Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates.

Understanding SARS-CoV-2 associated immune pathology is crucial to develop pan-effective vaccines and treatments. Here we investigate the immune events from the acute state up to four weeks post SARS-CoV-2 infection, in non-human primates (NHP) with heterogeneous pulmonary pathology. We show a robust migration of CD16 expressing monocytes to the lungs occurring during the acute phase, and we describe two subsets of interstitial macrophages (HLA-DR+CD206-): a transitional CD11c+CD16+ cell population directly associated with IL-6 levels in plasma, and a long-lasting CD11b+CD16+ cell population. Trafficking of monocytes is mediated by TARC (CCL17) and associates with viral load measured in bronchial brushes. We also describe associations between disease outcomes and high levels of cell infiltration in lungs including CD11b+CD16hi macrophages and CD11b+ neutrophils. Accumulation of macrophages is long-lasting and detectable even in animals with mild or no signs of disease. Interestingly, animals with anti-inflammatory responses including high IL-10:IL-6 and kynurenine to tryptophan ratios show less severe illness. Our results unravel cellular mechanisms of COVID-19 and suggest that NHP may be appropriate models to test immune therapies.

Azolla along a phosphorus gradient: biphasic growth response linked to diazotroph traits and phosphorus-induced iron chlorosis.

Azolla spp., a water fern often used for phytoremediation, is a strong phosphorus (P) accumulator due to its high growth rate and N2 fixing symbionts (diazotrophs). It is known that plant growth is stimulated by P, but the nature of the interactive response of both symbionts along a P gradient, and related changes in growth-limiting factors, are unclear. We determined growth, and N and P sequestration rates of Azolla filiculoides in N-free water at different P concentrations. The growth response appeared to be biphasic and highest at levels ≥10 P µmol l-1. Diazotrophic N sequestration increased upon P addition, and rates were three times higher at high P than at low P. At 10 µmol P l-1, N sequestration rates reached its maximum and A. filiculoides growth became saturated. Due to luxury consumption, P sequestration rates increased until 50 µmol P l-1. At higher P concentrations (≥50 µmol l-1), however, chlorosis occurred that seems to be caused by iron- (Fe-), and not by N-deficiency. We demonstrate that traits of the complete symbiosis in relation to P and Fe availability determine plant performance, stressing the role of nutrient stoichiometry. The results are discussed regarding Azolla's potential use in a bio-based economy.

Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana.

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 = 1.6 µg/kg) and resulted in a mean time from inoculation to death of 18 and 180 min when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 min post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication.

Mucosal vaccines for biodefense.

Bioterrorism is the deliberate release of biological toxins, pathogenic viruses, bacteria, parasites, or other infectious agents into the public sphere with the objective of causing panic, illness, and/or death on a local, regional, or possibly national scale. The list of potential biological agents compiled by the Centers for Disease Control and Prevention is long and diverse. However, a trait common to virtually all the potential bioterrorism agents is the fact that they are likely to be disseminated by either aerosol or in food/water supplies with the intention of targeting the mucosal surfaces of the respiratory or gastrointestinal tracts, respectively. In some instances, inhalation or ingestion would mimic the natural route by which humans are exposed to these agents. In other instances, (e.g., the inhalation of a toxin is normally associated with food borne illness), it would represent an unnatural route of exposure. For most potential bioterrorism agents, the respiratory or gastrointestinal mucosa may simply serve as a route of entry by which they gain access to the systemic compartment where intoxication/replication occurs. For others, however, the respiratory or gastrointestinal mucosa is the primary tissue associated with pathogenesis, and therefore, the tissue for which countermeasures must be developed.

Aerobiology and inhalation exposure to biological select agents and toxins.

Aerosol is the most likely route of dissemination of biological select agents and toxins in a bioterrorist attack, regardless of the natural route of exposure to the agent. The use of animal models for testing preventative and therapeutic countermeasures requires knowledge of the pathogenesis of disease after inhalation exposure. Factors that relate to outcome after respiratory exposure include the inherent infectivity and virulence and/or toxicity of the agent in the host under investigation, in addition to characteristics of the aerosol particle and host that affect the delivered dose of, and host response to, the inhaled material. This introductory article discusses the emerging science of aerobiology and the unique features of respiratory tract anatomy, physiology, and immunology that are relevant to the pathogenesis of aerosolized biothreat agents.

Growth mechanism of confined polyelectrolyte multilayers in nanoporous templates.

We investigate the mechanism of polyelectrolyte multilayer (PEM) assembly in nanoporous templates with a view to synthesizing nanotubes or nanowires under optimal conditions. For this purpose, we focus on the effect of parameters related to the geometrical constraints (pore diameter), the size of the macromolecules (their molar mass and the ionic strength), and the interaction between the pore walls and the adsorbed chains (modulated by the ionic strength). Our results reveal the existence of two regimes in the mechanism of PEM growth: (i) the first regime is comparable to that observed on flat substrates, including the influence of ionic strength and (ii) the second regime, which is slower in terms of kinetics, results from the interconnection established between polyelectrolyte chains across the pores and leads to the formation of a dense gel. As a consequence, the diffusion of polyelectrolytes in nanopores becomes the controlling factor of PEM growth in this second regime. The dense gel, owing to its peculiar structure, enhances the formation of nanowires or of partially occluded nanotubes in some cases, depending on initial pore dimensions.

Long term changes in atmospheric N and S throughfall deposition and effects on soil solution chemistry in a Scots pine forest in the Netherlands.

In a Scots pine forest the throughfall deposition and the chemical composition of the soil solution was monitored since 1984. (Inter)national legislation measures led to a reduction of the deposition of nitrogen and sulphur. The deposition of sulphur has decreased by approximately 65%. The total mineral-nitrogen deposition has decreased by ca. 25%, which is mainly due to a reduction in ammonium-N deposition (-40%), since nitrate-N deposition has increased (+50%). The nitrogen concentration in the upper mineral soil solution at 10 cm depth has decreased, leading to an improved nutritional balance, which may result in improved tree vitality. In the drainage water at 90 cm depth the fluxes of NO3(-) and SO4(2-) have decreased, resulting in a reduced leeching of accompanying base cations, thus preserving nutrients in the ecosystem. It may take still several years, however, before this will meet the prerequisite of a sustainable ecosystem.

Diverse TCRs recognize murine CD1.

Human and murine T cells that specifically recognize CD1d and produce IL-4 and IFN-gamma play a role in immunoregulation and tumor rejection. In the mouse, most CD1d1-reactive T cells described express an invariant Valpha14-Jalpha281 TCR associated with TCR beta-chains of limited diversity. Similarly, human CD1d-reactive T cells express a highly restricted TCR repertoire. Here we report the unexpected result that in mice immunized with CD1d1-bearing transfectant cells, a diverse repertoire of TCRs was expressed by CD1d1-reactive T cell clones isolated by limiting dilution without preselection for NK1 expression. Only 3 of 10 CD1d1-reactive T cell clones expressed the invariant Valpha14-Jalpha281 TCRalpha rearrangement. T cells expressing Valpha10, -11, -15, and -17, and having non-germline-encoded nucleotides resulting in diverse V-J junctions were identified. Like CD1d1-reactive T cells expressing the invariant Valpha14-Jalpha281 TCR alpha-chain, CD1d1-reactive clones with diverse TCRs produced both Type 1 (IFN-y) and Type 2 (IL-4, IL-10) cytokines. This establishes the existence of significant diversity in the TCRs directly reactive to the CD1d1 protein. Our findings reveal that CD1d interacts with a broad array of TCRs, suggesting substantial redundancy and flexibility of the immune system in providing T cells serving the role(s) mediated by CD1d reactivity.

Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation.

We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.