Chemotactic effect of urokinase-type plasminogen activator on mouse spermatozoa in vitro

The aim of this study is to investigate the chemotactic effect of urokinase-type plasminogen activator (uPA) on mouse spermatozoa. Capillary assays were applied to study the chemotactic activity of ascending and descending gradients of uPA. Firstly, the chemotactic effect of an ascending gradient of uPA on mouse spermatozoa was observed by counting the number of spermatozoa that migrated into the capillary after incubation with uPA for 5, 10, 20, and 30 min, respectively, compared with that after incubation with F10. Twenty minutes was a suitable incubation time to obtain a plateau of sperm accumulation. Meanwhile, to confirm the specific effect of uPA on mouse sperm chemotaxis, uPA inhibitor (PAI-1) and anti-uPAR rabbit IgG were added to the test solution containing 20 U/mL uPA, respectively. To exclude the possibility that PAI-1 and anti-uPAR rabbit IgG may affect sperm accumulation nonspecifically, PAI-1 and anti-uPAR rabbit IgG were added to F10, respectively. It was found that the chemotactic effect of uPA was neutralized completely by PAI-1 and anti-uPAR rabbit IgG. PAI-1 and anti-uPAR rabbit IgG had no neutralizing effect on the sperm chemotactic effect. Lastly, the sperm chemotaxis response to a descending gradient of uPA was also observed. Taken together, the results suggest that uPA can induce sperm chemotaxis in vitro by binding to its receptor on the sperm membrane and may act as a chemoattractant in precontacting sperm-egg communication thereby increasing the chance encounter of spermatozoa and eggs.     

Aqueous Extract of Rosmarinus officinalis L. Inhibits Neutrophil Influx and Cytokine Secretion

Rosmarinus officinalis L. phenolic compounds have attracted considerable attention because of their antioxidant and antimicrobial properties, including its ability to treat inflammatory disorders. In this work, we investigated the in vivo and in vitro effects of R. officinalis aqueous extract on neutrophil trafficking from the blood into an inflamed tissue, on cell‐derived secretion of chemical mediators, and on oxidative stress. Anti‐inflammatory activity was investigated using carrageenan‐induced inflammation in the subcutaneous tissue of male Wistar rats orally treated with the R. officinalis extract (100, 200, or 400 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (prostaglandin E2 (PGE2), TNF‐α, interleukin 6 (IL‐6), leukotriene B4 (LTB4), and cytokine‐induced neutrophil chemoattractant 1 by enzyme‐linked immunosorbent assay), and the anti‐oxidative profile (super oxide dismutase (SOD), glutathione peroxidase, and thiobarbituric acid reactive substance (TBARS) spectrophotometry) were quantified in the inflamed exudate. N‐Formyl‐methionine‐leucine‐phenylalanine‐induced chemotaxis, lipopolysaccharide‐induced NO₂^? production (Greiss reaction), and adhesion molecule expression (flow cytometry) were in vitro quantified using oyster glycogen recruited peritoneal neutrophils previous treated with the extract (1, 10, or 100 µg/mL). Animals orally treated with phosphate‐buffered saline and neutrophils incubated with Hank's balanced salt solution were used as control. R. officinalis extract oral treatment caused a dose‐dependent reduction in the neutrophil migration as well as decreased SOD, TBARS, LTB4, PGE2, IL‐6, and TNF‐α levels in the inflamed exudate. In vitro treatment with R. officinalis decreased neutrophil chemotaxis, NO₂^? production, and shedding of L‐selectin and β2 integrin expressions. Results here presented show that R. officinalis aqueous extract displays important in vivo and in vitro anti‐inflammatory actions by blocking pathways of neutrophil migration and secretion, suggesting its therapeutic application to acute inflammatory reactions. Copyright © 2014 John Wiley & Sons, Ltd.     

Enhanced Human Neutrophil Vitamin C Status,Chemotaxis and Oxidant Generation Following Dietary Supplementation with Vitamin C-Rich SunGold Kiwifruit

Neutrophils are the body’s primary defenders against invading pathogens. These cells migrate to loci of infection where they engulf micro-organisms and subject them to an array of reactive oxygen species and antimicrobial proteins to effect killing. Spent neutrophils subsequently undergo apoptosis and are cleared by macrophages, thereby resolving the inflammatory episode. Neutrophils contain high concentrations of vitamin C (ascorbate) and this is thought to be essential for their function. This may be one mechanism whereby vitamin C enhances immune function. The aim of our study was to assess the effect of dietary supplementation with vitamin C-rich SunGold kiwifruit on four important functions of neutrophils: chemotaxis, oxidant generation, extracellular trap formation, and apoptosis. Fourteen young men (aged 18–30 years) with suboptimal plasma vitamin C status (<50 μmol/L) were supplemented for four weeks with two SunGold kiwifruit/day. Plasma vitamin C status was monitored weekly and neutrophil vitamin C levels were assessed at baseline and post-intervention. Neutrophil function assays were carried out on cells isolated at baseline and post-intervention. Plasma vitamin C levels increased to >70 μmol/L (p < 0.001) within one week of supplementation and there was a significant increase in neutrophil vitamin C status following four weeks’ intervention (p = 0.016). We observed a significant 20% increase in neutrophil chemotaxis post-intervention (p = 0.041) and also a comparable increase in oxidant generation (p = 0.031). Supplementation did not affect neutrophil extracellular trap formation or spontaneous apoptosis. Our data indicate that supplementation with vitamin C-rich kiwifruit is associated with improvement of important neutrophil functions, which would be expected to translate into enhanced immunity.     

Pharmacological activity of a Bv8 analogue modified in position 24

BACKGROUND AND PURPOSE

The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK₁ and PK₂, in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation.

EXPERIMENTAL APPROACH

We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK₁ and PK₂ receptors in cell preparations and in affecting nociceptive thresholds in rodents.

KEY RESULTS

A-24 preferentially bound to PK₂ receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased β-endorphin levels in mouse brain.

CONCLUSIONS AND IMPLICATIONS

A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK₁ receptors and by activating the central opioid system and the descending pain control pathway through brain PK₂ receptors.     

神经内分泌多肽7B2对T淋巴细胞的影响

观察神经内分泌多肽7B2对T淋巴细胞增殖和趋化功能的影响，当其浓度在250pg／ml和500pg／ml时，可抑制10份正常人外周血的PHA淋转率，其平均抑制率分别为26．97％、61．04％。采用微量玻片T淋巴细胞趋化试验证实．当7B2浓度在3ng／ml和5ng／ml时可抑制以白细胞介素2（IL-2）作为起化剂的6份外周血经PHA刺激的T淋巴细胞趋化率，其抑制率分别为32.78％和91．78％。表明神经内分泌多肽7B2参与T淋巴细胞的调控。     

Partial characterization of the fibroblast chemotactic constituents of human aqueous humour

This study demonstrates that aqueous humour derived from patients undergoing cataract extraction contains chemoattractants for ocular fibroblasts. The chemoattractant activity is independent of the surgical approach to obtaining the specimen as well as the surgeon operating. Studies in rabbits demonstate that the chemoatractant activity is not artefactually produced by aqueous sampling and confirm the integrity of the blood-aqueous barrier. Substances resonsible for this activity have molecular weights greater than 30,000; they are deactivated by low pH but retain activity at high pH. Identification of the chemoattractants in aqueous humour may allow pharmacological manipulation of would healing in relation to trabeculectomies.     

FOLATE STATUS OF PATIENTS ON LOW-DOSE METHOTREXATE THERAPY

Methotrexate in small doses impairs the folate-dependent incorporation of formate into serine in lymphocytes.     

Demonstration of neutrophil chemotactic activity in the sputum of cystic fibrosis patients with Pseudomonas aeruginosa infection

The pathogenesis of tissue damage in the lungs of cystic fibrosis patients with chronic P. aeruginosa infection is quite complex and not well understood. Inflammatory cells, particularly neutrophils, are accumulated in the damaged tissues and in the sputum. This study demonstrates the presence of a heat-stable neutrophil chemotactic factor(s) in the sputum of CF patients. The chemotactic activity seems to be associated with the endotoxin present in the sputum. Chemotaxis of sol phase sputum was determined in a modified Boyden chamber assay. It was found that the sputum obtained from the patients showed strong chemotactic activity for peripheral blood neutrophils of normal individuals. The activity was greater than twice that of casein, a strong neutrophil chemo-attractant. Sputum at about dilution of 1:50 exhibited chemotactic activity equal to that of casein. Heat treatment of the sputum at 56 degrees C for 30 min, to inactivate complement, and at 100 degrees C for 15 min, to denature other proteins, somewhat reduced the chemotactic activity, but there was still strong chemotactic activity. The presence of endotoxin was demonstrated in both the non-heated and the heated samples by LAL and rocket immunoelectrophoresis. It is concluded that the sputum of CF patients contain chemotactic activity of heat-stable nature and most likely of bacterial origin endotoxin. These factors are involved in attraction of neutrophils to the lungs and sputum of these patients and contribute to the tissue damage of cystic fibrosis.     

Acteoside relieves mesangial cell injury by regulating Th22 cell chemotaxis and proliferation in IgA nephropathy

The existing therapies of IgA nephropathy are unsatisfying. Acteoside, the main component of Rehmannia glutinosa with anti-inflammatory and anti-immune effects, can improve urinary protein excretion and immune disorder. Th22 cell is involved in IgA nephropathy progression. This study was determined to explore the effect of acteoside on mesangial injury underlying Th22 cell disorder in IgA nephropathy. Serum Th22 cells and urine total protein of patients with IgA nephropathy were measured before and after six months treatment of Rehmannia glutinosa acteoside or valsartan. Chemotactic assay and co-culture assay were performed to investigate the effect of acteoside on Th22 cell chemotaxis and differentiation. The expression of CCL20, CCL22 and CCL27 were analyzed. To explore the effect of acteoside on mesangial cell injury induced by inflammation, IL-1, IL-6, TNF-α and TGF-β1 were tested. Results showed that the proteinuria and Th22 lymphocytosis of patients with IgA nephropathy significantly improved after combination treatment of Rehmannia glutinosa acteoside and valsartan, compared with valsartan monotherapy. In vitro study further demonstrated that acteoside inhibit Th22 cell chemotaxis by suppressing the production of Th22 cell attractive chemokines, i.e., CCL20, CCL22 and CCL27. In addition, acteoside inhibited the Th22 cell proliferation. Co-culture assay proved that acteoside could relieve the overexpression of pro-inflammatory cytokines, and prevent the synthesis of TGF-β1. TGF-β1 level in mesangial cells was positively correlated with the Th22 cell. This research demonstrated that acteoside can alleviate mesangial cell inflammatory injury by modulating Th22 lymphocytes chemotaxis and proliferation.     

A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells’ morphogenesis, and they range in a three‐dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffness. Copyright © 2014 John Wiley & Sons, Ltd.