CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.     

Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis

Background

Wear particle-induced periprosthetic osteolysis that results in aseptic loosening is the most common cause of long-term failure after total joint replacement.

Materials and methods

Icariin (ICA), a flavonoid isolated from Epimedium pubescens, inhibits osteoclast formation, but its effects on wear particle-induced inflammatory osteoclastogenesis remains unclear. We investigated the role of ICA in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which is stimulated by titanium (Ti) particles and the receptor activator of NF-κB ligand.

Results

ICA effectively inhibited osteoclast formation and bone resorption in the differentiation medium. ICA (10⁻⁷ mol/L) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells compared with the control, and significantly reduced the percentage of the surface covered by resorption lacunae. Quantitative real-time polymerase chain reaction analysis showed that ICA inhibited messenger RNA expression for the receptor activator of nuclear factor-κB, cathepsin K, tartrate-resistant acid phosphatase-positive, and matrix metalloproteinase-9 in RAW264.7 cells stimulated by Ti particles and receptor activator of NF-κB ligand. ICA also reduced pro-inflammatory cytokine expression of interleukin-1β and tumor necrosis factor-α in RAW264.7 cells cultured with Ti particles. In addition, incubation with cholecystokinin-8 showed that ICA had no toxic effects on RAW264.7 cells.

Conclusions

ICA possibly elicited inhibitory effects on inflammatory osteoclastogenesis induced by Ti particles, indicating that ICA may be useful for the prevention and treatment of wear particle-induced osteolysis.     

Aging aggravates long-term renal ischemia-reperfusion injury in a rat model

Aim

Ischemia-reperfusion injury (IRI) has been considered as the major cause of acute kidney injury and can result in poor long-term graft function. Functional recovery after IRI is impaired in the elderly. In the present study, we aimed to compare kidney morphology, function, oxidative stress, inflammation, and development of renal fibrosis in young and aged rats after renal IRI.

Materials and methods

Rat models of warm renal IRI were established by clamping left pedicles for 45 min after right nephrectomy, then the clamp was removed, and kidneys were reperfused for up to 12 wk. Biochemical and histologic renal damage were assessed at 12 wk after reperfusion. The immunohistochemical staining of monocyte macrophage antigen-1 (ED-1) and transforming growth factor beta 1 (TGF-β1) and messenger RNA level of TGF-β1 in the kidney were analyzed.

Results

Renal IRI caused significant increases of malondialdehyde and 8-hydroxydeoxyguanosine levels and a decrease of superoxide dismutase activity in young and aged IRI rats; however, these changes were more obvious in the aged rats. IRI resulted in severe inflammation and tubulointerstitial fibrosis with decreased creatinine (Cr) clearance and increased histologic damage in aged rats compared with young rats. Moreover, we measured the ratio of Cr clearance between young and aged IRI rats. It demonstrated that aged IRI rats did have poor Cr clearance compared with the young IRI rats. ED-1 and TGF-β1 expression levels in the kidney were significantly higher in aged rats than in young rats after IRI.

Conclusion

Aged rats are more susceptible to IRI-induced renal failure, which may associate with the increased oxidative stress, increased histologic damage, and increased inflammation and tubulointerstitial fibrosis. Targeting oxidative stress and inflammatory response should improve the kidney recovery after IRI.     

Synthesis of phenylazonaphtol-β-<Emphasis Type="SmallCaps">D</Emphasis>-<Emphasis Type="Italic">O</Emphasis>-glycosides,evaluation as substrates for beta-glycosidase activity and molecular studies

Background

Phenylazonaphtol-β-D-O-glycosides are alternative substrates for the detection of enzymatic activity of β-glycosidases which are involved in various important processes. These azoic compounds are currently exploited as prodrugs for colonic disease due the presence of β-glycosidase activity in the gut flora and therefore allowing the release of the drug at the specific site.

Results

Phenylazonaphtol-β-D-O-glucoside 3a and galactoside 3b were prepared via diazonium salt conditions under weak acidic conditions which do not compromise the O-glycosidic bond stability, by coupling reaction between 2-naphtol sodium salt with aminoglycosides 1a and 1b. The resulting phenylazonaphtol glycosides 2a and 2b were deprotected affording the phenylazonaphtol glycosides 3a and 3b in quantitative yield. The galactoside glycoside 3b was assayed as substrate for in vitro β-galactosidase enzymatic activity showing strong absorbance after releasing of the azoic chromophore. Also, docking studies were performed to determine the best pose as well as the interactions between the ligand and the residues located at the active site.

Conclusions

The methodology developed for synthesizing the phenylazonaphtol glycosides described proved to be convenient for generating azoic functionalities in the presence of glycosidic bonds and the glycosides suitable as alternative substrates and potentially useful prodrugs in the treatment of colonic diseases.

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Coagulopathy in COVID‐19

The COVID‐19 pandemic has become an urgent issue in every country. Based on recent reports, the most severely ill patients present with coagulopathy, and disseminated intravascular coagulation (DIC)‐like massive intravascular clot formation is frequently seen in this cohort. Therefore, coagulation tests may be considered useful to discriminate severe cases of COVID‐19. The clinical presentation of COVID‐19‐associated coagulopathy is organ dysfunction primarily, whereas hemorrhagic events are less frequent. Changes in hemostatic biomarkers represented by increase in D‐dimer and fibrin/fibrinogen degradation products indicate the essence of coagulopathy is massive fibrin formation. In comparison with bacterial‐sepsis‐associated coagulopathy/DIC, prolongation of prothrombin time, and activated partial thromboplastin time, and decrease in antithrombin activity is less frequent and thrombocytopenia is relatively uncommon in COVID‐19. The mechanisms of the coagulopathy are not fully elucidated, however. It is speculated that the dysregulated immune responses orchestrated by inflammatory cytokines, lymphocyte cell death, hypoxia, and endothelial damage are involved. Bleeding tendency is uncommon, but the incidence of thrombosis in COVID‐19 and the adequacy of current recommendations regarding standard venous thromboembolic dosing are uncertain.