Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia

Fungal infections have skyrocketed in immune-compromised patients lacking CD4⁺ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A⁺ CD8⁺ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4⁺ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ⁺ CD8⁺ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8⁺ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.     

Physiologically based pharmacokinetic/pharmacodynamic model for the prediction of morphine brain disposition and analgesia in adults and children

Morphine is a widely used opioid analgesic, which shows large differences in clinical response in children, even when aiming for equivalent plasma drug concentrations. Age-dependent brain disposition of morphine could contribute to this variability, as developmental increase in blood-brain barrier (BBB) P-glycoprotein (Pgp) expression has been reported. In addition, age-related pharmacodynamics might also explain the variability in effect. To assess the influence of these processes on morphine effectiveness, a multi-compartment brain physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) model was developed in R (Version 3.6.2). Active Pgp-mediated morphine transport was measured in MDCKII-Pgp cells grown on transwell filters and translated by an in vitro-in vivo extrapolation approach, which included developmental Pgp expression. Passive BBB permeability of morphine and its active metabolite morphine-6-glucuronide (M6G) and their pharmacodynamic parameters were derived from experiments reported in literature. Model simulations after single dose morphine were compared with measured and published concentrations of morphine and M6G in plasma, brain extracellular fluid (ECF) and cerebrospinal fluid (CSF), as well as published drug responses in children (1 day– 16 years) and adults. Visual predictive checks indicated acceptable overlays between simulated and measured morphine and M6G concentration-time profiles and prediction errors were between 1 and -1. Incorporation of active Pgp-mediated BBB transport into the PB-PK/PD model resulted in a 1.3-fold reduced brain exposure in adults, indicating only a modest contribution on brain disposition. Analgesic effect-time profiles could be described reasonably well for older children and adults, but were largely underpredicted for neonates. In summary, an age-appropriate morphine PB-PK/PD model was developed for the prediction of brain pharmacokinetics and analgesic effects. In the neonatal population, pharmacodynamic characteristics, but not brain drug disposition, appear to be altered compared to adults and older children, which may explain the reported differences in analgesic effect.     

Novel 3-nitro-1H-1,2,4-triazole-based piperazines and 2-amino-1,3-benzothiazoles as antichagasic agents

We have previously shown that 3-nitro-1H-1,2,4-triazole-based amines demonstrate significant trypanocidal activity, in particular against Trypanosoma cruzi, the causative parasite of Chagas disease. In the present work we further expanded our research by evaluating in vitro the trypanocidal activity of nitrotriazole-based piperazines and nitrotriazole-based 2-amino-1,3-benzothiazoles to establish additional SARs. All nitrotriazole-based derivatives were active or moderately active against T. cruzi; however two of them did not fulfill the selectivity criteria. Five derivatives were active or moderately active against Trypanosoma brucei rhodesiense while one derivative was moderately active against Leishmania donovani. Active compounds against T. cruzi demonstrated selectivity indexes (toxicity to host cells/toxicity to T. cruzi amastigotes) from 117 to 1725 and 12 of 13 compounds were up to 39-fold more potent than the reference compound benznidazole. Detailed SARs are discussed.     

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N₂) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N₂ fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N₂ fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N₂ fixation and photosynthesis commonly observed. However, N₂ fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.