Use of COVID-19 Convalescent Plasma for Treatment of Symptomatic SARS-CoV-2 Infection at a Children's Hospital: A Contribution to a Still Inadequate Body of Evidence.

Data on COVID-19 convalescent plasma (CCP) safety and efficacy in children and young adults are limited. This single-center prospective, open-label trial evaluates CCP safety, neutralizing antibody kinetics, and outcomes in children and young adults with moderate/severe COVID-19 (April 2020-March 2021). A total of 46 subjects received CCP; 43 were included in the safety analysis (SAS); 7.0% < 2 years old, 2.3% 2-<6, 27.9% 6-<12, 39.5% 12-<19, and 23.3% > 19 years old; 28 were included in the antibody kinetic analysis (AbKS); 10.7% < 2 years old, 10.7% 6-<12, 53.8% 12-<19, and 25.0% > 19 years old. No adverse events occurred. The median COVID-19 severity score improved (5.0 pre-CCP to 1.0 by day 7; p < 0.001). A rapid increase in the median percentage of inhibition was observed in AbKS (22.5% (13.0%, 41.5%) pre-infusion to 52% (23.7%, 72%) 24 h post-infusion); a similar increase was observed in nine immune-competent subjects (28% (23%, 35%) to 63% (53%, 72%)). The inhibition percentage increased until day 7 and persisted at 21 and 90 days. CCP is well tolerated in children and young adults, providing rapid and robust increased antibodies. CCP should remain a therapeutic option for this population for whom vaccines are not fully available and given that the safety and efficacy of existing monoclonal antibodies and antiviral agents have not been established.

Safety and Antibody Kinetics of COVID-19 Convalescent Plasma for the Treatment of Moderate to Severe Cases of SARS-CoV-2 Infection in Pediatric Patients.

BACKGROUND: Therapies against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its life-threatening respiratory infection coronavirus disease 2019 (COVID-19) have been evaluated, including COVID-19 convalescent plasma (CCP). Multiple large reports of CCP treatment in adults exist. Pediatric data on CCP safety and efficacy are limited. METHODS: Single-center prospective, open-label trial looking at safety, antibody kinetics and outcomes of CCP (10 mL/kg, max 1 unit) treatment for COVID-19 in hospitalized pediatric patients with moderate to severe disease or at high-risk for serious illness. RESULTS: Thirteen patients were enrolled. No infusion-related adverse events occurred. No hematologic or metabolic adverse events were noted during hospitalization or at 3-weeks. Ten patients had clinical improvement by day 7 (WHO eight-category ordinal severity scale for COVID-19). Following CCP, anti-SARS-CoV-2 anti-nucleocapsid IgG increased significantly at 24 hours and high levels were sustained at 7- and 21-days. Transient IgM response was noted. Twelve patients (92.3%) were discharged home, 9 (75%) by day 7 post-CCP. One remained on invasive ventilatory support 42 days after CCP and was eventually discharged to an intermediate care facility. The single patient death was retrospectively confirmed to have had brain death before CCP. CONCLUSION: CCP was well tolerated in pediatric patients, resulted in rapid antibody increase, and did not appear to interfere with immune responses measured at 21 days. More pediatric data are necessary to establish the efficacy of CCP, but our data suggest benefit in moderate to severe COVID-19 when used early. Other immunologic or antiviral interventions may be added as supported by emerging data.

Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells.

OBJECTIVE AND DESIGN: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC. RESULTS: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. CONCLUSIONS: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

Expression of cytokines by multipotent neural progenitor cells.

Recent work with mammalian neural stem cells has highlighted the role of cytokine signaling in the proliferation and differentiation of these multipotent cells. While the responsiveness of neural progenitors to exogenously applied growth factors has been demonstrated in vivo as well as in vitro, little attention has been given to the production of cytokines by these cells. Here we use immunocytochemistry, RT-PCR, and ELISA to show that under standard growth conditions multipotent neural progenitor cells from humans express multiple cytokines including IL-1alpha, IL-1beta, IL-6, TGF-beta1, TGF-beta2, TNF-alpha, but not IL-2, IL-4, or IFN-gamma. Neural progenitor cells from rat and mouse express some, but not all, of these cytokines under similar conditions. While the function of cytokine expression by neural progenitor cells remains to be elucidated, these signaling molecules are known to be involved in neural development and may play a role in the activation of quiescent stem cells by a variety of pathological processes.

The immunological properties of adult hippocampal progenitor cells.

Adult hippocampal progenitor cells (AHPCs) derived from mature rats were studied in mixed co-cultures and shown not to elicit a proliferative response from human peripheral blood mononuclear cells (PBMCs) or allogeneic spleen cells. FACS analysis revealed low class I and no detectable class II (Ia) MHC expression by these cells. RT-PCR showed that AHPCs express the anti-inflammatory cytokine TGF-beta1. AHPCs did not, however, significantly impede the proliferation of OKT3- or PHA-stimulated PBMCs. Taken together, these results indicate that AHPCs are non-immunogenic in vitro. This is consistent with their pattern of MHC expression and does not require an active immunosuppressive mechanism.

Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro.

To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n = 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P <.001) as compared with both the control and patient plasma with no detectable anti-GPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P <.001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.

Identification of a novel regulatory element in the human interleukin 1 alpha (IL-1alpha) gene promoter.

Human interleukin 1alpha (IL-1alpha) is secreted by a variety of cell types including epithelial cells, endothelial cells, keratinocytes, adipocytes and activated monocytes/macrophages. IL-1alpha is a major player in immune and inflammatory responses, yet very little is known about its regulation. We have identified a novel regulatory sequence at -65 to -41 of the human IL-1alpha promoter using DNase I footprint studies. A triple GCC repeat is present within the footprint region. In order to study the importance of this GCC motif in the regulation of IL-1alpha, we mutated the GCC to a CAA at positions -60 to -58 and -52 to -50. In transfection studies using the human epithelial (HEp-2) and human erythroleukemia (HEL/DAMI) cell lines, the mutated promoter fragment showed a 90% decrease in basal activity and was not inducible by external stimuli. To characterize transcription factor binding to the footprint sequence, we used both electrophoretic mobility shift assays (EMSA) and DNase I protection techniques. Both of these studies confirmed that the GCC motif is also essential for DNA/protein complex formation. The NCBI and TESS databases were utilized to search for known transcripiton factor binding sites with homolgy to the footprint sequence. The only element identified with any homology was an NFkappaB binding sequence. However, competition assays using cold NFkappaB consensus sequence DNA had no effect on the mobility shift signal. This data strongly suggests that a novel regulatory element associated with the GCC motif is located in the footprint sequence. These results will contribute to understanding of the regulatory mechanisms governing induction of the IL-1alpha gene and may lead to the isolation of a novel transcription factor.

Expression of melanoma antigen gene by cells from inflamed joints in juvenile rheumatoid arthritis.

OBJECTIVE: To determine if synovial fluid (SF) cells from inflamed joints of patients with juvenile rheumatoid arthritis (JRA) express melanoma antigen gene (MAGE) RNA and protein. METHODS: The pattern of MAGE-A1 expression was analyzed in inflammatory synovial tissue and peripheral blood mononuclear cells (PBMC) from patients with JRA by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry. RESULTS: MAGE-A1, previously detected only in tumor cells, is strongly expressed in SF cells from patients with JRA. Immunocytochemistry revealed strong staining of SF cells in all of 22 specimens tested. PBMC from patients (7 of 7) also expressed MAGE-A1, but not as strongly as SF cells. Two-color immunofluorescence showed colocalization with CD4 and CD14. Flow cytometry on 3 samples of SF cells confirmed the presence of MAGE-A1 on the cell surface and intracellularly. Five of 5 SF cell samples were positive for MAGE-A1 by RT-PCR. CONCLUSION: Mononuclear cells from inflamed joints and blood from patients with JRA express MAGE-A1. MAGE family proteins were previously thought to be expressed only by certain tumors and presented by HLA Class I, resulting in tumor cell lysis by cytotoxic T cells. The observation of MAGE-A1 expression in JRA suggests an association with autoimmune disease and a possible causal role for MAGE antigen in the chronic inflammation of JRA.