New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Reduced Proteolipid Protein 2 promotes endoplasmic reticulum stress-related apoptosis and increases drug sensitivity in acute myeloid leukemia.

BACKGROUND: The Proteolipid Protein 2 (PLP2), a protein in the Endoplasmic Reticulum (ER) membrane, has been reported to be highly expressed in various tumors. Previous studies have demonstrated that the reduced PLP2 can induce apoptosis and autophagy through ER stress-related pathways, leading to a decreased proliferation and aggressiveness. However, there is no research literature on the role of PLP2 in Acute Myeloid Leukemia (AML). METHODS: PLP2 expression, clinical data, genetic mutations, and karyotype changes from GEO, TCGA, and timer2.0 databases were analyzed through the R packages. The possible functions and pathways of cells were explored through GO, KEGG, and GSEA enrichment analysis using the clusterProfiler R package. Immuno-infiltration analysis was conducted using the Cibersort algorithm and the Xcell R package. RT-PCR and western blot techniques were employed to identify the PLP2 expression, examine the knockdown effects in THP-1 cells, and assess the expression of genes associated with endoplasmic reticulum stress and apoptosis. Flow cytometry was utilized to determine the apoptosis and survival rates of different groups. RESULTS: PLP2 expression was observed in different subsets of AML and other cancers. Enrichment analyses revealed that PLP2 was involved in various tumor-related biological processes, primarily apoptosis and lysosomal functions. Additionally, PLP2 expression showed a strong association with immune cell infiltration, particularly monocytes. In vitro, the knockdown of PLP2 enhanced endoplasmic reticulum stress-related apoptosis and increased drug sensitivity in THP-1 cells. CONCLUSIONS: PLP2 could be a novel therapeutic target in AML, in addition, PLP2 is a potential endoplasmic reticulum stress regulatory gene in AML.

Proteolipid protein 1 is involved in the regulation of intestinal motility and barrier function in the mouse.

Proteolipid protein 1 (Plp1) is highly expressed in enteric glia, labeling cells throughout the mucosa, muscularis, and the extrinsic innervation. Plp1 is a major constituent of myelin in the central and peripheral nervous systems, but the absence of myelin in the enteric nervous system (ENS) suggests another role for Plp1 in the gut. Although the functions of enteric glia are still being established, there is strong evidence that they regulate intestinal motility and permeability. To interrogate the role of Plp1 in enteric glia, we investigated gut motility, secretomotor function and permeability, and evaluated the ENS in mice lacking Plp1. We studied two time points: ∼3 mo (young) and >1 yr (old). Old Plp1 null mice exhibited increased fecal output, decreased fecal water content, faster whole gut transit times, reduced intestinal permeability, and faster colonic migrating motor complexes. Interestingly, in both young and old mice, the ENS exhibited normal glial and neuronal numbers as well as glial arborization density in the absence of Plp1. As Plp1-associated functions involve mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mapk/Erk1/2) signaling and Mapk/Erk1/2 are reported to have a regulatory role in intestinal motility, we measured protein expression of Erk1/2 and its active form in the small intestine. Old Plp1 null mice had reduced levels of phosphorylated-Erk1/2. Although Plp1 is not required for the normal appearance of enteric glial cells, it has a regulatory role in intestinal motility and barrier function. Our results suggest that functional changes mediated by Plp1-expressing enteric glia may involve Erk1/2 activation.NEW & NOTEWORTHY Here, we describe that Plp1 regulates gut motility and barrier function. The functional effects of Plp1 eradication are only seen in old mice, not young. The effects of Plp1 appear to be mediated through the Erk1/2 pathway.

Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A.

L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial infections.

PLP2-derived peptide Rb4 triggers PARP-1-mediated necrotic death in murine melanoma cells.

Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.

Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates.

Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.

Functional Characterization of Porcine NK-Lysin: A Novel Immunomodulator That Regulates Intestinal Inflammatory Response.

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.

Bovine NK-lysin-derived peptides have bactericidal effects against Mycobacterium avium subspecies paratuberculosis.

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) is complex, but little is known about the role that natural killer (NK) cells play. In the present study, four bovine NK-lysin peptides were synthesized to evaluate their bactericidal activity against MAP. The results demonstrated that bNK-lysin peptides were directly bactericidal against MAP, with bNK1 and bNK2A being more potent than bNK2B and bNK2C. Mechanistically, transmission electron microscopy revealed that the incubation of MAP with bNK2A resulted in extensive damage to cell membranes and cytosolic content leakage. Furthermore, the addition of bNK2A linked with a cell-penetrating peptide resulted in increased MAP killing in a macrophage model.

Effects of Fortetropin on the Rate of Muscle Protein Synthesis in Older Men and Women: A Randomized, Double-Blinded, Placebo-Controlled Study.

BACKGROUND: Fortetropin is a proteo-lipid complex made from fertilized egg yolk and, in young men, has been shown to increase lean body mass. METHODS: The purpose of this study was to examine the effects of 21 days of Fortetropin supplementation on the fractional synthetic rate (FSR) of muscle protein in 10 healthy, older men and 10 women (66.4 ± 4.5 y). We used 2H2O labeling to measure FSR of multiple muscle protein ontologies. D3-creatine dilution was used to determine muscle mass at baseline. Subjects ingested 70% 2H2O for 21 day and saliva samples were collected to determine body 2H2O enrichment. A microbiopsy was obtained from the m. vastus lateralis on Day 21. Subjects were randomly assigned to Fortetropin (19.8 g/d) or placebo (cheese powder, 19.8 g/d). RESULTS: Restricting kinetic data to proteins with ≥2 peptides measured in at least 4 subjects per group resulted in 117 proteins meeting these criteria. The mean FSR for a majority of proteins in several muscle gene ontologies was higher in the Fortetropin group compared to placebo (32/38 myofibril proteins, 33/44 sarcoplasmic proteins, and 12/17 mitochondrial proteins) and this proportion was significantly different between groups using a binomial test and were independent of sex or baseline muscle mass. CONCLUSIONS: The overall magnitude of the difference in muscle protein FSR of Fortetropin from placebo was 18%, with multiple gene ontologies affected. While these results should be confirmed in larger cohorts, they suggest that Fortetropin supplementation is effective for promoting muscle protein synthesis in older people.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

NKL-24: A novel antimicrobial peptide derived from zebrafish NK-lysin that inhibits bacterial growth and enhances resistance against Vibrio parahaemolyticus infection in Yesso scallop, Patinopecten yessoensis.

The extensive use of antibiotics in aquaculture has resulted in the prevalence of antibiotic-resistant bacteria and, consequently, new antibacterial strategies or drugs with clear modes of action are urgently needed. Antimicrobial peptides (AMPs) are currently widely considered as alternatives to antibiotics in the treatment of infections in aquatic animals. In this study, we aimed to evaluate the effects of NKL-24, a truncated peptide derived from zebrafish NK-lysin, against Yesso scallop (Patinopecten yessoensis) pathogen, Vibrio parahaemolyticus. The results showed that NKL-24 had a potent antibacterial effect against V. parahaemolyticus via a membrane active cell-killing mechanism. The in vitro study showed that sub-lethal levels of NKL-24 obviously reduced bacterial swimming ability and downregulated the transcription of the selected genes associated with V. parahaemolyticus virulence. Studies on NKL-24 biosafety in hemocytes and in Yesso scallop have shown no adverse effects from this peptide. Bacteria challenge test results demonstrated that NKL-24 significantly decreased the mortality and inhibited bacterial growth in the scallop infected with V. parahaemolyticus, while further in vivo examination revealed that NKL-24 could enhance non-specific immune parameters. Moreover, NKL-24 was capable of modulating a series of V. parahaemolyticus-responsive genes in the scallop. These results suggest the protective action of NKL-24 against V. parahaemolyticus and the potential of this peptide as a promising candidate for aquaculture applications.

Fortetropin inhibits disuse muscle atrophy in dogs after tibial plateau leveling osteotomy.

OBJECTIVE: To determine if a commercial myostatin reducer (Fortetropin®) would inhibit disuse muscle atrophy in dogs after a tibial plateau leveling osteotomy. DESIGN: A prospective randomized, double-blinded, placebo-controlled clinical trial. ANIMALS: One hundred client-owned dogs presenting for surgical correction of cranial cruciate ligament rupture by tibial plateau leveling osteotomy. PROCEDURES: Patients were randomly assigned into the Fortetropin® or placebo group and clients were instructed to add the assigned supplement to the dog's normal diet once daily for twelve weeks. Enrolled patients had ultrasound measurements of muscle thickness, tape measure measurements of thigh circumference, serum myostatin level assays, and static stance analysis evaluated at weeks 0, 8, and 12. RESULTS: From week 0 to week 8, there was no change for thigh circumference in the Fortetropin® group for the affected limb (-0.54cm, P = 0.31), but a significant decrease in thigh circumference for the placebo group (-1.21cm, P = 0.03). There was no significant change in serum myostatin levels of dogs in the Fortetropin® group at any time point (P>0.05), while there was a significant rise of serum myostatin levels of dogs in placebo group during the period of forced exercise restriction (week 0 to week 8; +2,892 pg/ml, P = 0.02). The percent of body weight supported by the affected limb increased in dogs treated with Fortetropin® (+7.0%, P<0.01) and the placebo group (+4.9%, P<0.01) at the end of the period of forced exercise restriction. The difference in weight bearing between the Fortetropin® and placebo groups was not statistically significant (P = 0.10). CONCLUSION: Dogs receiving Fortetropin® had a similar increase in stance force on the affected limb, no significant increase in serum myostatin levels, and no significant reduction in thigh circumference at the end of the period of forced exercise restriction compared to the placebo. These findings support the feeding of Fortetropin® to prevent disuse muscle atrophy in canine patients undergoing a tibial plateau leveling osteotomy.

Effect of sarcolipin-mediated cell transdifferentiation in sarcopenia-associated skeletal muscle fibrosis.

Fibrosis is a key pathological event during muscle aging that accelerates the development of sarcopenia. We show that sarcolipin (SLN) is highly expressed during aging, promotes intracellular calcium overload and participates in impaired myogenic differentiation. d-Galactose (D-gal) was used to induce senescence in C2C12 myoblasts. Conventional AAV-mediated SLN knockdown cells were used to study the role of SLN in muscle physiology and pathophysiology. C2C12 cells were treated with D-gal, which promoted fibrosis and SLN upregulation. The expression of TGF-ß1 and α-SMA, which participate in myogenic transdifferentiation, were also elevated. C2C12 cells with reduced sarcolipin expression produced decreased amounts of collagen. Our study identified an unrecognized role of SLN in regulating myogenic transdifferentiation during aging-associated skeletal muscle cell fibrosis. Targeting SLN may be a novel therapeutic strategy to relieve sarcopenia-associated muscle fibrosis.

Membrane disruptive antimicrobial potential of NK-lysin from yellow catfish (Pelteobagrus fulvidraco).

NK-lysins, a type of broad-spectrum antimicrobial peptide (AMP), act as an essential effector of innate defense against microbial attack in higher vertebrates and so in fish. The present study delineates the structural and functional characterization of NK-lysin from yellow catfish (Pelteobagrus fulvidrac) (Pelteobagrus fulvidraco). PfNK-lysin encodes a 153-residue peptide, which displays the hallmark features of other known NK-lysins with the ordered array of six well-conserved cysteine residues and five-exon/four-intron structure. It was found to be ubiquitous in tissues, being detected most abundantly in gill and head kidney. In vivo exposure to stimuli (LPS, PolyI:C, and Edwardsiella ictaluri) induced PfNK-lysin expression in head kidney and spleen. Synthetic PfNK-lysin-derived peptide exhibited in vitro bactericidal potency against both Gram-positive and Gram-negative bacteria, with the highest inhibitory effect on pathogen Edwardsiella ictaluri. Fluorescence microscopy and scanning electron microscopy further confirmed its capacity to cause damage to the bacterial plasma membrane. Taken together, these data suggest that PfNK-lysin might participate in antimicrobial defense of yellow catfish by membrane-disruptive action.

Exploring small cationic peptides of different origin as potential antimicrobial agents in aquaculture.

Antimicrobial peptides (AMPs) form part of the innate immune response, which is of vital importance in fish, especially in eggs and early larval stages. Compared to antibiotics, AMPs show action against a wider spectrum of pathogens, including viruses, fungi and parasites, are more friendly to the environment, and do not seem to generate resistance in bacteria. Thus, we have tested in vitro the potential use of several synthetic peptides as antimicrobial agents in aquaculture: frog Caerin1.1, European sea bass Dicentracin (Dic) and NK-lysin peptides (NKLPs) and sole NKLP27. Our results demonstrate that the highest bactericidal activity against both human and fish pathogens was obtained with Caerin1.1 followed by sea bass Dic and NKLPs, having the sea bass NKLP20.2 none to negligible activity. Interestingly, Aeromonas salmonicida was refractory to all the fish peptides tested. Regarding the antiviral activity, synthetic peptides were able to inhibit the viral infection of nodavirus (NNV), viral septicaemia haemorrhagic virus (VHSV), infectious pancreatic necrosis virus (IPNV) and spring viremia carp virus (SVCV), which are some of the most devastating virus for aquaculture. However, their effectiveness was highly dependent on the type of virus. Strikingly, IPNV resulted the most resistant virus since Caeerin1.1 and sea bass NKLP20.2 were unable to reduce its titre and the other peptides tested only reduced it to values in the 43-78% range. These data demonstrate that synthetic peptides have great antibacterial and antiviral in vitro activity against important fish pathogens and point to their use as potential therapeutic agents in aquaculture.

Recombinant porcine NK-lysin inhibits the invasion of hepatocellular carcinoma cells in vitro.

The therapeutics having ability to target cancer cells specifically and exhibit nominal cytopathic effect on normal healthy cells are highly significant for cancer therapeutic applications. Recombinant porcine natural killer lysin (rpNK-lysin) has proven cationic anti-bacterial and anti-tumor peptide. Herein, we report its anti-invasion and anti-metastasis effects on hepatocellular carcinoma (HCC) cells in vitro. We first investigate the maximum non-toxic concentration (MNTC) of rpNK-lysin for the normal hepato cells (L-02). Using MNTC rpNK-lysin, we explore anti-proliferative, anti-adhesive, anti-invasive and anti-metastatic effect of rpNK-lysin on three different HCC cells lines (SMMC-7721, 97-H and HepG2) through MTT, wound-healing, adhesion and invasion assay along with mRNA and protein expression. The results reveal that rpNK-lysin has potential to specifically inhibit HCC cells growth in a dose and time-dependent manner with a little cytopathic effect on the L-02 cells, effectively reduce migration, adhesion and invasion ability of HCC cells. rpNK-lysin significantly reduce Fascin1 expression, which subsequently decrease ß-catenin expression and metaloproteinases (MMP-2 and MMP9). This study suggest that MNTC rpNK-lysin has an anti-invasion and anti-metastasis effect on HCC cells in vitro through inhibition of Fascin 1 expression which regulates Wnt/ß-catenin signaling pathway by inducing ß-catenin degradation and subsequently results in suppression of MMP-2 and MMP9 expression.

NK Cell Induced T Cell Anergy Depends on GRAIL Expression.

NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.

Anti-Invasion and Antimetastatic Effects of Porcine Recombinant NK-lysin on SMMC-7721 Human Hepatocellular Carcinoma Cells.

The high invasion and metastasizing abilities of hepatocellular carcinoma (HCC) are the primary reasons for the high mortality rate of patients. Therefore, identification of agents to inhibit invasion and metastasis is very important for treatment of HCC. We analyzed the anti-invasion and antimetastatic effects of porcine recombinant NK-lysin, which was designed and expressed in vitro by our research group, on SMMC-7721 hepatocellular carcinoma cells via wound-healing assays, adhesion assays, invasion assays, real-time polymerase chain reaction (PCR), and Western blot analysis. MTT assay results indicated that NK-lysin inhibited the growth of SMMC-7721 cells in a dose- and time-dependent manner. NK-lysin reduced the ability of cell migration, adhesion, and invasion. Based on gene and protein expression analysis, NK-lysin decreased ß-catenin and MMP-2 expression. These results suggested that NK-lysin has anti-invasion and antimetastatic effects on hepatocellular carcinoma cells in vitro by reducing the level of the ß-catenin and MMP-2.

Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion.

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl71⁻100 is shown as a promising broad-spectrum antiviral candidate.

A Combination of the Aerosolized PPAR-γ Agonist Pioglitazone and a Synthetic Surfactant Protein B Peptide Mimic Prevents Hyperoxia-Induced Neonatal Lung Injury in Rats.

BACKGROUND: Despite improvements in perinatal care, bronchopulmonary dysplasia (BPD) in extremely premature infants has not decreased. Postnatal surfactant therapy provides symptomatic relief from respiratory distress syndrome, but does not translate into a reduction in BPD. Therefore, the search for effective interventions to prevent BPD continues. OBJECTIVES: Since PPAR-γ agonists have been demonstrated to promote neonatal lung maturation and injury repair, we hypothesized that a formulation of a PPAR-γ agonist, pioglitazone (PGZ) and a synthetic lung surfactant (a surfactant protein B peptide mimic, B-YL) combined would stimulate lung maturation and block hyperoxia-induced neonatal lung injury more effectively than either modality alone. METHODS: One-day-old Sprague-Dawley rat pups were administered PGZ + B-YL via nebulization every 24 h for up to 72 h. The pups were exposed to either 21 or 95% O2, and then sacrificed. Their lungs were examined for markers of lung maturation (levels of PPAR-γ, SP-C and choline-phosphate cytidylyltransferase [CCT-α] and [3H]triolein uptake) and injury repair (bronchoalveolar lavage cell count and protein content, and levels of LEF-1, fibronectin, ALK5, and ß-catenin) by Western blot analysis. RESULTS: Markers of alveolar epithelial/mesenchymal maturation (PPAR-γ, SP-C, CCT-α, and triolein uptake) increased significantly in the PGZ + B-YL group, more than with either drug alone. Similarly, markers of hyperoxia-induced lung injury were blocked effectively with PGZ + B-YL treatment. CONCLUSIONS: Nebulized PPAR-γ agonist PGZ with a synthetic lung surfactant accelerates lung maturation and prevents neonatal hyperoxia-induced lung injury more than either modality alone, with the potential to provide more effective prevention of BPD.