Novel nano-carriers with N-formylmethionyl-leucyl-phenylalanine-modified liposomes improve effects of C16-angiopoietin 1 in acute animal model of multiple sclerosis.

Gradual loss of neuronal structure and function due to impaired blood-brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

A computer vision approach for analyzing label free leukocyte trafficking dynamics on a microvascular mimetic.

High-content imaging techniques in conjunction with in vitro microphysiological systems (MPS) allow for novel explorations of physiological phenomena with a high degree of translational relevance due to the usage of human cell lines. MPS featuring ultrathin and nanoporous silicon nitride membranes (µSiM) have been utilized in the past to facilitate high magnification phase contrast microscopy recordings of leukocyte trafficking events in a living mimetic of the human vascular microenvironment. Notably, the imaging plane can be set directly at the endothelial interface in a µSiM device, resulting in a high-resolution capture of an endothelial cell (EC) and leukocyte coculture reacting to different stimulatory conditions. The abundance of data generated from recording observations at this interface can be used to elucidate disease mechanisms related to vascular barrier dysfunction, such as sepsis. The appearance of leukocytes in these recordings is dynamic, changing in character, location and time. Consequently, conventional image processing techniques are incapable of extracting the spatiotemporal profiles and bulk statistics of numerous leukocytes responding to a disease state, necessitating labor-intensive manual processing, a significant limitation of this approach. Here we describe a machine learning pipeline that uses a semantic segmentation algorithm and classification script that, in combination, is capable of automated and label-free leukocyte trafficking analysis in a coculture mimetic. The developed computational toolset has demonstrable parity with manually tabulated datasets when characterizing leukocyte spatiotemporal behavior, is computationally efficient and capable of managing large imaging datasets in a semi-automated manner.

The study of genetic predisposition on periodontitis and peri-implantitis.

Background: Peri-implant mucositis and peri-implantitis cases increase in number with the increase of implant applications. Peri-implant mucositis and peri-implantitis are defined as inflammatory diseases with inflammation and loss in soft and hard tissue, similar to the other periodontal diseases. As observed in many diseases, genetic predisposition factors also affect the progress of periodontitis and peri-implantitis. Aim: This study examines if there is any solid genetic predisposition causing periodontitis and peri-implantitis formation in Turkish patients. Patients & Methods: In order to evaluate single nucleotide polymorphism (SNP), Interleukin-8 (IL-8) and N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP), playing a role in the chemotaxis of neutrophils, and Fc Gamma Receptor IIA (FcγRIIA) and Fc Gamma Receptor IIIA (FcγRIIIA), playing a role in the antigen-antibody complexes and phagocytosis, were selected. Thirty-two Turkish non-smoking subjects, having periodontitis, thirty-three Turkish non-smoking subjects, having peri-implantitis and thirty-three Turkish non-smoking healthy subjects were selected. In total 98 adults participated in our study. Collected saliva samples from the participants were used for DNA isolation. SNPs were determined in these subgroups of the study by means of genotype-specific polymerase chain reactions. Results: When IL-8 A-251T, FcγRIIa -H131 and FcγRIIIa -V158 polymorphism were evaluated, no significant difference was found between periodontitis, peri-implantitis and healthy groups. However, this study observed that fMLP Receptor (FPR1) gene polymorphism creates a significant difference in individuals at higher risk of periodontitis or peri-implantitis. Conclusion: Results show that individuals with the G genotype have a higher risk of periodontitis, while individuals with G / C genotype have higher risk of peri-implantitis.

In vitro neutrophil migration is associated with inhaled corticosteroid treatment and serum cytokines in pediatric asthma.

Background: Different asthma phenotypes are driven by molecular endotypes. A Th1-high phenotype is linked to severe, therapy-refractory asthma, subclinical infections and neutrophil inflammation. Previously, we found neutrophil granulocytes (NGs) from asthmatics exhibit decreased chemotaxis towards leukotriene B4 (LTB4), a chemoattractant involved in inflammation response. We hypothesized that this pattern is driven by asthma in general and aggravated in a Th1-high phenotype. Methods: NGs from asthmatic nd healthy children were stimulated with 10 nM LTB4/100 nM N-formylmethionine-leucyl-phenylalanine and neutrophil migration was documented following our prior SiMA (simplified migration assay) workflow, capturing morphologic and dynamic parameters from single-cell tracking in the images. Demographic, clinical and serum cytokine data were determined in the ALLIANCE cohort. Results: A reduced chemotactic response towards LTB4 was confirmed in asthmatic donors regardless of inhaled corticosteroid (ICS) treatment. By contrast, only NGs from ICS-treated asthmatic children migrate similarly to controls with the exception of Th1-high donors, whose NGs presented a reduced and less directed migration towards the chemokines. ICS-treated and Th1-high asthmatic donors present an altered surface receptor profile, which partly correlates with migration. Conclusions: Neutrophil migration in vitro may be affected by ICS-therapy or a Th1-high phenotype. This may be explained by alteration of receptor expression and could be used as a tool to monitor asthma treatment.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the ßγ subunit of Gi-protein of fMLP receptor in human neutrophils.

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein ßγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 µM)-induced superoxide anion production (IC50:1.98 ± 0.14 µM), the release of cathepsin G (IC50:2.76 ± 0.15 µM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the ßγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCß by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the ßγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

Optimization of the Transwell® assay for the analysis of neutrophil chemotaxis using flow cytometry to refine the clinical investigation of immunodeficient patients.

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women.

Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics.

Serum of limb remote ischemic postconditioning inhibits fMLP-triggered activation and reactive oxygen species releasing of rat neutrophils.

OBJECTIVES: The study explores the protective role of the peripheral serum of limb remote ischemic postconditioning (LRIP) in reducing the reactive oxygen species (ROS) levels and neutrophil activation, which are responsible for the deleterious reperfusion injury. METHODS: LRIP was induced in Sprague-Dawley rats by three cycles of 5 min occlusion /5 min reperfusion on the left hind limb. The blood samples were collected before LRIP or 0 and 1 h after LRIP (named SerumSham, SerumLRIP0, SerumLRIP1, respectively). The effects of LRIP serum on ROS level and neutrophils activation were determined. The expression of MyD88-TRAF6-MAPKs and PI3K/AKT pathways in neutrophils were examined. RESULTS: When compared with SerumSham, SerumLRIP0 and SerumLRIP1 significantly reduced the ROS released from neutrophils activated by fMLP. Meanwhile, the mRNA expression levels of NADPH oxidase subunit p22phox and multiple ROS-producing related key proteins, such as NADPH oxidase subunit p47phox ser 304, ser 345. MyD88, p-ERK, p-JNK and p-P38 expression of neutrophils were downregulated by SerumLRIP0 and SerumLRIP1. SerumLRIP1 also downregulated p47phox mRNA expression and tumor necrosis factor receptor-associated factor 6 (TRAF6) protein expression. CONCLUSION: LRIP serum protects against ROS level and neutrophils activation involving the MyD88-TRAF6-MAPKs. This finding provides new insight into the understanding of LRIP mechanisms.

[Mitoxantrone Inhibits FMLP-Induced Degenerative Changes in Human Neutrophils].

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.

The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb.

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.

Severe Traumatic Injury Induces Phenotypic and Functional Changes of Neutrophils and Monocytes.

BACKGROUND: Severe traumatic injury has been associated with high susceptibility for the development of secondary complications caused by dysbalanced immune response. As the first line of the cellular immune response, neutrophils and monocytes recruited to the site of tissue damage and/or infection, are divided into three different subsets according to their CD16/CD62L and CD16/CD14 expression, respectively. Their differential functions have not yet been clearly understood. Thus, we evaluated the phenotypic changes of neutrophil and monocyte subsets among their functionality regarding oxidative burst and the phagocytic capacity in severely traumatized patients. METHODS: Peripheral blood was withdrawn from severely injured trauma patients (TP; n = 15, ISS ≥ 16) within the first 12 h post-trauma and from healthy volunteers (HV; n = 15) and stimulated with fMLP and PMA. CD16dimCD62Lbright (immature), CD16brightCD62Lbright (mature) and CD16brightCD62Ldim (CD62Llow) neutrophil subsets and CD14brightCD16- (classical), CD14brightCD16+ (intermediate) and CD14dimCD16+ (non-classical) monocyte subsets of HV and TP were either directly analyzed by flow cytometry or the examined subsets of HV were sorted first by fluorescence-activated cell sorting and subsequently analyzed. Subset-specific generation of reactive oxygen species (ROS) and of E. coli bioparticle phagocytosis were evaluated. RESULTS: In TP, the counts of immature neutrophils were significantly increased vs. HV. The numbers of mature and CD62Ldim neutrophils remained unchanged but the production of ROS was significantly enhanced in TP vs. HV and the stimulation with fMLP significantly increased the generation of ROS in the mature and CD62Ldim neutrophils of HV. The counts of phagocyting neutrophils did not change but the mean phagocytic capacity showed an increasing trend in TP. In TP, the monocytes shifted toward the intermediate phenotype, whereas the classical and non-classical monocytes became less abundant. ROS generation was significantly increased in all monocyte subsets in TP vs. HV and PMA stimulation significantly increased those level in both, HV and TP. However, the PMA-induced mean ROS generation was significantly lower in intermediate monocytes of TP vs. HV. Sorting of monocyte and neutrophil subsets revealed a significant increase of ROS and decrease of phagocytic capacity vs. whole blood analysis. CONCLUSIONS: Neutrophils and monocytes display a phenotypic shift following severe injury. The increased functional abnormalities of certain subsets may contribute to the dysbalanced immune response and attenuate the antimicrobial function and thus, may represent a potential therapeutic target. Further studies on isolated subsets are necessary for evaluation of their physiological role after severe traumatic injury.

Prolyl-Isomerase Pin1 Controls Key fMLP-Induced Neutrophil Functions.

Neutrophils are key cells of the innate immune and inflammatory responses. They are the first blood cells to migrate to the infection site where they release high amounts of reactive oxygen species (ROS) and several peptides and enzymes required for microbial killing. However, excessive neutrophil activation can induce tissue injury participating in inflammation, thus the characterization of the enzymes involved in neutrophil activation could help to identify new pharmacological targets to treat inflammation. The prolyl-isomerase Pin1 is a ubiquitous enzyme involved in several functions, however, its role in neutrophil functions is less known. In this study, we show that the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP or fMLF), a G-protein coupled receptor (GPCR) agonist-induced Pin1 activation in human neutrophils. PiB and juglone, two Pin1 inhibitors inhibited Pin1 activity in neutrophils and consequently inhibited fMLP-induced chemotaxis and -degranulation of azurophil and specific granules as measured by myeloperoxidase and neutrophil gelatinase-associated lipocalin (NGAL) release respectively. We also showed that PiB inhibited TNFα + fMLP-induced superoxide production, confirming the effect of juglone. These data show that inhibitors of Pin1 impaired key pro-inflammatory neutrophil functions elicited by GPCR activation and suggest that Pin1 could control neutrophil inflammatory functions.

The anti-inflammatory effect of ε-viniferin by specifically targeting formyl peptide receptor 1 on human neutrophils.

The uncontrol respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of ε-viniferin, a lignan from the root of Vitis thunbergii var. thunbergii, inhibits N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP) induced respiratory burst by antagonizing formyl peptide receptor 1 in human neutrophils. Briefly, ε-viniferin specifically inhibited fMLP (0.1 µM: formyl peptide receptor 1 agonist or 1 µM: formyl peptide receptor 1, 2 agonist)-induced superoxide anion production in a concentration-dependent manner (IC50 = 2.30 ± 0.96 or 9.80 ± 0.21 µM, respectively) without affecting this induced by formyl peptide receptor 2 agonist (WKYMVM). ε-viniferin inhibited fMLP (0.1 µM)-induced phosphorylation of ERK, Akt, Src or intracellular calcium mobilization without affecting these caused by WKYMVM. The synergistic suppression of fMLP (1 µM)-induced superoxide anion production was observed only in the combination of ε-viniferin and formyl peptide receptor 2 antagonist (WRW4) but not in combination of ε-viniferin and formyl peptide receptor 1 antagonist (cyclosporine H). ε-viniferin inhibited FITC-fMLP binding to formyl peptide receptors. Moreover, the synergistic suppression of FITC-fMLP binding was observation only in the combination of ε-viniferin and WRW4 but not in other combinations. ATPγS induced superoxide anion production through formyl peptide receptor 1 in fMLP desensitized neutrophils and this effect was inhibited by ε-viniferin. The concentration-response curve of fMLP-induced superoxide anion was not parallel shifted by ε-viniferin. Furthermore, the inhibiting effect of ε-viniferin on fMLP-induced superoxide anion production was reversible. These results suggest that ε-viniferin is an antagonist of formyl peptide receptor 1 in a reversible and non-competitive manner.

Targeting Neprilysin (NEP) pathways: A potential new hope to defeat COVID-19 ghost.

COVID-19 is an ongoing viral pandemic disease that is caused by SARS-CoV2, inducing severe pneumonia in humans. However, several classes of repurposed drugs have been recommended, no specific vaccines or effective therapeutic interventions for COVID-19 are developed till now. Viral dependence on ACE-2, as entry receptors, drove the researchers into RAS impact on COVID-19 pathogenesis. Several evidences have pointed at Neprilysin (NEP) as one of pulmonary RAS components. Considering the protective effect of NEP against pulmonary inflammatory reactions and fibrosis, it is suggested to direct the future efforts towards its potential role in COVID-19 pathophysiology. Thus, the review aimed to shed light on the potential beneficial effects of NEP pathways as a novel target for COVID-19 therapy by summarizing its possible molecular mechanisms. Additional experimental and clinical studies explaining more the relationships between NEP and COVID-19 will greatly benefit in designing the future treatment approaches.

The vascular glycocalyx is not a mechanosensor in conduit arteries in the anesthetized pig.

BACKGROUND: The role of the glycocalyx as the endothelial sensor of an increase in blood flow was assessed in the iliac artery in vivo. METHODS: Acetylcholine-induced flow mediated dilation was evaluated before and after vascular glycocalyx disruption. This was accomplished by exposing the iliac lumen to the chemotactic agent fMLP (1 µM; n = 6 pigs), concomitant heparinase III (100 mU ml-1) and hyaluronidase (14 mg ml-1) (n = 4), and neuraminidase (140 mU ml-1; n = 5), for 20 min in separate iliac artery preparations. Only one lumen intervention per iliac was conducted. RESULTS: For the heparinase III + hyaluronidase experiment, the iliac diameter increased by an average of 0.54 ± 0.11 mm before and 0.45 ± 0.03 mm after the enzymes (P = 0.42; paired Student's t test). The iliac diameter increased by 0.31 ± 0.02 mm before and 0.29 ± 0.07 mm after fMLP exposure (P = 0.7) and the diameter increased by 0.54 ± 0.11 mm before and 0.54 ± 0.09 mm after neuraminidase exposure (P = 0.98). In all cases, the shear stress changes before and after lumen exposure were not significantly different to each other. CONCLUSION: There was no significant reduction in flow mediated dilation of the iliac in response to any of the interventions conducted. Therefore, the vascular endothelial glycocalyx as whole is not required for flow mediated dilation in conduit arteries in the intact animal.

Galectin-1 modulation of neutrophil reactive oxygen species production depends on the cell activation state.

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10-9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.

Evaluation of the in vitro effects of commercial herbal preparations significant in African traditional medicine on platelets.

BACKGROUND: Commercial herbal medicines (CHMs) marketed as immune boosters are gaining wide popularity in South Africa, in the absence of control and regulatory guidelines. These commercially packaged and labelled herbal preparations, acquired in various retail outlets, are used without consulting either a conventional health provider or a traditional health practitioner. Although they are indicated for immune-boosting purposes, they might exert many other beneficial and unwanted effects on physiological systems. Platelets are crucial in haemostasis and important for the immunological system. The aim was to investigate the effect of the CHMs used to strengthen the immune system on the activity of human platelets. METHODS: Six CHMs commonly used as African traditional medicines in Pretoria, South Africa, were tested for their effects on healthy, isolated human platelets, using a bioluminescence method. The tested herbal medicines were Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Ngoma™ Herbal Tonic Immune Booster, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster and serial-diluted standards of each from 10 to 10,000 times. The luminol-enhanced luminescence activity of the platelets was measured after incubation with the herbal medicines and activation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). RESULTS: Five herbal medicines, namely Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster exerted comparable weak inhibitory effects on both PMA and fMLP-induced platelets, which were concentration dependent at high doses, and inversely related to concentration at low doses. Intlamba Zifo™, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ exhibited weak, but non-systematic stimulatory effects at low doses, which were not statistically significant. Ngoma™ Herbal Tonic Immune Booster had weak, inhibitory effects at high doses and weak stimulatory effects that were inversely related to concentration at low doses. CONCLUSION: The findings suggest a potential beneficial role of the CHMs in the suppression of platelets' reactivity and in enhancing the immune system. Caution, however, should be exercised as platelet inhibition and stimulation predispose to the risk of bleeding and thrombosis, respectively.

A review of the fate of inhaled α-quartz in the lungs of rats.

The distribution of dust particles within the lungs and their excretion are highly associated with their pulmonary toxicity. Literature was reviewed to discern pulmonary translocation pathways for inhaled α-quartz compared to those for inhaled TiO2. Accordingly, it was hypothesized α-quartz particles in the alveoli were phagocytized by alveolar macrophages but silica-containing macrophages remained in the alveoli for longer time in contrast to the rapid elimination from the alveoli seen for TiO2-containing macrophages. In addition, it was presumed that free silica particles are translocated in the interstitium, possibly through the cytoplasm of Type I epithelial cells, as observed with TiO2. Free silica particles are presumed to be phagocytized by interstitial macrophages soon after the particles penetrate the interstitium; these dust cells are then translocated to the ciliated airway regions in the lumen through bronchus-associated lymphoid tissue (BALT). The pulmonary retention half-time of dust particles in rats exposed to α-quartz is several times longer than that of rats exposed to TiO2, as long as the lung dust burden is ≈ 3 mg. The reduced pulmonary particle clearance ability in rats exposed to α-quartz aerosol is presumably attributed to the long-term retention of dust cells both in the alveoli and in the interstitium; this retention may be caused by the reduced chemotactic abilities of α-quartz-containing dust cells. However, the accumulation of α-quartz-containing dust cells in the lungs is not associated with the occurrence of pulmonary inflammation.

Hydroxyethyl Starch 130/0.4 Binds to Neutrophils Impairing Their Chemotaxis through a Mac-1 Dependent Interaction.

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP (p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.

Mitochondrial Damage-Associated Molecular Patterns of Injured Axons Induce Outgrowth of Schwann Cell Processes.

Activated Schwann cells put out cytoplasmic processes that play a significant role in cell migration and axon regeneration. Following nerve injury, axonal mitochondria release mitochondrial damage-associated molecular patterns (mtDAMPs), including formylated peptides and mitochondrial DNA (mtDNA). We hypothesize that mtDAMPs released from disintegrated axonal mitochondria may stimulate Schwann cells to put out cytoplasmic processes. We investigated RT4-D6P2T schwannoma cells (RT4) in vitro treated with N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) or cytosine-phospho-guanine oligodeoxynucleotide (CpG ODN) for 1, 6 and 24 h. We also used immunohistochemical detection to monitor the expression of formylpeptide receptor 2 (FPR2) and toll-like receptor 9 (TLR9), the canonical receptors for formylated peptides and mtDNA, in RT4 cells and Schwann cells distal to nerve injury. RT4 cells treated with fMLP put out a significantly higher number of cytoplasmic processes compared to control cells. Preincubation with PBP10, a selective inhibitor of FPR2 resulted in a significant reduction of cytoplasmic process outgrowth. A significantly higher number of cytoplasmic processes was also found after treatment with CpG ODN compared to control cells. Pretreatment with inhibitory ODN (INH ODN) resulted in a reduced number of cytoplasmic processes after subsequent treatment with CpG ODN only at 6 h, but 1 and 24 h treatment with CpG ODN demonstrated an additive effect of INH ODN on the development of cytoplasmic processes. Immunohistochemistry and western blot detected increased levels of tyrosine-phosphorylated paxillin in RT4 cells associated with cytoplasmic process outgrowth after fMLP or CpG ODN treatment. We found increased immunofluorescence of FPR2 and TLR9 in RT4 cells treated with fMLP or CpG ODN as well as in activated Schwann cells distal to the nerve injury. In addition, activated Schwann cells displayed FPR2 and TLR9 immunostaining close to GAP43-immunopositive regenerated axons and their growth cones after nerve crush. Increased FPR2 and TLR9 immunoreaction was associated with activation of p38 and NFkB, respectively. Surprisingly, the growth cones displayed also FPR2 and TLR9 immunostaining. These results present the first evidence that potential mtDAMPs may play a key role in the induction of Schwann cell processes. This reaction of Schwann cells can be mediated via FPR2 and TLR9 that are canonical receptors for formylated peptides and mtDNA. The possible role for FPR2 and TLR9 in growth cones is also discussed.