Peripheral Immune Profile of Children With Talaromyces Marneffei Infections: A Retrospective Analysis of 21 Cases

BackgroundTalaromyces (formerly Penicillium) marneffei (T. marneffei) is an opportunistic pathogen that infects immunodeficient and immunocompromised children. The aim of the study is to determine the clinical features and peripheral immune state of Talaromyces marneffei (T. marneffei) infections in children for early detection and diagnosis.MethodsWe retrospectively reviewed 21 pediatric patients who were diagnosed with T. marneffei infections and were followed up in the Guangzhou Women and Children’s Medical Center from January 2010 to January 2020. For each patient, we collected and analyzed clinical characteristics, peripheral immunological results, genetic tests, complications and prognosis.ResultsCommon clinical features of the patients included fever (20/21, 95.24%), cough (17/21, 80.95%) and hepatomegaly (17/21, 80.95%). Severe complications included septic shock (12/21, 57.14%), hemophagocytic lymphohistiocytosis (HLH) (11/21, 52.38%), acute respiratory distress syndrome (ARDS) (10/21, 47.62%), multiple organ dysfunction syndrome (MODS) (9/21, 42.86%), and disseminated intravascular coagulation (DIC) (7/21, 33.33%). Eleven children (11/21, 52.38%) eventually died of T. marneffei infections. All patients were HIV negative. Seven cases revealed reduced antibody levels, especially IgG. Higher levels of IgE were detected in 9 cases with an obvious increase in two patients. Ten patients showed decreased complement C3 levels, some of whom had low C4 levels. Three patients displayed decreased absolute T lymphocyte counts, including the CD 4+ and CD 8+ subsets. A reduction in NK cells was present in most patients. No patient had positive nitro blue tetrazolium (NBT) test results. Nine patients were assessed using gene tests. Of the cases, one case had no disease-specific gene mutation. Four children had confirmed hyperimmunoglobulin M syndrome (HIGM) with CD40LG variation, one case had severe combined immunodeficiency (SCID), and one case had hyper-IgE syndrome (HIES). One patient was identified as having a heterozygous mutation in STAT3 gene; however, he showed no typical clinical manifestations of HIES at his age. One patient had a mutated COPA gene with uncertain pathogenic potential. Another patient was diagnosed with HIES that depended on her clinical features and the National Institutes of Health (NIH) scoring system.ConclusionT. marneffei infections in HIV-negative children induced severe systemic complications and poor prognosis. Children with T. marneffei infections commonly exhibited abnormal immunoglobulin levels in peripheral blood, particularly decreased IgG or increased IgE levels, further suggesting possible underlying PIDs in these patients.

80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage

Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.

CopR of Sulfolobus solfataricus represents a novel class of archaeal-specific copper-responsive activators of transcription

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)–1. Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.