Population structure and genetic diversity of Polish Arabian horses based on pedigree data.

With a history tracing back to at least the 18th century and a substantial global influence on various breeds, Polish Arabian horse population is of paramount importance for both breeders and conservationists. However, its genetic makeup and the population dynamics are still not well understood. This study presents an analysis of the modern Polish Arabian horse population using pedigree data, focusing on the breed's genetic diversity and population structure. Our analysis encompassed 1 498 individuals defined as the reference population (RP) and their 11 065 ancestors, which resulted in the dataset of 12 254 individuals (total population). We traced their genealogy to assess inbreeding coefficients (F), founder effects, and genetic variability measures such as the effective number of founders (fe), ancestors (fa), or founder genome equivalents (fge). The results indicated a good pedigree quality with an average of 28.1 maximum traced generations, revealing high pedigree completeness for initial generations with a decline beyond the seventh generation. The genetic diversity parameters showed a considerable bottleneck effect, with an effective number of founders at 73, which reflects a substantial loss of genetic diversity over time. Despite the vast total number of founders (852), only a few have had a lasting impact on the current population, signaling the need for revised breeding strategies to maintain diversity. The study identified a slight but consistent rise in inbreeding over the last century, with a marginal recent decline, and a significant difference in the contribution of various founders. The average F was 5.8%, with 99.6% of the reference population being inbred. The analysis of effective population size (Ne) highlighted potential risks for genetic diversity, urging for revision of breeding goals to consider a wider array of founder lineages. The study indicated that stallions belonging to RP can be attributed to 15 distinct sirelines, whereas mares to 45 unique damlines, more than considered in the current breeding program (8 and 15, respectively). Conclusively, the study underlines the need for ongoing monitoring and strategic breeding to maintain and enhance the genetic diversity of Polish Arabians, considering the breed's historical significance and contemporary genetic challenges.

Comparison of mouse, rat and rabbit models for adipose - Derived stem cells (ASC) research.

PURPOSE OF THE STUDY: Cellular therapies are becoming more popular and there is a big demand for suitable animal model for research in field of tissue engineering. Both the small (rodents) and large animals have their advantages for cellular therapy experiments. Appropriate animal research model would be a bridge between basic research and clinical medicine. The aim of this study was to compare mouse, rat and rabbit as animal models useful for adipose - derived stem cell research. MATERIALS AND METHODS: Quantity, phenotype, clonogenic and differentiation potential of cells isolated from different localizations of adipose tissue from WAG and LEW/W rat strains, rabbit and mouse were analysed. RESULTS: The highest number of cells from 1 g of tissue were isolated from cervical white fat of LEW/W rat. ASCs isolated from rat had also the highest clonogenic potential. Phenotype and capability to differentiate into osteogenic, adipogenic and chondrogenic lineages are at the same level for rat and rabbit. CONCLUSIONS: Rat as a research model can be a rational solution between large animal models and typical laboratory mice because of their size, genetic homogenity, availability of genetically modified stains and possibility to perform research mimicking clinical applications.

HDAC3 Regulates Gingival Fibroblast Inflammatory Responses in Periodontitis.

Histone deacetylases (HDACs) are important regulators of gene expression that are aberrantly regulated in several inflammatory and infectious diseases. HDAC inhibitors (HDACi) suppress inflammatory activation of various cell types through epigenetic and non-epigenetic mechanisms, and ameliorate pathology in a mouse model of periodontitis. Activation of gingival fibroblasts (GFs) significantly contributes to the development of periodontitis and the anaerobic bacterium Porphyromonas gingivalis plays a key role in driving chronic inflammation. Here, we analyzed the role of HDACs in inflammatory responses of GFs. Pan-HDACi suberoylanilide hydroxamic acid (SAHA) and/or ITF2357 (givinostat) significantly reduced TNFα- and P. gingivalis-inducible expression and/or production of a cluster of inflammatory mediators in healthy donor GFs (IL1B, CCL2, CCL5, CXCL10, COX2, and MMP3) without affecting cell viability. Selective inhibition of HDAC3/6, but not specific HDAC1, HDAC6, or HDAC8 inhibition, reproduced the suppressive effects of pan-HDACi on the inflammatory gene expression profile induced by TNFα and P. gingivalis, suggesting a critical role for HDAC3 in GF inflammatory activation. Consistently, silencing of HDAC3 expression with siRNA largely recapitulated the effects of HDAC3/6i on mRNA levels of inflammatory mediators in P. gingivalis-infected GFs. In contrast, P. gingivalis internalization and intracellular survival in GFs remained unaffected by HDACi. Activation of mitogen-activated protein kinases and NFκB signaling was unaffected by global or HDAC3/6-selective HDACi, and new protein synthesis was not required for gene suppression by HDACi. Finally, pan-HDACi and HDAC3/6i suppressed P. gingivalis-induced expression of IL1B, CCL2, CCL5, CXCL10, MMP1, and MMP3 in GFs from patients with periodontitis. Our results identify HDAC3 as an important regulator of inflammatory gene expression in GFs and suggest that therapeutic targeting of HDAC activity, in particular HDAC3, may be clinically beneficial in suppressing inflammation in periodontal disease.

Nutrient loss from three small-size watersheds in the southern Baltic Sea in relation to agricultural practices and policy.

Agriculture is the major contributor of waterborne nutrient fluxes into the Baltic Sea, one of the world's most eutrophication-sensitive areas. Poland, as a large, densely populated state ohf the Baltic Region, with dominating agricultural land use, largely contributes to riverborne loads of N and P. The aim of our study was to examine the input of nutrients from three small first-order agricultural watersheds (Bladzikowski Stream, Gizdepka river and Mrzezino canal) in the Pomerania region, into the Bay of Puck, inner part of the Gulf of Gdansk. This study attempts to give a partial answer as to the question if inputs of nutrients from the 3 analysed watersheds comply with the targets of the Baltic Sea Action Plan (BSAP) and Country Allocated Reduction Targets (CART). The impact of agricultural practices was assessed on the basis of farm questionnaires and calculations of nutrient balances for the examined farms. The nutrient concentrations in the soil and drainage ditches were examined, followed by an assessment of nutrient concentrations in the watercourses at the sampling points located close to the estuaries. The average mineral N fertiliser consumption (109 kg N/ha) in the analysed watersheds was higher than Poland's average. The average N and P surpluses for surveyed farms (96.4 kg/ha and 4.4 kg/ha, respectively) were higher than the EU mean in case of N and markedly lower in case of P. We used Principal Component Analysis which confirmed that there were correlations between nutrient surpluses and nutrient concentrations in streams and/or drainage ditches. The N-NO3 and Pmin concentrations were also correlated to precipitation. The average N concentrations in the analysed watercourses were equal to 1.53 mg/L for Gizdepka, 1.88 mg/L for Mrzezino canal and 3.52 mg/L for Bladzikowski Stream. The mean P concentrations observed in the investigated watercourses were markedly higher than 0.1 mg/L. With regard to BSAP objectives, as well as CART set for Poland, the average nutrient concentrations in rivers should be approximately at the level of 2.5 mg N/L and 0.07 mg P/L.

The effects of 1st and 2nd generation biodiesel exhaust exposure on hematological and biochemical blood indices of Fisher344 male rats - The FuelHealth project.

Diesel exhaust emissions (DEE), being one of the main causes of ambient air pollution, exert a detrimental effect on human health and increase morbidity and mortality related to cardiovascular and pulmonary diseases. Therefore, the objective of the present study was to investigate potential adverse effects of exhausts emissions from B7 fuel, the first-generation biofuel containing 7% of fatty acid methyl esters (FAME), and SHB20 fuel, the second-generation biofuel containing 20% FAME/hydrotreated vegetable oil (HVO), after a whole-body exposure with and without diesel particle filter (DPF). The experiment was performed on 95 male Fischer 344 rats, divided into 10 groups (8 experimental, 2 control). Animals were exposed to DEE (diluted with charcoal-filtered room air to 2.1-2.2% (v/v)) for 7 or 28 days (6 h/day, 5 days/week) in an inhalation chamber. DEE originated from Euro 5 engine with or without DPF treatment, run on B7 or SHB20 fuel. Animals in the control groups were exposed to clean air. Our results showed that the majority of haematological and biochemical parameters examined in blood were at a similar level in the exposed and control animals. However, exposure to DEE from the SHB20 fuel caused an increase in the number of red blood cells (RBC) and haemoglobin concentration. Moreover, 7 days exposure to DEE from SHB20 fuel induced genotoxic effects manifested by increased levels of DNA single-strand breaks in peripheral blood lymphocytes. Furthermore, inhalation of both types of DEE induced oxidative stress and caused imbalance of anti-oxidant defence enzymes. In conclusion, exposure to DEE from B7, which was associated with higher exposure to polycyclic aromatic hydrocarbons, resulted in decreased number of T and NK lymphocytes, while DEE from SHB20 induced a higher level of DNA single-strand breaks, oxidative stress and increased red blood cells parameters. Additionally, DPF technology generated increased number of smaller PM and made the DEE more reactive and more harmful, manifested as deregulation of redox balance.

Endometriosis resembling endometrial cancer in a postmenopausal patient.

Endometriosis occurs in 2-4% of postmenopausal women. There have been a few reports of endometriosis in women in whom neither history nor diagnostic imaging indicated the presence of this disease, either at reproductive age or after menopause. A case is described of an 84-year-old patient with extensive deep pelvic endometriosis imitating advanced neoplastic process.

Analysis of mutations in the SOS-1 gene in two Polish families with hereditary gingival fibromatosis.

OBJECTIVES: To establish whether two families from Malopolska and Mazovia provinces in Poland are affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. MATERIAL AND METHODS: Six subjects with hereditary gingival fibromatosis and five healthy subjects were enrolled in the study. Gingival biopsies were collected during gingivectomy or tooth extraction and used for histopathological evaluation. Total RNA and genomic DNA were purified from cultured gingival fibroblasts followed by cDNA and genomic DNA sequencing and analysis. RESULTS: Hereditary gingival fibromatosis was confirmed by periodontal examination, X-ray, and laboratory tests. Histopathological evaluation showed hyperplastic epithelium, numerous collagen bundles, and abundant-to-moderate fibroblasts in subepithelial and connective tissue. Sequencing of exons 19-22 of the Son-of-Sevenless-1 gene did not reveal a single-cytosine insertion nor other mutations. CONCLUSIONS: Patients from two Polish families under study had not been affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. Further studies of the remaining regions of this gene as well as of other genes are needed to identify disease-related mutations in these patients. This will help to unravel the pathogenic mechanism of gingival overgrowth.

The influence of enteral obestatin administration to suckling rats on intestinal contractility.

This study investigated the effect of enteral administration of obestatin on the contractility of whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated with: C-saline solution; LO-obestatin (125nmol/kgb.wt); HO-obestatin (250nmol/kgb.wt). Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life. Sections were studied in an organ bath, for isometric recording in the presence of acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was performed in the intestinal segments. In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction compared to that observed in the C group. In the middle jejunum, the LO treatment also decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous contraction in the jejunum of LO and HO-treated animals. Compared to the C group, duodenal sections from HO animals and middle jejunum sections from LO and HO groups displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in the thickness of the muscularis layer was observed in the duodenum of LO and HO groups whereas the number ICC did not change significantly after treatment with obestatin. Moreover, the enteral administration of obestatin did not effect significantly on the cytoplasmic expression of M2 receptor in the jejunum. Our study demonstrated that enteral administration of obestatin to suckling rats influences small intestine contractility in the segment specific manner.

Endometriotic lesions mimicking advanced ovarian cancer - A case report and a review of the literature.

BACKGROUND: Approximately 2-5% of women affected by endometriosis are postmenopausal. The disease may simulate various malignancies. A case of endometriosis in a postmenopausal woman, spreading similarly to ovarian cancer, is reported. A broad review of existing literature on postmenopausal endometriosis, endometriosis involving the urinary tract, and the links between endometriosis and cancer are also presented. CASE DESCRIPTION: A 51-year-old woman was diagnosed with a pelvic mass. She complained of pain in the lower abdomen that began three weeks prior, and had no history of dysmenorrhea, acyclic pelvic pain, or infertility. CT scan revealed a solid and cystic tumor in the region of the right adnexa, infiltrating the surrounding tissues, with possible infiltration of the urinary bladder, as well as soft-tissue lesions of the small intestinal mesentery. Bilateral hydronephrosis and distension of the ureters were also present. A malignant neoplasm of the ovary or the corpus uteri was suspected. Total abdominal hysterectomy and appendectomy were performed. A superficial infiltration of the urinary bladder was also excised. The pathology report revealed endometriotic foci in the tumor and in the bladder infiltration, as well as an endometriotic cyst in the right ovary. Two years postoperatively the patient is disease-free and in good condition. CONCLUSIONS: Clinicians should remain conscious of the possibility of endometriosis mimicking advanced ovarian cancer and infiltrating the peritoneum and internal organs of the abdominal cavity, including the urinary tract. Imaging techniques, including CT and MRI, are not always effective in establishing the correct diagnosis preoperatively.

Tumor necrosis factor-α alters integrins and metalloprotease ADAM12 levels and signaling in differentiating myoblasts.

The extracellular matrix (ECM) is important in the regulation of myogenesis. We hypothesized that tumor necrosis factor-α (TNF-α) modifies ECM during differentiation of mouse C2C12 myoblasts. Exogenous TNF-α (1 ng/ml) stimulated myoblast fusion on the 3rd day (by 160% vs control) but not on the 5th day of myogenesis. The level of integrin α5 was significantly augmented by TNF-α during 5 day-differentiation; however, integrin ß1 was higher than control only on the 3rd day of cytokine treatment. Both the abundance of integrin α5 bound to actin and the level of integrin ß1 complexed with integrin α5 increased in the presence of TNF-α, especially on the 3rd day of differentiation. Similarly, the stimulatory effects of TNF-α on integrin α3, metalloprotease ADAM12 and kinases related to integrins, FAK and ILK, were limited to the 3rd day of differentiation. We concluded that TNF-α-induced changes in ECM components in differentiating myogenic cells, i.e. i) increased expression of integrin α5, ß1, α3, and metalloprotease ADAM12, ii) enhanced formation of α5ß1 integrin receptors and interaction of integrin α5-cytoskeleton, and iii) increased expression of kinases associated with integrin signaling, FAK and ILK, were temporarily associated with the onset of myocyte fusion.

Ovarian cancer diagnosed accidentally during treatment for ruptured ectopic pregnancy: is fertility-sparing surgery a viable alternative? Case report and review of the literature.

According to cancer incidence statistics, it is estimated that 226,000 women are diagnosed annually with epithelial ovarian cancer (EOC) and 140,000 die of the disease worldwide. Ovarian cancer represents the fourth leading cause of all cancer-related deaths in women, and the first cause of death among all gynecological malignancies. With the constant shift towards later parenthood, the growing incidence of EOC in women of reproductive age is noted. Most young EOC women are concerned with preserving their fertility despite oncological outcomes. Nowadays gynecologic oncologists are being asked to include into their decision-making processes the patients' desire for fertility preserving alternatives. The question remains whether it is possible to use fertility-sparing surgery (FSS) without compromising the survival. In the present report, the authors present a case of a 27-year-old patient with ovarian cancer accidentally diagnosed during surgical treatment of an ectopic pregnancy. In this paper, the proper selection of the patients for the conservative management, oncological safety, indications for subsequent chemotherapy, the risk of relapses, obstetrical outcomes, and further oncological control were analyzed based on the largest and most relevant series outcomes data and recommendations. Numerous recent studies have confirm that FSS in young women with early stage of epithelial ovarian cancer, who wish to preserve their childbearing potential, after appropriate selection, appears a viable and safe option. However, there is still a possibility of relapse and regular oncological control is strongly recommended.

Salivary oxidative status in patients with oral lichen planus.

Reactive oxygen species (ROS) are involved in the pathogenesis of many inflammatory diseases, including oral lichen planus. Therefore, determining the salivary markers of oxidative stress is an excellent alternative approach to diagnosing oral cavity diseases. The objective of our study was to provide preliminary validation and determination of the salivary markers of oxidative stress in both patients with reticular and erosive forms of oral lichen planus as well as in healthy individuals without any oral lesions. In total, 62 patients with oral lichen planus (OLP) were enrolled in the study, including 31 with the reticular form of lichen planus (44.63 ± 11.05 years) and 31 with erosive forms (40.43 ± 10.05 years), who had never been treated for their disease. The control group comprised 30 individuals without any oral lesions (42.12 ± 12.22 years). We determined the saliva levels in glutathione (GSH), total antioxidant capacity (TAC), and thiobarbituric acid reactive substances (TBARS). The mean saliva levels of GSH and TAC were significantly lower (P < 0.01) in OLP patients compared to the control group. The mean levels of salivary TBARS were higher in both OLP groups (reticular and erosive) compared to the control group (P = 0.01). The lower saliva levels of GSH and TAC in patients with OLP indicate that free radicals and the resulting oxidative damage may play an important role in the pathogenesis of OLP lesions. In conclusion, monitoring the oxidant-antioxidant status of saliva may serve as an efficient and less intrusive marker for determining stages of disease development in patients with OLP.

Effect of biologically active substances present in water extracts of white mustard and coriander on antioxidant status and lipid peroxidation of mouse C2C12 skeletal muscle cells.

Coriander and white mustard, an annual plants originated in the Mediterranean region, have been cultivated and used as spices for a long time. Recent studies have shown that they may constitute a potential source of phenolic compounds. The aim of this study was to evaluate the content of polyphenols in coriander and white mustard water extracts and to investigate their antioxidant activity in C2C12 mouse skeletal muscle cells, which serve as a good model of cells with intensive metabolism. HPLC analysis showed that polyphenols were able to permeate from the water extracts of studied plants into the undifferentiated myoblasts as well as myocytes undergoing differentiation, increasing the concentration of reduced glutathione and upregulating glutathione reductase and peroxidase activity. White mustard and coriander extracts also decreased the levels of oxysterols and sum of tiobarbituric acid reactive substances (TBARS) in both: myoblasts and differentiating myocytes, demonstrating protective effect on cell membranes. The obtained results indicate that polyphenols synthesized by both herbs may have beneficial effects on muscle tissue.

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.

In vitro-to-in vivo correlation of the skin penetration, liver clearance and hepatotoxicity of caffeine.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to-in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.

Postradiation carcinosarcoma of the corpus uteri--a case report.

INTRODUCTION: Radiation therapy is a very effective treatment modality, commonly used for numerous gynecological malignancies, e.g. cervical cancer. Unfortunately, ionizing radiation is associated with numerous side effects, including secondary cancer formation. A case of carcinosarcoma of the corpus uteri in a woman with a history of pelvic irradiation for cervical carcinoma is reported. The literature has been reviewed to present the incidence, optimal management, and prognosis in cases of postradiation uterine carcinosarcoma. CASE: A 55-year-old woman with a history of pelvic radiotherapy for cervical cancer five years earlier was diagnosed with a pelvic mass. Endovaginal ultrasound examination revealed a solid and cystic tumor, 12.5 cm in diameter. The patient was scheduled for surgery. Gross examination revealed an enlarged, plain corpus uteri, 12 cm in diameter. Both adnexa were normal. Pelvic and abdominal peritoneum were macroscopically normal and normal on palpation. Iliac and obturatory lymph nodes were enlarged on both sides. Radical hysterectomy, omentectomy, and ilio-obturator lymph node dissection were performed. The pathology report revealed carcinosarcoma of the corpus uteri with lymph nodes metastases--FIGO IIIC1. No adjuvant treatment was given. The patient is still alive and disease-free one year after surgery. CONCLUSIONS: Clinicians should.remain conscious of the potential carcinogenic effect of radiation therapy. Uterine carcinosarcoma may occur years after radiotherapy applied for cervical cancer. Therefore, long-term control following pelvic irradiation is always necessary.

The significance of motivation in periodontal treatment: the influence of adult patients' motivation on the clinical periodontal status.

UNLABELLED: Motivation plays an important role in the treatment process of chronic diseases, as treatment requires behavioural change and lifelong adherence to medical recommendations. Periodontitis is a good example of such health condition as to maintain good periodontal health patients have to adhere to a strict oral hygiene regimen. OBJECTIVE: To examine whether the motivation of patients suffering from chronic periodontitis influences their clinical periodontal condition. BASIC RESEARCH DESIGN: Cross sectional study. CLINICAL SETTING: Department of Periodontology and Oral Medicine, Dental University Clinic, Jagiellonian University, Krakow, Poland. PARTICIPANTS: 199 adult periodontal patients, aged 20-78 years. INTERVENTIONS: Questionnaire concerning patients' medical and dental history, modified Zychlinscy motivation assessment questionnaire, clinical periodontal examination. MAIN OUTCOME MEASURES: The extent of motivation. Periodontal status evaluated with the use of periodontal indices (API, BOP, CPITN). RESULTS: The mean motivation score was 57.4. The mean API and BOP values were 55.7% and 46.4%, respectively. For most of the patients the recorded CPITN value was 3. Correlations were observed between motivation and both API and BOP, and between API and BOP. CONCLUSION: Periodontal patients with greater motivation having better oral health (lower API and BOP) suggests an influence on the quality of their self-management of the disease (i.e. adherence to their oral hygiene regimen).

Peptidylarginine deiminase from Porphyromonas gingivalis contributes to infection of gingival fibroblasts and induction of prostaglandin E2 -signaling pathway.

Porphyromonas gingivalis (P. gingivalis) expres-ses the enzyme peptidylarginine deiminase (PPAD), which has a strong preference for C-terminal arginines. Due to the combined activity of PPAD and Arg-specific gingipains, P. gingivalis on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of P. gingivalis with primary human gingival fibroblasts (PHGF) and P. gingivalis-induced synthesis of prostaglandin E2 (PGE2 ), PHGF were infected with wild-type P. gingivalis ATCC 33277, an isogenic PPAD-knockout strain (∆ppad) or a mutated strain (C351A) expressing an inactive enzyme in which the catalytic cysteine has been mutated to alanine (PPAD(C351A) ). Cells were infected in medium containing the mutants alone or in medium supplemented with purified, active PPAD. PHGF infection was assessed by colony-forming assay, microscopic analysis and flow cytometry. Expression of cyclo-oxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1), key factors in the prostaglandin synthesis pathway, was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while PGE2 synthesis was evaluated by enzyme immunoassay. PHGF were infected more efficiently by wild-type P. gingivalis than by the ∆ppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wild-type P. gingivalis, but not by the PPAD activity-null mutant strains (Δppad and C351A). The impaired ability of the Δppad strain to adhere to and/or invade PHGF and both Δppad and C351A to stimulate the PGE2 -synthesis pathway was fully restored by the addition of purified PPAD. The latter effect was strongly inhibited by aspirin. Collectively, our results implicate PPAD activity, but not PPAD itself, as an important factor for gingival fibroblast infection and activation of PGE2 synthesis, the latter of which may strongly contribute to bone resorption and eventual tooth loss.

In vitro testing the potential of a novel chimeric IgG variant for inhibiting collagen fibrils formation in recurrent hereditary gingival fibromatosis: chimeric antibody in a gingival model.

Gingival fibromatosis is a progressive enlargement of the gingiva. It may hinder oral cavity hygiene and result in underlying bone loss. The long-term benefits of surgery cannot be predicted. On the other hand, alternative, efficient and non-invasive methods are not available at present. The aim of this study was to test the inhibitory effects of a chimeric IgG variant on collagen fibril formation in the cell culture of gingival fibroblasts taken from a patient with hereditary gingival fibromatosis with a high propensity for recurrence. Gingival biopsies were collected from the mandibular gingiva and used for histological evaluation as well as to establish a fibroblast culture. A histological evaluation was made in haematoxylin-eosin and Heidenhain's trichrome stained tissue sections. The inhibitory effect of a chimeric antibody on collagen fibril formation was determined in fibroblast cultures by using a collagen-specific Western blot and immunofluorescent staining. A histological evaluation revealed epithelial acanthosis with singular elongated rete pegs extending into the underlying connective tissue stroma that consisted of locally abundant, irregular collagen bundles. Based on observations with an in vitro model we conclude that a chimeric anti-collagen antibody efficiently inhibits collagen fibril accumulation in cell culture derived from diffuse, hereditary gingival fibromatosis that is characterized by a high propensity for recurrence (high proliferation index). Employing cell cultures from standardized group of patients with recurrent hereditary gingival fibromatosis as well as standarizing relevant 3D (tissue-like) models will be crucial for further tests of the antibody.

Expression of microRNAs and their target genes and pathways associated with ovarian follicle development in cattle.

Development of ovarian follicles is controlled at the molecular level by several gene products whose precise expression leads to regression or ovulation of follicles. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs) causing translation repression or mRNA degradation. The aim of this study was to identify miRNAs expressed in theca and/or granulosa layers and their putative target genes/pathways that are involved in bovine ovarian follicle development. By using miRCURY microarray (Exiqon) we identified 14 and 49 differentially expressed miRNAs (P < 0.01) between dominant and subordinate follicles in theca and granulosa cells, respectively. The expression levels of four selected miRNAs were confirmed by qRT-PCR. To identify target prediction and pathways of differentially expressed miRNAs we used Union of Genes option in DIANA miRPath v.2.0 software. The predicted targets for these miRNAs were enriched for pathways involving oocyte meiosis, Wnt, TGF-beta, ErbB, insulin, P13K-Akt, and MAPK signaling pathways. This study identified differentially expressed miRNAs in the theca and granulosa cells of dominant and subordinate follicles and implicates them in having important roles in regulating known molecular pathways that determine the fate of ovarian follicle development.