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Cystic endometrial hyperplasia (CEH)—pyometra syndrome is one of the most common diseases of noncastrated female dogs. However, determination of etiological mechanisms and differential diagnosis of CEH—pyometra syndrome are undefined. The aim of this study is to compare immunohistochemically the expression of cyclooxygenase-2 (COX-2) inflammatory mediator, Ki-67 antigen proliferation marker, vascular endothelial growth factor (VEGF-A) angiogenesis mediator and its FLT-1 and KDR receptors, and correlate with Doppler velocimetry of uterine artery and endometrial vascularization in bitches with CEH—pyometra syndrome. Bitches were allocated into CEH-mucometra Group (n = 13), Pyometra Group (n = 11), and Control Group (n = 8). Pyometra Group presented cytoplasmatic staining intensity for COX-2, VEGF-A, and FLT-1 and KDR receptors in luminal epithelium cells significantly higher compared to CEH-mucometra and Control groups. For the glandular epithelium, Pyometra Group had higher immunostaining score for VEGF-A and its receptors (FLT-1 and KDR). Hemodynamic indexes showed negative correlation with VEGF-A and its receptors as well as with COX-2. On the other hand, uterine vascularization score showed positive correlation in relation to immunostaining of COX-2, VEGF-A, and receptors in the endometrium luminal epithelium. In conclusion, uterus of bitches with CEH—pyometra syndrome show inflammatory process characterized by COX-2 expression, resulting in greater expression of proliferative Ki-67 marker as tissue response against the infectious agent. Furthermore, the increased VEGF-A expression and its receptors in CEH—pyometra reflect the increased blood flow and lower vascular resistance. Therefore, canine pyometra is characterized by an inflammatory, proliferative, and vascular disorder. Summary Sentence Canine CEH—pyometra syndrome is characterized by an inflammatory, proliferative and vascular disorder, with higher uterine blood flow and vascularization.

Canine pyometra frequently occurs in middle-aged to older intact bitches, which seriously affects the life of dogs and brings an economic loss to their owners. Hence, finding a key metabolite is very important for the diagnosis and development of a new safe and effective therapy for the disease. In this study, dogs with pyometra were identified by blood examinations, laboratory analyses and diagnostic imaging, and fifteen endometrium tissues of sick dogs with pyometra and fifteen controls were collected and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The results indicated that the elevated inflammatory cells were observed in dogs with pyometra, suggesting that sick dogs suffered systemic inflammation. In the untargeted metabolic profile, 705 ion features in the positive polarity mode and 414 ion features in the negative polarity mode were obtained in endometrium tissues of sick dogs with pyometra, with a total of 275 differential metabolites (173 in positive and 102 in negative polarity modes). Moreover, the multivariate statistical analyses such as PCA and PLS-DA also showed that the metabolites were significantly different between the two groups. Then, these differential metabolites were subjected to pathway analysis using Metaboanalyst 4.0, and Galactose metabolism, cAMP signaling pathway and Glycerophospholipid metabolism were enriched, proving some insights into the metabolic changes during pyometra. Moreover, the receiver operating characteristic curves further confirmed kynurenic acid was expected to be a candidate biomarker of canine pyometra. In conclusion, this study provided a new idea for exploring early diagnosis methods and a safe and effective therapy for canine pyometra.

Failures in hypothalamic kisspeptin/Kiss1r signaling are associated with infertility, and in vitro studies have shown that kisspeptin can modulate angiogenesis and immune activity. Because there is no in vivo research on the functional relationship between these factors in the reproductive system, especially in domestic cats, we evaluated the expression profile of kisspeptin/Kiss1r and angiogenic and immunological mediators in the genital tract of cyclic cats and of those with pyometra. The uterus of cats in diestrus exhibited greater gene and protein expression of Kiss1, as well as Vegf, Pigf, Mif, and Il6. In contrast, Kiss1r presented greater expression in proestrus/estrus, similarly to that observed for the immunostaining of INFγ, MIF, TNFα, and IL10. These factors were positively correlated with Kiss1 and/or Kiss1r, and a positive correlation between Kiss1 and Kiss1r was also observed in the uterus of cats during the estrous cycle. Cats with pyometra showed greater immunostaining of Kiss1 and Kiss1r on the endometrial surface and reduced immunostaining of Kiss1 in deep glands, whereas there was a significant reduction in Vegf, Pigf, Mif, and Il6 mRNA, and an increase in Tnf mRNA. The findings reveal that there is a gene correlation between kisspeptin/Kiss1r and angiogenic and immune mediators in the uterus of the domestic cat, which is modulated by the estrous cycle, and that pyometra affects the expression of these mediators. This study suggests, for the first time, a functional relationship between the Kiss/Kiss1r system and angiogenic and immune mediators in the female genital tract. Summary sentence Kisspeptin/Kiss1R system and angiogenic and immune mediators in the uterus of domestic cats are modulated by the estrous cycle and affected by pyometra, being that these mediators have a gene correlation in the uterus of these animals.

The pathogenesis of canine pyometra is multifactorial, involving hormonal imbalances, aberrant immune responses, and metabolic dysregulation includes lipid metabolism and oxidative stress. This study focuses on lipid metabolism and oxidative stress, revealing the key regulatory role of AMPK and PLIN2 in canine pyometra. Bitches with open cervix pyometra (n:8) and healthy bitches undergoing elective ovariohysterectomy (n:4) were enrolled to the study. In experiment one, the serum and tissue levels of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity were assessed. Additionally, uterine histopathological analysis, AMPK and PLIN2 expressions were determined through immunohistochemistry. Furthermore, inflammation, oxidative stress, and lipid metabolism-related factors were evaluated using Western blot analysis. In experiment two, primary cell cultures were prepared from healthy uterine endometrial cells of the dogs in control group. Cultured canine endometrial epithelial cells were treated with lipopolysaccharide (LPS) along with oleic acid (OA) to induce an inflammatory response. Tissue and serum MDA and SOD levels were greater in dogs with pyometra. Accumulated lipid droplets were observed in the uterine tissue of bitches with pyometra. The phosphorylation of AMPK and the expression of PLIN2 significantly increased in the pyometra group. The expression of related lipid synthesis proteins such as ACC1, FASN, SREBP-1c, and PLIN2 was upregulated, while PPARα and PGC1α were downregulated in bitches with pyometra. In experiment two, activation of AMPK and PLIN2 not only restores the expression of PGC1α, but also effectively alleviates inflammation and oxidative stress. The role of lipid metabolism and oxidative stress in canine pyometra is elucidated, thus contributing to the pathogenesis of pyometra in dogs.

Background Nesfatin-1 is a neuropeptide that regulates the hypothalamic-pituitary-gonadal axis and may play a role in uterus function. It is co-expressed with other peptides, such as phoenixin, which can influence sex hormone secretion. Our previous research has confirmed that phoenixin-14 is involved in the development of cystic endometrial hyperplasia (CEH) and pyometra in dogs. Therefore, based on the similarities and interactions between these neuropeptides, we hypothesized that nesfatin-1 might also regulate the reproductive system in dogs. This study aimed to determine the expression of nesfatin-1 and its interaction with phoenixin-14 in dogs with CEH or pyometra compared to healthy females, and concerning animals’ body condition score (BCS 4–5/9 vs. BCS > 5/9). Results The analysis of nesfatin-1 in the uterus of bitches consisted of qPCR, western blot and immunofluorescence assays, and ELISAs. The results showed significantly higher nesfatin-1 encoding gene, nucleobindin-2 mRNA ( Nucb2 ) and nesfatin-1 protein expression in overweight females and those suffering from CEH or pyometra compared to healthy animals. The immunoreactivity of nesfatin-1 was elevated in the uteri of bitches with higher BCS > 5/9. Moreover, nesfatin-1 blood concentrations increased in all examined overweight bitches. In the case of phoenixin signals, we found opposite results, regardless of the female body condition score. Conclusion The etiology of CEH and pyometra are not fully known, although we have expanded the level of knowledge with respect to the possible interaction of nesfatin-1 and phoenixin in female dogs’ uteri. They interact oppositely. With increasing female body weight, the expression of nesfatin-1 in the uterus and its peripheral blood concentration increased. However, for female dogs affected by CEH and pyometra, a decreased level of phoenixin-14, irrespective of their body condition score is characteristic. This knowledge could be crucial in the development of biomarkers for these conditions, which may lead to earlier recognition.

Background Calprotectin (S100A8/A9) is a protein related to innate immunity that is considered a biomarker of inflammation. Currently, there is a commercially available automated assay for the measurement of calprotectin concentration (Bühlmann fCal Turbo ® assay), which has been previously validated for saliva and serum of swine and for saliva of horses, and in the canine species it has been validated for use with fecal samples, but it has not been previously validated in canine saliva or serum. Thus, the aim of this study was to perform an analytical validation of an automated assay for the measurement of calprotectin in the saliva and serum of dogs. In addition, changes in this protein in saliva and serum of three diseases with different pathogenic mechanisms – leishmaniasis, pyometra and hyperadrenocorticism – were evaluated. Finally, in these diseases, the correlation of salivary and serum calprotectin with the serum levels of three acute phase proteins (APPs), including C-reactive protein (CRP), haptoglobin (Hp) and ferritin, was also assessed. Results The analytical validation results showed that the assay was precise (coefficients of variation < 15% in all cases), accurate (the dilutional parallelism for serum and salivary calprotectin showed observed-to-expected ratios with a mean of 96.9% and 97.2%, respectively), and presented a limit of detection of 0.038 mg/L. When this assay was applied to the different diseases, a significant increase in the concentration of salivary calprotectin in dogs with leishmaniasis ( p  = 0.0002) and in those with pyometra ( p  = 0.002), compared to healthy ones, was observed, whereas no significant differences were found in serum. Furthermore, a significant positive moderate correlation was obtained between salivary calprotectin and serum CRP ( r  = 0.5; p  = 0.001) and haptoglobin ( r  = 0.5; p  = 0.002), and between calprotectin and CRP ( r  = 0.67; p  < 0.001) in serum. Conclusions Calprotectin (S100A8/A9) can be measured in dog saliva and serum samples by the automated method validated in this study, and when measured in saliva it could be used as a potential biomarker of inflammation and immune activation in the dog.

Toll-like receptors (TLRs) are essential components of both innate and adaptive immunity, influencing various physiological and pathological processes. It is also known that immune system components are present in the corpus luteum (CL) and regulate its functions. This study aimed to, for the first time, examine the mRNA expression levels of TLR2, TLR4, TLR7, and interleukin-8 (IL-8) in canine luteal tissue samples collected during early diestrus (EDI) (n=5), early pregnancy (EPR) (n=5), and pyometra (PYO) (n=5), a pathological condition in which the CL remains active. The luteal tissue samples were obtained via ovariohysterectomy and analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). It was found that the mRNA expression levels of TLR2, TLR7, and IL-8 were highest in the PYO group (p<0.05). However, no significant difference was observed in TLR4 mRNA expression between groups (p>0.05). This study reveals distinct expression profiles of TLRs and IL-8 in canine luteal tissue under different physiological and pathological states. In conclusion, pyometra may induce changes in the mRNA expression patterns of TLRs and IL-8 in canine luteal tissues. Further research involving protein-level analyses with larger sample sizes is necessary to better understand the roles of these molecules in the regulation of luteal function.

Objectives To determine T helper (Th)1 and Th2 cytokine polarization, as well as high‐sensitive cardiac troponin I (hs‐cTnI) levels, in cats with pyometra. Methods We used 40 queens in the study. A total of 20 out of these 40 queens were diagnosed with the pyometra group (PYO) and the other 20 made up the healthy group (control; CTR). We measured concentrations of hs‐cTnI, aspartate aminotransferase (AST), creatine kinase (CK) and l‐lactate in queens from both groups. Additionally, we measured cytokine concentrations in all queens. Results The hs‐cTnI concentration in the PYO group (26.95 ± 5.08 ng/L) was significantly higher than that of the CTR group (7.00 ± 0.82 ng/L) (p < 0.000). Furthermore, the PYO group had a higher CK concentration (344.50 ± 39.63 U/L) than the CTR group (191.00 ± 15.44 U/L) (p = 0.002). The PYO group also demonstrated higher concentrations of TNF‐α (9.77 ± 0.81 ng/mL), IFN‐γ (25.37 ± 2.09 ng/mL), IL‐2 (4.37 ± 0.39 ng/mL), IL‐4 (245.64 ± 15.83 pg/mL), IL‐5 (63.13 ± 1.65 pg/mL) and IL‐10 (123.58 ± 4.30 ng/mL) compared to the CTR group (p < 0.000). Conclusions Overall, it is suggested that changes in cytokine concentrations increase in queens with pyometra, potentially causing harm to the heart muscle. It is crucial to consider that the heart muscle may also be affected in queens with pyometra during the treatment process. • This study investigates the relationship between hs‐cTnI and Th1/Th2 cytokines polarization in queens suffering from pyometra. • A correlation was found between hs‐cTnI and CK concentrations and cytokines in queens with pyometra. • hs‐cTnI is extremely important in developing treatment methods against pyometra, protective measures and monitoring heart muscle damage in cats with pyometra.

The aim of the present study was to determine the concentrations of glutathione (GSH), vitamin C, copper (Cu) and zinc (Zn) in the uterine tissues in diagnosis of canine pyometra. Fourteen samples of uterine tissues from female dogs with pyometra and twelve samples of healthy uteruses (control) were used. The concentrations of GSH and vitamin C were determined in the uterine tissue homogenates using spectrophotometric methods. The concentrations of Cu and Zn were measured using atomic absorption spectrometer. The results obtained showed the significantly lower (p<0.05) concentration of GSH and the trend towards lower concentration of vitamin C in the pyometra samples compared to the control. The concentrations of Cu and Zn were similar in the uterine tissues from female dogs with pyometra and those from healthy female dogs. The lower GSH and vitamin C concentrations in the uterine tissues of female dogs with pyometra indicate that the non-enzymatic antioxidant mechanisms are impaired in the uterus of dogs with pyometra. These findings suggest that the imbalance of oxidative-antioxidative can play an important role in pathogenesis of canine pyometra.

BACKGROUND: Pyometra often induces systemic inflammatory response syndrome (SIRS) and early diagnosis is crucial for survival. Chromogranin A (CgA) is a neuroendocrine secretory protein that is co-released with catecholamines from the adrenal medulla and sympathetic nerve endings. A prognostic value of CgA has been found in humans that are critically ill or that have SIRS associated with infection. CgA has not yet been studied in dogs with bacterial infection. The aim of the study was to investigate CgA, measured by Chromogranin A361-372 (Catestatin; Cst) and Chromogranin A17-38 (Vasostatin; VS) in healthy dogs and in dogs with pyometra. RESULTS: Fifty dogs with pyometra, sampled prior to surgery and 64 healthy female dogs were included. In 19 pyometra cases, blood samples were also collected postoperatively. Concentrations of Cst and VS were measured in heparinised plasma and Cst also measured in EDTA plasma, by in-house radioimmunoassays. Student’s t-test and Wilcoxon two-sample test was used to test for differences between dog groups. Pre- and postoperative samples in dogs with pyometra were analysed by paired t-test. Pearson correlation was used to investigate associations of laboratory variables and hospitalization. P < 0.05 was considered significant. Concentrations of Cst were decreased in pyometra dogs (mean ± SE, 1.01 ± 0.05 nmol/L) compared to healthy dogs (mean ± SE, 1.70 ± 0.03 nmol/L) (p ≤ 0.0001). VS concentrations did not differ significantly between dogs with pyometra (0.40 ± 0.04 nmol/L) and healthy dogs (0.42 ± 0.03 nmol/L). Mean ± SE pre- and postoperative concentration of Cst (1.0 ± 0.04 nmol/L and 0.9 ± 0.2 nmol/L) and VS (0.36 ± 0.04 nmol/L and 0.36 ± 0.04 nmol/L) in dogs with pyometra did not differ significantly. Neither Cst nor VS concentrations were associated with duration of hospitalization and were not significantly different in the four dogs with pyometra that had prolonged (≥3 d) postoperative hospitalization. CONCLUSION: Concentrations of Cst, but not VS, were decreased in pyometra. Cst and VS concentrations before and after ovariohysterectomy did not differ significantly and were not associated with duration of hospitalization. Further studies are warranted to evaluate a possible diagnostic or prognostic value for Cst and VS.