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Article ; Online: Molecular Mechanisms of Lipopolysaccharide (LPS) Induced Inflammation in an Immortalized Ovine Luteal Endothelial Cell Line (OLENDO)

Aykut Gram / Mariusz P. Kowalewski

Veterinary Sciences, Vol 9, Iss 99, p

2022 Volume 99

Abstract: Escherichia coli ( E. coli ) is the most common Gram-negative bacterium causing infection of the uterus or mammary gland and is one of the major causes of infertility in livestock. In those animals affected by E. coli driven LPS-mediated infections, ... ...

Abstract	Escherichia coli ( E. coli ) is the most common Gram-negative bacterium causing infection of the uterus or mammary gland and is one of the major causes of infertility in livestock. In those animals affected by E. coli driven LPS-mediated infections, fertility problems occur in part due to disrupted follicular and luteal functionality. However, the molecular mechanisms by which LPS induces inflammation, and specifically, the role of LPS in the disruption of capillary morphogenesis and endothelial barrier function remain unclear. Here, we hypothesized that LPS may lead to alterations in luteal angiogenesis and vascular function by inducing inflammatory reactions in endothelial cells. Accordingly, OLENDO cells were treated with LPS followed by evaluation of the expression of selected representative proinflammatory cytokines: NF-kB , IL6 , IL8 , TNFα , and ICAM 1 . While TNFα was not affected by treatment with LPS, transcripts of NF-kB , IL6 , and IL8 were affected in a dosage-dependent manner. Additionally, the activity of TLR2 and TLR4 was blocked, resulting in suppression of the LPS-induced expression of ICAM 1 , NF-kB , IL6 , and IL8 . Inhibition of the PKA or MAPK/ERK pathways suppressed the LPS-stimulated expression of NF-kB , IL6 , and IL8 , whereas blocking the PKC pathway had the opposite effect. Furthermore, LPS-induced phosphorylation of Erk1 and Erk2 was inhibited when the TLR4 or MAPK/ERK pathways were blocked. Finally, LPS seems to induce inflammatory processes in OLENDO cells via TLR2 and TLR4, utilizing different signaling pathways.
Keywords	toll-like receptor ; ovary ; interleukins ; PKA ; PKC ; MAPKs ; Veterinary medicine ; SF600-1100
Subject code	630
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Antigestagens Mediate the Expression of Decidualization Markers, Extracellular Matrix Factors and Connexin 43 in Decidualized Dog Uterine Stromal (DUS) Cells

Ali Kazemian / Miguel Tavares Pereira / Bernd Hoffmann / Mariusz P. Kowalewski

Animals, Vol 12, Iss 798, p

2022 Volume 798

Abstract: Feto-maternal communication in the dog involves the differentiation of stromal cells into decidual cells. As the only placental cells expressing the nuclear progesterone (P4) receptor (PGR), decidual cells play crucial roles in the maintenance and ... ...

Abstract	Feto-maternal communication in the dog involves the differentiation of stromal cells into decidual cells. As the only placental cells expressing the nuclear progesterone (P4) receptor (PGR), decidual cells play crucial roles in the maintenance and termination of pregnancy. Accordingly, to investigate possible PGR-mediated mechanisms in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with functional PGR-blockers, mifepristone and aglepristone. Effects on decidualization markers, epithelial and mesenchymal factors, and markers of cellular viability were assessed. Decidualization increased the expression of PTGES , PGR , IGF1 , and PRLR, along with ECM1 , COL4 and CX43, but downregulated IGF2 . DUS cells retained their mesenchymal character, and the expression of COL4 indicated the mesenchymal-epithelial transformation. Antigestagen treatment decreased the availability of PTGES , PRLR , IGF1 and PGR. Furthermore, antigestagens decreased the mRNA and protein expression of CX43, and transcriptional levels of ECM1 and COL4 . Additionally, antigestagens increased levels of activated-CASP3 (a proapoptotic factor), associated with lowered levels of PCNA (a proliferation marker). These data reveal important aspects of the functional involvement of PGR in canine decidual cells, regarding the expression of decidualization markers and acquisition of epithelial-like characteristics. Some of these mechanisms may be crucial for the maintenance and/or termination of canine pregnancy.
Keywords	dog ( Canis lupus familiaris ) ; dog uterine stromal (DUS) cells ; decidualization ; nuclear progesterone receptor (PGR) ; antigestagens ; Veterinary medicine ; SF600-1100 ; Zoology ; QL1-991
Subject code	570
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues – a multistudy analysis

Marta Nowak / Selim Aslan / Mariusz P. Kowalewski

BMC Veterinary Research, Vol 16, Iss 1, Pp 1-

2020 Volume 12

Abstract: Abstract Background Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other ...

Abstract	Abstract Background Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other species, the reproductive tract is continuously undergoing hormonal and cycle stage-dependent morphological changes, which are associated with altered gene expression. However, there have been few attempts published so far targeted to the dog aimed at determining optimal reference genes for the reproductive organs. Most of these approaches relied on genes previously described in other species. Large-scale transcriptome-based experiments are promising tools for defining potential candidate reference genes, but were never considered in this context in canine research. Results Here, using available microarray and RNA-seq datasets derived from reproductive organs (corpus luteum, placenta, healthy and diseased uteri) of dogs, we have performed multistudy analysis to identify the most stably expressed genes for expression studies, in each tissue separately and collectively for different tissues. The stability of newly identified reference genes (EIF4H, KDELR2, KDM4A and PTK2) has been determined and ranked relative to previously used reference genes, i.e., GAPDH, β-actin and cyclophillin A/PPIA, using RefFinder and NormFinder algorithms. Finally, expression of selected target genes (luteal IL-1b and MHCII, placental COX2 and VEGFA, and uterine IGF2 and LHR) was re-evaluated and normalized. All proposed candidate reference genes were more stable, ranked higher and introduced less variation than previously used genes. Conclusions Based on our analyses, we recommend applying KDM4A and PTK2 for normalization of gene expression in the canine CL and placenta. The inclusion of a third reference gene, EIF4H, is suggested for healthy uteri. With this, the interpretation of qPCR data will be more reliable, allowing better understanding of canine reproductive physiology.
Keywords	Reference genes ; Dog (Canis lupus familiaris) ; Reproductive tract ; Veterinary medicine ; SF600-1100
Subject code	572 ; 612
Language	English
Publishing date	2020-11-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Utero-Placental Immune Milieu during Normal and Aglepristone-Induced Parturition in the Dog

Miguel Tavares Pereira / Renata Nowaczyk / Selim Aslan / Serhan S. Ay / Mariusz P. Kowalewski

Animals, Vol 11, Iss 3598, p

2021 Volume 3598

Abstract: Maternal immunotolerance is required for the maintenance of pregnancy, in sharp contrast with the uterine pro-inflammatory activity observed during parturition in several species. Correspondingly, in the dog, increased immune signaling at term has been ... ...

Abstract	Maternal immunotolerance is required for the maintenance of pregnancy, in sharp contrast with the uterine pro-inflammatory activity observed during parturition in several species. Correspondingly, in the dog, increased immune signaling at term has been suggested, but a deeper understanding of the uterine immune milieu is still missing. Thus, the availability of 30 immune-related factors was assessed in utero-placental samples collected during post-implantation (days 18–25 of pregnancy) and mid-gestation (days 35–40) stages, and at the time of prepartum luteolysis. Gene expression and/or protein localization studies were employed. Samples collected from antigestagen (aglepristone)-treated dogs were further analyzed. Progression of pregnancy was associated with the downregulation of IL1β and upregulation of IL10 ( p < 0.05) at mid-gestation. When compared with mid-gestation, a higher availability of several factors was observed at term (e.g., CD206 , CD4 , TLR4 ). However, in contrast with natural parturition, MHCII , CD25 , CCR7 , TNFα , IDO1 and AIF1 were upregulated after aglepristone treatment ( p < 0.05), but not TNFR1 or CCL13 ( p > 0.05). Altogether, these results show an increased immune activity during canine parturition, involving, i.a., M2 macrophages, Treg and Th cells, with strong support for progesterone-mediated immunomodulation. Furthermore, differences between term and induced parturition/abortion could relate to differences in placental maturation towards parturition and/or functional traits of antigestagens.
Keywords	dog ( Canis lupus familiaris ) ; immune system ; utero-placental ; parturition ; aglepristone ; Veterinary medicine ; SF600-1100 ; Zoology ; QL1-991
Subject code	630
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Luteal and hypophyseal expression of the canine relaxin (RLN) system during pregnancy

Marta Nowak / Alois Boos / Mariusz P Kowalewski

PLoS ONE, Vol 13, Iss 1, p e

Implications for luteotropic function.

2018 Volume 0191374

Abstract: By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) ... ...

Abstract	By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) serum PRL increases in pregnant bitches shortly after RLN appears in the circulation, and a possible functional relationship between the RLN and the PRL systems in regulating progesterone secretion has been implied. Therefore, here (Study 1) the luteal expression and localization of the RLN system was investigated by immunohistochemistry using custom-made antibodies and semi-quantitative PCR, at selected time points during gestation: pre-implantation (d. 8-12), post-implantation (d. 18-25), mid-gestation (d. 35-40) and at normal and antigestagen-induced luteolysis. Further, (Study 2) hypophyseal expression of the RLN system and its spatial association with PRL was assessed. Luteal expression of RLN, but not of its receptors, was time-dependent: it increased significantly following implantation towards mid-gestation and decreased at prepartum. Antigestagen treatment resulted in downregulation of RLN and RXFP2. Whereas RLN was localized in steroidogenic cells, RXFP1 and RXFP2 also stained strongly in macrophages and vascular endothelial cells. The RLN system was detected in the canine adenohypophysis and was co-localized with PRL in hypophyseal lactotrophs. The intraluteal RLN seems to be involved in regulating the canine corpus luteum (CL) in a time-dependent manner. The presence of RLN family members in the adenohypophysis implies their possible involvement in regulating the availability of PRL and other pituitary hormones.
Keywords	Medicine ; R ; Science ; Q
Subject code	630
Language	English
Publishing date	2018-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Effects of ACTH-Induced Long-Term Hypercortisolism on the Transcriptome of Canine Visceral Adipose Tissue

Miguel Tavares Pereira / Isabelle Martin / Hubert Rehrauer / Mariusz P. Kowalewski / Felicitas S. Boretti / Nadja S. Sieber-Ruckstuhl

Veterinary Sciences, Vol 9, Iss 250, p

2022 Volume 250

Abstract: Cushing’s syndrome, or hypercortisolism (HC), a common endocrinopathy in adult dogs, is caused by chronic hypercortisolemia. Among different metabolic disorders, this syndrome is associated with enhanced subcutaneous lipolysis and visceral adiposity. ... ...

Abstract	Cushing’s syndrome, or hypercortisolism (HC), a common endocrinopathy in adult dogs, is caused by chronic hypercortisolemia. Among different metabolic disorders, this syndrome is associated with enhanced subcutaneous lipolysis and visceral adiposity. However, effects of HC in adipose tissue, especially regarding visceral adipose tissue (VAT), are still poorly understood. Herein, the transcriptomic effects of chronic HC on VAT of dogs were evaluated. For this, subcutaneously implanted ACTH-releasing pumps were used, followed by deep RNA sequencing of the canine VAT. Prolonged HC seems to affect a plethora of regulatory mechanisms in VAT of treated dogs, with 1190 differentially expressed genes (DEGs, p and FDR < 0.01) being found. The 691 downregulated DEGs were mostly associated with functional terms like cell adhesion and migration, intracellular signaling, immune response, extracellular matrix and angiogenesis. Treatment also appeared to modulate local glucocorticoid and insulin signaling and hormonal sensitivity, and several factors, e.g., TIMP4, FGF1, CCR2, CXCR4 and HSD11B1/2, were identified as possible important players in the glucocorticoid-related expansion of VAT. Modulation of their function during chronic HC might present interesting targets for further clinical studies. Similarities in the effects of chronic HC on VAT of dogs and humans are highlighted.
Keywords	dog ( Canis lupus familiaris ) ; Cushing’s syndrome ; atherosclerosis ; animal model ; Veterinary medicine ; SF600-1100
Subject code	630
Language	English
Publishing date	2022-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog

Miguel Tavares Pereira / Renata Nowaczyk / Rita Payan-Carreira / Sonia Miranda / Selim Aslan / Duygu Kaya / Mariusz P. Kowalewski

Frontiers in Veterinary Science, Vol

2021 Volume 7

Abstract: In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- ... ...

Abstract	In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10–12, E+) and corresponding non-pregnant controls (E–), implantation (day 17, Imp) and post-implantation (day 18–25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E– samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1β. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.
Keywords	dog (Canis lupus familiaris) ; immune system ; uterus ; early pregnancy ; embryo-maternal communication ; Veterinary medicine ; SF600-1100
Subject code	630
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	Frontiers Media S.A.
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Prostaglandin-mediated effects in early canine corpus luteum: In vivo effects on vascular and immune factors

Tavares Pereira, Miguel / Aykut Gram / Renata Nowaczyk / Alois Boos / Bernd Hoffmann / Tomasz Janowski / Mariusz P. Kowalewski

Reproductive Biology. 2019 Mar., v. 19, no. 1

2019

Abstract: Prostaglandins (PGs) are important regulators of the early corpus luteum (CL) in the dog. Whereas, initially, CL is gonadotropin independent, in the second half of its lifespan, hypophyseal support is required. The transition period is marked by ... ...

Abstract	Prostaglandins (PGs) are important regulators of the early corpus luteum (CL) in the dog. Whereas, initially, CL is gonadotropin independent, in the second half of its lifespan, hypophyseal support is required. The transition period is marked by decreased availability of PGs, in particular of PGE2. We previously reported that inhibition of COX2/PTGS2 in vivo suppressed luteal production of PGE2, lowered circulating progesterone and negatively affected luteal development. Therefore, bitches were treated with a COX2-specific blocker, firocoxib, for 5, 10, 20 and 30 days after ovulation, leading to suppression of the steroidogenic machinery. Control groups received a placebo for the same periods. Considering the wide range of possible modulatory roles of PGs shown in different organ systems, this follow-up project aimed to understand further possible PG-mediated effects in early canine CL. Thirty-four (34) factors related predominantly to vascularization and immune response were screened (mRNAs and proteins) on samples from the above described in vivo study. Most of the effects were observed during the transitional period (days 20 and 30). The inhibition of COX2 diminished the expression of angiopoietin family members ANGPT1, -2, Tie1 and -2 receptors. The expression of endothelin (ET)-1 was increased. Concerning the immune system, increased expression of the pro-inflammatory cytokines, IL1β, IL6 and IL12a, and elevated expression levels of CD4, was observed. Cumulatively, besides its involvement in regulating steroidogenesis, our results indicate a broader role of PGs in the canine CL, including modulation of angiogenesis, vascular stabilization and local immunomodulation. Possible cross-species translational effects are strongly implied.
Keywords	angiogenesis ; bitches ; corpus luteum ; immune response ; immune system ; immunomodulation ; in vivo studies ; interleukin-12 ; interleukin-1beta ; interleukin-6 ; longevity ; messenger RNA ; ovulation ; placebos ; progesterone ; prostaglandins ; receptors ; steroidogenesis
Language	English
Dates of publication	2019-03
Size	p. 100-111.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	2189316-0
ISSN	1642-431X
ISSN	1642-431X
DOI	10.1016/j.repbio.2019.02.001
Database	NAL-Catalogue (AGRICOLA)

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Zs.A 6095: Show issues

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Article: Cells expressing CD4, CD8, MHCII and endoglin in the canine corpus luteum of pregnancy, and prepartum activation of the luteal TNFα system

Nowaczyk, Renata M / Alois Boos / Aykut Gram / Ewelina Jursza-Piotrowska / Mariusz P. Kowalewski / Marta J. Siemieniuch

Theriogenology. 2017 Aug., v. 98

2017

Abstract: In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study ... ...

Abstract	In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study investigated the presence of CD4-, CD8-, MHCII- and endoglin-expressing cells in CL collected throughout pregnancy from pre-implantation until prepartum luteolysis. Immunohistochemistry and semi-quantitative RT-PCR were applied. The time-dependent expression of CD4, CD8 and endoglin was more strongly related to formation of the CL, whereas MHCII was induced during luteolysis. Next, the luteal expression of TNFα and its receptors, TNFR1 and TNFR2, was analyzed in non-pregnant dogs between days 5–65 after ovulation and during pregnancy. Moreover, the effects of progesterone withdrawal were investigated in mid-pregnant dogs treated with an antigestagen aglepristone. The TNFα system was induced in the early CL of non-pregnant dogs. In pregnant dogs, expression of TNFα did not vary much, contrasting with increased expression of both receptors in the post-implantation period and significantly decreased expression at mid-gestation; prepartum luteolysis was characterized by increased TNFR2 expression. Apart from the downregulated expression of TNFR1, the changes observed following antigestagen treatment resembled those observed during normal prepartum luteolysis. A modulatory function of the TNFα system during formation of the canine CL is suggested, possibly related to the strong accompanying vascularization and luteal infiltration with activated macrophages. Contrasting with the slow luteal regression in non-pregnant dogs, in pregnant animals the upregulation of TNFR2 expression during prepartum luteolysis implies functional involvement of the TNFα system during that time.
Keywords	bitches ; corpus luteum ; immunohistochemistry ; luteolysis ; macrophages ; ovulation ; pregnancy ; progesterone ; receptors ; reverse transcriptase polymerase chain reaction ; tumor necrosis factor-alpha
Language	English
Dates of publication	2017-08
Size	p. 123-132.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	189232-0
ISSN	1879-3231 ; 0093-691X
ISSN (online)	1879-3231
ISSN	0093-691X
DOI	10.1016/j.theriogenology.2017.05.003
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Placental Origin of Prostaglandin F2α in the Domestic Cat

Marta J. Siemieniuch / Ewelina Jursza / Anna Z. Szóstek / Lina Zschockelt / Alois Boos / Mariusz P. Kowalewski

Mediators of Inflammation, Vol

2014 Volume 2014

Keywords	Pathology ; RB1-214 ; Medicine ; R
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Hindawi Publishing Corporation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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