Coronavirus disease 2019 (COVID-19) was first identified in China in late 2019 and is caused by newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies had ...reported the stability of SARS-CoV-2 in cell culture media and deposited onto surfaces under a limited set of environmental conditions. Here, we broadly investigated the effects of relative humidity, temperature, and droplet size on the stability of SARS-CoV-2 in a simulated clinically relevant matrix dried on nonporous surfaces. The results show that SARS-CoV-2 decayed more rapidly when either humidity or temperature was increased but that droplet volume (1 to 50 μl) and surface type (stainless steel, plastic, or nitrile glove) did not significantly impact decay rate. At room temperature (24°C), virus half-life ranged from 6.3 to 18.6 h depending on the relative humidity but was reduced to 1.0 to 8.9 h when the temperature was increased to 35°C. These findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and have implications for assessment of the risk posed by surface contamination in indoor environments.
Mitigating the transmission of SARS-CoV-2 in clinical settings and public spaces is critically important to reduce the number of COVID-19 cases while effective vaccines and therapeutics are under development. SARS-CoV-2 transmission is thought to primarily occur through direct person-to-person transfer of infectious respiratory droplets or through aerosol-generating medical procedures. However, contact with contaminated surfaces may also play a significant role. In this context, understanding the factors contributing to SARS-CoV-2 persistence on surfaces will enable a more accurate estimation of the risk of contact transmission and inform mitigation strategies. To this end, we have developed a simple mathematical model that can be used to estimate virus decay on nonporous surfaces under a range of conditions and which may be utilized operationally to identify indoor environments in which the virus is most persistent.
Background & Aims Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal ...adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor’s immunosuppressive mechanisms, or both. Methods Kras G12D/+ ;Trp53 R172H/+ ;Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. Results Gemcitabine in combination with a CD40 agonist induced T-cell−dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4+ and CD8+ T cells infiltrated subcutaneous tumors, but only CD4+ T cells infiltrated spontaneous pancreatic tumors (not CD8+ T cells). In mice depleted of Ly6Clow F4/80+ extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8+ T cells and induced tumor regression, mediated by CD8+ T cells. Conclusions Ly6Clow F4/80+ macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.
Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We ...investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor's immunosuppressive mechanisms, or both.
Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry.
Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells.
Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various ...epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)
. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
A recombinant formulation of silk fibroin containing the arginine–glycine–aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, ...we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal–epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.
Patients with prostate cancer with tumors harboring defects in DNA-repair genes (DRD) generally do not respond well to AR-directed therapy. Furthermore, canonical pathways evolve during disease ...progression and may affect treatment with existing therapies. Due to the limited treatment options after failure of hormonal and taxane therapy, and the tumor heterogeneity induced by DRD, we sought to characterize the alterations in primary and metastatic prostate cancer. Tumors from 1,027 patients with advanced prostate cancer that underwent comprehensive genomic profiling for routine clinical care were reviewed to assess DRD mutation rates (27-gene panel) and co-occurring mutations in select canonical prostate cancer pathways. DRD alterations were identified in 20 genes and in 17% of patients (BRCA2 and ATM most common) occurring with slightly higher frequency in specimens from metastatic biopsy sites and men older than 50 years of age. Microsatellite instability-high (MSI-H) and tumor mutational burden-high occurred with 3% frequency in the overall cohort but were not enriched in metastatic disease. Biomarkers previously associated with antitumor immunity are found at high frequencies in MSI-H patients, including JAK1 (68%) and PTEN (32%). Lastly, mutations in TP53, AR, PTEN, APC, CTNNB1, and PIK3CA were all significantly enriched in metastatic samples. We identified clinically significant subgroups of patients demonstrating (1) defects in DNA-repair pathways, (2) intrinsic prostate cancer signaling pathways that may prevent antitumor immunity, and (3) distinct genomic differences between localized and metastatic prostate cancer. These results lend support that genomic profiling for advanced prostate cancer may identify actionable targets not routinely used in the current metastatic paradigm.
While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore ...examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone (n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM (n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment (n = 3) or treatment with HLE alone on HAM (n = 3). Animals treated with RLMSC (n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE (p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20–30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.
When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for ...biomaterials is therefore a significant and common problem. In this review, we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance, and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of 4 experimental designs developed within our respective laboratories in Australia, the United Kingdom, and Belgium.
In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P-selectin (CD62P) in human corneal epithelial ...cells. The SV40 immortalised cell line, HCE-T (validated by STR profiling), along with multiple donor corneal-limbal tissue samples, were examined for P-selectin expression using a combination of immunocytochemistry, Western blotting, RT-PCR and immunohistochemistry. Potential expression of the major ligand for P-selectin (P-selectin glycoprotein ligand-1; PSGL-1; CD162) was also examined by immunocytochemistry and RT-PCR. A selective inhibitor of P-selectin-PSGL-1 binding (KF38789) was subsequently tested for effects on HCE-T cells using a cell culture gap-closure assay. HCE-T cells as well as primary epithelial cultures derived from donor corneal-limbal tissue, displayed positive immunostaining for P-selectin. Staining was particularly evident at cell-cell boundaries and at the outer edge of expanding epithelial islands. P-selectin expression was confirmed by Western blotting and RT-PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal-limbal tissue stained for P-selectin, keratin 3 and p63. PSGL-1 was detected by RT-PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 μM) significantly reduced closure of a 500-μm gap between confluent sheets of HCE-T cells over an 8-hr period (by ∼40%, p < 0.01; paired two-tailed T test), but had no effect on culture gap-closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P-selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells.
•Human corneal epithelial (HCE) cells express P-selectin in vitro and in situ.•HCE and limbal fibroblasts both appear to express the P-selectin ligand, PSGL-1.•An inhibitor of P-selectin binding to PSGL-1 reduces HCE migration in vitro.•These findings suggest a potential new role for P-selectin within the cornea.
Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280–320 nm), in particular, causes DNA damage in ...epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB‐induced damage. To investigate these processes, established two and three‐dimensional culture models were utilized to study the impact of fibroblast–keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase‐3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast‐produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation‐induced damage.
Cellular cross‐talk between dermal fibroblasts and epidermal keratinocytes is critical for orchestrating cutaneous repair and regeneration. Herein, we describe the influence of keratinocyte–fibroblast interactions on key processes in the ultraviolet B photoresponse. Importantly, fibroblast‐produced factors were observed to enhance keratinocyte survival and DNA repair by regulating the expression of specific apoptotic and cell cycle regulation proteins.
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