Investigation of SARS-CoV-2 individual proteins reveals the in vitro and in vivo immunogenicity of membrane protein.

Evidence in SARS-CoV-2 patients have identified that viral infection is accompanied by the expression of inflammatory mediators by both immune and stromal cells within the pulmonary system. However, the immunogenicity of individual SARS-CoV-2 proteins has yet to be evaluated. The SARS-CoV-2 virus consists of 29 proteins, categorized either as nonstructural proteins (NSP's), structural proteins (SP's) or accessory proteins. Here we sought to evaluate the immunogenicity of NSP 1, 7, 8, 9, 10, 12, 14, 16 and the SP's spike protein (full length, S1, S2 and receptor binding domain subunits), nucleocapsid and membrane SARS-CoV-2 proteins against THP-1 and human peripheral blood mononuclear cells (PBMCs). Our results indicate that various SARS-CoV-2 proteins elicit a proinflammatory immune response indicated by increases in cytokines TNF, IL-6 and IL-1ß. Our results support that SARS-CoV-2 membrane protein induced a robust increase of TNF, IL-6, IL-1ß and IL-10 expression in both THP-1 and human PBMC's. Further evaluation of intranasal membrane protein challenge in male and female BALB/c mice show increases in BALF (bronchalveolar lavage fluid) proinflammatory cytokine expression, elevated cellularity, predominantly neutrophilic, and concomitant peribronchiolar and perivascular lymphomononuclear and neutrophilic inflammation. Our results suggest that individual membrane associated SARS-CoV-2 proteins induce a robust immune response that may contribute to viral induced cytokine release syndrome (CRS) in the lungs of moderate to severe COVID-19 patients. We posit that SARS-CoV-2 membrane challenges in immune-competent mice can serve as an adequate surrogate for the development of novel treatments for SARS-CoV-2 induced pulmonary inflammation, thereby avoiding expensive live virus studies under BSL-3 conditions.

Implementation of a brief anxiety assessment and evaluation in a Department of Veterans Affairs geriatric primary care clinic.

Anxiety disorders are common and debilitating in older individuals, yet anxiety is often not formally assessed in primary care. We conducted a quality improvement project to examine the feasibility of implementing a brief anxiety assessment, the Geriatric Anxiety Inventory (GAI), in a Department of Veterans Affairs geriatric primary care clinic. We compared the GAI with a depression assessment, the 15-item Geriatric Depression Scale (GDS-15). Fifty older Veterans (mean = 78.5 +/- 7.4 yr) completed the GAI and GDS-15. Mean completion time and feedback to patients was brief (6.20 min; n = 10). Good internal consistency (alpha = 0.82) was found for GAI scores. Patients with psychiatric diagnoses obtained significantly higher GAI scores (mean = 4.73 +/- 1.15) compared with patients without psychiatric diagnoses (mean = 1.15 +/- 1.86, t(11.46) = -3.10, p = 0.01). Findings suggest that the GAI is acceptable to patients but may not be suitable for differentiating anxiety symptoms or disorders from depression. Interdisciplinary team members continued to implement the GAI after project completion to screen for and track anxiety symptoms in our geriatric primary care patients. Detecting anxiety with the GAI had the benefit of allowing providers to initiate conversations about available treatments and track symptoms as part of noting treatment progress.

Uridine Cytidine Kinase Like-1 Enhances Tumor Cell Proliferation and Mediates Protection from Natural Killer-Mediated Killing.

Uridine cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein over-expressed in many tumor cells, especially in highly malignant, aggressive tumors. Sequence analysis indicates that UCKL-1 has homology to uridine kinases, enzymes that play a role in DNA and RNA synthesis and that are often up-regulated in tumor cells. Previous studies have shown that UCKL-1 is a substrate for natural killer lytic-associated molecule (NKLAM), an E3 ubiquitin ligase found in NK cell cytolytic granules. Ubiquitination of UCKL-1 by NKLAM leads to its degradation. Increased expression of NKLAM enhances NK-mediated tumoricidal activity. The fact that UCKL-1 is a substrate for NKLAM suggests that UCKL-1 may provide resistance to NK killing in tumor cells. Here we show that UCKL-1 over-expression protects tumor cells from NK killing and enhances tumor survival in vivo. UCKL-1 also has a much broader role, protecting tumor cells from spontaneous and drug-induced apoptosis and increasing tumor cell proliferation. Nuclear factor-kappa B (NF-κB) activity is higher in tumor cells transfected with UCKL-1 compared to control transfected cells, suggesting at least one possible mechanism by which UCKL-1 influences tumor growth and survival.

E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated NKLAM-KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNß). The JAK/STAT pathway is critical for the production of IFNß, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis.

Natural killer lytic-associated molecule (NKLAM) is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO) mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild-type (WT) mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP), which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow (BM) of NKLAM KO mice 2 weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were 4-5-fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice 2 weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

Platelet-activating factor and metastasis: calcium-independent phospholipase A2ß deficiency protects against breast cancer metastasis to the lung.

We determined the contribution of calcium-independent phospholipase A(2)ß (iPLA(2)ß) to lung metastasis development following breast cancer injection into wild-type (WT) and iPLA(2)ß-knockout (iPLA(2)ß-KO) mice. WT and iPLA(2)ß-KO mice were injected in the mammary pad with 200,000 E0771 breast cancer cells. There was no difference in primary tumor size between WT and iPLA(2)ß-KO mice at 27 days postinjection. However, we observed an 11-fold greater number of breast cancer cells in the lungs of WT mice compared with iPLA(2)ß-KO animals (P < 0.05). Isolated WT lung endothelial cells demonstrated a significant increase in platelet-activating factor (PAF) production when stimulated with thrombin [1 IU/ml, 10 min, 4,330 ± 555 vs. 15,227 ± 1,043 disintegrations per minute (dpm), P < 0.01] or TNF-α (10 ng/ml, 2 h, 16,532 ± 538 dpm, P < 0.01). Adherence of E0771 cells to WT endothelial cells was increased by thrombin (4.8 ± 0.3% vs. 70.9 ± 6.3, P < 0.01) or TNF-α (60.5 ± 4.3, P < 0.01). These responses were blocked by pretreatment with the iPLA(2)ß-selective inhibitor (S)-bromoenol lactone and absent in lung endothelial cells from iPLA(2)ß-KO mice. These data indicate that endothelial cell iPLA(2)ß is responsible for PAF production and adherence of E0771 cells and may play a role in cancer cell migration to distal locations.

Impaired NK cytolytic activity and enhanced tumor growth in NK lytic-associated molecule-deficient mice.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells. It is weakly expressed in resting NK cells but upon target cell stimulation or after incubation with cytokines that enhance NK killing, NKLAM mRNA levels increase and protein is synthesized and is targeted to cytoplasmic granule membranes. We have previously shown that NKLAM plays a role in perforin/granzyme-mediated cytolysis in vitro. To further investigate the function of NKLAM in NK cell-mediated cytotoxicity, we generated, by gene targeting, NKLAM-deficient mice. These mice have normal numbers of NK cells and other lymphoid populations in the spleen. They also have no alterations in NK maturation or NK receptor repertoire. NK cells from NKLAM-deficient and WT mice have comparable amounts of perforin, granzyme B, and lysosomal membrane-associated protein 1 (CD107a) in their cytotoxic granules and comparable levels of granule exocytosis are induced by PMA and calcium ionophore A23187. However, NKLAM-deficient NK cells display significantly less NK cytotoxic activity in vitro than WT NK cells. They also secrete less IFN-gamma upon target cell stimulation, In addition, NKLAM-deficient mice exhibit greater numbers of pulmonary metastases after i.v. injection with B16 melanoma cells. These studies indicate that NKLAM-deficient mice have diminished capacity to control tumor metastases and support the role for NKLAM in NK function both in vitro and in vivo.