Probes for TNF

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

The severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus whose sequence is known. COVID-19 is associated with a heterogeneous clinical phenotype ranging from asymptomatic to fatal disease. It appears that access to nasopharyngeal respiratory epithelia expressing angiotensin-converting enzyme (ACE) 2, the receptor for SARS CoV-2, is followed by viral replication in the pulmonary alveolar septal capillary bed. We have shown in prior studies that incomplete viral particles, termed pseudovirions, dock to deep subcutaneous and other vascular beds potentially contributing to the prothrombotic state and systemic complement activation that characterizes severe and critical COVID-19. A variety of skin rashes have been described in the setting of SARS-CoV-2 infection and more recently, following COVID-19 vaccination. The vaccines deliver a laboratory synthesized mRNA that encodes a protein that is identical to the spike glycoprotein of SARS-COV-2 allowing the production of immunogenic spike glycoprotein that will then elicit T cell and B cell adaptive immune responses. In this paper we review an array of cutaneous manifestations of COVID-19 that provide an opportunity to study critical pathophysiologic mechanisms that underlie all clinical facets of COVID-19 ranging from asymptomatic/mild to severe and critical COVID-19. We classify cutaneous COVID-19 according to underlying pathophysiologic principles. In this regard we propose two main pathways: 1) complement mediated thrombotic vascular injury syndromes deploying the alternative and mannan binding lectin pathways in the setting of severe and critical COVID-19 and 2) the robust T cell and type I interferon driven inflammatory and humoral driven immune complex mediated vasculitic cutaneous reactions seen with mild and moderate COVID-19. Novel data on cutaneous vaccine reactions are presented that manifest a clinical and morphologic parallel with similar eruptions seen in patients suffering from mild and moderate COVID-19 and in most cases represent systemic eczematoid hypersensitivity reactions to a putative vaccine based antigen. Finally, we show for the first time the localization of human synthesized spike glycoprotein following the COVID-19 vaccine to the cutaneous and subcutaneous vasculature confirming the ability of SARS CoV-2 spike glycoprotein to bind endothelium in the absence of intact virus.

Immunological characterization of the Fiebig-equivalent stages of SHIVAD8-EO infection showed that, despite the observed differences in progression of infection between the challenge groups, the timing of virus-specific CD8+ T cell responses, as well as the viral load and virus distribution in the LNs, was concordant between the groups when assessed by Fiebig-equivalent staging. LN SHIVAD8-EO RNA+ cells were initially detected in both follicular and extrafollicular areas and mostly preceding peak plasma viremia at Fiebig-equivalent stage II, in agreement with previous studies (31, 41-44). Their levels increased proportionally to viral load in plasma, and seemed to decrease at Fiebig-equivalent stage VI when strong LN SIV-specific CD8+ T cell responses were detected. These responses, which appeared to develop at later stages than in the peripheral blood of acutely HIV-1-infected subjects (45), were predominantly characterized by production of the highly proinflammatory cytokine TNF and expression of CD107a, indicative of degranulation. In addition, levels of fCD8+ T cells, previously shown to be highly cytolytic ex vivo and able to mediate killing of HIV-infected target cells in vitro (31, 46), were higher at Fiebig-equivalent stage VI than stage V. LN NK cell levels also transiently increased at Fiebig-equivalent stage V. Thus, our data are consistent with the notion that, after peak plasma viremia during Fiebig-equivalent stages V and VI of SHIVAD8-EO infection, cytotoxic immune responses develop in the LNs and may lead to lysis of virus-infected cells and release of virions. In fact, LN SHIVAD8-EO RNA+ virions were almost absent at Fiebig-equivalent stage II when SHIVAD8-EO RNA+ cells, likely producing virions (47), were already present but CD8+ T cell responses were undetectable. However, virions were highly abundant in the follicles during Fiebig-equivalent stage VI when CD8+ T cell responses were stronger. Although there was no significant correlation between the levels of LN SHIVAD8-EO RNA+ cells and the virus-specific function of LN CD8+ T cells in our study, possibly because of limited sample size and data variation between animals, previous studies reported in situ observation of CD8+ T cells expressing T cell intracellular antigen-1 (TIA-1) or perforin (48, 49) and increased activation of cytotoxic cells (50) in the LNs of HIV-1-infected subjects. Furthermore, CD8+ T cells with HIV- or SIV-specific killing activity in vitro were detected in the LNs, splenic white pulp, and tonsil GCs of HIV-1-infected subjects (46, 51), and in the LNs of SIV-infected NHPs (31, 52, 53). Also, NK cells were shown to accumulate in the LNs of African green monkeys during nonpathogenic SIV infection, where they played an important role in the control of viral replication (54).