Dietary Supplementation with Pomegranate and Onion Affects Lipid and Protein Oxidation in the Breast Meat, Thigh, and Liver, Cellular Stress Protein Responses, and Gene Expression of Liver Enzymes Involved in Protein Synthesis in Broilers.

The present study examined the effects of dietary supplementation with extracts of pomegranate (Punica granatum) and onion (Allium cepa), either encapsulated in cyclodextrin (POMALCD group) or in an aqueous (POMALAQ group) form, on breast meat, thigh meat, and liver composition, oxidative stability, cellular signaling pathways, and the gene expression of certain hepatic genes. The results showed that breast and thigh meat contained significantly (p < 0.05) higher moisture content in the group with the aqueous extract, compared to the control and POMALCD groups. Moreover, the protein content was significantly (p < 0.05) higher in the thigh and liver samples of the treated groups in comparison to the control. The iron-induced challenge deteriorated (p < 0.001) the lipid and protein oxidative status of the control group, whereas both supplemented groups showed considerable tolerance in all tissues. The supplementation of pomegranate and onion extracts mitigated or maintained heat shock protein (HSP) levels and elevated (p < 0.05) the Bcl-2/Bad ratio in thigh and breast meat, whereas mitogen-activated protein kinase (MAPK) activation was modulated at a lower rate. After normalization to ß-actin expression, quantitative real-time PCR analysis revealed a significant (p < 0.05) induction in the expression of MTR and MSRB1 genes in the liver of the supplemented groups. No differences were observed for the TAT, SMS, and BHMT genes. In conclusion, dietary mixtures of herbal extracts with pomegranate and onion improved protein and lipid oxidative stability in meat, enhanced the hepatic energy status, and exerted ameliorative effects on stress-related proteins. The encapsulated extract of pomegranate and onion, using cyclodextrin as a carrier, appeared to reduce lipid oxidation to a greater extent than the aqueous extract. In contrast, the aqueous extract exhibited higher total antioxidant capacity (TAC) values and provided better protection against protein carbonyl formation.

Synergistic, bacteriostatic effect of propolis and glycerol against Listeria monocytogenes in chocolate milk under refrigerated storage.

Propolis ethanolic extracts, with or without glycerol, were added into pasteurized, non-fat chocolate milk, which was artificially contaminated with Listeria monocytogenes. The addition of propolis ethanolic extracts dissolved into glycerol led to a definite anti-listerial effect in milk stored at 4 ℃, with both propolis concentrations tested (2 or 4 mg of dry propolis ethanolic extract per milliliter of chocolate milk) leading to inhibition of L. monocytogenes growth throughout 20 days of storage. The combined addition of propolis ethanolic extracts with glycerol was also effective in significantly reducing the rate of growth of L. monocytogenes in chocolate milk stored under improper (10 ℃) refrigeration storage conditions (more than five-fold increase in the generation time of L. monocytogenes compared to control trials). Finally, the combined addition of a deodorized propolis ethanolic extract with glycerol resulted in a significant anti-listerial effect upon storage of contaminated milk at 4 ℃ (more than three-fold increase in the generation time of L. monocytogenes compared to controls) and in a smaller anti-listerial effect upon milk storage at 10 ℃ (two-fold increase in the generation time of the pathogen compared to controls). Of note, chocolate milk containing deodorized propolis ethanolic extract and glycerol received a positive consumer acceptability score on the nine-point hedonic scale (median acceptability score of "7"). Hence, propolis may possess a promising role as a natural anti-listerial preservative in dairy drinks.

A Ptolemaic mummy reveals evidence of invasive dentistry in ancient Egypt.

Over the last decades, it has been confirmed that computerized tomography (CT) is a valuable tool for studying mummies. In joint efforts put forth by the Mummy Research Project of the Hellenic Institute of Egyptology, the National Archaeological Museum, and the Athens Medical Center, a mummy was transported to the Radiology Department of the Athens Medical Center for study. Thus, a complete CT scanning was performed of this Ptolemaic mummy (AIG 3343: Sekhem, male, 150-30 BCE), belonging to the Egyptian Collection of the National Archaeological Museum of Athens. The most significant finding is an interproximal carious cavity packed with protective material. This is the second case of dental packing in the literature among ancient Egyptian mummies studied to date. Its remarkable resemblance to the previously published study may indicate a common dental intervention performed by ancient Egyptians. Despite the well-known early medical traditions of ancient Egypt, spanning from the Old Kingdom to the Ptolemaic and Roman Periods, little evidence remains of their practices in dentistry. Our finding represents a rare perspective on the origins of what remains today a major allied health field discipline.

Chronic maxillary atelectasis under the wrappings of an Egyptian mummy.

OBJECTIVES: In the context of a joint Mummy Research Project of the National Archaeological Museum, the Hellenic Institute of Egyptology and the Athens Medical Centre, an Egyptian mummy of the mid-Ptolemaic Period was transferred to our hospital and was thoroughly investigated with Computed Tomography. METHODS: The mummy was carefully removed from its coffin and scanned in a 64-detector row computed tomographic scanner. Multiplanar and anthropometric measurements were obtained using advanced software. RESULTS: The mummy appeared to be well-preserved and belonged to a young male adult. Among the findings, the most interesting and uncommon one was the asymmetry of the maxillary sinuses and the orbits. There were no signs of trauma. CONCLUSIONS: Computed Tomography revealed in a non-destructive way a rare, based on the published data, facial deformity in an Egyptian mummy attributed to chronic maxillary atelectasis.

Inhibitory activity of propolis against Listeria monocytogenes in milk stored under refrigeration.

Propolis is a natural bee-product with documented antimicrobial properties in vitro. The objective of this study was to develop a protocol for adding propolis into milk and to determine whether the addition of propolis can confer anti-listerial activity during the storage of milk under optimal or improper refrigeration conditions. Upon dissolving propolis ethanolic extract (PEE) into glycerol, the PEE-glycerol mixture contained no visible insoluble particles and could be dispersed evenly into milk, without leaving any insoluble material. PEE, with or without glycerol, was added into extended shelf-life milk, artificially contaminated with Listeria monocytogenes. The addition of PEE dissolved into glycerol resulted in a pronounced and dose-dependent anti-listerial effect in milk stored at 4 °C, with the higher concentration tested (4 mg of dry PEE per mL of milk) resulting in complete inhibition of L. monocytogenes growth throughout 30 days of storage. The combination of PEE with glycerol was also effective in significantly reducing the growth rate of the pathogen in milk stored under improper refrigeration (10 °C). Based on a patented PEE-deodorization protocol, the addition of deodorized PEE into milk resulted in a product with average consumer acceptability. However, the PEE deodorization process resulted in reduction or even complete removal of propolis constituents with known antibacterial activity, with a concomitant significant reduction in its anti-listerial effect. Nonetheless, the data presented in this manuscript highlight the strong anti-listerial potential of propolis in milk and suggest that, upon further research on its deodorization and standardization, there may be room for the application of propolis as a natural preservative in dairy beverages.

Study of Toll-like receptor and B-defensins genes expression pattern in porcine reproductive organs.

Toll-like receptors (TLRs) and b-defensins (BD) molecules are group of molecules that recognize various microbial components and play a crucial role in the activation of the innate immune system in vertebrate species. Although TLRs gene expression has been studied in various pig tissues, little is known about their expression in porcine reproductive tract. Concerning b-defensins genes, only BD1, 2 and 3 counterparts have been well studied in pigs' reproductive organs. The aim of this study was to investigate the expression pattern of both gene families in pigs' male and female reproductive organs, and embryos, as potential tool for further association studies in respect to immunity and disease resistance. RT-PCR analysis revealed that all of the examined TLR genes were expressed in the reproductive organs of male and female pigs, with TLR3 and TLR5 showing the higher levels and TLR9 the lowest, in all analyzed tissues. BD genes showed a different expression pattern in respect to the examined tissue. In embryos, TLR1 revealed high expression levels, while only BD3, BD108, and BD123 were found to be expressed.

Activation of innate immune system in response to lipopolysaccharide in chicken Sertoli cells.

Sertoli cells (SCs) play an important physiological role in the testis, as they support, nourish, and protect the germ cells. As protection of the developing spermatozoa is an emerging aspect of reproductive physiology, this study examined the expression pattern of innate immune-related genes, including avian ß-defensins (AvBDs), Toll-like receptors (TLRs), and cytokines, and investigated the time course of an inflammatory response in rooster SCs triggered by exposure to the bacterial endotoxin lipopolysaccharide (LPS). SCs were isolated from 6-week-old chicken, cultured in vitro, and stimulated with 1 µg/ml LPS at different time courses (0, 6, 12, 24, and 48  h). Data on expression analysis revealed that all ten members of the chicken TLR family, nine members of the AvBD family, as well as eight cytokine genes were expressed in SCs. Quantitative real-time PCR analysis revealed that LPS treatment resulted in significant induction of the expression levels of six TLRs, six AvBDs, and four cytokine genes, while two cytokine genes were downregulated and two other genes were unchanged. The increasing interleukin 1ß (IL1ß) production was confirmed in the conditioned medium. Furthermore, the phagocytosis of SCs was increased after LPS treatment. In conclusion, these findings provide evidence that SCs express innate immune-related genes and respond directly to bacterial ligands. These genes represent an important component of the immune system, which could be integrated into semen, and present a distinctive constituent of the protective repertoire of the testis against ascending infections.

Vascular and parenchymal lesions along with enhanced neurogenesis characterize the brain of asymptomatic stroke-prone spontaneous hypertensive rats.

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.

Temporal changes in the expression of avian ß-defensins in the chicken vagina during sexual maturation and Salmonella infection.

Avian ß-defensins (AvßDs) constitute a family of antimicrobial peptides that are critical to innate immunity in chickens, providing protection against microbial pathogens including Salmonella Enteritidis (SE). As apart from the digestive tract another main route of SE colonization in birds is via infection of the oviduct and specifically of the vagina, the aim of this study was to investigate the expression of the complete family of AvßDs, in the chicken vagina in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the vaginal AvßDs expression. Expression analysis revealed that 11 members of the AvßD family were expressed in the chicken vagina. Quantitative real-time PCR analysis revealed that the mRNA abundance of five AvßDs was up regulated and of one AvßD was down regulated with respect to sexual maturation. In addition SE infection resulted in a significant induction of AvßD5, 7, 10, 11, 12 and 14 in the vagina of sexually mature birds, and in a significant induction of AvßD5 and 11 in the vagina of aged birds. These findings provide strong evidence to suggest that an AvßD-mediated immune response mechanism exists in the chicken vagina providing protection against bacterial pathogens including Salmonella species.

Characterization of PROFILIN genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors and expression analysis in cotton genotypes differing in fiber characteristics.

The actin-binding protein profilin (PRF) plays an important role in cell growth and expansion by regulating the organization of the actin filaments. Recent studies have reported association between fiber elongation in cultivated cotton (Gossypium hirsutum) and PRF expression. In the present study, we cloned four genomic clones from allotetraploid cotton (G. hirsutum) and its putative diploid progenitors (G. arboreum and G. raimondii) designated GhPRF1_A, GhPRF1_D, GaPRF1, and GrPRF1 encoding cotton PRF and characterized their genomic structure, phylogenetic relationships and promoter structure. Sequence analysis of the coding regions of all clones resulted in a single protein product which revealed more than 80% similarity to most plant PRFs and a typical organization with an actin-binding and a polybasic phospholipid binding motif at the carboxy terminus. DNA blot hybridization suggested that PRF gene is present with more than one copy in the allotetraploid species G. hirsutum. Expression analysis performed in various organs of cultivated cotton revealed that the PRF gene was preferentially expressed in cotton fibers. Very low levels of expression were observed in whole flowers, while PRF transcripts were not detected in other organs examined. Furthermore, higher levels of expression were observed at the early stages of cotton fiber development (at 10 days post anthesis), indicative that this gene may play a major role in the early stages of cotton fiber development. Quantitation of the expression by real-time PCR revealed higher expression levels in a G. hirsutum variety with higher fiber percentage compared to a variety with lower percentage. In addition, higher levels of expression were found in cultivated allotetraploid G. barbadense cotton species with higher fiber length in comparison to cultivated allotetraploid G. hirsutum.

Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to Salmonella enterica infection.

In recent years host antimicrobial peptides and proteins have been recognised as key mediators of the innate immune response in many vertebrate species, providing the first line of defense against potential pathogens. In chickens a number of cationic antimicrobial peptides have been recently identified. However, although these peptides have been studied extensively in the avian gastrointestinal tract, little is known about their function in the chicken reproductive organs and embryos. Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) has been previously reported to function in protecting birds against microbial attack. The aim of this study was to investigate the expression of cLEAP-2 gene in the chicken reproductive organs, as well as in chicken embryos during embryonic development, and to determine whether cLEAP-2 expression in the chicken reproductive organs was constitutive or induced as a response to Salmonella enteritidis infection. RNA was extracted from ovary, oviduct, testis and epididymis of sexually mature healthy and Salmonella infected birds, as well as from chicken embryos until day ten of embryonic development. Expression analysis data revealed that cLEAP-2 was expressed in the chicken ovary, testis and epididymis as well as in embryos during early embryonic development. Quantitative real-time PCR analysis revealed that cLEAP-2 expression was constitutive in the chicken epididymis, but was significantly up regulated in the chicken gonads, following Salmonella infection. In addition, expression of cLEAP-2 during chicken embryogenesis appeared to be developmentally regulated. These data provide evidence to suggest a key role of cLEAP-2 in the protection of the chicken reproductive organs and the developing embryos from Salmonella colonization.

Expression of chicken zygote arrest 1 (Zar1) and Zar1-like genes during sexual maturation and embryogenesis.

Maternal mRNAs, which are expressed in oocytes, play an important role in the success of early embryo development, as they allow the first cleavages to occur. Zygote arrest 1 (Zar1) is an oocyte-specific maternal-effect gene that functions at the oocyte-to-embryo transition in many vertebrate species including human, pig, cattle, sheep, mouse, rat, frog and zebrafish. Recently, through in silico studies, a gene structurally related to Zar1, called Zar1-like has been identified in many vertebrates, including the chicken. The objectives of this study were to investigate the expression of the chicken Zar1 and Zar1-like genes in chicken tissues and embryos and to determine whether sexual maturation affects their mRNA abundance. RNA was extracted from various organs of chickens aged from one month up to two years old and from chicken embryos until day ten of embryonic development. Expression analysis of the genes was performed using RT-PCR and real-time PCR. RT-PCR analysis revealed that both genes were preferentially expressed in chicken oocytes, ovary and testes and in embryos during embryonic development. Quantitative real-time PCR analysis revealed a significant up regulation of Zar1 in the mature ovary, and also a significant up regulation of Zar1 and Zar1-like genes in the testes of sexually mature roosters, suggesting a key role of these genes in the chicken fertility. In contrast, expression of Zar1-like was not affected by age in the chicken ovary. Our results indicate that the chicken Zar1 and Zar1-like transcripts are co-expressed in high levels in the chicken gonads. In addition their expression beyond the stage of embryonic genome activation suggests an embryonic and not only a maternal origin of these transcripts.

The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane.

Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) is known to have killing activities against Salmonella spp., but the mechanism by which killing occurs remains to be elucidated. The ability of cLEAP-2 to disrupt the outer membrane of several Salmonella spp. was assessed using the fluorescent probe N-phenyl-1-naphthylamine (NPN). A rapid dose-dependent permeabilization of the outer membranes of Salmonella enterica serovar Typhimurium phoP, and S. enterica serovar Typhimurium SL1344 was observed although no significant permeabilisation of the S. enteriditis membrane was detected. These data suggested that the ability of the mature cLEAP-2 peptide to permeabilise the Salmonella outer membrane is important in mediating its killing activities. The ability of the peptide to kill Gram-positive bacteria, specifically Streptococcus spp. and Staphylococcus spp. was also investigated using recombinant peptide and a time-kill assay. Of the strains analysed the Streptococcus pyogenes M1 strain appeared the most resistant to LEAP-2 killing although S. pyogenes mutants deficient in Sortase and M1 activities showed increased sensitivity to the mature peptide. This suggested the involvement of specific Streptococcus cell wall proteins including M1 in the resistance of the bacteria to cLEAP-2 killing. cLEAP-2 showed no significant toxicity towards mammalian erythrocytes indicating selectivity for bacterial over eukaryote cell membranes. These data provide further support for mature cLEAP-2 functioning in protecting the chicken against microbial attack.

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors expressed during fiber elongation.

Multiple cellular pathways have been shown to be involved during fiber initiation and elongation stages in the cultivated allotetraploid cotton (Gossypium hirsutum). The cell wall enzymes xyloglucan endotransglycosylase/hydrolases (XTH) have been reported to be associated with the biosynthesis of the cell wall and the growth of cotton fibers, probably regulating the plasticity of the primary cell wall. Among various cotton fiber cDNAs found to be preferentially expressed in cotton fibers, a xyloglucan endotransglycosylase (XTH) cDNA was significantly up-regulated during the elongation stage of cotton fiber development. In the present study, we isolated and characterized genomic clones encoding cotton XTH from cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii), designated GhXTH1-1, GhXTH1-2, GaXTH1 and GrXTH, respectively. In addition, we isolated and characterized, by in silico methods, the putative promoter of XTH1 from Gossypium hirsutum. Sequence analysis revealed more than 50% homology to XTH's at the protein level. DNA gel blot hybridization indicated that at least two copies of GhXTH1 are present in Gossypium hirsutum whereas the diploid progenitor species Gossypium arboreum and Gossypium raimondii has only a single copy. Quantitative real-time PCR and high-resolution melting experiments indicated that in Gossypium hirsutum cultivars, in cotton fibers during early stages of fiber elongation specifically expressing only the GhXTH1-1 gene and expression levels of GhXTH1-1 in fibers varies among cultivars differing in fiber percentage and fiber length.

Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors.

The seasonal cycle and persistence of a plant is governed by a combination of the determinate or indeterminate status of shoot and root apical meristems. A perennial plant is one in which the apical meristem of at least one of its shoot axes remains indeterminate beyond the first growth season. TERMINAL FLOWER1 (TFL1) genes play important roles in regulating flowering time, the fate of inflorescence meristem and perenniality. To investigate the role of TFL1-like genes in the determination of the apical meristems in an industrially important crop cultivated for its fibers, we isolated and characterized two TFL1 homologs (TFL1a and TFL1b) from tetraploid cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii). All isolated genes maintain the same exon-intron organization. Their phylogenetic analysis at the amino acid level confirmed that the isolated sequences are TFL1-like genes and collocate in the TFL1 clade of the PEBP protein family. Expression analysis revealed that the genes TFL1a and TFL1b have slightly different expression patterns, suggesting different functional roles in the determination of the meristems. Additionally, promoter analysis by computational methods revealed the presence of common binding motifs in TFL1-like promoters. These are the first reported TFL1-like genes isolated from cotton, the most important crop for the textile industry.

Effects of treatment with somatostatin analogues on QT interval duration in acromegalic patients.

OBJECTIVE: Cardiovascular disease is a major contributor to the increased mortality of acromegalic patients. Prolongation of the QT interval is considered an established risk factor for potentially fatal cardiac arrhythmias, an event frequently observed in acromegaly. Changes in ventricular repolarization have been observed with the use of octreotide, one of the somatostatin analogues (SSA) currently used for the medical treatment of this disease. Furthermore, octreotide is listed among the drugs able to prolong the QT interval. Thus, we elected to study the effects of long-term SSA administration on QT duration and left ventricular mass (LVM) in a group of acromegalic patients. DESIGN AND PATIENTS: In a retrospective study, 30 acromegalic patients (19 women and 11 men, aged 25-77 years) were studied under basal conditions; 24 of them (15 women and nine men, aged 25-77 years) were studied again after 3-63 months of treatment (median 18 months) with SSA. Twenty-four healthy volunteers served as controls. MEASUREMENTS: Patients and controls underwent electrocardiographic (ECG) analysis, and QT interval duration corrected for heart rate (QTc) was established according to the Bazett formula. In 17 of the SSA-treated patients, M- and B-mode echocardiography for the assessment of LVM index (LVMi) was performed. RESULTS: Baseline QTc was significantly longer in patients than in controls. SSA administration was followed by a significant decrease in QTc, which reached a mean value similar to that measured in the controls. In particular, treatment with SSA normalized QTc in three out of the six patients with abnormally elevated values at baseline. After treatment, a significant reduction in heart rate was recorded, while LVMi displayed a slight but not significant decrease. CONCLUSIONS: Acromegalic patients frequently display an abnormally prolonged QT interval, a known risk factor for potentially fatal arrhythmias. Treatment of these patients with SSA is able to improve and even normalize this alteration, probably contributing to the beneficial effects of these drugs on cardiac rhythm in this endocrine disorder. The inclusion of octreotide in the list of drugs that may increase QTc should be reconsidered as regards its indication in acromegaly.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts.

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.

Induction of cationic chicken liver-expressed antimicrobial peptide 2 in response to Salmonella enterica infection.

Cationic antimicrobial peptides constitute part of the innate immune system and provide an essential role in the defense against infection. At present there is a paucity of information regarding the antimicrobial profile of the chicken (Gallus gallus). Using in silico studies, an expressed sequence tag (EST) clone was identified which encodes a novel cationic antimicrobial peptide, chicken liver-expressed antimicrobial peptide 2 (cLEAP-2). The predicted amino acid sequence composed a prepropeptide, and the active peptide contained four conserved cysteine amino acids. The gene was localized to chromosome 13, and analysis of the genome revealed three exons separated by two introns. The cLEAP-2 gene was expressed in a number of chicken epithelial tissues including the small intestine, liver, lung, and kidney. Northern analysis identified liver-specific cLEAP-2 splice variants, suggesting some degree of tissue-specific regulation. To investigate whether cLEAP-2 expression was constitutive or induced in response to microbial infection, 4-day-old birds were orally infected with Salmonella. Analyses of cLEAP-2 expression by semiquantitative reverse transcription-PCR indicated that cLEAP-2 mRNA was upregulated significantly in the small intestinal tissues and the liver, indicative of direct and systemic responses. The antimicrobial activity of cLEAP-2 against Salmonella was analyzed in vitro with a time-kill assay and recombinant cLEAP-2. Interestingly Salmonella enterica serovar Typhimurium SL1344 showed increased susceptibility to the active cationic peptide (amino acids 37 to 76) compared to S. enterica serovar Typhimurium C5 and Salmonella enteritidis. Taken together, these data suggest that cationic cLEAP-2 is part of the innate host defense mechanisms of the chicken.