Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

Monitoring cytosolic calcium in the dinoflagellate Crypthecodinium cohnii with calcium orange-AM

Calcium plays several important roles in the signal transduction pathways of dinoflagellates. We describe here the development of calcium orange-AM as an intracellular calcium reporter for the heterotrophic dinoflagellate Crypthecodinium cohnii. We demonstrated with confocal microscopy that by restricting the incubation period to 3045 min, no compartmentalization of the dye occurs in the mitochondria or endoplasmic reticulum. The dye fluorescence responded well to the effects of calcium ionophores and calcium chelators. By calibrating the dye with known calcium concentrations, we determined the intracellular calcium concentration of C. cohnii to be 158 +/- 56 nM, which rose to about 550 nM upon mechanical stimulation

cAMP in the cell cycle of the dinoflagellate Crypthecodinium cohnii (Dinophyta)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler, Zn the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii. cAMP peaked during the G(1) phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G(1) and G(2)+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O-2'-dibutyryl (Bt(2))-cAMP directly stimulated the growth of C. cohnii. Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis, The size of Bt(2)-cAMP treated cells at G(1) was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates

A dinoflagellate mutant with higher frequency of multiple fission

The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G(1) phase with a corresponding delay of the G(2)+M phase

A Neuromuscular Junction-like Calcium Release Mechanism In A Unicellular Organism

pp. 111–156 of this Free journal issue entitled: Abstracts of the 24th and the 25th Scientific Meeting of the Hong KongPoster Presentations: no. P-29/25When a myocyte is depolarized by an action potential, calcium ions enter the cell through L-type calcium channels located on the sarcolemma. This calcium, or the physical interaction between the activated L-type calcium channel, triggers a subsequent release of calcium that is stored in the sarcoplasmic reticulum (SR) through calcium-release channels ryanodine receptors. Crypthecodium cohnii is an unicellular dinoflagellate (phyloge-netically related to malarial parasite) with cortical multi-membranous structures apparently similar to the SR. In the present study, both fluorescence-conjugated ryanodine and dihydropyridine gave positive labellings on the cortical area of dinoflagellate cells in where the chlortetracycline-stained calcium stores were located. Either mechanical stimulations or potassium ions could induce membrane potential changes and calcium mobilizations. In addition, cytosolic calcium mobilizations induced by L-type calcium channel agonist (Bay K) was inhibited by ryanodine receptor antagonist (dantrolene). When the membrane potentials were disrupted by sodium ionophore, both mechanically-induced cytosolic calcium mobilizations and membrane potential changes were reduced. This indicated that the dihydropyridine receptor-like protein was upstream of the ryanodine receptor-like channel. Accumulated data, therefore, are consistent with a neuromuscular type excitation-contraction coupling-like mechanism in the dinoflagellates. Acknowledgement: Part of this research was supported by HKUST’s EHIA05/06.SC04 in Molecular Medicine awarded to J.T.Y.W. and S.Y.W.S).link_to_OA_fulltex

Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G(1) phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii

cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate Crypthecodinium cohnii (CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized C. cohnii cells, the transcript level peaked at early G, and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (Saccharomyces cerevisiae) and certain mammalian cell types suggest a role for eIF-5A in the G(1)/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while failing within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor D-difluoromethylornithine and the specific hypusination inhibitor N-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinotlagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G(2) + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G(2) + M phase can accumulate to over 95\%. Even after a 48-h treatment with nocodazole (2.5 mg L-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible

Frequent loss of heterozygosity on multiple chromosomes in Chinese esophageal squamous cell carcinomas

Analysis of the loss of heterozygosity (LOH) detected by polymerase chain reaction techniques using 18 polymorphic markers localized to chromosomes 3p, 5, 17, and 18q in 40 Hong Kong. Chinese esophageal squamous cell carcinoma (ESC) patients showed that multiple alterations on several chromosomes are involved in ESC development. The LOH rates detected for markers on chromosome 3 ranged from 44.0 to 85.7\%, for chromosome 5 from 40.9 to 61.9\%, for chromosome 17 from 40.0 to 100\%, and for chromosome 18 from 38.9 to 58.3\%. No significant association was observed between LOH and the clinical and histopathological parameters. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved

p53 mutations detected in colorectal carcinoma patients in Hong Kong

A mutational spectrum for exons 5-8 of the p53 tumor suppressor gene in colorectal carcinomas in Hong Kong Chinese was established, Ninety-nine colorectal carcinomas from Dong Kong patients were analyzed for mutations in p53 gene by PCR-single-strand conformation polymorphism analysis and direct DNA sequencing, Thirty-five of the 99 tumors (35.4\%) contained mutations, Point mutations accounted for 80\% of all genetic changes and were predominantly base transitions at CpG dinucleotide sites, mutations that were also predominant in Caucasian carcinomas. The major hot spots at codons 175 and 248 of p53 in Caucasians are also hot spots in the Chinese gene, Identical mutations in codons 152 and 306 were detected in two independent tumors in the Chinese, which were reported only rarely in Caucasians, Moreover, a significantly higher frequency (20\%) of deletion and insertion mutations was observed in Dong Kong colorectal cancer patients, Distinct genetic and/or environmental factors may contribute to these findings

Ki-ras codon 12 point mutational activation in Hong Kong colorectal carcinoma patients

This study investigated the frequency and importance of Ki-ras codon 12 mutations in 99 Hong Kong Chinese colorectal carcinoma specimens by allele-specific oligonucleotide hybridization. The frequency of mutations detected was 30\% and the most common mutation observed resulted in aspartic acid substitutions. Previous studies showed that specific Ki-ras mutations have been significantly associated with prognosis. Ki-ras codon 12 point mutational activation in CRC was significantly associated with the differentiation status of tumors in this study. Ethnic differences in the patterns of Ki-ras codon 12 point mutations were observed. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved