Pyrogenic and inflammatory mediators are produced by polarized M1 and M2 macrophages activated with D-dimer and SARS-CoV-2 spike immune complexes.

Lung macrophages are the first line of defense against invading respiratory pathogens including SARS-CoV-2, yet activation of macrophage in the lungs can lead to hyperinflammatory immune response seen in severe COVID-19. Here we used human M1 and M2 polarized macrophages as a surrogate model of inflammatory and regulatory macrophages and explored whether immune complexes (IC) containing spike-specific IgG can trigger aberrant cytokine responses in macrophages in the lungs and associated lymph nodes. We show that IC of SARS-CoV-2 recombinant S protein coated with spike-specific monoclonal antibody induced production of Prostaglandin E2 (PGE2) in non-polarized (M0) and in M1 and M2-type polarized human macrophages only in the presence of D-dimer (DD), a fibrinogen degradation product, associated with coagulopathy in COVID-19.

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8.

Production of fever mediator PGE in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor.

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE production in vitro.

T cell-derived soluble glycoprotein GPIbα mediates PGE production in human monocytes activated with the vaccine adjuvant MDP.

Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E (PGE) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE followed by fever.

Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 10 pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification.

What Is the Predictive Value of Animal Models for Vaccine Efficacy in Humans? The Importance of Bridging Studies and Species-Independent Correlates of Protection.

Animal models have played a pivotal role in all stages of vaccine development. Their predictive value for vaccine effectiveness depends on the pathogen, the robustness of the animal challenge model, and the correlates of protection (if known). This article will cover key questions regarding bridging animal studies to efficacy trials in humans.

An evaluation of novel real-time technology as a tool for measurement of radiobiological and radiation-induced bystander effects.

The xCELLigence real-time cell impedance system uses a non-invasive and label-free method to create a cell index that is a composite measure of cell proliferation. The aim of this study was to evaluate xCELLigence against clonogenic assay (gold standard) for measuring radiobiological effects and radiation-induced bystander effects (RIBE). A radiobiological study was conducted by irradiating EMT6.

Postchallenge administration of brincidofovir protects healthy and immune-deficient mice reconstituted with limited numbers of T cells from lethal challenge with IHD-J-Luc vaccinia virus.

Unlabelled: Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 10(5) PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads.

An evaluation of dose equivalence between synchrotron microbeam radiation therapy and conventional broad beam radiation using clonogenic and cell impedance assays.

Background: High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments.

Aim: To develop an in vitro approach to determine biological dose equivalence between MRT and BB using two different cell-based assays.

Differences in PGE2 production between primary human monocytes and differentiated macrophages: role of IL-1β and TRIF/IRF3.

Prostaglandin E2 (PGE2) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE2 is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE2 in macrophages exclusively via IL-1β-independent mechanisms.

Common fibroid-associated genes are differentially expressed in phenotypically dissimilar cell populations isolated from within human fibroids and myometrium.

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium.

Clonality of smooth muscle and fibroblast cell populations isolated from human fibroid and myometrial tissues.

Uterine fibroids are conventionally defined as clonally derived benign tumours from the proliferation of a single smooth muscle cell (SMC). We have previously identified fibroblast-like cells in fibroids, the presence of which raises the question as to whether all cells within the fibroid have the same clonal origin. The first aim of this study was to develop a fluorescence-activated cell sorting (FACS)-based method to isolate different cell types from human myometrium and fibroid tissues.

Aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids.

Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal.

Effects of postchallenge administration of ST-246 on dissemination of IHD-J-Luc vaccinia virus in normal mice and in immune-deficient mice reconstituted with T cells.

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu(-)/nu(-)) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads.

Fibroid-associated heavy menstrual bleeding: correlation between clinical features, Doppler ultrasound assessment of vasculature, and tissue gene expression profiles.

Despite the prevalence of uterine fibroids (Fs), few studies have investigated the links between clinical features and the cellular or molecular mechanisms that drive F growth and development. Such knowledge will ultimately help to differentiate symptomatic from asymptomatic Fs and could result in the development of more effective and individualized treatments. The aim of this study was to investigate the relationship between ultrasound appearance, blood flow, and angiogenic gene expression in F, perifibroid (PM), and distant myometrial (DM) tissues.

Genome-wide transcription responses to synchrotron microbeam radiotherapy.

The majority of cancer patients achieve benefit from radiotherapy. A significant limitation of radiotherapy is its relatively low therapeutic index, defined as the maximum radiation dose that causes acceptable normal tissue damage to the minimum dose required to achieve tumor control. Recently, a new radiotherapy modality using synchrotron-generated X-ray microbeam radiotherapy has been demonstrated in animal models to ablate tumors with concurrent sparing of normal tissue.

Measurements of vaccinia virus dissemination using whole body imaging: approaches for predicting of lethality in challenge models and testing of vaccines and antiviral treatments.

Preclinical evaluation of novel anti-smallpox vaccines and antiviral treatments often rely on mouse -challenge models using pathogenic vaccinia virus, such as Western Reserve (WR) strain or other orthopoxviruses. Traditionally, efficacy of treatment is evaluated using various readouts, such as lethality (rare), measurements of body weight loss, pox lesion scoring, and determination of viral loads in internal organs by enumerating plaques in sensitive cell lines. These methodologies provide valuable information about the contribution of the treatment to protection from infection, yet all have similar limitations: they do not evaluate dissemination of the virus within the same animal and require large numbers of animals.

Use of human MonoMac6 cells for development of in vitro assay predictive of adjuvant safety in vivo.

Subunit vaccines composed of recombinant or purified antigens have a good safety record but are poorly immunogenic and require adjuvants to activate innate immunity and facilitate antigen specific immune response. Of the many adjuvant formulations that are under development, very few are licensed mainly due to concerns about adverse side effects. The goal of our study was to develop in vitro assays that could predict toxicity of adjuvants in vivo.

Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease.

Background: Previously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV).

Results: Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection.

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs.

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins.

Conformation and thermostability of oligonucleotide d(GGTTGGTGTGGTTGG) containing thiophosphoryl internucleotide bonds at different positions.

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (TBA) is an efficient tool for the inhibition of thrombin function. We have studied conformations and thermodynamic stability of a number of modified TBA oligonucleotides containing thiophosphoryl substitution at different internucleotide sites. Using circular dichroism such modifications were found not to disrupt the antiparallel intramolecular quadruplex specific for TBA.

Application of bioluminescence imaging to the prediction of lethality in vaccinia virus-infected mice.

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence.

DNA modification of live cell surface.

We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface.

Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells.

Background: Despite the fact that uterine fibroids are the most common benign tumors in women, their etiology is poorly understood. We have previously shown that multiple members of the retinoic acid (RA) pathway have altered expression in fibroids compared with normal myometrium. The aims of the present study were: to investigate regulation of genes involved in the RA pathway in vitro; and to identify genes that can be used as markers to distinguish myometrial and fibroid smooth muscle cells in culture.

Regeneration of the adult thymus is preceded by the expansion of K5+K8+ epithelial cell progenitors and by increased expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma.

Studies of HIV-1-infected individuals on anti-retroviral therapies and of patients receiving lymphoablating treatments indicate that the thymus retains restorative capacity even in adults. The contributions of the thymic epithelial cells (TECs) to the regeneration of the thymus and the identity of epithelial cell progenitors were evaluated in murine models of transient thymic atrophy followed by a complete regeneration. Using microarray approach, we analyzed the pattern of gene expression in TECs sorted from mice that were depleted of thymocytes by steroid treatment or by irradiation.