Correlation of IL-12, IL-22, and IL-23 in patients with psoriasis and metabolic syndrome. Preliminary report.

BACKGROUND: Psoriasis is an autoimmune skin disease characterised by proliferation of keratinocytes, primarily due to cytokines Th1 and Th17. This profile is involved in pathogenesis of metabolic syndrome, a frequently found comorbidity in patients with psoriasis. OBJECTIVE: In this study we determine the correlation of levels of pro-inflammatory cytokines TNF-α, IL-23, IL-12, and IL-22 in patients with psoriasis with and without metabolic syndrome and clinically healthy controls. METHODS: We included 55 patients with plaque psoriasis: 30 with metabolic syndrome (PPMS), 25 without metabolic syndrome (PP), 15 healthy subjects (HS) and 15 with metabolic syndrome (MS). Quantification of serum levels of IL-12, TNF-α, IL-22, and IL-23 was done by ELISA. RESULTS: We observed that serum levels of IL-12 were more elevated in PP group, while the lowest levels of TNF-α were seen in HS group. IL-22 was found to be higher in PP than in PPMS (p<0.05). PP patients with PASI scores rating as severe showed higher levels of IL-12. TNF-α level analysis showed significant differences in HS group compared with the others; levels of this cytokine were lower in patients with PP and moderate PASI scores than in MS group (p<0.05). We found no correlation between cytokine levels and psoriasis or between cytokines and PASI scores. In PP group, a positive correlation was observed between IL-23 and fasting glucose (r=0.432, p<0.05), as well as a negative correlation between IL-23, IL-22, and IL-12 versus waist circumference (r=-0.504, r=-0.556 and r=-0.511, respectively; p<0.05). CONCLUSIONS: Psoriasis is not just a skin disorder, but rather a condition with systemic implications, with intervention of pro-inflammatory cytokines that contribute to metabolic syndrome and other comorbidities, which in turn increases the risk of developing cardiovascular disease.

5-HT Receptor Antagonism Attenuates the Ischemia-Reperfusion Injury After Rabbit Lung Preservation.

The success of lung transplantation is threatened by the appearance of ischemia-reperfusion injury, which is characterized by increased vascular permeability. 5-Hydroxytryptamine (5-HT; serotonin) is known to produce microvascular leakage in the systemic circulation, but its possible role in ischemia-reperfusion injury after lung preservation has not been reported. In this work we measured the release of 5-HT during a 24-hour rabbit lung preservation, and the effect of methiothepin (antagonist of the majority of 5-HT receptors) and SB204741 (antagonist of 5-HT2B/2C receptors) on the modified capillary filtration coefficient (mKf,c) was evaluated at the end of this period. Our results showed that the highest release rate of 5-HT occurred during the first 15 minutes after the lung harvesting and progressively decreased in the following time intervals. The baseline mKf,c greatly increased after 24 hours of lung preservation, and this increment was partially reduced by methiothepin and even more by SB204741. We concluded that 5-HT may play an important role in the ischemia-reperfusion process after lung preservation.

Associations of killer cell immunoglobulin- like receptor genes with rheumatoid arthritis.

OBJECTIVE: Rheumatoid Arthritis (RA) is an autoimmune and chronic inflammatory disease of unknown etiology. Killer cell immunoglobulin-like receptors are expressed on the surface of natural killer cells and CD28null T-cells, both present in synovial membrane of RA. Therefore we evaluated the associations of KIR genes with RA. METHODS: 16 KIR genes were genotyped in 100 healthy subjects (HS) and 100 RA patients from Western Mexico using PCR-SSP. Differences in KIR genotypes and gene frequencies were assessed using the X^{2} test. RESULTS: Gene frequency of KIR2DL3 was lower in RA than in HS (p= 0.0019), whereas KIR2DL2 and KIR2DS2 were higher in RA than HS (p =0.0004 and p = 0.0487, respectively). In addition were identified 38 genotypes (from G1-G38) in both studied groups, and the genotype frequencies of G1, G6 and G14 showed significant differences (p =0.0001, p =0.0208 and p =0.0300, respectively). CONCLUSIONS: The presence of KIR2DL2, KIR2DS2 and absence of KIR2DL3 are associated with RA. Moreover, two genotypes BX are associated with RA. These results suggest that KIRs can be involved in RA susceptibility.

Association of CD28 IVS3 +17T/C polymorphism with soluble CD28 in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology in which inflammatory pathology involves T cell activation and the CD28 costimulatory molecule involved in T cell presentation. The gene includes the CD28 IVS3 +17T/C polymorphism that could be associated with susceptibility to RA whereas the soluble concentrations of CD28 (sCD28) could be related to clinical activity. METHODS: We investigated the CD28 IVS3 +17T/C polymorphism in 200 RA patients and 200 healthy subjects (HS). Furthermore, we quantified the sCD28 concentrations in 77 samples of each group. We applied indexes focused to determine the activity and disability (DAS28 and Spanish HAQ-DI, respectively) in RA patients. RESULTS: RA patients had significantly higher frequencies of the CD28 T allele compared to HS (p = 0.032 OR = 1.59, C.I. 1.02-2.49). In addition, the IVS3 +17 T/T genotype frequency was also increased in RA vs. HS (p = 0.026). The RA patients showed higher sCD28 serum levels than HS (p = 0.001). Carriers of the T/T genotype in RA patients showed higher sCD28 levels than C/C carriers (p =0.047). In addition, a correlation between sCD28 and Spanish HAQ-DI (correlation, 0.272; p =0.016), was found. CONCLUSION: The T allele in CD28 IVS3 +17T/C polymorphism is associated with a susceptibility to RA in Western Mexico. In addition, increased sCD28 levels are related to T/T genotype in RA patients.

IL-12 and IL-18 levels in serum and gingival tissue in aggressive and chronic periodontitis.

OBJECTIVE: The aim of this study was to compare the levels of interleukin-12 (IL-12) and IL-18 in gingival tissue and serum between patients with chronic (n = 18) or aggressive periodontitis (n = 12) and healthy subjects (HS) (n = 9). METHODS: Gingival tissue biopsies and serum were obtained from all study subjects. The tissue was homogenized and cytokines IL-12 and IL-18 were quantified by enzyme-linked immunosorbent assay. RESULTS: Interleukin-12 levels in gingival tissue were significantly higher in aggressive periodontitis patients than in HS; serum IL-12 was significantly elevated in aggressive periodontitis relative to both chronic periodontitis (CP) and HS. IL-18 levels in gingival tissue showed no significant differences between the groups. Patients with CP showed significantly elevated levels of serum IL-18 compared with HS; however, the aggressive periodontitis group showed no significant differences with either the CP group or the HS. CONCLUSIONS: Our results showed higher levels of IL-12 in gingival tissue and serum of patients with aggressive periodontitis, and IL-18 was elevated in the serum of CP patients. The patterns of IL-12 and IL-18 are different in chronic and aggressive periodontitis; this finding suggests distinctive mechanisms of immunopathogenesis between these forms of periodontitis.

Mitochondrial and nuclear markers for the authentication of partridge meat and the specific identification of red-legged partridge meat products by polymerase chain reaction.

Two PCR assays for the identification of partridge meat (red-legged partridge, chukar partridge, barbary partridge, and gray partridge species) and the specific identification of red-legged partridge meat products were developed based on species-specific primers targeting the 12S ribosomal RNA mitochondrial gene. Moreover, various PCR techniques based on the use of random amplified polymorphic DNA markers and nuclear growth hormone and rhodopsin genes were tested to find a method for the differentiation between pure and hybrid red-legged partridges. Among these techniques, the PCR method based on the amplification and sequencing of a nuclear rhodopsin gene fragment was selected as a suitable tool for the discrimination among meats from pure and hybrid red-legged partridge individuals. The PCR assays reported in this work could be useful in inspection programs to verify the correct labeling of raw and heat-treated partridge meat products.

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal.

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.

Polymerase chain reaction assay for verifying the labeling of meat and commercial meat products from game birds targeting specific sequences from the mitochondrial D-loop region.

A PCR assay was developed for the identification of meats and commercial meat products from quail (Coturnix coturnix), pheasant (Phasianus colchicus), partridge (Alectoris spp.), guinea fowl (Numida meleagris), pigeon (Columba spp.), Eurasian woodcock (Scolopax rusticola), and song thrush (Turdus philomelos) based on oligonucleotide primers targeting specific sequences from the mitochondrial D-loop region. The primers designed generated specific fragments of 96, 100, 104, 106, 147, 127, and 154 bp in length for quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock, and song thrush tissues, respectively. The specificity of each primer pair was tested against DNA from various game and domestic species. In this work, satisfactory amplification was accomplished in the analysis of experimentally pasteurized (72 degrees C for 30 min) and sterilized (121 degrees C for 20 min) meats, as well as in commercial meat products from the target species. The technique was also applied to raw and sterilized muscular binary mixtures, with a detection limit of 0.1% (wt/wt) for each of the targeted species. The proposed PCR assay represents a rapid and straightforward method for the detection of possible mislabeling in game bird meat products.

Identification of raw and heat-processed meats from game bird species by polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial D-loop region.

Polymerase chain reaction-RFLP analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), chukar partridge (Alectoris chukar), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), and woodpigeon (Columba palumbus). Polymerase chain reaction amplification was carried out using a set of primers flanking a conserved region of approximately 310 bp from the mitochondrial D-loop region. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of HinfI, MboII, and Hpy188III endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. Consistent results were obtained with both raw and heat-processed meats.

Molecular analysis of the replication region of the pCIZ2 plasmid from the multiple bacteriocin producer strain Enterococcus faecium L50.

The sequence analysis of the 7383 bp plasmid pCIZ2 from Enterococcus faecium L50 enabled the identification of a DNA region involved in its replication. The structural organization of the pCIZ2 replication region is highly similar to those of well-known theta-replicating plasmids. It contains an untranslated region, the putative replication origin (ori), constituted by two sets of direct repeats of 12 and 22 bp (iterons), and followed by three open-reading frames (orf8 to orf10). orf8 encodes the replication initiation protein (RepE). The transcriptional start site of the replication locus was identified 13 nucleotides upstream of the repE start codon. A two-dimensional agarose gel electrophoresis analysis revealed pCIZ2 intermediates profile typical of the theta-type replication mechanism. Subcloning of different DNA fragments of the pCIZ2 replication region in Escherichia coli and, subsequently, in the plasmidless E. faecium L50/14-2 allowed the determination of the minimal replicon on a 1.2kb DNA fragment containing only the overall ori and repE which also act in trans. The involvement of orf9 in the plasmid copy number and in the plasmid stability was investigated. The pCIZ2 recombinant plasmids constitute narrow-host range shuttle cloning vectors (E. coli-E. faecium) that could be very useful for enterococcal genes studies, allowing an easy identification due to their histochemical recognition.

Technical note: Detection of cat, dog, and rat or mouse tissues in food and animal feed using species-specific polymerase chain reaction.

A PCR method based on the nucleotide sequence variation in the 12S ribosomal RNA, mitochondrial gene has been developed for the specific and qualitative detection and identification of cat, dog, and rat or mouse tissue in food and feedstuffs. The primers designed generated specific fragments of 108, 101, and 96 bp in length for cat, dog, and rat or mouse tissues, respectively. Specificity of the primers was tested against 32 nontarget species including mammals, birds, fish, and plant species. This PCR method allowed detection of raw and heated cat, dog, and rat or mouse tissues in meat/oats mixtures even when the concentration of the target species was reduced to 0.1%. Furthermore, the performance of the method was not affected by prolonged heat-treatment (up to 133 degrees C for 20 min at 300 kPa), and consequently, it could be very useful to verify the origin of raw materials in food and feedstuffs submitted to denaturing technologies, for which other methods cannot be applied.

Antimicrobial and safety aspects, and biotechnological potential of bacteriocinogenic enterococci isolated from mallard ducks (Anas platyrhynchos).

Samples from the intestinal content and carcasses of mallard ducks (Anas platyrhynchos) were evaluated for enterococci with antimicrobial activity, presence of genes coding bacteriocins and their expression, and potential virulence factors. Enterococcus faecalis comprised the largest enterococcal species with antagonistic activity followed by E. faecium, E. hirae, Enterococcus spp., and the non-enterococci. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of most producer cultures. However, all E. faecium isolates showed antimicrobial activity in their supernatants and encoded bacteriocins, although the occurrence in the isolates of several enterocin genes did not always correlate with a higher antagonistic activity in supernatants. The efaAfm determinant was the only virulence gene detected in E. faecium, while E. faecalis showed a larger number of virulence determinants, and E. hirae did not carry any of the virulence genes examined. The rapid identification of genes coding described bacteriocins permits recognition of isolates that are potentially producers of novel bacteriocins. Purification of the antimicrobial activity of E. hirae DCH5 and Lactococcus garvieae DCC43 revealed unique chromatographic fragments after MALDI-TOF mass spectrometry analysis, suggesting the antagonistic peptides were purified to homogeneity. Bacteriocinogenic E. faecium and E. hirae isolates may be considered hygienic for production of bacteriocins, and potentially safe due to their low incidence of potential virulence genes and susceptibility to most clinically relevant antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors, raises concerns regarding their potential pathogenicity to consumers.

Technical note: detection of chicken, turkey, duck, and goose tissues in feedstuffs using species-specific polymerase chain reaction.

PCR method was applied for the qualitative identification of chicken (Gallus gallus), turkey (Meleagris gallipavo), duck (Anas platyrhynchos x Cairina muschata), and goose (Anser anser) tissues in feed-stuffs, on an individual basis. The assay uses oligonucleotide primers that are specific for each avian species, targeting the 12S rRNA mitochondrial gene. The primers designed generated amplicons of 95, 122, 64, and 98 bp length for chicken, turkey, duck, and goose, respectively. The specificity of the primers was tested against 29 animal species including mammals, birds, and fish, as well as 8 plant species. Analysis of experimental feedstuffs demonstrated the detection of each target species in the range of 0.1 to 100%. The performance of this method was not affected by prolonged heat-treatment (up to 133 degrees C for 20 min at 300 kPa), and consequently, it could be very useful for the accurate identification of tissues from these 4 avian species in products submitted to denaturing technologies, for which other methods cannot be applied.

PCR identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) targeting specific sequences from the mitochondrial D-loop region.

A polymerase chain reaction (PCR) assay was developed for the identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by using oligonucleotides targeting mitochondrial D-loop sequences. A D-loop region (∼700-1000 bp) was firstly amplified and sequenced from various game and domestic meat DNAs, and three primer sets were then designed on the basis of nucleotide multialignment of the generated D-loop sequences. As expected from sequence analysis, PCR amplification of the targeted D-loop fragments was successfully achieved from chamois (88 bp), pyrenean ibex (178 bp), and mouflon (155 bp) meats, showing adequate specificity and reproducibility against a number of game and domestic meats. Mouflon and sheep meats were amplified together in accordance to the high nucleotide identity of their mt D-loop sequences. In this work, satisfactory amplification was also accomplished in the analysis of experimentally pasteurized (72°C for 30min) and sterilized (121°C for 20min) meats, with a detection limit of ∼0.1% for each of the targeted species. The proposed PCR assay represents a rapid and straightforward method for the detection of possible adulterations in game meat products.

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene.

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.

Application of polymerase chain reaction to detect adulteration of sheep's milk with goats' milk.

The polymerase chain reaction has been applied for the specific detection of goats' milk in sheep's milk using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of goat-specific primers yielded a 122-bp fragment from goats' milk DNA, whereas no amplification signal was obtained in sheep's, cows', and water buffaloes' milk DNA. Polymerase chain reaction analysis of raw and heat-treated milk binary mixtures of sheep/goat enabled the specific detection of goats' milk with a sensitivity threshold of 0.1%. This study demonstrates the usefulness of the proposed polymerase chain reaction assay for authentication of milk products in routine analysis.

Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli.

The cloning and expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, was studied in Escherichia coli. PCR-amplified products of the preenterocin P gene (entP) or entP plus the putative EntP immunity gene (entiP), were cloned in plasmid pETBlue-1 under the control of the inducible T7lac promoter. Although target genes in derivative plasmids pJG01 (entP) and pJG02 (entP plus entiP) did not generate products with antimicrobial activity after an in vitro combined transcription/translation reaction, they were expressed as biologically active products following transformation and induction in the E. coli Tuner(DE3)pLacI host. The use of specific antibodies and an ELISA permitted the detection and quantification of EntP in the supernatant (SN), cellular soluble protein fraction (CSF), and inclusion bodies (IB) of E. coli Tuner(DE3)pLacI cells transformed with either pJG01 or pJG02. Functional EntP from the supernatants of E. coli Tuner(DE3)pLacI (pJG01) cultures grown in a complex medium was recovered, at a high efficiency, by immunoaffinity chromatography in a single step. A purification method based on hydrophobic adsorption and reverse-phase chromatographies also permitted the recovery of active EntP from the supernatants of the same cultures grown in a minimally defined medium. The E. coli Tuner(DE3)pLacI (pJG01) cells would merit consideration as an alternative experimental model for the heterologous production and functional expression of EntP, as well as for the fast and efficient recovery of this bacteriocin from the supernatant of this recombinant producer.

Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique.

A polymerase chain reaction (PCR) assay was developed for the specific identification of cows' milk in sheep's and goats' milk by using primers targeting the mitochondrial 12S rRNA gene. The use of a forward primer complementary to a conserved DNA sequence, along with a reverse primer specific for cow, yielded a 223-bp fragment from cows' milk DNA, whereas no amplification signal was obtained in sheep's and goats' milk DNA. The technique was applied to raw, pasteurized, and sterilized milk binary mixtures of cow-sheep and cow-goat, enabling the specific detection of cows' milk with a good sensitivity threshold (0.1%). The proposed PCR assay represents a rapid and straightforward method applicable to the authentication of milk and other dairy products in routine analysis.

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH.

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.