Prednisolone Targets Claudins in Mouse Brain Blood Vessels

Endothelial cells in brain capillaries are crucial for the function of the blood–brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB’s permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood–brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid–base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure

SARS-CoV-2-Induced Pathology—Relevance to COVID-19 Pathophysiology

In spite of intensive studies of different aspects of a new coronavirus infection, many issues still remain unclear. In a screening analysis of histopathology in l200 lethal cases, authors succeeded in performing a wide spectrum of immune histochemical reactions (CD2, CD 3, CD 4, CD 5, CD 7, CD 8, CD14, CD 20, CD 31, CD 34, CD 56, CD 57, CD 68, CD 163, collagen 1,3, spike protein SARS-CoV-2, caspase-3, MLCM; ACE2 receptor, occludin, and claudin-1 and -3) and electron microscopy. The results of the histological and IHC studies of deceased people with varying degrees of severity of coronavirus infection confirmed the ability of these pathogens to cause cytoproliferative changes, primarily in epithelial and endothelial cells. Lesions of various organs are possible, while the reasons for significant differences in organotropy remain unclear. Severe respiratory failure in COVID-19 in humans is associated with a very peculiar viral pneumonia. In the pathogenesis of COVID-19, the most important role is played by lesions of the microcirculatory bed, the genesis of which requires further study, but direct viral damage is most likely. Endothelial damage can be associated with both thrombosis in vessels of various calibers, leading to characteristic complications, and the development of DIC syndrome with maximal kidney damage. Such lesions can be the basis of clinically diagnosed septic shock, while usually there are no morphological data in favor of classical sepsis caused by bacteria or fungi. A massive infiltration of the lung tissue and other organs, mainly by T lymphocytes, including those with suppressor properties, makes it necessary to conduct a differential diagnosis between the morphological manifestation of the protective cellular immune response and direct viral lesions but does not exclude the hypothesis of an immunopathological component of pathogenesis. In many of the deceased, even in the absence of clear clinical symptoms, a variety of extrapulmonary lesions were also detected. The mechanism of their development probably has a complex nature: direct lesions associated with the generalization of viral infection and vascular disorders associated with endothelial damage and having an autoimmune nature. Many aspects of the pathogenesis of coronavirus infection require further comprehensive study

Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP)

<div><p>Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein expressed exclusively in lymphocytes. LPAP is a component of a supramolecular complex composed of the phosphatase CD45, the co-receptor CD4, and the kinase Lck. In contrast to its immunologically important partners, the function of LPAP is unknown. We hypothesized that the biological role of LPAP may be determined by analyzing LPAP phosphorylation. In the present study, we identified LPAP phosphorylation sites by site-directed mutagenesis, phospho-specific antibodies, and protein immunoprecipitation coupled to mass spectrometry analysis. Our results confirmed previous reports that Ser-99, Ser-153, and Ser-163 are phosphorylated, as well as provided evidence for the phosphorylation of Ser-172. Using various SDS-PAGE techniques, we detected and quantified a set of LPAP phosphoforms that were assigned to a combination of particular phosphorylation events. The phosphorylation of LPAP appears to be a tightly regulated process. Our results support the model: following phorbol 12-myristate 13-acetate (PMA) or TCR/CD3 activation of T cells, LPAP is rapidly dephosphorylated at Ser-99 and Ser-172 and slowly phosphorylated at Ser-163. Ser-153 exhibited a high basal level of phosphorylation in both resting and activated cells. Therefore, we suggest that LPAP may function as a co-regulator of T-cell signaling.</p></div

LPAP phosphorylation in resting CEM cells.

<p>LPAP-deficient CEM cells stably transfected with LPAP mutants were lysed in TX-100. Immunoprecipitated LPAP was either treated (+) or untreated (-) with the phosphatase CIP as indicated and subjected to 2D-DIGE or phosphate-affinity SDS-PAGE. For 2D-DIGE analysis LPAP was Cy3-labeled (green, CIP-treated) or Cy5-labeled (red, CIP-untreated). (A) 2D-DIGE analysis of LPAP Δ163–206 and Δ153–206 deletion mutants. Arrows indicate the positions of Δ163–206 and Δ153–206 deletion mutants, which were run on the same gel. (B) phosphorylation of S99A LPAP mutant (first column), S153A (second column), triple mutant S99A/S153A/S172A (third column), and triple mutant S99A/S155A/S172A (fourth column) detected by 2D-DIGE. (C) LPAP phosphorylation detected by Phos-tag SDS-PAGE. LPAP point mutants S99A (lanes 3–4), S153A (lanes 5–6, 9–10), S172A (lanes 7–8) or LPAP from parental CEM (lanes 1–2) were subjected to Phos-tag SDS-PAGE and blotted with anti-LPAP antibody. Lanes 9–10 were exposed for longer time to show minor bands (right panel). LPAP bands were named P0, P1, P2, P3, and P4 according to their relative mobility from fast to slow and assigned to the phosphorylation sites. The phosphorylation site(s) of each band were assigned as described on the left side: P0, unphosphorylated; P1, Ser-153; P2, Ser-99/Ser-153 and Ser-99; P3, Ser-172/Ser-153 and Ser-172; P4, Ser-99/Ser172/Ser-153 and Ser-99/Ser-172. (D) graph of band intensities ± s.d. (n = 5) in Phos-tag SDS-PAGE for resting CEM cells.</p

LPAP mutations that affect phosphorylation-dependent mobility shift.

<p>LPAP-deficient CEM cells stably transfected with LPAP were lysed in TX-100. Cell lysates were immunoprecipitated with anti-LPAP antibody. Samples were either treated (+) or untreated with the phosphatase CIP (-) as indicated and subjected to 18% SDS-PAGE or 2D-PAGE. (A) Western blot analysis of LPAP alanine point mutants. LPAP from parental CEM cells (lanes 1–2) was included for comparison. (B) 2D-DIGE analysis of S172A LPAP mutant. Cy3-labeled LPAP (green) was dephosporylated (indicated as +CIP), whereas Cy5-labeled LPAP (red) was left untreated (-CIP).</p

Schematic of LPAP structure with phosphorylation site prediction and representation of LPAP deletion and point mutants.

<p>(A) schematic representation of LPAP domains: SP, signal peptide; EX, extracellular; TR, transmembrane; Lck, Lck binding domain; LCR, low complexity region domain; a.a., amino acids; CL3, CL7, epitopes of anti-LPAP mAbs. Numbers indicate the position of amino acids. (B) phosphosites predicted by NetPhos 2.0. C, point and deletion LPAP mutants used in this work.</p

LPAP phosphorylation in activated CEM and Jurkat cells.

<p>(A) CEM cells were activated with PMA (10 ng/ml) for 30 min, lysed and immunoprecipitated with anti-LPAP antibody. Samples of phosphatase-treated (+CIP) Cy3-labeled (green) and untreated (-CIP) Cy5-labeled (red) were mixed, run on 2D-PAGE and visualized with a fluorescent gel scanner. (B) CEM cells were incubated for different times at 37°C with 10 ng/ml PMA and lysed. Immunoprecipitated protein was resolved on Phos-tag SDS-PAGE (upper panel) or conventional 18% SDS-PAGE (lower panel) and blotted with anti-LPAP antibody. Samples in lane 5 were not PMA-activated but treated with phosphatase (indicated as 0 + CIP). (C) phosphorylation of LPAP from PMA-activated parental CEM (lanes 3–4) or cells stably transfected with mutant S163A (lanes 5–6) or double mutant S99A/S153A (lanes 7–8) detected by phosphate-affinity SDS-PAGE. LPAP from resting parental CEM cells is included as a control (lanes 1–2). Arrows show activation-dependent bands in CEM-wt and S99A/S153A mutant, but not in S163A mutant. (D) Wild type Jurkat cells (wt) or Lck-deficient JCaM 1.6 cells (KO) were activated with PMA (10 ng/ml) for 30 min or OKT3 (1 μg/ml) mAb for 15 min, lysed and immunoprecipitated with anti-LPAP antibody. Immunopurified protein was resolved on 18% SDS-PAGE and blotted with anti-LPAP antibody.</p