Short- and long-term outcomes of pulmonary exposure to a sublethal dose of ricin in mice.

Ricin, an extremely potent toxin produced from the seeds of castor plant, Ricinus communis, is ribosome-inactivating protein that blocks cell-protein synthesis. It is considered a biological threat due to worldwide availability of castor beans, massive quantities as a by-product of castor oil production, high stability and ease of production. The consequence of exposure to lethal dose of ricin was extensively described in various animal models. However, it is assumed that in case of aerosolized ricin bioterror attack, the majority of individuals would be exposed to sublethal doses rather than to lethal ones. Therefore, the purpose of current study was to assess short- and long-term effects on physiological parameters and function following sublethal pulmonary exposure. We show that in the short-term, sublethal exposure of mice to ricin resulted in acute lung injury, including interstitial pneumonia, cytokine storm, neutrophil influx, edema and cellular death. This damage was manifested in reduced lung performance and physiological function. Interestingly, although in the long-term, mice recovered from acute lung damage and restored pulmonary and physiological functionality, the reparative process was associated with lasting fibrotic lesions. Therefore, restriction of short-term acute phase of the disease and management of long-term pulmonary fibrosis by medical countermeasures is expected to facilitate the quality of life of exposed survivors.

Preliminary nonclinical safety and immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Mice with induced pulmonary morbidities display severe lung inflammation and mortality following exposure to SARS-CoV-2.

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.

Astrocytes facilitate melanoma brain metastasis via secretion of IL-23.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

Tumor-derived osteopontin reprograms normal mammary fibroblasts to promote inflammation and tumor growth in breast cancer.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell-secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell-secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVß3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs.

c-Kit is suppressed in human colon cancer tissue and contributes to L1-mediated metastasis.

The transmembrane neural cell adhesion receptor L1 is a Wnt/ß-catenin target gene expressed in many tumor types. In human colorectal cancer, L1 localizes preferentially to the invasive front of tumors and when overexpressed in colorectal cancer cells, it facilitates their metastasis to the liver. In this study, we investigated genes that are regulated in human colorectal cancer and by the L1-NF-κB pathway that has been implicated in liver metastasis. c-Kit was the most highly suppressed gene in both colorectal cancer tissue and the L1-NF-κB pathway. c-Kit suppression that resulted from L1-mediated signaling relied upon NF-κB, which directly inhibited the transcription of SP1, a major activator of the c-Kit gene promoter. Reconstituting c-Kit expression in L1-transfected cells blocked the biological effects conferred by L1 overexpression in driving motility and liver metastasis. We found that c-Kit expression in colorectal cancer cells is associated with a more pronounced epithelial morphology, along with increased expression of E-cadherin and decreased expression of Slug. Although c-Kit overexpression inhibited the motility and metastasis of L1-expressing colorectal cancer cells, it enhanced colorectal cancer cell proliferation and tumorigenesis, arguing that separate pathways mediate tumorigenicity and metastasis by c-Kit. Our findings provide insights into how colorectal cancer metastasizes to the liver, the most common site of dissemination in this cancer.

Nuclear factor-kappaB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells.

Hyperactivation of beta-catenin-T-cell-factor (TCF)-regulated gene transcription is a hallmark of colorectal cancer (CRC). The cell-neural adhesion molecule L1CAM (hereafter referred to as L1) is a target of beta-catenin-TCF, exclusively expressed at the CRC invasive front in humans. L1 overexpression in CRC cells increases cell growth and motility, and promotes liver metastasis. Genes induced by L1 are also expressed in human CRC tissue but the mechanisms by which L1 confers metastasis are still unknown. We found that signaling by the nuclear factor kappaB (NF-kappaB) is essential, because inhibition of signaling by the inhibitor of kappaB super repressor (IkappaB-SR) blocked L1-mediated metastasis. Overexpression of the NF-kappaB p65 subunit was sufficient to increase CRC cell proliferation, motility and metastasis. Binding of the L1 cytodomain to ezrin - a cytoskeleton-crosslinking protein - is necessary for metastasis because when binding to L1 was interrupted or ezrin gene expression was suppressed with specific shRNA, metastasis did not occur. L1 and ezrin bound to and mediated the phosphorylation of IkappaB. We also observed a complex containing IkappaB, L1 and ezrin in the juxtamembrane region of CRC cells. Furthermore, we found that L1, ezrin and phosphorylated p65 are co-expressed at the invasive front in human CRC tissue, indicating that L1-mediated activation of NF-kappaB signaling involving ezrin is a major route of CRC progression.

L1-CAM in cancerous tissues.

BACKGROUND: L1-cell adhesion molecule (L1-CAM) is a cell adhesion receptor of the immunoglobulin superfamily, known for its roles in nerve cell function. While originally believed to be present only in brain cells, in recent years L1-CAM has been detected in other tissues, and in a variety of cancer cells, including some common types of human cancer. OBJECTIVE/METHODS: We review the prevalence of L1-CAM in human cancer, the possible mechanisms involved in L1-CAM-mediated tumorigenesis, and cancer therapies based upon L1-CAM antibody treatment. RESULTS/CONCLUSIONS: In colon cancer cells, the L1-CAM gene was identified as a target of the Wnt/beta-catenin-TCF signaling pathway, and L1-CAM was exclusively detected at the invasive front of colon and ovarian cancer tissue. The expression of L1-CAM in normal and cancer cells enhanced tumorigenesis and conferred metastasis in colon cancer cells. Antibodies against the L1-CAM ectodomain severely inhibited the proliferation of a variety of cancer cells in culture and reduced tumor burden when injected into mice harboring cancer cells expressing L1-CAM. These results, in addition to the presence of L1-CAM on the cell surface and its restricted distribution in normal tissues, make it an ideal target for tumor therapy.