Responses of Mast Cells to Pathogens: Beneficial and Detrimental Roles.

Mast cells (MCs) are strategically located in tissues close to the external environment, being one of the first immune cells to interact with invading pathogens. They are long living effector cells equipped with different receptors that allow microbial recognition. Once activated, MCs release numerous biologically active mediators in the site of pathogen contact, which induce vascular endothelium modification, inflammation development and extracellular matrix remodeling. Efficient and direct antimicrobial mechanisms of MCs involve phagocytosis with oxidative and non-oxidative microbial destruction, extracellular trap formation, and the release of antimicrobial substances. MCs also contribute to host defense through the attraction and activation of phagocytic and inflammatory cells, shaping the innate and adaptive immune responses. However, as part of their response to pathogens and under an impaired, sustained, or systemic activation, MCs may contribute to tissue damage. This review will focus on the current knowledge about direct and indirect contribution of MCs to pathogen clearance. Antimicrobial mechanisms of MCs are addressed with special attention to signaling pathways involved and molecular weapons implicated. The role of MCs in a dysregulated host response that can increase morbidity and mortality is also reviewed and discussed, highlighting the complexity of MCs biology in the context of host-pathogen interactions.

Steroid hormone modulates the production of cathelicidin and human ß-defensins in lung epithelial cells and macrophages promoting Mycobacterium tuberculosis killing.

Several studies have documented the interaction between the immune and endocrine systems as an effective defense strategy against tuberculosis, involving the production of several molecules and immunological processes. In this study, we determined the effect of cortisol and dehydroepiandrosterone (DHEA) on the production of antimicrobial peptides such as cathelicidin and human ß-defensin (HBD) -2, and HBD-3 and their effect on intracellular growth of Mycobacterium tuberculosis (Mtb) in lung epithelial cells and macrophages. Our results showed that DHEA promotes the production of these antimicrobial peptides in infected cells, correlating with the decrease of Mtb bacilli loads. These results suggest the use of exogenous DHEA as an adjuvant for tuberculosis therapy.

Inhibition of defensin A and cecropin A responses to dengue virus 1 infection in Aedes aegypti. / Inhibición de las respuestas de defensina A y cecropina A contra la infección del virus dengue 1 en Aedes aegypti

INTRODUCTION: It is essential to determine the interactions between viruses and mosquitoes to diminish dengue viral transmission. These interactions constitute a very complex system of highly regulated pathways known as the innate immune system of the mosquito, which produces antimicrobial peptides that act as effector molecules against bacterial and fungal infections. There is less information about such effects on virus infections. OBJECTIVE: To determine the expression of two antimicrobial peptide genes, defensin A and cecropin A, in Aedes aegypti mosquitoes infected with DENV-1. MATERIALS AND METHODS: We used the F1 generation of mosquitoes orally infected with DENV-1 and real-time PCR analysis to determine whether the defensin A and cecropin A genes played a role in controlling DENV-1 replication in Ae. aegypti. As a reference, we conducted similar experiments with the bacteria Escherichia coli. RESULTS: Basal levels of defensin A and cecropin A mRNA were expressed in uninfected mosquitoes at different times post-blood feeding. The infected mosquitoes experienced reduced expression of these mRNA by at least eightfold when compared to uninfected control mosquitoes at all times post-infection. In contrast with the behavior of DENV-1, results showed that bacterial infection produced up-regulation of defensin and cecropin genes; however, the induction of transcripts occurred at later times (15 days). CONCLUSION: DENV-1 virus inhibited the expression of defensin A and cecropin A genes in a wild Ae. aegypti population from Venezuela.

Introducción. Es esencial determinar las interacciones entre los virus y los mosquitos para disminuir la transmisión viral. Estas interacciones constituyen un sistema muy complejo y muy regulado conocido como sistema inmunitario innato del mosquito, el cual produce péptidos antimicrobianos, moléculas efectoras que funcionan contra las infecciones bacterianas y fúngicas; se tiene poca información de su acción sobre los virus. Objetivo. Determinar la expresión de dos genes AMP (defensina A y cecropina A) en mosquitos Aedes aegypti infectados con el virus DENV-1. Materiales y métodos. Se infectaron oralmente mosquitos de generación F1 con DENV-1 y mediante el análisis con PCR en tiempo real se determinó el potencial papel de los genes defensina A y cecropina A en el control de la replicación del DENV-1 en Ae. aegypti. Como referencia, se infectaron mosquitos con Escherichia coli. Resultados: Los mosquitos no infectados expresaron niveles basales de los ARNm de los genes defensina A y cecropina A en diversos momentos después de la alimentación. Los mosquitos infectados experimentaron una reducción, por lo menos, de ocho veces en la expresión de estos ARNm con respecto a los mosquitos de control en todo el periodo posterior a la alimentación. En contraste con el comportamiento del virus DENV-1, los resultados mostraron que la infección bacteriana produjo una regulación positiva de los genes defensina y cecropina; sin embargo, la inducción de los transcritos ocurrió tardíamente (15 días). Conclusión. El virus DENV-1 inhibió la expresión de los genes defensina A y cecropina A en una población silvestre de Ae. aegypti en Venezuela.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations.

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

Sarconesin II, a New Antimicrobial Peptide Isolated from Sarconesiopsis magellanica Excretions and Secretions.

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.

Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Draft genome sequence of Enterococcus faecium E86, a strain producing broad-spectrum antimicrobial peptides: Description of a novel bacteriocin immunity protein and a novel sequence type.

OBJECTIVES: The aim of this study was to report the draft genome sequence of the bacteriocinogenic strain Enterococcus faecium E86. Bacteriocins are prokaryotic peptides or proteins with antimicrobial activity. The genome information may contribute to the identification of enterocins produced by this strain that exhibit inhibitory activity against the foodborne pathogen Listeria monocytogenes and vancomycin-resistant enterococci (VRE) involved in human infections, among other bacterial genera and species. METHODS: An Illumina MiSeq platform was used for genome sequencing. De novo assembly of 5 735 838 paired-end reads was done using the A5-miseq pipeline, yielding >300-fold average genome coverage. Genome annotation was performed by the RAST server, and mining of the bacteriocinogenic gene clusters was done using the BAGEL3 and antiSMASH v.4 platforms. RESULTS: The total scaffold size was determined to be 2 689 107 bp, approximately 2.7 Mbp, featuring a G + C content of 38.1%. The genome contains 2858 coding sequences and 74 RNA genes. Genome analyses revealed the presence of: 30 genes involved in drug resistance; 2 bacteriocinogenic gene clusters (for enterocin P and enterocin TW21); EntiTW21, a novel bacteriocin immunity protein and a novel multilocus sequence type (ST1500). CONCLUSION: This work highlights the potential biotechnological application of this strain for the production of enterocin P, a bacteriocin that can be employed in the food industry as a biopreservative against L. monocytogenes and as an alternative to classical antibiotics against VRE.

Molecular farming of antimicrobial peptides: available platforms and strategies for improving protein biosynthesis using modified virus vectors.

The constant demand for new antibiotic drugs has driven efforts by the scientific community to prospect for peptides with a broad spectrum of action. In this context, antimicrobial peptides (AMPs) have acquired great scientific importance in recent years due to their ability to possess antimicrobial and immunomodulatory activity. In the last two decades, plants have attracted the interest of the scientific community and industry as regards their potential as biofactories of heterologous proteins. One of the most promising approaches is the use of viral vectors to maximize the transient expression of drugs in the leaves of the plant Nicotiana benthamiana. Recently, the MagnifectionTM expression system was launched. This sophisticated commercial platform allows the assembly of the viral particle in leaf cells and the systemic spread of heterologous protein biosynthesis in green tissues caused by Agrobacterium tumefaciens "gene delivery method". The system also presents increased gene expression levels mediated by potent viral expression machinery. These characteristics allow the mass recovery of heterologous proteins in the leaves of N. benthamiana in 8 to 10 days. This system was highly efficient for the synthesis of different classes of pharmacological proteins and contains enormous potential for the rapid and abundant biosynthesis of AMPs.

In silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design.

Plants are extensively used in traditional medicine, and several plant antimicrobial peptides have been described as potential alternatives to conventional antibiotics. However, after more than four decades of research no plant antimicrobial peptide is currently used for treating bacterial infections, due to their length, post-translational modifications or  high dose requirement for a therapeutic effect . Here we report the design of antimicrobial peptides derived from a guava glycine-rich peptide using a genetic algorithm. This approach yields guavanin peptides, arginine-rich α-helical peptides that possess an unusual hydrophobic counterpart mainly composed of tyrosine residues. Guavanin 2 is characterized as a prototype peptide in terms of structure and activity. Nuclear magnetic resonance analysis indicates that the peptide adopts an α-helical structure in hydrophobic environments. Guavanin 2 is bactericidal at low concentrations, causing membrane disruption and triggering hyperpolarization. This computational approach for the exploration of natural products could be used to design effective peptide antibiotics.

Evaluation of Caenorhabditis elegans as a host model for Paracoccidioides brasiliensis and Paracoccidioides lutzii.

Paracoccidioidomycosis is a systemic fungal infection affecting mainly Latin American countries that is caused by Paracoccidioides brasiliensis and Paracoccidioides lutzii. During the study of fungal pathogenesis, in vivo studies are crucial to understand the overall mechanisms involving the infection as well as to search for new therapeutic treatments and diagnosis. Caenorhabditis elegans is described as an infection model for different fungi species and a well-characterized organism to study the innate immune response. This study evaluates C. elegans as an infection model for Paracoccidioides spp. It was observed that both species do not cause infection in C. elegans, as occurs with Candida albicans, and one possible explanation is that the irregular size and shape of Paracoccidioides spp. difficult the ingestion of these fungi by the nematode. Besides this difficulty in the infection, we could observe that the simple exposition of C. elegans to Paracoccidioides species was able to trigger a distinct pattern of expression of antimicrobial peptide genes. The expression of cnc-4, nlpl-27 and nlp-31 was superior after the exposure to P. brasiliensis in comparison to P. lutzii (P < 0.05), and these findings demonstrate important differences regarding innate immune response activation caused by the two species of the Paracoccidioides genus.

Heterologous expression of abaecin peptide from Apis mellifera in Pichia pastoris.

BACKGROUND: Antimicrobial peptides (AMPs) are the first line of host immune defense against pathogens. Among AMPs from the honeybee Apis mellifera, abaecin is a major broad-spectrum antibacterial proline-enriched cationic peptide. RESULTS: For heterologous expression of abaecin in Pichia pastoris, we designed an ORF with HisTag, and the codon usage was optimized. The gene was chemically synthetized and cloned in the pUC57 vector. The new ORF was sub-cloned in the pPIC9 expression vector and transformed into P. pastoris. After selection of positive clones, the expression was induced by methanol. The supernatant was analyzed at different times to determine the optimal time for the recombinant peptide expression. As a proof-of-concept, Escherichia coli was co-incubated with the recombinant peptide to verify its antimicrobial potential. DISCUSSION: Briefly, the recombinant Abaecin (rAbaecin) has efficiently decreased E. coli growth (P < 0.05) through an in vitro assay, and may be considered as a novel therapeutic agent that may complement other conventional antibiotic therapies.

Developmental expression of LTßR and differential expression in Escherichia coli F18 resistant/sensitive piglets.

We analyzed LTßR mRNA expression in piglets from birth to weaning and compared the differential expression between Escherichia coli F18-resistant and sensitive populations to determine whether this gene could be used as a genetic marker for E. coli F18 resistance. Sutai piglets of different age groups (8, 18, 30, and 35 days; N = 4 each) and piglets demonstrating resistance/sensitivity to E. coli F18 were used. LTßR expression levels were determined by real-time PCR. The LTßR expression levels in the lymph node, duodenum, and jejunum were significantly higher in 8-day-old piglets than in the other age groups (P < 0.01), and the expression levels were significantly higher in the lungs of 8-day-old piglets than in 35-day-old piglets (P < 0.01) and 30 day-old piglets (P < 0.05). In liver tissue, the expression level was significantly higher in the 35-day-old piglets than in other age groups (P < 0.01). In the stomach tissue, the expression level was significantly higher in 35-day-old piglets than in 18-day-old piglets (P < 0.05). LTßR expression in the lymph nodes was significantly higher in the resistant group than in the sensitive group (P < 0.01), but there was no significant difference in the other tissues (P > 0.05). These results indicate that 8 days after birth is a crucial stage in the formation of mesentery lymph nodes and immune barriers in pigs, and increased expression of LTßR may be beneficial for developing resistance to E. coli F18.

Reflections About Inflammatory Bowel Disease and Vitamins A and D.

Ulcerative colitis and Crohn's disease are two major forms of the inflammatory bowel diseases (IBDs). Vitamin A (VA) and vitamin D (VD) may be associated with reduction in inflammation in these disorders. The aim of this review was to show the current evidence that may associate VA and VD with IBDs. Data linking VA, VD, and IBDs were studied. Both VA and VD may be related to the immune system in different manners. The active form of VA, retinoic acid, may be related to the growth factor-ß and release of interleukin-10 (IL-10), thus involved with the resolution of the inflammation. Its deficiency is associated with the increase of disease activity. The active form of VD is 1,25(OH)2D3 that produces biological effects via the nuclear hormone receptor named VD receptor (VDR), which may interfere with the immune cells and macrophages leading to the suppression of the inflammatory process by decreasing the release of TNF-α, IL-1, IL-6, and IL-8, IL-12, and IL-23. VDR may also activate nucleotide-binding oligomerization domain 2 expression and stimulate the production of the defensin and cathelicidin that are important to the homeostasis of the mucosal immune barrier. The use of VA and VD could be helpful in the treatment and prevention of IBDs but more studies are necessary to establish the precise role of these compounds in the prevention or remission of these inflammatory processes.

Impact of Trypanosoma cruzi on antimicrobial peptide gene expression and activity in the fat body and midgut of Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In natural habitats, these insects are in contact with a variety of bacteria, fungi, virus and parasites that they acquire from both their environments and the blood of their hosts. Microorganism ingestion may trigger the synthesis of humoral immune factors, including antimicrobial peptides (AMPs). The objective of this study was to compare the expression levels of AMPs (defensins and prolixicin) in the different midgut compartments and the fat body of R. prolixus infected with different T. cruzi strains. The T. cruzi Dm 28c clone (TcI) successfully develops whereas Y strain (TcII) does not complete its life- cycle in R. prolixus. The relative AMP gene expressions were evaluated in the insect midgut and fat body infected on different days with the T. cruzi Dm 28c clone and the Y strain. The influence of the antibacterial activity on the intestinal microbiota was taken into account. METHODS: The presence of T. cruzi in the midgut of R. prolixus was analysed by optical microscope. The relative expression of the antimicrobial peptides encoding genes defensin (defA, defB, defC) and prolixicin (prol) was quantified by RT-qPCR. The antimicrobial activity of the AMPs against Staphylococcus aureus, Escherichia coli and Serratia marcescens were evaluated in vitro using turbidimetric tests with haemolymph, anterior and posterior midgut samples. Midgut bacteria were quantified using colony forming unit (CFU) assays and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Our results showed that the infection of R. prolixus by the two different T. cruzi strains exhibited different temporal AMP induction profiles in the anterior and posterior midgut. Insects infected with T. cruzi Dm 28c exhibited an increase in defC and prol transcripts and a simultaneous reduction in the midgut cultivable bacteria population, Serratia marcescens and Rhodococcus rhodnii. In contrast, the T. cruzi Y strain neither induced AMP gene expression in the gut nor reduced the number of colony formation units in the anterior midgut. Beside the induction of a local immune response in the midgut after feeding R. prolixus with T. cruzi, a simultaneous systemic response was also detected in the fat body. CONCLUSIONS: R. prolixus AMP gene expressions and the cultivable midgut bacterial microbiota were modulated in distinct patterns, which depend on the T. cruzi genotype used for infection.

Expression of essential genes for biosynthesis of antimicrobial peptides of Bacillus is modulated by inactivated cells of target microorganisms.

Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms.

The activation of the TLR2/p38 pathway by sodium butyrate in bovine mammary epithelial cells is involved in the reduction of Staphylococcus aureus internalization.

Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1ß and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen invasion, and NaB may exert anti-inflammatory effects during infection.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Ascorbic acid, ultraviolet C rays, and glucose but not hyperthermia are elicitors of human ß-defensin 1 mRNA in normal keratinocytes.

Hosts' innate defense systems are upregulated by antimicrobial peptide elicitors (APEs). Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA), and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulation of human ß-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes (NHK). The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C) and hyperthermia (39°C) are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections.

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Diversity and potential application of endophytic bacteria in ginger.

Here, 248 endophytic bacterial strains were isolated to assess the distribution and population diversity of endophytic bacteria in ginger plants. A total of 10.4 x 10(4) to 20.2 x 10(4) CFU/g fresh weight endophytic bacteria of different growth stages were isolated. Maximum bacterium numbers were obtained at the seedling stage. A total of 107 functional strains were screened, including 17 antibacterial strains and 90 indole acetic acid-producing strains. Based on 16S rDNA sequence restriction fragment length polymorphism and 16S rDNA sequences, these 107 strains were mapped and grouped into 16 genera. Bacillus and Pseudomonas were the dominant genera; however, the bacteria belonged to a tremendous range of genera, with the highest species richness being observed at the seedling stage. Sixteen strains exhibited antimicrobial activity against Pythium myriotylum Drechsler, while 7 strains exhibited antimicrobial activity against Phyllosticta zingiberi Hori. Bacillus was the dominant antibacterial strain. Pseudomonas fluorescens, B. megaterium, and Enterobacter ludwigii produced remarkably high levels of IAA. Only a few endophytic bacterial strains were inhibited in fresh ginger juice. Most of these strains were present during seedling stage, including Roseateles depolymerans, Chryseobacterium taiwanense, E. ludwigii, Agrobacterium larrymoorei, P. fluorescens, and Bacillus amyloliquefaciens. This study indicates that the community of endophytic bacteria in ginger changes with the synthesis of antibacterial substances.