Analysis of key markers: IL-10/sHLA-G in psoriasis patients and the identification of 14-bp INDEL in the HLA-G gene.

BACKGROUND: Psoriasis is a chronic inflammation resulting from interactions between immunological and genetic factors. An important tolerogenic role in this autoimmunological disease is played by HLA-G, which is modulated by IL-10. Therefore, this study (N.=80) aimed to evaluate changes in the serum sHLA-G and IL-10 levels in active psoriasis vulgaris and in the early stages of treatment with Methotrexate (MTX) compared to healthy controls. The 14-bp INDEL of the HLA-G gene was evaluated to find possible associations with clinical and laboratory variables. METHODS: The level of sHLA-G and IL-10 in serum was evaluated (ELISA tests) in patients before the first dose of MTX and at week 12 of treatment, compared to healthy control donors. The 14-bp INDEL in 3'UTR of the HLA-G gene was identified using gDNA templates isolated from full blood. HLA-G amplicons were obtained by PCR, separated by electrophoresis and sequenced. RESULTS: The mean serum IL-10 level was 4.653±3.33 pg/mL in psoriatic patients, 13.3±9.64 pg/mL after short MTX treatment, compared to 6.23 pg/mL in healthy controls. In addition, the serum level of sHLA-G was 0.275±0.03 ng/mL and 0.332±0.06 ng/mL in patients before and after MTX treatment, respectively, and 0.302±0.08 ng/mL in the control group. A correlation was found (r=-0.43; P<0.005) between the IL-10 and BSA serum levels in psoriasis patients after MTX treatment, indicating health improvement. The three genotypes identified in the 3'UTR of the HLA-G revealed no association with sHLA-G level in serum. CONCLUSIONS: The mean levels of sHLA-G and the key anti-inflammatory cytokine IL-10 in the blood of pretreatment psoriasis patients are low and indicate that the immunotolerance mechanisms have failed. Treatment of psoriasis patients with low systemic levels of sHLA-G and IL-10 brings them to the same or higher protein levels, respectively, as in healthy donors. Higher sHLA-G levels in healthy donors and after MTX treatment, compared to the sHLA-G levels in the acute phase of psoriasis, indicates its immune system surveillance function.

Identification of pregnancy-associated glycoprotein family (PAG) in the brown bear (Ursus arctos L.).

Pregnancy-associated glycoproteins (PAGs) are abundant embryo-originated products expressed in the pre-placental trophoblast and, later, in the post-implantational chorionic epithelium of some mammalian species. This paper describes the identification and cellular immunolocalization of the chorionic PAG family in the discoidal-type placenta of the brown bear (Ursus arctos L. - Ua), in which the PAGs were named 'UaPAG-Ls'. The study used: 1) Western blot for total placental glycoproteins; and 2) cross-species heterologous double fluorescent immunohistochemistry (IHC) for cellular immune-localization of the PAGs. This is the first study reporting the identification and immunolocalization of the UaPAG-L family in placental cells during early pregnancy in the brown bear. Our Western analysis revealed a dominant mature 72 kDa UaPAG-L isoform was expressed in all Ua placentas during early pregnancy. Various other UaPAG-L isoforms (16-66 kDa) were also identified. Using IHC, the UaPAG-L proteins were localized to trophectodermal cells (TRD), where signal intensity resembled intense TRD proliferation within developing placenta. The data increases our general knowledge of PAG proteins localized in discoidal-type placenta during early pregnancy in the brown bear.

Identification of Placental Aspartic Proteinase in the Eurasian Beaver (Castor fiber L.).

Aspartic proteinases (AP) form a multigenic group widely distributed in various organisms and includes pepsins (pep), cathepsins D and E, pregnancy associated glycoproteins (PAGs) as well as plant, fungal, and retroviral proteinases. This study describes the transcript identification and expression localization of the AP within the discoid placenta of the Castor fiber. We identified 1257 bp of the AP cDNA sequence, encoding 391 amino acids (aa) of the polypeptide precursor composed of 16 aa signal peptide, 46 aa pro-piece, and 329 aa of the mature protein. Within the AP precursor, one site of potential N-glycosylation (NPS119–121) and two Asp residues (D) specific for the catalytic cleft of AP were identified (VLFDTGSSNLWV91–102 and GIVDTGTSLLTV277–288). The highest homology of the identified placental AP nucleotide and aa sequence was to mouse pepsinogen C (75.8% and 70.1%, respectively). Identified AP also shared high homology with other superfamily members: PAGs, cathepsins, and napsins. The AP identified in this study was named as pepsinogen/PAG-Like (pep/PAG-L). Diversified pep/PAG-L protein profiles with a dominant 58 kDa isoform were identified. Immune reactive signals of the pep/PAG-L were localized within the trophectodermal cells of the beaver placenta. This is the first report describing the placental AP (pep/PAG-L) in the C. fiber.

Novel effects of identified SNPs within the porcine Pregnancy-Associated Glycoprotein gene family (pPAGs) on the major reproductive traits in Hirschmann hybrid-line sows.

This is the first study describing identification of SNPs within the multiple and polymorphic Pregnancy-Associated Glycoprotein gene family (PAGs) in the genome of the domestic pig (pPAGs). We identified pPAG-like (pPAG-L) genotypes in primiparous and multiparous farmed hybrid-line JSR Hirschmann (Hrn) sows (N=159), in which various novel associations with their phenotypes for the major reproductive traits have been discovered. Genomic DNA templates were isolated from the blood and different pPAG-L primers were used to amplify various regions by PCR. Electrophoretically-separated amplicons were selected, purified and sequenced. All identified SNPs were verified for possible pPAG2-L genotype associations with the major reproductive traits. In total, 196 SNPs were identified within the entire structure of the pPAG2-Ls, encompassing 9 exons and 8 (A-H) introns, resembling all aspartic proteinases. It was discovered that among all SNPs, one diplotype localized in exon 6 (657C>T/749G>C; pPAG2 ORF cDNA numbering; L34361) caused amino acid substitutions (Asp220→Asn and Ser250→Thr) in the polypeptide precursors and was associated with an increase in the number of live-born piglets (P≤0.05) in Hrn sows. In turn, co-localized SNP (504g>a; KF537535 numbering) in the intron F of the pPAG2-Ls, but only in the homozygotic genotype (gg), was associated with an increased number of live-born (P≤0.01) and weaned (P≤0.05) piglets in the Hrn sows. These results qualify the pPAG2-Ls as candidate genes of the main QTLs. The novel pPAG SNP profiles provide the basis for a diagnostic genotyping test required for early pre-selection of female/male piglets, presumably mainly useful in various breeding herds.

Gene structure of the pregnancy-associated glycoprotein-like (PAG-L) in the Eurasian beaver (Castor fiber L.).

The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1-9) and eight introns (A-H). The length of the CfPAG-L exons (59-200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288-1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3-72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.

Novel SNPs and InDels discovered in two promoter regions of porcine pregnancy-associated glycoprotein 2-like subfamily (pPAG2-Ls) in crossbreed pigs.

This is a pioneer study of single nucleotide polymorphisms (SNPs) within the entire promoter region (1204 bp) of the dominant pPAG2-L subfamily in the pig. The pPAG2-L subfamily was sequenced/examined using genomic deoxyribonucleic acid (gDNA) templates of crossbreed pigs (Landrace x Large White), and compared to two bacterial artificial chromosome (BAC) clones containing gDNA of the Duroc breed (as the positive controls). Our analysis of the pPAG2-L promoter identified 31 SNPs and one InDel mutation in crossbreed pigs. Among 42 SNPs identified in two BAC clones, 24 SNPs had not been previously detected in crossbreed pigs. The sequence alignment of pPAG2-L promoter, performed with Lasagne-Search 2.0, Cluster Bluster and MatInspector software, revealed a total of 28 transcription factor binding sites (TFBS) and 10 TFBS (AP-1, CCAAT, CHOP:C, FOXP1, LSF, MRF-2, Myc, NF1, NF-Y, TGIF) within SNPs in the core sequences. It was noted that TFBS (NF1) was found to be unique to the pPAG2 promoter sequence containing SNPs: g.-1100G>A(R), g.-1101T>C(Y), represented by GA and TC genotypes (p x = 0.12). Our broad-based novel database thus provides an SNP PAG2-L pattern for modern genotyping of female and male progenitors. This is required for further studies of various potential correlations between guiding SNP genotypes of the pPAG2-L subfamily in the sows of many breeds, in which the most economically important reproductive traits are properly documented on each farm.

Identification of the pregnancy-associated glycoprotein family (PAGs) and some aspects of placenta development in the European moose (Alces alces L.).

This study describes the identification and a broad-based characterization of the pregnancy-associated glycoprotein (PAG) genes expressed in the synepitheliochorial placenta of the Alces alces (Aa; N = 51). We used: (1) both size measurements (cm) of various Aa embryos/fetuses (crown-rump length) and placentomes (PLCs); (2) PCR, Southern and sequencing; (3) Western-blot for total placental glycoproteins; (4) deglycosylation of total cotyledonary proteins; and (5) double heterologous IHC for cellular immune-localization of the PAGs as pregnancy advanced (50-200 days post coitum). The crown-rump length and PLC size measurements permitted a novel pattern estimation of various pregnancy stages in wild Aa. The PLC number varied (5-21) and was the greatest at the mid and late stages of gestation in females bearing singletons or twins. The genomic existence of the identified PAG-like family was named AaPAG-L. Amplicon profiles of the AaPAG-L varied in the number and length (118-2000 bp). Southern with porcine cDNA probes confirmed specificity and revealed dominant AaPAG-L amplicons in males and females. Nucleotide sequences of the AaPAG-L amplicons shared 86.27% homology with the bovine PAG1 (bPAG1) gene. Amino acid AaPAG sequences revealed in silico 88.23% to 100% homology with the bPAG1 precursor. Western-blots revealed a dominant mature 55 kDa AaPAG fraction, and the major ∼48 kDa glycosylated form that was deglycosylated to ∼44 kDa. The AaPAG-Ls was immuno-localized to mono- and bi-nucleated trophectodermal cells (TRD-chorionic epithelium), where signal intensity resembled intense TRD proliferation within developing PLCs as pregnancy advanced. This is the first study identifying the AaPAG-L family in the largest representative among the Cervidae.