COVID-19 convalescent plasma (CCP) has been the only specific anti-viral therapy against SARS-CoV-2 available for more than one year. Following the negative results from most randomized controlled ...trials on its efficacy in COVID-19 hospitalized patients and the availability of anti-spike monoclonal antibodies (mAbs), the use of CCP has subsequently rapidly faded. However, the continuous appearance of new variants of concern (VOCs), most of which escape mAbs and vaccine-elicited neutralizing antibodies (nAbs), has renewed the interest towards CCP, at least in seronegative immunocompetent patients, and in immunocompromised patients not able to mount a protective immune response. We report here the experience of a single Italian hospital in collecting and transfusing CCP in immunocompromised patients hospitalized for severe COVID-19 between October 2021 and March 2022. During this 6-month period, we collected CCP from 32 vaccinated and convalescent regular blood donors, and infused high nAb-titer CCP units (titered against the specific VOC affecting the recipient) to 21 hospitalized patients with severe COVID-19, all of them seronegative at the time of CCP transfusion. Patients’ median age was 66 years (IQR 50–74 years) and approximately half of them (47.6%, 10/21) were immunocompromised. Two patients were rescued after previous failure of mAbs. No adverse reactions following CCP transfusion were recorded. A 28-day mortality rate of 14.3 percent (3/21) was reported, with age, advanced disease stage and late CCP transfusion associated with a worse outcome. This real-life experience also supports the use of CCP in seronegative hospitalized COVID-19 patients during the Delta and Omicron waves.
Congenital Factor XI (FXI) deficiency shows a high variability in clinical phenotype. To date, many allele variants have been shown to cause this bleeding disorder. However, the genotype-phenotype ...relationship is difficult to establish. This report provides insights into this bleeding disorder. Sixteen unrelated Italian index cases with congenital FXI deficiency and their relatives were investigated. After the identification of the deficiency, we obtained DNA from each subject and analyzed the FXI gene using direct sequencing. We identified 5 and 11 individuals with severe and moderate deficiency of FXI activity, respectively. Most patients (8/16) carried mutations in the Apple 2 domain and 4 patients showed c.403G>T (p.Glu135*; type II mutation). Four novel compound heterozygosities were identified. Bleeding symptoms were present in two severely deficient subjects carrying the combinations c.901T>C (p.Phe301Leu)/c.1556G>A (p.Trp519*) and c.943G>A (p.Glu315)/c.1556G>A (p.Trp519*), respectively. Bleeding episodes were also observed in the presence of a moderate deficiency in two individuals heterozygous for c.449C>T (p.Thr150Met) and c.1253G>T (p.Gly418Val), respectively. One novel mutation, c.1682C>A (p.Ala561Asp), was identified as potentially deleterious in an asymptomatic individual. We confirm an unclear prediction of phenotype from mutational data. The FXI levels should be coupled with FXI analysis for a more comprehensive prediction of the bleeding phenotype in FXI deficiency.
: Acquired hemophilia A (AHA) is a rare autoimmune disease caused by autoantibodies that bind and inactivate factor VIII (FVIII), predisposing to a potentially life-threatening bleeding.
: The main ...epidemiological, clinical, laboratory and therapeutic features of AHA are critically discussed. In particular, we focus on the hemostatic management of AHA patients analyzing the currently available treatment options and showing the latest data on the innovative hemostatic agents still under investigation. Authors searched the Medline and PubMed electronic databases for publication on AHA in the last twenty years.
: While a rapid recognition of suspected cases of AHA is essential to make a correct diagnosis and appropriately and timely treat the hemorrhagic manifestations, the multidisciplinary approach to this challenging, rare and life-threatening bleeding disorder is
equal importance to improve patients' outcome. Although promising, the safety and efficacy of the clinical use of emicizumab in AHA needs to be validated by trials including an adequate number of patients, before registering the drug also for this indication.
PUPs A-LONG evaluated safety and efficacy of recombinant factor VIII Fc fusion protein (rFVIIIFc) in previously untreated patients (PUPs) with hemophilia A. This open-label, Phase 3 study enrolled ...male PUPs (<6 years) with severe hemophilia A to receive rFVIIIFc. The primary endpoint was occurrence of inhibitor development. Secondary endpoints included annualized bleed rate (ABR). Of 103 subjects receiving ≥1 dose of rFVIIIFc, 80 (78%) were aged <1 year at study start, 20 (19%) had family history of inhibitors, and 82 (80%) had high-risk F8 mutations. Twenty subjects began on prophylaxis, while 81 began an on-demand regimen (69 later switched to prophylaxis). Eighty-seven (81%) subjects completed the study. Inhibitor incidence was 31.1% (95% confidence interval CI: 21.8%–41.7%) in subjects with ≥10 exposure days (or inhibitor); high-titer inhibitor incidence was 15.6% (95% CI: 8.8%–24.7%). The median (range) time to high-titer inhibitor development was 9 (4–14) exposure days. Twenty-eight (27%) subjects experienced 32 rFVIIIFc treatment-related adverse events; most were inhibitor development. There was 1 non–treatment-related death due to intracranial hemorrhage (onset prior to first rFVIIIFc dose). The overall median (interquartile range) ABR was 1.49 (0.00–4.40) for subjects on variable prophylaxis dosing regimens. In this study of rFVIIIFc in pediatric PUPs with severe hemophilia A, overall inhibitor development was within expected range, although high-titer inhibitor development was on the low end of the range reported in literature. rFVIIIFc was well-tolerated and effective as prophylaxis and for treatment of bleeds. This trial is registered at www.clinicaltrials.gov (NCT02234323).
Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly ...increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia.
MicroRNA (miR) modulation to potentiate the functionality vascular regenerative cells may represent a successful strategy to optimize cell therapies for ischemic diseases. Spinetti, Martelli, Emanueli, et al. here demonstrate that the master hypoxamiR miR-210 improves human circulating proangiogenic cells functions and their therapeutic potential in limb ischemia.
•This report provides a post hoc analysis of males with severe hemophilia A who developed inhibitors in the PUPs A-LONG trial.•High-risk F8 variants and factor dosing were associated with high-titer ...inhibitors (HTI) and low-titer to HTI progression, respectively.
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Inhibitor development is a major therapeutic complication for people with hemophilia. The phase 3 PUPs A-LONG study evaluated the safety and efficacy of efmoroctocog alfa (a recombinant factor VIII Fc fusion protein, herein referred to as rFVIIIFc) in previously untreated patients (PUPs) with severe hemophilia A. Male PUPs <6 years old were enrolled and received rFVIIIFc; inhibitor development was the primary end point. Post hoc analyses, including patient treatment regimen patterns and timing of inhibitor development, descriptive and Kaplan-Meier analyses of time to first inhibitor-positive test by treatment regimen and by titer, and consumption, were performed to describe patients who developed inhibitors during PUPs A-LONG. We investigated patient characteristics (eg, demographics and genotype) and nongenetic risk factors (eg, intense factor exposure and central venous access device CVAD placement) that may predict inhibitor development and characteristics of inhibitor development (low-titer vs high-titer inhibitor). Baseline characteristics were similarly distributed for age, race, and ethnicity across both patients who were inhibitor-positive and those who were inhibitor-negative (all P > .05). High-risk F8 variants were associated with development of high-titer inhibitors (P = .028). High-titer inhibitor development was often preceded by the presence of a low-titer inhibitor. Patients whose low-titer inhibitor progressed to a high-titer inhibitor received a higher mean dose per infusion (98.4 IU/kg, n = 5) compared with those whose low-titer inhibitor resolved spontaneously (59.2 IU/kg, n = 7; P = .033) or persisted (45.0 IU/kg, n = 5; P = .047). There was no association between CVAD placement surgery and inhibitor development. Post hoc analyses suggest that F8 genotype and dose of factor are as important as inhibitor risk factors and require further investigation. This study was registered at ClinicalTrials.gov as #NCT02234323.
rVIII-SingleChain is indicated for treatment and prophylaxis of bleeding in patients with haemophilia A (HA). The safety and efficacy of rVIII-SingleChain have previously been shown in the AFFINITY ...clinical trial programme. This survey evaluated clinical experience following a switch to rVIII-SingleChain from the perspective of both physicians and patients. A web-based survey (July–September 2019) involving 14 Haemophilia Treatment Centres (HTCs) collected data about HA patients who were under treatment with rVIII-SingleChain for ≥ 12 months, as reported by their physicians. In addition, about half of these patients were separately interviewed. Out of 91 patients receiving rVIII-SingleChain in the 14 participating HTCs, 48 had been treated for ≥ 12 months; among those 48, 38% were ≤ 18 years, 37% 19–40 years and 25 % ≥ 41 years; 73% of them had severe HA and 85% were being treated with prophylactic therapy. Twenty-six patients accepted to be separately interviewed: mean age was 30 years; 62% had severe HA and 85% were receiving prophylaxis. Focusing on those patients who were already in prophylaxis with prior FVIII (all but one with recombinant factors), infusion frequency was significantly reduced from 3–2 per week following the switch to rVIII-SingleChain (mean, 2.74 vs. 2.44, respectively; p=0.013), as reported by physicians; the rate of patients needing 3 infusions per week dropped from 74% with previous products to 44% with rFVIII-SingleChain. The annual mean factor consumption was 4740 IU/Kg (median, 4500 IU/Kg; min, 2.215 IU/Kg; max, 7.200 IU/Kg) with prior product and 4320 IU/Kg (median, 4320 IU/Kg; min, 2.215 IU/Kg; max, 6.646 IU/Kg) with rVIII-SingleChain. Both physicians and patients reported a significant reduction in annual total bleeding rates with rVIII-SingleChain compared with prior product (mean 2.15–0.96 and 2.46–0.71 events/year, p = 0.031 and p = 0.018, respectively). Mean satisfaction ratings (from 1; dissatisfied, to 5; very satisfied) for rVIII-SingleChain were quite high for both physicians (4.14, 86% satisfied/very satisfied) and patients (4.18, 86% satisfied/very satisfied). This survey suggested that switching to rVIII-SingleChain allowed patients to reduce their injection frequency without increasing factor consumption or compromising clinical results. Both physicians and patients reported a positive experience with rVIII-SingleChain after 1 year of treatment.
Background
The development of an immunogenic response to FVIII and the appearance of neutralizing antibodies - “inhibitors” - against FVIII is the most important adverse event in hemophilia ...treatment. Immune Tolerance Induction (ITI) via the long-term, intravenous administrations of high-dose FVIII is the only proven strategy to eradicate inhibitors. The success rate is about 60-70% and no formal demonstration of its mechanism of action has been provided yet. Indeed, the mechanisms underlying successful ITI have been unclear, and not much is known about the major factors determinant of success vs. failure.
Our aim is to understand the process of activation and immune regulation in response to FVIII in patients undergoing ITI, and comparing the immunological events in patients who successfully eradicate anti-FVIII antibody to those in patients failing to achieve FVIII tolerance.
Methods
This is a multicenter, observational, prospective cohort study enrolling severe HA patients who developed high-titer inhibitors, candidate to ITI. A blood sample has been collected before starting and during the course of ITI, until ITI ends (5 blood samples during the first year, 3 during the second year if ITI treatment is not already ended, plus a final sample 30 days after ITI conclusion). Total PBMCs have been used to establish cell cultures where cells are re-stimulated with the same FVIII used during the ITI or medium alone.
To identify candidate genes, key proteins and cell subsets associated to differential outcomes to ITI, immune cells are used for gene expression profiling (Human Inflammation & Immunity Transcriptome Targeted RNA Panel, Qiagen) together with extensive phenotypic characterization of immune cells via the CyTOF (Cytometry by Time Of Flight mass spectrometry) technology. To identify potential biomarkers predictors of ITI outcomes metabolomics analysis and multiplex cytokine arrays are performed in plasma collected at each time point during ITI and in supernatants from cells cultures. Plasma samples, are collected at each time point and stored at -80°C for cytokine determination and metabolomic analysis. Total anti-FVIII antibody (and their isotypes) and inhibitor titer are also determined in plasma samples by Nijmegen methodology in the central laboratory as described previously (1).
Results
Currently, 18 subjects have been enrolled among 8 Hemophilia Centers. Among these 5 subjects, two patients reached the study endpoint of ITI success by study criteria (negative inhibitor titer, a normal FVIII recovery, a normal FVIII half-life and the absence of anamnesis upon further FVIII exposure) while three were assessed as ITI failure. The analysis of anti-FVIII antibody isotypes revealed the presence of IgG4 as the most relevant component of the total anti-FVIII antibodies. The inhibitor clearance was also accompanied by the disappearance of IgG4 anti-FVIII antibodies in both tolerized patients, while those who failed to eradicate the inhibitor showed a sustained IgG4 anti-FVIII response (Fig. 1). The evaluation of the cytokines in the supernatants from in vitro cultured cells showed a consistent increase in production of pro-inflammatory cytokines in response to FVIII (IL-1β, IL-6, IL-12, IL-17A, IL-15) at all time points only in patients who continued to produce high-titer inhibitors.
Conclusions
Here we report an interim analysis of a prospective study of the immunological mechanisms of immune tolerance induction. The preliminary results obtained so far suggest that anti-FVIII IgG4 are a major component of the total anti-FVIII antibodies and their persistence is associated with unfavourable ITI outcome. We expect that the ongoing immune gene expression profiling, immune-phenotypic characterization and determination of soluble marker of activation/regulation of the immune system in supernatants and plasma in this cohort of patients will provide substantial knowledge to increase our current understanding of the complex immunological pathways involved in the development of tolerance to FVIII. Ultimately, this could lead to the discovery of biomarkers of ITI success/failure and to the generation of focused and strategic intervention to modulate the immune system during this treatment.
References
1. Matino D, Gargaro M, et al. “IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII”. J Clin Invest. 2015 Oct 1;125(10):3766-81. doi: 10.1172/JCI81859.
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Matino:Sobi: Speakers Bureau. Peyvandi:Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Roche: Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Octapharma US: Honoraria; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy. Santagostino:Pfizer: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Iorio:NovoNordisk: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Novo Nordisk; Shire: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Shire; CSL: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with CSL; Grifols: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Grifols; Octapharma: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Octapharma; Bayer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Bayer; Pfizer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Pfizer; Roche: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Roche.
Abstract
Background
The aims of this study were: to investigate the capacity of the rare disease healthcare network in Campania to diagnose patients with rare diseases during the outbreak of ...Covid-19; and to shed light on problematic diagnoses during this period.
Methods
To describe the impact of the Covid-19 pandemic on the diagnosis of patients with rare diseases, a retrospective analysis of the Campania Region Rare Disease Registry was performed. A tailored questionnaire was sent to rare disease experts to investigate major issues during the emergency period.
Results
Prevalence of new diagnoses of rare disease in March and April 2020 was significantly lower than in 2019 (117 versus 317, P < 0.001 and 37 versus 349, P < 0.001, respectively) and 2018 (117 versus 389, P < 0.001 and 37 versus 282, P < 0.001, respectively). Eighty-two among 98 rare disease experts completed the questionnaire. Diagnostic success (95%), access to diagnosis (80%) and follow-up (72%), lack of Personal Protective Equipment (60%), lack of Covid-19 guidelines (50%) and the need for home therapy (78%) were the most important issues raised during Covid-19 outbreak.
Conclusions
This study describes the effects of the Covid-19 outbreak on the diagnosis of rare disease in a single Italian region and investigates potential issues of diagnosis and management during this period.
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