = 0.046, Figure 1B). One patient who did not experience complications had a positive Ham test and sC5b-9, while 42 patients who did not present complications had only elevated C5b-9. Since VOC was the most common complication, significant results were replicated in patients with VOC (data not shown). ADAMTS13 was similar and within normal limits at steady state and follow-up (Figure 2B). One other parameter studied as a marker of thromboinflammation was NETs. We were able to measure NETS on 60 of our 81 patients. NETs at steady state were significantly increased in patients who then developed complications related to the disease. Additionally, there was a significant increase of NETs measured at follow-up compared to steady state (p < 0.001, Figure 2B). Patients with a positive modified Ham test had increased C5b9 (p < 0.001). No significant association was found between NETs and C5b-9 in our population. We started our genetic analysis from rare variants with a minor allele frequency (MAF) less than 1%, since rare variants have been commonly described in patients with complement-related disorders10. We detected 23 rare variants, as shown in detail in Supporting Information S1: Table 1. Almost all rare variants were documented in unique patients. Only rs35836460 in CFH, rs186530184 in CFHR1, rs183647515 in THBD, and rs202206149 in ADAMTS13 were found in two patients, with rs3176136 in THBD detected in three patients. Among 23 rare variants, only three were characterized as pathogenic by bioinformatic tools as described in the next paragraph. To further understand the clinical significance of the remaining detected variants, we used four bioinformatic tools. We found 8 missense variants characterized as deleterious by these tools, as presented in detail in Supporting Information S1: Table 2. Among them, three were also rare variants (rs117793540 in C3, rs1800579 in THBD, and rs143568784 in ADAMTS13), potentially confirming its pathogenic role. Each rare variant was found in one patient. Additionally, rs144082872 in CFI was also detected in one patient. The remaining deleterious variants were detected in 2–15 patients each. Patients were heterozygous, except for one homozygous for rs28647808 (ADAMTS13). Next, we searched for detected variants in the database of complement gene variants. As shown in Supporting Information S1: Table 3, 21 variants have been described in this complement database. In agreement with the bioinformatic tools, rs144082872 in CFI has also been characterized as pathogenic in this database. To better characterize the phenotype of SCD patients, we investigated associations of functional and genetic assays with clinical characteristics. All patients with pathogenic or deleterious variants had increased complement activation by functional assays (modified Ham test or soluble C5b9), except for two patients with CFB and two with ADAMTS13 variants. Interestingly, patients with a combination of pathogenic or deleterious variants had a significantly higher transfusion dependency rate (65% vs. 35%, p = 0.031). Finally, we sought to determine predictors of complications. Among studied baseline patient characteristics (Supporting Information S1: Table 4), there was no univariate association with complications. Therefore, we included steady-state soluble C5b-9 and NETs, as well as the presence of a pathogenic or deleterious variant in multivariate analysis. The only independent predictor was C5b-9 levels. In our study, we reveal activation of complement and thromboinflammation, even in the steady state of SCD patients who later develop complications. Notably, these markers experience a significant surge during the onset of complications. Germline genetic variants may contribute to the predisposition to complement activation, linked to clinical characteristics. Importantly, C5b-9 independently predicts complications in our multivariate analysis. The inflammatory response becomes a key driver in SCD's pathophysiology, fueled by recurrent ischemic reperfusion events. Besides direct tissue damage, chronic inflammation contributes to cumulative damage in SCD. Complement activation plays a crucial role in both inflammation and the prominent hemolysis seen in SCD. Given the disease's multifactorial nature, markers such as NETs and ADAMTS13 are explored as potential treatment targets. Assessing complement activation in clinical labs faces challenges due to limited access to robust assays.9 Our study addresses this by introducing a modified Ham test for early and robust detection of complement activation, alongside confirming soluble C5b-9 as a practical marker for complications' evaluation. The increase in Bb fragments supports alternative pathway activation. Previous research using less precise assays indicated alternative pathway activation in SCD.11 Exploring the genomic landscape of complement-related variants in SCD is a novel aspect of our study. Genetic analysis using multiple tools identifies pathogenic variants, highlighting an association between variants and clinical characteristics.12 Our study has approached genetic analysis with multiple tools, while previous studies in complement-related disorders have reported only rare variants.13 As our group has shown in thrombotic microangiopathies (TMA),14 additional variants might also harbor clinical significance. In an effort to better understand the clinical significance of rare variants, novel databases have been created, such as the Database of Complement Gene Variants.12 These databases are considerably helpful in linking the genotype with the phenotype in these patients. However, this approach does not take into account the majority of detected variants, whose functional and clinical significance remains to be studied. Variants such as rs1047286, rs2230199, rs800292 for C3, as well as rs1061147, rs1061170 for CFH, and the deleterious variant rs12614 for CFB have been implicated in the development of age-related macular degeneration (AMD).15, 16 Current SCD pharmacological treatments, including hydroxyurea and voxelotor, show positive effects, but the need for novel therapeutic approaches persists.17 Crizanlizumab, initially promising, was discontinued by the European Medicines Agency (EMA) due to efficacy issues.18 Eculizumab, a complement inhibitor, shows encouraging results, but challenges hinder wider study.19 Preclinical data suggest that MASP-2 or MASP-3 inhibition may prevent SCD complications.20 The CROSSWALK trial is investigating the benefits of a C5 inhibitor in VOC prevention.21 Our study has limitations, including varying patient treatments across centers and limited population size. Long-term observations in a larger cohort could provide additional insights. Data gaps on ADAMTS13Ag hinder a full understanding of its role in SCD. The predominantly S/β genotype in our population limits generalizability, and the impact of hydroxyurea on complement profiles is explored but inconclusive. The study's timing during the COVID-19 pandemic affected sample collection and patient visits. One other limitation is that 20 of our patients were on a chronic transfusion schedule. Moreover, our study's cohort is rather small, and further studies with bigger cohorts are needed to determine whether or not genetic variants play a role. In conclusion, complement activation and thromboinflammation are evident in SCD patients, especially during complications. Considering the safety and efficacy of complement inhibitors in other complementopathies and preliminary data from SCD, our study highlights useful tools to early detect patients that might benefit from complement inhibition. The authors would like to thank the immunobiologist Maria Spachidou for their technical assistance. Eleni Gavriilaki was supported by the ASH Global Research Award. Christos Varelas and Eleni Gavriilaki designed research. Christos Varelas, Efthymia Vlachaki, Philippos Klonizakis, Despoina Pantelidou, Michael Diamantidis, Nikolaos Sabanis, Ioanna Christodoulou, Despina Papadopoulou, Evdoxia Koravou, Ioanna Sakellari, Stamatia Theodoridou, and Ioannis Mitroulis performed research. Christos Varelas, Fani Minti, Apostolia Papalexandri, Tasoula Touloumenidou, and Eleni Gavriilaki analyzed data and wrote the paper. Efthymia Vlachaki, Sofia Vakalopoulou, Vasilis Perifanis, George Vassilopoulos, and Eleni Gavriilaki contributed to the research design and edited and approved the paper. Eleni Gavriilaki has consulted for Alexion, Omeros, and Sanofi Cooperation. The other authors declare no competing financial interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. The work was in part funded by the General Secretariat for Research and Technology Management and Implementation Authority for Research, Technological Development and Innovation Actions (MIA-RTDI) (grant T2EDK-02288, MDS-TARGET). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Preeclampsia is a progressive multi-systemic disorder characterized by proteinuria, critical organ damage, and new-onset hypertension. It can be further complicated by HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), resulting in critical liver or renal damage, disseminated coagulation, and grand mal seizures. This study aimed to examine the involvement of ADAMTS13, von Willebrand, and the complement system in the pathogenesis of preeclampsia/HELLP syndrome. We studied 30 Caucasian preeclamptic pregnant women and a control group of 15 healthy pregnancies. Genetic sequencing of ADAMTS13 and complement regulatory genes (MiniSeq System, Illumina) was performed. The modified Ham test was used to check for complement activation, ADAMTS13 activity, von Willebrand antigen (vWFAg) levels, and soluble C5b-9 levels were measured. Patients with preeclampsia had a decreased ADAMTS13 activity and increased C5b-9 levels. The vWFAg was significantly correlated with ADAMTS13 activity (r = 0.497, p = 0.003). Risk-factor variants were found in the genes of ADAMTS13, C3, thrombomodulin, CFB, CFH, MBL2, and, finally, MASP2. A portion of pregnant women with preeclampsia showed a decline in ADAMTS13 activity, correlated with vWFAg levels. These patients also exhibited an elevated complement activation and high-risk genetic variants in regulatory genes. Further research is needed to determine if these factors can serve as reliable biomarkers.

Introduction: Human leukocyte antigen (HLA) polymorphisms have been associated with the development of various autoimmune diseases, as well as malignant neoplasms. Non-Hodgkin lymphomas (NHLs) are a heterogenous group of lymphoid malignancies in which a genetic substrate has been established and is deemed to play a crucial role in disease pathogenesis. This study aimed to identify whether variations in the HLA gene region were associated with diffuse large B-cell lymphoma (DLBCL) risk and prognosis. Methods: We defined HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1) alleles in 60 patients with DLBCL and compared the results to those found by 236 healthy adult donors from the bone marrow bank of Northern Greece. HLA typing was performed by two molecular methods, Sequence - Specific Oligonucleotide HLA typing (SSO) and Sequence - Specific Primer HLA typing (SSP), from white blood cells recovered from peripheral blood. The phenotypic frequencies of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 between patients and controls were compared with the 2-sided Fisher's exact test. Results with p-value <0.05 were considered statistically significant. Odds Ratios with 95% Confidence Intervals were calculated to further strengthen the results. The 2-sided Fisher's exact test was also applied to alleles found only in one of the two groups, while the odds ratios together with the confidence intervals were corrected with Haldane-Anscombe method. Results: Among the studied HLA polymorphisms, the frequency HLA-C*12 allele was significantly lower in patients with DLBCL compared with control subjects (6.7% vs. 34.7%, OR = 0.16, 95% CI: 0.04-0.44). Frequency of HLA-B*39 was significantly lower in patients with DLBCL compared with controls, but due to the low frequency of this polymorphism in the studied population and small sample size, determinations regarding the significance of this findings were limited. Survival analysis revealed that the presence of HLA-C*12 was not associated with improved or worsened overall and progression-free survival. No statistically significant associations were observed in the phenotypic frequencies of HLA-A, HLA-DQB1, HLA-DRB1 and the rest of HLA-B alleles between the control and DLBCL groups. Discussion: Collectively, our results provide valuable insight regarding the role of HLA variations on DLBCL risk. Further studies are required to consolidate our findings and ascertain the clinical implications of these genetic variations on DLBCL management and prognosis.

Changes in gut microbiome composition have been implicated in the pathogenesis of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our objective was to explore the microbial abundance in patients with GvHD after allo-HSCT. We conducted a single-center, prospective study in patients who underwent allo-HSCT and developed grade II or higher acute GvHD and/or moderate or severe chronic GvHD, to explore the microbial abundance of taxa at the phylum, family, genus, and species level, and we utilized alpha and beta diversity indices to further describe our findings. We collected fecal specimens at −2 to +2 (T1), +11 to +17 (T2), +25 to +30 (T3), +90 (T4), and +180 (T5) days to assess changes in gut microbiota, with day 0 being the day of allo-HSCT. We included 20 allo-HSCT recipients in the study. Compared with timepoint T1, at timepoint T4 we found a significant decrease in the abundance of Proteobacteria phylum (14.22% at T1 vs. 4.07% at T4, p = 0.01) and Enterobacteriaceae family (13.3% at T1 vs. <0.05% at T4, p < 0.05), as well as a significant increase in Enterococcus species (0.1% at T1 vs. 12.8% at T4, p < 0.05) in patients who developed acute GvHD. Regarding patients who developed chronic GvHD after allo-HSCT, there was a significant reduction in the abundance of Eurobactereaceae family (1.32% at T1 vs. 0.53% at T4, p < 0.05) and Roseruria genus (3.97% at T1 vs. 0.09% at T4, p < 0.05) at T4 compared with T1. Alpha and beta diversity analyses did not reveal a difference in the abundance of bacteria at the genus level in GvHD patients at T4 compared with T1. Our study reinforces results from previous studies regarding changes in gut microbiota in patients with acute GvHD and provides new data regarding the gut microbiome changes in chronic GvHD. Future studies will need to incorporate clinical parameters in their analyses to establish their association with specific changes in gut microbiota in patients with GvHD after allo-HSCT.

Background: Despite advances in the therapeutics of Sickle Cell Disease (SCD), vaso-occlusion, painful crisis, and other complications (such as delayed hemolytic transfusion reaction/DHTR) remain a major cause of morbidity and mortality. Recent research data suggest complement activation, especially during SCD complications. These advances led to recommendation of complement inhibition in patients with DHTR and ongoing hyperhemolysis. However, limited data exist about other SCD complications. Hypothesis: We hypothesized that novel functional assays will be useful in detecting complement activation in SCD, especially during complications, and that these patients present distinct clinical or genetic features that have not been investigated yet. Methods: We enrolled consecutive SCD patients from four Thalassemia Units, meeting the eligibility criteria: (i) age > 16 years, (ii) established SCD diagnosis and (iii) willingness to participate in the study and sign a written informed consent. We collected samples at enrolment and at any updates of disease status during 1-year follow-up (initiation of hydroxyurea, vaso-occlusive crisis, hemolytic reactions, nephropathy, or TMA) or at last follow-up if no update was observed. Complement activation was detected using soluble C5b-9 (Quidel kit), and the modified Ham test. Values above 245 ng/ml have been established in our laboratory as above normal limits for C5b-9, and 20% for modified Ham test, respectively. The latter is a cell proliferation assay based on the susceptibility of a PNH (paroxysmal nocturnal hemoglobinuria)-like cell line to complement-activated serum. We also utilized next generation sequencing (NGS) analysis with a customized panel (complement factor H/CFH, CFH-related, CFI, CFB, CFD, C3, CD55, C5, CD46, thombomodulin/THBD, ADAMTS13, MiniSeq, Illumina). Identified variants were evaluated by using 4 bioinformatic tools. Variants predicted to be deleterious from at least 2 tools (50%), were assumed as pathogenic and were compared with the 2018 database of complement gene variants. Results: We studied 81 adult SCD patients (Table 1). Among them, 23 presented a disease update during follow-up (17 vaso-occlusive crises and 6 proteinuria). Patients with SCD complications during follow-up had excessive complement activation detected even from steady state with increased C5b-9 (7/23, 30%, p=0.028) and positive modified Ham test (5/23, 21%, p=0.001). During follow-up, C5b-9 significantly increased (p<0.001), particularly in patients with complications (p=0.046). Modified Ham test positivity remained the same. Regarding genetics, we detected 25 rare variants (Minor Allele Frequency < 1%). Only 3 of them found in 3 patients were also characterized as pathogenic: rs117793540 in C3, rs1800579 in THBD, and rs143568784 in ADAMTS13. We also documented 5 additional deleterious variants in 12 patients: rs144082872 in CFI, rs12614 in CFB, rs28503257, rs145825553 and rs28647808 in ADAMTS13. Patients were heterozygous, except for 1 homozygous for rs28647808. Patients with deleterious variants had increased complement activation by functional assays, except for 2 patients with CFB and 2 with ADAMTS13 variants. Interestingly, patients with a combination of a deleterious variant had a significantly higher transfusion dependency rate (65% versus 35%, p=0.031). Conclusion We have shown for the first time prospectively that complement is activated even from steady state in patients that develop SCD complication, with significant increase during complications. Complement-related genetic variants undeline this predisposition to complement activation in certain patients. Considering safety and efficacy of complement inhibitors in other complementopathies and preliminary data from SCD, our study highlights useful tools to early detect patients that might benefit from complement inhibition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

While the impact of oral microbiome dysbiosis on autoimmune diseases has been partially investigated, its role on bullous diseases like Pemphigus Vulgaris (PV) is a totally unexplored field. This study aims to present the composition and relative abundance of microbial communities in both healthy individuals and patients with oral PV lesions. Ion Torrent was used to apply deep sequencing of the bacterial 16S rRNA gene to oral smear samples of 15 healthy subjects and 15 patients. The results showed that the most dominant phyla were Firmicutes (55.88% controls-c vs 61.27% patients-p, p value = 0.002), Proteobacteria (9.17%c vs 12.33%p, p value = 0.007) and Fusobacteria (3.39%c vs 4.09%p, p value = 0.03). Alpha diversity showed a significant difference in the number of genera between patients and controls (p value = 0.04). Beta diversity showed statistical differences in the microbial community composition between two groups. Fusobacterium nucleatum, Gemella haemolysans and Parvimonas micra were statistically abundant in patients. We noticed the characteristic fetor coming out of oral PV lesions. Most of anaerobic bacteria responsible for oral halitosis are periopathogenic. Though, only F. nucleatum and P. micra were differentially abundant in our patients. Especially, F. nucleatum has been reported many times as responsible for bad breath. Furthermore, Streptococcus salivarius and Rothia mucilaginosa, species mostly associated with clean breath, were found in relative abundance in the healthy group. Consequently, the distinct malodor observed in PV patients might be attributed either to the abundance of F. nucleatum and P. micra and/or to the lower levels of S. salivarius and R. mucilanginosa in oral lesions

Tick-borne bacteria pose a significant threat to human and veterinary public health. Greece is a Mediterranean country with rich tick fauna and the most commonly detected tick-borne bacterial pathogens are members of the Rickettsia and Anaplasma species. The variable V2–V4 and V6–V9 regions of 16S rRNA gene of seven ticks belonging to four genera representative in Greece (Ixodes, Rhipicephalus, Dermacentor, Haemophyssalis) were analysed using multiple primer pairs by next generation sequencing (NGS). Nine bacterial phyla corresponding to 95 families, 116 genera and 172 species were identified. Proteobacteria was the predominant phylum in five of the seven ticks, followed by Actinobacteria, which predominated in two ticks. The tick-borne bacteria included Rickettsia and Anaplasma species, while “Candidatus Midichloria mitochondrii” were detected in high abundance in I. ricinus ticks and less in Rhipicephalus bursa; Coxiella-like endosymbionts were detected in Rh. sanguineus, H. parva, and less in Rh. bursa ticks. Co-infections with Rickettsia and Anaplasma were also observed. 16S rRNA NGS is a powerful tool to investigate the tick bacteriome and can improve the strategies for prevention and control of tick-borne diseases.

Background: Pseudoexfoliation syndrome (PEX) and glaucoma (pseudoexfoliative glaucoma; PEXG, primary open-angle glaucoma; POAG) have mainly been studied for their associations with genes' polymorphisms. The purpose of this exploratory study was to investigate the role of polymorphisms in genes encoding for micro RNAs (miRNAs) and in genes related to miRNA biogenesis. Material and Methods: In the present genetic association study, ninety-two polymorphisms were investigated for their contribution to PEX (n = 203), PEXG (n = 38), and POAG (n = 40) pathogenesis compared to a control group (n = 188). The next generation sequencing (NGS) genotypic analysis revealed data for additional 28 variants. Results: A protective association was found between PEX and polymorphism 11382316 (mir-3161) [odds ratio (OR) = 0.64, 95% confidence interval (CI): 0.47-0.86, p = 0.003], rs2155248 (mir-1304) [OR = 0.66, 95%CI: 0.47-0.94, p = 0.019], and rs28635903 (mir-1268a) [OR = 0.30, 95%CI: 0.10-0.94, p = 0.029]. Polymorphism rs113297757 (mir-3196) was associated with an increased risk of POAG [OR = 7.75, 95%CI: 2.13-28.76, p = 3 x 10(-4)]. Polymorphism rs1057035 (DICER) and rs55671916 (XPO5) in the 3'-UTR of genes related to miRNA biogenesis was associated with decreased risk of PEX [OR = 0.65, 95%CI: 0.46-0.92, p = 0.014] and increased risk of PEXG [OR = 2.84, 95%CI: 1.02-7.94, p = 0.038], respectively. The aforementioned associations according to the allelic model were further supported by the genotypic models of statistical analyses. Conclusions: This is the first study to report distinct associations of PEX, PEXG, and POAG in the same population with variants of genes involved in miRNA biogenesis and with miRNA genes' polymorphisms. Further studies in larger groups of patients of various origins are needed to confirm the reported preliminary results.

Neural precursor cells (NPCs) located in the subventricular zone (SVZ), a well-defined NPC niche, play a crucial role in central nervous system (CNS) homeostasis. Moreover, NPCs are involved in the endogenous reparative process both in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the possibility that NPCs may be vulnerable to immune-related components may not be ruled out. Therefore, we investigated the potential affinity of myelin oligodendrocyte glycoprotein (MOG)-induced humoral response(s) to NPCs.