BACKGROUND Hyperimmune plasma from Covid-19 convalescent is a potential treatment for severe Covid-19. METHODS We conducted a multicenter one arm proof of concept interventional study. Patients with Covid-19 disease with moderate-to-severe Acute Respiratory Distress Syndrome, elevated C-reactive Protein and need for mechanical ventilation and/or CPAP were enrolled. One to three 250-300 ml unit of hyperimmune plasma (neutralizing antibodies titer ≥1:160) were administered. Primary outcome was 7-days hospital mortality. Secondary outcomes were PaO2/FiO2, laboratory and radiologic changes, as well as weaning from mechanical ventilation and safety. RESULTS The study observed 46 patients from March, 25 to April, 21 2020. Patients were aged 63, 61% male, 30 on CPAP and 7 intubated. PaO2/FiO2 was 128 (SD 47). Symptoms and ARDS duration were 14 (SD 7) and 6 days (SD 3). Three patients (6.5%) died within 7 days. The upper one-sided 90%CI was 13.9%, allowing to reject the null hypothesis of a 15% mortality. PaO2/FiO2 increased by 112 units (95%CI 82 to142) in survivors, the chest radiogram severity decreased in 23% (95%CI 5% to 42%); CRP, Ferritin and LDH decreased by 60, 36 and 20% respectively. Weaning from CPAP was obtained in 26/30 patients and 3/7 were extubated. Five serious adverse events occurred in 4 patients (2 likely, 2 possible treatment related). CONCLUSIONS Hyperimmune plasma in Covid-19 shows promising benefits, to be confirmed in a randomized controlled trial. This proof of concept study could open to future developments including hyperimmune plasma banking, development of standardized pharmaceutical products and monoclonal antibodies.

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood. (Blood. 2011;117(8):2476-2483) IntroductionHemopoietic stem cells reside in bone marrow-specialized niches that dictate how they differentiate, proliferate, mature, and enter the peripheral circulation. [1][2][3][4] Megakaryocyte (MK) maturation and platelet generation are consequent to MK migration from the osteoblastic to the vascular niche, where MKs extend pro-platelets and newly generated platelets are released into the bloodstream. 5,6 The characteristics of the microenvironment surrounding MKs play an important role in the regulation of platelet production within the bone marrow. 3,7 In particular, the interaction of MKs with different extracellular matrices (ECMs) that fill the bone marrow spaces seems to orchestrate their maturation in specific sites. 8 It has been demonstrated that interactions of primary human MKs with matrices thought to fill the vascular niche, such as fibrinogen or von Willebrand factor, are able to sustain MK maturation and pro-platelets, whereas type I collagen totally suppresses these events and prevents premature platelet release in the osteoblastic niche. 7,9 The negative regulation of pro-platelets by type I collagen is mediated by the interaction with the integrin ␣2␤1 and involves the Rho/ROCK pathway. 10,11 However, the exact sequence of events that determines the interaction of MKs with the ECM, and therefore their regulation, is not completely understood. 12 Recent studies 13 have demonstrated that the encounter between a cell and an adhesive substrate involves an initial passive interaction characterized by cell adhesion and spreading, followed by an active stage that involves actin polymerization and...

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A 16-year-old boy was admitted to our emergency department, in Lombardy, complaining of intense pain in his chest-radiating to his left arm-which had started 1 h earlier. The day before he had a fever of 38•3°C that decreased after 100 mg of nimesulide. He reported no other symptoms, no medical history, and no contact with anyone with confirmed COVID-19.We found his vital signs to be normal apart from his temperature which was raised at 38•5°C. On auscultation of the patient's chest, we heard normal heart sounds, no pericardial rub, and no abnormal respiratory signs. We found no lymphadenopathy, no rash, and no areas of localised tenderness on the chest wall. An electrocardiogram (ECG) showed inferolateral ST-segment elevation (figure) and a transthoracic echocardiography showed hypokinesia of the inferior and inferolateral segments of the left ventricle, with a preserved ejection fraction of 52%; no pericardial effusion was seen. Investigations showed raised high-sensitivity cardiac troponin I (9449 ng/L), creatine phosphokinase (671•0 U/L), C-reactive protein (32•5 mg/L), and lactate dehydrogenase (276•0 U/L) concentrations (appendix). The leucocyte count was 12•75 × 10⁹ per L, the neutrophil count was 10•04 × 10⁹ per L, and the lymphocyte count was 0•78 × 10⁹ per L.We gave the boy aspirin to relieve his pain and transferred him to the coronary care unit with a working diagnosis of acute myocarditis. The patient's pain gradually improved and after 2 h had completely resolved.However, during the first night, he reported further chest pain; the ECG was repeated but no significant changes were seen (appendix). We started him on intravenous ibuprofen 600 mg three times a day and both his symptoms and raised temperature resolved. Tests for autoantibodies and cardiotropic viruses were negative (appendix). On day 3, a nasopharyngeal swab test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was positive, so we started hydroxychloroquine and antiviral therapy. Serial measurements of the patient's troponin concentration showed a gradual reduction from a peak of 16 862 ng/L on day 1, to 39 ng/L on day 8. The inflammatory markers also returned to normal and the ST-segment elevation on ECG resolved (appendix).On day 11-after nasopharyngeal swabs taken on 2 consecutive days were negative-MRI T2-weighted short-tau inversion recovery sequences showed changes supporting the diagnosis of acute myocarditis (figure; appendix). On day 12, he was well, asymptomatic, and allowed home.Notably, throughout the entire time he was in hospital, our patient did not have any of the signs or symptomsapart from a fever-typically reported in COVID-19; his peripheral oxygen saturation levels remained within normal limits and two chest x-rays, on days 3 and 6, were clear (appendix). Paediatric patients reporting chest pain and other features suggestive of acute myocarditis-with or without respiratory symptoms-should, we believe, also be tested for SARS-CoV-2 (video). ContributorsWe were all involved in the care and management of ...

Since the end of 2019, a new coronavirus strain has been reported in the Chinese province of Wuhan, indicated as 2019-nCoV or SARS-CoV-2. In February 2020, the first case of transmission on Italian soil was reported. On March 09, 2020, at the time of protocol design, the Italian Ministry of Health reported 10,149 people who had contracted the virus; of these, 8514 were positive, of which 5038 were hospitalized with symptoms (59.2%) and 877 in intensive care (10.3%), while the remaining 2599 were in home isolation; 631 were deceased (6.2%) and 1004 healed (9.9%). To date there are no studies in the literature that demonstrate its feasibility and efficacy in the context of the worldwide SARS-CoV-2 epidemic. Based upon the little existing evidence, we planned to assess the efficacy of the infusion of hyperimmune plasma in COVID-19 patients in a one-arm proof-of-concept clinical trial. The primary objective of our study is to evaluate the efficacy of the administration of plasma taken from convalescent donors of COVID-19 to critically ill patients with COVID-19 in terms of their survival. Death from any cause will be considered. The main limit of this study is its one-arm proof-of-concept design with only 43 patients enrolled. However, in the absence of previous evidence, larger and/or randomized trials did not appear to be ethically acceptable. Moreover, the results from this study, if encouraging, will allow us to plan further informed large clinical trials. Trial registration: NCT 04321421 March 23, 2020. Keywords COVID-19 • Hyperimmune plasma • Plasmapheresis Background and rationale* Claudia Del Fante

Hyperimmune plasma from Covid-19 convalescent is a potential treatment for severe Covid-19. We conducted a multicenter one arm proof of concept interventional study. Patients with Covid-19 disease with moderate-to-severe Acute Respiratory Distress Syndrome, elevated C-reactive Protein and need for mechanical ventilation and/or CPAP were enrolled. One to three 250-300 ml unit of hyperimmune plasma (neutralizing antibodies titer ≥1:160) were administered. Primary outcome was 7-days hospital mortality. Secondary outcomes were PaO2/FiO2, laboratory and radiologic changes, as well as weaning from mechanical ventilation and safety. The study observed 46 patients from March, 25 to April, 21 2020. Patients were aged 63, 61% male, of them, 30 were on CPAP and 7 intubated. PaO2/FiO2 was 128 (SD 47). Bilateral infiltrates on chest X-ray was present in 36 patients (84%). Symptoms and ARDS duration were 14 (SD 7) and 6 days (SD 3). Three patients (6.5%) died within 7 days as compared to an expected 15% from the National Statistics and 30% from a small concurrent cohort of 23 patients. The upper one-sided 90%CI was 13.9%, allowing to reject the null hypothesis of a 15% mortality. PaO2/FiO2 increased by 112 units (95%CI 82 to142) in survivors, the chest radiogram severity decreased in 23% (95%CI 5% to 42%); CRP, Ferritin and LDH decreased by 60, 36 and 20% respectively. Weaning from CPAP was obtained in 26/30 patients and 3/7 were extubated. Five serious adverse events occurred in 4 patients (2 likely, 2 possible treatment related). In conclusion, Hyperimmune plasma in Covid-19 shows promising benefits, to be confirmed in a randomized controlled trial. This proof of concept study could open to future developments including hyperimmune plasma banking, development of standardized pharmaceutical products and monoclonal antibodies.

Background Although many trials are currently investigating the safety and efficacy of convalescent plasma (CP) in critically ill COVID‐19 patients, there is a paucity of ongoing and published studies evaluating the CP donors' side. This retrospective study reports the first Italian experience on CP donors' selection and donations. Methods Patients aged 18‐68 years who had recovered from COVID‐19 at least 2 weeks previously were recruited between March 18 and June 30, 2020 in a study protocol at the Italian hospitals of Pavia and Mantova. Results During the study period, 494 of 512 donors recruited were judged eligible and underwent 504 plasmapheresis procedures. Eighty‐five percent (437/512) of the CP donors were males. The average time between symptom recovery and CP donation was 36.6 (±20.0) days. Four hundred and eighty‐eight plasmapheresis procedures (96.8%) were concluded and each unit was divided into two subunits (total 976) with an average volume of 316.2 (±22.7) mL. Ninety‐three percent (460/494) of CP donors at the time of plasma donation had a neutralizing IgG titer ≥1:80. Plasmapheresis‐related adverse reactions occurred in 2.6% (13/504) of cases; all the reactions were mild and none required therapeutic intervention. Donors' age and COVID‐19 severity were positively associated with greater antibody responses. Conclusion This study demonstrates the feasibility and safety of a pilot CP program conducted in Italy. The identification of factors (ie, age and severity of COVID‐19) positively associated with higher neutralizing antibody titers at the time of donation may help to optimize the selection of CP donors.