SARS-CoV-2 infection inducing severe flare up of Deficiency of Interleukin Thirty-six (IL-36) Receptor Antagonist (DITRA) resulting from a mutation invalidating the activating cleavage site of the IL-36 receptor antagonist

Journal of Clinical Immunology -     

Distinct roles of the photosystem II protein PsbS and zeaxanthin in the regulation of light harvesting in plants revealed by fluorescence lifetime snapshots

The photosystem II (PSII) protein PsbS and the enzyme violaxanthin deepoxidase (VDE) are known to influence the dynamics of energy-dependent quenching (qE), the component of nonphotochemical quenching (NPQ) that allows plants to respond to fast fluctuations in light intensity. Although the absence of PsbS and VDE has been shown to change the amount of quenching, there have not been any measurements that can detect whether the presence of these proteins alters the type of quenching that occurs. The chlorophyll fluorescence lifetime probes the excited-state chlorophyll relaxation dynamics and can be used to determine the amount of quenching as well as whether two different genotypes with the same amount of NPQ have similar dynamics of excited-state chlorophyll relaxation. We measured the fluorescence lifetimes on whole leaves of Arabidopsis thaliana throughout the induction and relaxation of NPQ for wild type and the qE mutants, npq4, which lacks PsbS; npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin; and npq1 npq4, which lacks both VDE and PsbS. These measurements show that although PsbS changes the amount of quenching and the rate at which quenching turns on, it does not affect the relaxation dynamics of excited chlorophyll during quenching. In addition, the data suggest that PsbS responds not only to ΔpH but also to the Δψ across the thylakoid membrane. In contrast, the presence of VDE, which is necessary for the accumulation of zeaxanthin, affects the excited-state chlorophyll relaxation dynamics.Plants regulate light harvesting by photosystem II (PSII) in response to changes in light intensity. One way that plants are able to regulate light harvesting is through turning on and off mechanisms that dissipate excess energy. This energy dissipation is assessed via nonphotochemical quenching (NPQ) measurements of chlorophyll fluorescence. Energy-dependent quenching (qE) is the NPQ process with the fastest kinetics. It turns on and off in seconds to minutes, allowing plants to respond to rapid fluctuations in light intensity, which is thought to reduce photodamage (1, 2).Illumination causes the formation of gradients of electrical potential (Δψ) and of proton concentration (ΔpH) across the thylakoid membrane. Although it has been suggested that Δψ may play a role in qE (3), only ΔpH is thought to trigger different proteins and enzymes to induce qE (4). The major known factors involved in induction of qE are the enzyme violaxanthin deepoxidase (VDE) (5) and the PSII protein PsbS (6). The mutant npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin, has a phenotype with lower qE compared with the wild type (7). Transient absorption measurements suggest that zeaxanthin may quench excited chlorophyll (8). The npq4 mutant, which lacks PsbS, shows no rapidly reversible quenching of chlorophyll fluorescence, suggesting that PsbS is required for qE in vivo (6). PsbS is pH sensitive (9) but is not thought to bind pigments, and thus is likely not the site of quenching (10). It has therefore been hypothesized that PsbS plays an indirect role in quenching, perhaps facilitating a rearrangement of proteins within the grana (11–13). In this paper, we examine the fluorescence lifetime of chlorophyll throughout the induction and relaxation of quenching in intact leaves with and without PsbS and zeaxanthin to examine whether PsbS and zeaxanthin change the type of quenching that occurs in plants.The amount and dynamics of qE are generally measured by changes in the chlorophyll fluorescence yield. One limitation of the chlorophyll fluorescence yield is that it can only inform on the amount of quenching, not on excited-state chlorophyll relaxation dynamics, which reflect how chlorophyll is quenched. Despite this issue, the amount of quenching is commonly used as a proxy for the type of quenching by separating components of quenching based on kinetics, mutants, and the effects of chemical inhibitors. By artificially increasing ΔpH in isolated chloroplasts from npq4, Johnson and Ruban (14, 15) have been able to increase the amount of quenching in npq4 plants to levels observed in wild type plants, suggesting that PsbS may catalyze qE. One potential complication with these studies is that the use of the chemical mediators of cyclic electron transport often necessitates studying isolated chloroplasts rather than intact leaves. In addition, the observation of equivalent amounts of quenching still does not prove that the type of quenching in npq4 is the same as in wild type.In contrast with fluorescence yield measurements, fluorescence lifetime measurements can be used to determine whether the relaxation dynamics of excited chlorophyll are modified by different mutations, informing on the role of a protein or molecule during quenching. The relaxation dynamics of excited chlorophyll during NPQ depends on many variables, including the distance to a quencher, the interactions between the orbitals of chlorophyll and the quencher, and the number of quenchers (16). The shape of the fluorescence lifetime decay curve can be used to determine whether two samples have similar excited chlorophyll relaxation dynamics. Our results show that, although the presence of PsbS does not alter excited chlorophyll relaxation dynamics, the absence of VDE does. These measurements are performed in intact leaves without any chemical treatments, and the data strongly suggest that PsbS plays a catalytic role in vivo.     

Investigation of the Possible Functions of PACAP in Human Trophoblast Cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.     

HLA-G,pre-eclampsia,immunity and vascular events

Pre-eclampsia, one of the main complications in pregnancy, is characterised by shallow cytotrophoblast invasion of decidua as well as by vascular endothelial cell dysfunction, leading to a poor perfusion of placenta. A striking feature of pre-eclamptic pregnancies is that expression of HLA-G protein is reduced in term placentas compared with normal pregnancy. How such HLA-G deficient expression may be related to the pre-eclamptic pathology is unknown. Here, we review the major structural characteristics of HLA-G and some of its functions that have been recently characterised. Soluble HLA-G1 isoform down-regulates both CD8(+) and CD4(+) T cell reactivity. HLA-G also modulates innate immunity by binding to several NK and/or decidual receptors, inducing particular cytokine secretion. HLA-G was shown to be less susceptible to human cytomegalovirus-derived US protein down-modulation. Finally, soluble HLA-G1 down-regulates endothelial cell proliferation and migration. In view of these different HLA-G properties, we will briefly discuss how defective HLA-G function may contribute to the low trophoblast invasion and vascular abnormalities observed in pre-eclamptic placentas.     

Effects of PACAP on Mitochondrial Apoptotic Pathways and Cytokine Expression in Rats Subjected to Renal Ischemia/Reperfusion

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly efficient cytoprotective actions. Its neuroprotective effects are well-known, but PACAP is able to exert similar actions in non-neuronal cells. Recently, we have shown that PACAP prolongs renal ischemic time, decreases mortality, and attenuates tubular degeneration in a rat model of renal ischemia/reperfusion, but the mechanism of renoprotection is not yet known. Therefore, the aim of the present study was to obtain further insight into the renoprotective effects of PACAP by examining its direct effects of PACAP on mitochondrial permeability transition in vitro and on the expression of the anti-apoptotic Bcl-2 and cytokines/chemokines in kidney tissues following 45 and 60 min renal ischemia and reperfusion in vivo. We found that PACAP did not have any direct effect on mitochondrial permeability transition. Cytokine array revealed that the expression of a few cytokines/chemokines was strongly increased after ischemia/reperfusion, which was ameliorated by PACAP treatment. Furthermore, in rats subjected to renal ischemia, PACAP treatment counteracted the ischemia/reperfusion-induced decrease of the anti-apoptotic Bcl-2, both after 45 and 60 min ischemia, as analyzed by Western blot. In summary, we showed that PACAP could attenuate tissue injury involving both anti-inflammatory and anti-apoptotic effects, but not directly acting on mitochondrial permeability transition.     

Changes in the Expression of Pituitary Adenylate Cyclase-Activating Polypeptide in the Human Placenta during Pregnancy and Its Effects on the Survival of JAR Choriocarcinoma Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.     

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity

We used well-characterized and positively charged nanoparticles (NP⁺) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP⁺ physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP⁺ surface charge was significantly reduced, proportionally to NP⁺/serum and NP⁺/BSA ratios, while NP⁺ size was not modified. Microscopy studies showed high endocytosis of NP⁺ in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP⁺. No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP⁺ genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP⁺ might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.