'We're taught green is good': Perspectives on time in range and time in tight range from youth with type 1 diabetes, and parents of youth with type 1 diabetes.

AIMS: Continuous glucose monitoring (CGM) systems are standard of care for youth with type 1 diabetes with the goal of spending >70% time in range (TIR; 70-180 mg/dL, 3.9-10 mmol/L). We aimed to understand paediatric CGM user experiences with TIR metrics considering recent discussion of shifting to time in tight range (TITR; >50% time between 70 and 140 mg/dL, 3.9 and 7.8 mmol/L). METHODS: Semi-structured interviews and focus groups with adolescents with type 1 diabetes and parents of youth with type 1 diabetes focused on experiences with TIR goals and reactions to TITR. Groups and interviews were audio-recorded, transcribed and analysed using content analysis. RESULTS: Thirty participants (N = 19 parents: age 43.6 ± 5.3 years, 79% female, 47% non-Hispanic White, 20 ± 5 months since child's diagnosis; N = 11 adolescents: age 15.3 ± 2 years, 55% female, 55% non-Hispanic White, 16 ± 3 months since diagnosis) attended. Participants had varying levels of understanding of TIR. Some developed personally preferred glucose ranges. Parents often aimed to surpass 70% TIR. Many described feelings of stress and disappointment when they did not meet a TIR goal. Concerns about TITR included increased stress and burden; risk of hypoglycaemia; and family conflict. Some participants said TITR would not change their daily lives; others said it would improve their diabetes management. Families requested care team support and a clear scientific rationale for TITR. CONCLUSIONS: The wealth of CGM data creates frequent opportunities for assessing diabetes management and carries implications for management burden. Input from people with type 1 diabetes and their families will be critical in considering a shift in glycaemic goals and targets.

Human CLEC16A regulates autophagy through modulating mTOR activity.

CLEC16A is genetically linked with multiple autoimmune disorders but its functional relevance in autoimmunity remains obscure. Recent evidence has signposted the emerging role of autophagy in autoimmune disease development. Here, by ectopic expression and siRNA silencing, we show that CLEC16A has an inhibitory role in starvation-induced autophagy in human cells. Combining quantitative proteomics and immunoblotting analyses, we found that CLEC16A likely regulates autophagy by activating mTOR pathway. Overexpression of CLEC16A was found to sensitize cells towards the availability of nutrients, resulting in a heightened mTOR activity, which in turn diminished LC3 autophagic activity following nutrient deprivation. CLEC16A deficiency, on the other hand, delayed mTOR activity in response to nutrient sensing, thereby resulted in an augmented autophagic response. CLEC16A was found residing in cytosolic vesicles and the Golgi, and nutrient removal promoted a stronger clustering within the Golgi, where it was possibly in a vantage position to activate mTOR upon nutrient replenishment. These findings suggest that Golgi-associated CLEC16A negatively regulates autophagy via modulation of mTOR activity, and may provide support for a functional link between CLEC16A and autoimmunity.

MicroRNA-155 Mediates Augmented CD40 Expression in Bone Marrow Derived Plasmacytoid Dendritic Cells in Symptomatic Lupus-Prone NZB/W F1 Mice.

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease characterized by hyperactivated immune responses to self-antigens and persistent systemic inflammation. Previously, we reported abnormalities in circulating and bone marrow (BM)-derived plasmacytoid dendritic cells (pDCs) from SLE patients. Here, we aim to seek for potential regulators that mediate functional aberrations of pDCs in SLE. BM-derived pDCs from NZB/W F1 mice before and after the disease onset were compared for toll-like receptor (TLR) induced responses and microRNA profile changes. While pDCs derived from symptomatic mice were phenotypically comparable to pre-symptomatic ones, functionally they exhibited hypersensitivity to TLR7 but not TLR9 stimulation, as represented by the elevated upregulation of CD40, CD86 and MHC class II molecules upon R837 stimulation. Upregulated induction of miR-155 in symptomatic pDCs following TLR7 stimulation was observed. Transfection of miR-155 mimics in pre-symptomatic pDCs induced an augmented expression of Cd40, which is consistent with the increased CD40 expression in symptomatic pDCs. Overall, our results provide evidence for miR-155-mediated regulation in pDC functional abnormalities in SLE. Findings from this study contribute to a better understanding of SLE pathogenesis and ignite future interests in evaluating the molecular regulation in autoimmunity.

Systemic Lupus Erythematosus Patients Exhibit Reduced Expression of CLEC16A Isoforms in Peripheral Leukocytes.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with multiple etiological factors. The SLE susceptibility locus on chromosome 16p13 encodes a novel gene CLEC16A and its functional relationship with SLE is unclear. This study aimed to investigate the expression correlation of the two major CLEC16A spliced transcripts with SLE development. Expressions of the long (V1) and short (V2) CLEC16A isoforms in the peripheral blood mononuclear cells (PBMCs) were assayed by quantitative real time PCR and compared between healthy individuals and SLE patients. Correlation of CLEC16A isoform expression levels with SLE susceptibility, disease severity and twelve clinical parameters were also evaluated. Full length transcripts of CLEC16A V1 and V2 isoforms were readily amplified from PBMCs of healthy controls and patients at varying abundance. Compared with healthy controls (n = 86), expression levels of V1 and V2 were significantly reduced by ~two- and four-fold respectively in SLE patients (n = 181). The relative V2/V1 ratio was also significantly reduced by approximately two-fold. With regard to SLE disease parameters, only a weak positive correlation was found between CLEC16A V1 expression levels and SLE disease activity index (SLEDAI) score. Taken together, CLEC16A was found to be a susceptibility factor for SLE, with possible contribution to the development of the disease.

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

SARS-CoV-2 variants divergently infect and damage cardiomyocytes in vitro and in vivo.

BACKGROUND: COVID-19 can cause cardiac complications and the latter are associated with poor prognosis and increased mortality. SARS-CoV-2 variants differ in their infectivity and pathogenicity, but how they affect cardiomyocytes (CMs) is unclear. METHODS: The effects of SARS-CoV-2 variants were investigated using human induced pluripotent stem cell-derived (hiPSC-) CMs in vitro and Golden Syrian hamsters in vivo. RESULTS: Different variants exhibited distinct tropism, mechanism of viral entry and pathology in the heart. Omicron BA.2 most efficiently infected and injured CMs in vitro and in vivo, and induced expression changes consistent with increased cardiac dysfunction, compared to other variants tested. Bioinformatics and upstream regulator analyses identified transcription factors and network predicted to control the unique transcriptome of Omicron BA.2 infected CMs. Increased infectivity of Omicron BA.2 is attributed to its ability to infect via endocytosis, independently of TMPRSS2, which is absent in CMs. CONCLUSIONS: In this study, we reveal previously unknown differences in how different SARS-CoV-2 variants affect CMs. Omicron BA.2, which is generally thought to cause mild disease, can damage CMs in vitro and in vivo. Our study highlights the need for further investigations to define the pathogenesis of cardiac complications arising from different SARS-CoV-2 variants.

Nivolumab with or without ipilimumab in pediatric patients with high-grade CNS malignancies: Safety, efficacy, biomarker, and pharmacokinetics-CheckMate 908.

BACKGROUND: Therapeutic options are limited in pediatric CNS malignancies. CheckMate 908 (NCT03130959) is an open-label, sequential-arm, phase 1b/2 study investigating nivolumab (NIVO) and NIVO + ipilimumab (IPI) in pediatric patients with high-grade CNS malignancies. METHODS: Patients (N = 166) in 5 cohorts received NIVO 3 mg/kg every 2 weeks (Q2W) or NIVO 3 mg/kg + IPI 1 mg/kg every 3 weeks (4 doses) followed by NIVO 3 mg/kg Q2W. Primary endpoints included overall survival (OS; newly diagnosed diffuse intrinsic pontine glioma [DIPG]) and progression-free survival (PFS; other recurrent/progressive or relapsed/resistant CNS cohorts). Secondary endpoints included other efficacy metrics and safety. Exploratory endpoints included pharmacokinetics and biomarker analyses. RESULTS: As of January 13, 2021, median OS (80% CI) was 11.7 (10.3-16.5) and 10.8 (9.1-15.8) months with NIVO and NIVO + IPI, respectively, in newly diagnosed DIPG. Median PFS (80% CI) with NIVO and NIVO + IPI was 1.7 (1.4-2.7) and 1.3 (1.2-1.5) months, respectively, in recurrent/progressive high-grade glioma; 1.4 (1.2-1.4) and 2.8 (1.5-4.5) months in relapsed/resistant medulloblastoma; and 1.4 (1.4-2.6) and 4.6 (1.4-5.4) months in relapsed/resistant ependymoma. In patients with other recurrent/progressive CNS tumors, median PFS (95% CI) was 1.2 (1.1-1.3) and 1.6 (1.3-3.5) months, respectively. Grade 3/4 treatment-related adverse-event rates were 14.1% (NIVO) and 27.2% (NIVO + IPI). NIVO and IPI first-dose trough concentrations were lower in youngest and lowest-weight patients. Baseline tumor programmed death ligand 1 expression was not associated with survival. CONCLUSIONS: NIVO ± IPI did not demonstrate clinical benefit relative to historical data. The overall safety profiles were manageable with no new safety signals.

An intranasal influenza virus-vectored vaccine prevents SARS-CoV-2 replication in respiratory tissues of mice and hamsters.

Current available vaccines for COVID-19 are effective in reducing severe diseases and deaths caused by SARS-CoV-2 infection but less optimal in preventing infection. Next-generation vaccines which are able to induce mucosal immunity in the upper respiratory to prevent or reduce infections caused by highly transmissible variants of SARS-CoV-2 are urgently needed. We have developed an intranasal vaccine candidate based on a live attenuated influenza virus (LAIV) with a deleted NS1 gene that encodes cell surface expression of the receptor-binding-domain (RBD) of the SARS-CoV-2 spike protein, designated DelNS1-RBD4N-DAF. Immune responses and protection against virus challenge following intranasal administration of DelNS1-RBD4N-DAF vaccines were analyzed in mice and compared with intramuscular injection of the BioNTech BNT162b2 mRNA vaccine in hamsters. DelNS1-RBD4N-DAF LAIVs induced high levels of neutralizing antibodies against various SARS-CoV-2 variants in mice and hamsters and stimulated robust T cell responses in mice. Notably, vaccination with DelNS1-RBD4N-DAF LAIVs, but not BNT162b2 mRNA, prevented replication of SARS-CoV-2 variants, including Delta and Omicron BA.2, in the respiratory tissues of animals. The DelNS1-RBD4N-DAF LAIV system warrants further evaluation in humans for the control of SARS-CoV-2 transmission and, more significantly, for creating dual function vaccines against both influenza and COVID-19 for use in annual vaccination strategies.

Regorafenib plus nivolumab in patients with mismatch repair-proficient/microsatellite stable metastatic colorectal cancer: a single-arm, open-label, multicentre phase 2 study.

Background: Anti-programmed cell death protein 1 antibodies plus multikinase inhibitors have shown encouraging activity in several tumour types, including colorectal cancer. This study assessed regorafenib plus nivolumab in patients with microsatellite stable/mismatch repair-proficient metastatic colorectal cancer. Methods: This single-arm, open-label, multicentre phase 2 study enrolled adults from 13 sites in the USA with previously treated advanced microsatellite stable/mismatch repair-proficient metastatic colorectal cancer. Eligible patients had known extended RAS and BRAF status, progression or intolerance to no more than two (for extended RAS mutant) or three (for extended RAS wild type) lines of systemic chemotherapy and an Eastern Cooperative Oncology Group performance status of 0 or 1. Regorafenib 80 mg/day was administered orally for 3 weeks on/1 week off (increased to 120 mg/day if 80 mg/day was well tolerated) with intravenous nivolumab 480 mg every 4 weeks. Primary endpoint was objective response rate. Secondary endpoints included safety, overall survival, and progression-free survival. Exploratory endpoints included biomarkers associated with antitumour activity. Patients who received at least one dose of study intervention were included in the efficacy and safety analyses. Tumour assessments were carried out every 8 weeks for the first year, and every 12 weeks thereafter until progressive disease/end of the study, and objective response rate was analysed after all patients had met the criteria for primary completion of five post-baseline scans and either 10-months' follow-up or drop out. This trial is registered with ClinicalTrials.gov, number NCT04126733. Findings: Between 14 October 2019 and 14 January 2020, 94 patients were enrolled, 70 received treatment. Five patients had a partial response, yielding an objective response rate of 7% (95% CI 2.4-15.9; p = 0.27). All responders had no liver metastases at baseline. Median overall survival (data immature) and progression-free survival were 11.9 months (95% CI 7.0-not evaluable) and 1.8 months (95% CI 1.8-2.4), respectively. Most patients (97%, 68/70) experienced a treatment-related adverse event; 51% were grade 1 or 2, 40% were grade 3, 3% were grade 4, and 3% were grade 5. The most common (≥20%) events were fatigue (26/70), palmar-plantar erythrodysesthesia syndrome (19/70), maculopapular rash (17/70), increased blood bilirubin (14/70), and decreased appetite (14/70). Higher baseline expression of tumour biomarkers of immune sensitivity correlated with antitumour activity. Interpretation: Further studies are warranted to identify subgroups of patients with clinical characteristics or biomarkers that would benefit most from treatment with regorafenib plus nivolumab. Funding: Bayer/Bristol Myers Squibb.

A Phase 1, Randomized, Double-Blinded, Placebo-Controlled and Dose-Escalation Study to Evaluate the Safety and Immunogenicity of the Intranasal DelNS1-nCoV-RBD LAIV for COVID-19 in Healthy Adults.

An intranasal COVID-19 vaccine, DelNS1-based RBD vaccines composed of H1N1 subtype (DelNS1-nCoV-RBD LAIV) was developed to evaluate the safety and immunogenicity in healthy adults. We conducted a phase 1 randomized, double-blinded, placebo-controlled study on healthy participants, age 18-55 and COVID-19 vaccines naïve, between March and September 2021. Participants were enrolled and randomly assigned (2:2:1) into the low and high dose DelNS1-nCoV-RBD LAIV manufactured in chicken embryonated eggs or placebo groups. The low and high-dose vaccine were composed of 1 × 107 EID50/ dose and 1 × 107.7 EID50/ dose in 0.2 mL respectively. The placebo vaccine was composed of inert excipients/dose in 0.2 mL. Recruited participants were administered the vaccine intranasally on day 0 and day 28. The primary end-point was the safety of the vaccine. The secondary endpoints included cellular, humoral, and mucosal immune responses post-vaccination at pre-specified time-points. The cellular response was measured by the T-cell ELISpot assay. The humoral response was measured by the serum anti-RBD IgG and live-virus neutralizing antibody against SARS-CoV-2. The saliva total Ig antibody responses in mucosal secretion against SARS-CoV-2 RBD was also assessed. Twenty-nine healthy Chinese participants were vaccinated (low-dose: 11; high-dose: 12 and placebo: 6). The median age was 26 years. Twenty participants (69%) were male. No participant was discontinued due to an adverse event or COVID-19 infection during the clinical trial. There was no significant difference in the incidence of adverse events (p = 0.620). For the T-cell response elicited after full vaccination, the positive PBMC in the high-dose group increased to 12.5 SFU/106 PMBC (day 42) from 0 (baseline), while it increased to 5 SFU/106 PBMC (day 42) from 2.5 SFU/106 PBMC (baseline) in the placebo group. The high-dose group showed a slightly higher level of mucosal Ig than the control group after receiving two doses of the vaccine (day 31, 0.24 vs. 0.21, p = 0.046; day 56 0.31 vs. 0.15, p = 0.45). There was no difference in the T-cell and saliva Ig response between the low-dose and placebo groups. The serum anti-RBD IgG and live virus neutralizing antibody against SARS-CoV-2 were undetectable in all samples. The high-dose intranasal DelNS1-nCoV-RBD LAIV is safe with moderate mucosal immunogenicity. A phase-2 booster trial with a two-dose regimen of the high-dose intranasal DelNS1-nCoV-RBD LAIV is warranted.

Identification of the MBF1 heat-response regulon of Arabidopsis thaliana.

Brief periods of heat stress of even a few days can have a detrimental effect on yield production worldwide, causing devastating economic and societal impacts. Here we report on the identification of a new heat-response regulon in plants controlled by the multiprotein bridging factor 1c (MBF1c) protein of Arabidopsis thaliana. Members of the highly conserved MBF1 protein family function as non-DNA-binding transcriptional co-activators involved in regulating metabolic and development pathways in different organisms from yeast to humans. Nonetheless, our studies suggest that MBF1c from Arabidopsis functions as a transcriptional regulator which binds DNA and controls the expression of 36 different transcripts during heat stress, including the important transcriptional regulator DRE-binding protein 2A (DREB2A), two heat shock transcription factors (HSFs), and several zinc finger proteins. We further identify CTAGA as a putative response element for MBF1c, demonstrate that the DNA-binding domain of MBF1c has a dominant-negative effect on heat tolerance when constitutively expressed in plants, and show that constitutive expression of MBF1c in soybean enhances yield production in plants grown under controlled growth conditions without causing adverse effects on growth. Our findings could have a significant impact on improving heat tolerance and yield of different crops subjected to heat stress.

What Do We Know about the Microbiome in Cystic Fibrosis? Is There a Role for Probiotics and Prebiotics?

Cystic fibrosis (CF) is a life-shortening genetic disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the gastrointestinal (GI) tract, CFTR dysfunction results in low intestinal pH, thick and inspissated mucus, a lack of endogenous pancreatic enzymes, and reduced motility. These mechanisms, combined with antibiotic therapies, drive GI inflammation and significant alteration of the GI microbiota (dysbiosis). Dysbiosis and inflammation are key factors in systemic inflammation and GI complications including malignancy. The following review examines the potential for probiotic and prebiotic therapies to provide clinical benefits through modulation of the microbiome. Evidence from randomised control trials suggest probiotics are likely to improve GI inflammation and reduce the incidence of CF pulmonary exacerbations. However, the highly variable, low-quality data is a barrier to the implementation of probiotics into routine CF care. Epidemiological studies and clinical trials support the potential of dietary fibre and prebiotic supplements to beneficially modulate the microbiome in gastrointestinal conditions. To date, limited evidence is available on their safety and efficacy in CF. Variable responses to probiotics and prebiotics highlight the need for personalised approaches that consider an individual's underlying microbiota, diet, and existing medications against the backdrop of the complex nutritional needs in CF.

Intestinal Inflammation and Alterations in the Gut Microbiota in Cystic Fibrosis: A Review of the Current Evidence, Pathophysiology and Future Directions.

Cystic fibrosis (CF) is a life-limiting autosomal recessive multisystem disease. While its burden of morbidity and mortality is classically associated with pulmonary disease, CF also profoundly affects the gastrointestinal (GI) tract. Chronic low-grade inflammation and alterations to the gut microbiota are hallmarks of the CF intestine. The etiology of these manifestations is likely multifactorial, resulting from cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, a high-fat CF diet, and the use of antibiotics. There may also be a bidirectional pathophysiological link between intestinal inflammation and changes to the gut microbiome. Additionally, a growing body of evidence suggests that these GI manifestations may have significant clinical associations with growth and nutrition, quality of life, and respiratory function in CF. As such, the potential utility of GI therapies and long-term GI outcomes are areas of interest in CF. Further research involving microbial modulation and multi-omics techniques may reveal novel insights. This article provides an overview of the current evidence, pathophysiology, and future research and therapeutic considerations pertaining to intestinal inflammation and alterations in the gut microbiota in CF.

Immune contexture of paediatric cancers.

BACKGROUND: The clinical development of immune checkpoint-targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies. METHODS: We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival. RESULTS: NB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff. CONCLUSIONS: Our results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.

Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination in mouse models.

BACKGROUND: Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal. METHODS: Since mucosal immunity is critical for nasal prevention, we investigated the efficacy of an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2. FINDINGS: Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity. INTERPRETATION: Our results demonstrated that intranasal influenza-based boost vaccination induces mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems. FUNDING: This study was supported by the Research Grants Council Collaborative Research Fund, General Research Fund and Health and Medical Research Fund in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program and matching fund from Shenzhen Immuno Cure BioTech Limited; the Health@InnoHK, Innovation and Technology Commission of Hong Kong; National Program on Key Research Project of China; donations from the Friends of Hope Education Fund; the Theme-Based Research Scheme.

Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein.

SARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.

Low dose inocula of SARS-CoV-2 Alpha variant transmits more efficiently than earlier variants in hamsters.

Emerging variants of SARS-CoV-2 have been shown to rapidly replace original circulating strains in humans soon after they emerged. There is a lack of experimental evidence to explain how these natural occurring variants spread more efficiently than existing strains of SARS-CoV-2 in transmission. We found that the Alpha variant (B.1.1.7) increased competitive fitness over earlier parental D614G lineages in in-vitro and in-vivo systems. Using hamster transmission model, we further demonstrated that the Alpha variant is able to replicate and shed more efficiently in the nasal cavity of hamsters than other variants with low dose and short duration of exposure. The capability to initiate effective infection with low inocula may be one of the key factors leading to the rapid transmission of emerging variants of SARS-CoV-2.

Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner.

The neonatal mouse heart is capable of transiently regenerating after injury from postnatal day (P) 0-7 and macrophages are found important in this process. However, whether macrophages alone are sufficient to orchestrate this regeneration; what regulates cardiomyocyte proliferation; why cardiomyocytes do not proliferate after P7; and whether adaptive immune cells such as regulatory T-cells (Treg) influence neonatal heart regeneration have less studied. Methods: We employed both loss- and gain-of-function transgenic mouse models to study the role of Treg in neonatal heart regeneration. In loss-of-function studies, we treated mice with the lytic anti-CD25 antibody that specifically depletes Treg; or we treated FOXP3DTR with diphtheria toxin that specifically ablates Treg. In gain-of-function studies, we adoptively transferred hCD2+ Treg from NOD.Foxp3hCD2 to NOD/SCID that contain Treg as the only T-cell population. Furthermore, we performed single-cell RNA-sequencing of Treg to uncover paracrine factors essential for cardiomyocyte proliferation. Results: Unlike their wild type counterparts, NOD/SCID mice that are deficient in T-cells but harbor macrophages fail to regenerate their injured myocardium at as early as P3. During the first week of injury, Treg are recruited to the injured cardiac muscle but their depletion contributes to more severe cardiac fibrosis. On the other hand, adoptive transfer of Treg results in mitigated fibrosis and enhanced proliferation and function of the injured cardiac muscle. Mechanistically, single-cell transcriptomic profiling reveals that Treg could be a source of regenerative factors. Treg directly promote proliferation of both mouse and human cardiomyocytes in a paracrine manner; and their secreted factors such as CCL24, GAS6 or AREG potentiate neonatal cardiomyocyte proliferation. By comparing the regenerating P3 and non-regenerating P8 heart, there is a significant increase in the absolute number of intracardiac Treg but the whole transcriptomes of these Treg do not differ regardless of whether the neonatal heart regenerates. Furthermore, even adult Treg, given sufficient quantity, possess the same regenerative capability. Conclusion: Our results demonstrate a regenerative role of Treg in neonatal heart regeneration. Treg can directly facilitate cardiomyocyte proliferation in a paracrine manner.

Mutational Analysis of Patients With Colorectal Cancer in CALGB/SWOG 80405 Identifies New Roles of Microsatellite Instability and Tumor Mutational Burden for Patient Outcome.

PURPOSE: CALGB/SWOG 80405 was a randomized phase III trial that found no statistically significant difference in overall survival (OS) in patients with first-line metastatic colorectal cancer treated with chemotherapy plus either bevacizumab or cetuximab. Primary tumor DNA from 843 patients has been used to discover genetic markers of OS. PATIENTS AND METHODS: Gene mutations were determined by polymerase chain reaction. Microsatellite status was determined by genotyping of microsatellites. Tumor mutational burden (TMB) was determined by next-generation sequencing. Cox proportional hazard models were used, with adjusting factors. Interaction of molecular alterations with either the bevacizumab or the cetuximab arms was tested. RESULTS: Patients with high TMB in their tumors had longer OS than did patients with low TMB (hazard ratio [HR], 0.73 [95% CI, 0.57 to 0.95]; P = .02). In patients with microsatellite instability-high (MSI-H) tumors, longer OS was observed in the bevacizumab arm than in the cetuximab arm (HR, 0.13 [95% CI, 0.06 to 0.30]; interaction P < .001 for interaction between microsatellite status and the two arms). Patients with BRAF mutant tumors had shorter OS than did patients with wild-type (WT) tumors (HR, 2.01 [95% CI, 1.49 to 2.71]; P < .001). Patients with extended RAS mutant tumors had shorter OS than did patients with WT tumors (HR, 1.52 [95% CI, 1.26 to 1.84]; P < .001). Patients with triple-negative tumors (WT for NRAS/KRAS/BRAF) had a median OS of 35.9 months (95% CI, 33.0 to 38.8 months) versus 22.2 months (95% CI, 19.6 to 24.4 months ) in patients with at least one mutated gene in their tumors (P < .001). CONCLUSION: In patients with metastatic colorectal cancer treated in first line, low TMB, and BRAF and RAS mutations are negative prognostic factors. Patients with MSI-H tumors benefited more from bevacizumab than from cetuximab, and studies to confirm this effect of MSI-H are warranted.

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

PURPOSE: Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. RESULTS: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. CONCLUSIONS: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.