Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

BACKGROUND: DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. OBJECTIVES: In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. METHODS: We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. RESULTS: We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. CONCLUSIONS: The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

A novel approach in the prevention of mastitis: electrical teat dipping.

Teat dipping is widely used in dairy cattle, especially to protect against contagious mastitis. Here we determine the effect of the device called 'Electrical Teat Dipping' (ETD), which was developed by combining teat dipping application and electrical field stimulation technique on teats. For this purpose, the front teats of 100 Holstein breed milking cows were evaluated in two groups, with ETD being applied once to the left front teat of these cows, and conventional teat dipping (CTD) being applied once to the right front teat, both after milking. Ultrasonographic measurements of the teats were made before milking and after teat dipping. We found that the width of the teat canal (1.88 ± 0.07 mm) in the teat using ETD was narrower after the application compared to those with CTD (2.28 ± 0.05 mm). Based on our findings, we conclude that the effects of ETD on the teat are very positive and can potentially be used as a new approach in the preventative control of mastitis in cows.

Pulsed-wave Doppler ultrasonographical measurements of supra-mammary lymph nodes for diagnosis of bovine mastitis.

The aim of this study was to investigate the relationship between mastitis and supra-mammary lymph nodes in lactating cows in terms of Pulsed-wave (PW) Doppler ultrasonographical measurements. A total of 102 head cows in lactation period were divided into three groups. The cows in which all mammary lobes were California mastitis test (CMT)-negative (n = 27) formed the 1st group; those with CMT-positive mammary lobe (n = 43) formed the 2nd group and the cows with clinical mastitis in at least one mammary lobe (n = 32) formed the 3rd group. In PW Doppler ultrasonography, end-diastolic velocity, systolic peak velocity and time-averaged maximum velocity were measured at the most prominent artery of the lymph node. A quantitative scaling was performed by determining the pulsatile index and resistance index scales based on blood flow parameters. There was no statistically significant difference between the study groups in terms of PW Doppler ultrasonographical measurements of supra-mammary lymph nodes. In conclusion, the use of PW Doppler ultrasonographic data of the supra-mammary lymph nodes will not provide useful information about the current condition of mastitis in cows.

Changes in somatic cell count, composition and cytokine levels in milk from cows with mastitis due to mixed infections

Mastitis is a mammary gland inflammation that is very common worldwide, mostly caused by bacteria, and causes enormous economic losses. Many microorganisms cause this disease. The most common causes of mastitis by these microorganisms are Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Streptococcus agalactiae (S. agalactiae). The anti-inflammatory properties of transforming growth factor (TGF)-ß include: 1) limiting interferon (IFN)-γ production; 2) increasing the expression of the interleukine (IL)-1 receptor antagonist; 3) inhibiting macrophage production of chemokines, pro-inflammatory cytokines, nitric oxide, and reactive oxygen intermediates; and 4) increasing macrophage clearance of bacterial debris and damaged parenchymal cells. It is stated that cytokines and milk composition change in case of mastitis. In this study, it was aimed to reveal the changes in milk TGF-ß1 and Tumor necrosis factor (TNF)-α concentrations and milk composition in mixed infections caused by three pathogens causing mastitis. In this study, milk samples from 90 cows were divided into 5 groups. Tumor necrosis factor (TNF)-α and TGF-ß1 concentrations and milk composition were determined in these milk samples. The California Mastitis Test (CMT) was applied to the cows included in the study and scoring was done. According to the CMT results of the milk samples taken, CMT(-) cows were included in group 1 (n = 22). Those with the CMT(+) were sent to the microbiology laboratory for analysis within 2 h. After the bacteria was determined, combination groupings were formed. Group 2 (n = 17), in which S. aureus and E. coli grew together, group 3 (n = 21), in which S. aureus and S. agalactiae grew together, group 4 (n = 8), in which S. agalactiae and E. coli grew together in milk samples, and milk samples without any bacterial growth in CMT (+) formed group 5 (n = 22), respectively. Somatic cell count was measured with the DeLaval Cell Counter® (Cell Counter DCC) device. Mineral matter, fat, protein, lactose, electrical conductivity and specific gravity were measured in milk samples using Lactoscan Milk Analyzer (Milkotronic/EUROPE). Milk samples were then stored at -80°C to measure TGF-ß1 and TNF-α. Tumor necrosis factor-α and TGF-ß1 concentrations in milk samples were measured using ELISA kits (Sunred Biological Technology). Changes in milk TNF-α and TGF-ß1 concentration and milk composition were determined in milk samples with mastitis caused by mixed infection. The TNF-α concentration of group 4 was higher than the other groups. On the other hand, the highest concentration of TGF-ß1 was found in group 2. While the number of somatic cells in group 1 was lower than in groups 2, 3, and 4, there was no statistical difference between groups 1 and 5. The lowest milk fat ratio was found in group 1, and it was found to be statistically lower than groups 2, 3, and 4. While the rate of solid-non-fat of group 1 increased compared to groups 2 and 3, the highest protein ratio was found in groups 1 and 5. There was no difference between the 5 groups in terms of mineral matter ratios. While the specific gravity was highest in group 1, there was no statistical difference between the other 4 groups. Overall, it was concluded that there was an increase in TNF-α and TGF-ß1 concentrations and a change in milk composition in samples with bacterial growth.(AU)

Effects of Ovarian Transplant on Anti-Müllerian Hormone, Kisspeptin-1, and Kisspeptin-1 Receptor Levels in Rats.

OBJECTIVES: Information is scarce on levels of kisspeptin-1 and the kisspeptin-1 receptorin females after ovarian transplant. In this study, our aim was to explore serum estradiol, anti-Müllerian hormone, kisspeptin-1, and kisspeptin receptor levels, along with kisspeptin-1-positive cell density, in ovaries from rats after ovarian transplant. MATERIALS AND METHODS: For this study, 28 female Sprague Dawley rats were divided into 4 groups, with sham surgery performed on rats in group 1 (control group). Group 2 rats had ovaries transplanted under the peritoneum, and group 3 rats had their own ovaries transplanted subcutaneously. Group 4 rats were maintained in an estrous state. Serum anti-Müllerian hormone, kisspeptin-1, estradiol, and ovarian kisspeptin receptor levels were determined using commercial enzyme-linked immunosorbent assay kits. Kisspeptin-1-positive cell densities in the ovaries were determined immunohistochemically.The ovaries were also examined histopathologically. RESULTS: Our statistical analyses showed that levels of kisspeptin receptors in the ovaries were lowest in the subcutaneously transplanted group (group 3; 628.57 ± 35.69 pg/mL). The highest serum anti-Müllerian hormone levels were found in the estrous group (group 4; 16.91 ± 2.12 ng/mL). Differences between groups in terms of serum kisspeptin-1 and estradiol concentrations were not statistically significant. CONCLUSIONS: Our findings suggest that, in rats, results were better in the peritoneum transplant group than in the subcutaneous transplant group. We also found that serum anti-Müllerian hormone levels were lower in the transplant groups than in the estrous group, although levels were not completely decreased to zero. These results support the finding that ovarian activities continue after transplant.

Expression of the anti-Mullerian hormone, kisspeptin 1, and kisspeptin 1 receptor in polycystic ovary syndrome and controlled ovarian stimulation rat models.

Polycystic ovary syndrome represents a significant cause of female infertility. The aim of this study was to investigate the expression of anti-Mul-lerian hormone (AMH), kisspeptin 1 (KISS-1), and kisspeptin 1 receptor (KISS1r) in rat models of polycystic ovary syndrome (PCOS) and controlled ovarian stimulation (COS). For this purpose, 28 rats were assigned into four groups. Estrus and Diestrus groups consisted of rats in estrus and diestrus phases, respectively, while COS and PCOS groups consisted of rats with induced COS and PCOS, respectively. The serum AMH, KISS-1, and estradiol levels, and ovarian KISS1r levels were analyzed by enzyme-linked immunosorbent assay. Furthermore, histopathological analysis of the ovary tissue was done and ovarian KISS-1 expression was determined by immunohistochemical assay. The results revealed that ovarian KISS1r levels were higher in the Estrus (1271.43±51.98 pg/mL) and COS (1191.43±85.67 pg/mL) groups, compared to Diestrus and PCOS groups. The highest level of AMH was found in the Estrus group (16.91±2.12 ng/mL). The results indicate that AMH had no effect on the development of COS and PCOS, while KISS-1 was found to affect the development of COS in rats.

The relationship between mastitis and the B-mode, colour Doppler ultrasonography measurements of supramammary lymph nodes in cows.

This research communication aims to test the hypothesis that B-Mode, colour Doppler ultrasonographic measurements and characteristics can identify mastitis in dairy cows. A total of 102 lactating cows were divided into 3 groups: cows in which all mammary quarters were CMT-negative, cows with CMT-positive mammary quarters and cows with clinical mastitis in at least one quarter. Colour Doppler ultrasonography measurements of the supramammary lymph nodes revealed that distortion-type vascular morphology, the rate of type 4 vascular densities and the incidence of mixed-type vascular distributions were highest in the clinical mastitis group, whereas the frequency of avascularity in supramammary lymph nodes was highest in the CMT-negative group. All differences were significant. In conclusion, the use of B-mode and colour Doppler ultrasonographic measurements of the supramammary lymph nodes can provide useful information about the current condition of mastitis in cows, although its diagnostic potential remains to be determined.

The Effects of Post-Mating Administration of Anti-IL-10 and Anti-TGFß on Conception Rates in Mice.

BACKGROUND: In fertility studies, it has been shown that transforming growth factor ß (TGFß) and interlukin 10 (IL-10) play very important roles in implantation, maternal immune tolerance, placentation and fetal development, and the release beginning of release for fetal and postnatal death. The present study aims to determine the effects of the postmating administration of neutralizing antibodies against IL-10 and TGFß, which significantly impact pregnancy in females and the conception rates in mice via assessments of blood serum and uterine fluid concentrations of IL-2, IL-4, IL-6, IL-10, IL-17, interferon γ (IFNγ), Tumor necrosis factor α (TNFα), and TGFß. MATERIALS AND METHODS: In this experimental study, 21 BALB/c strain female mice were mated and randomly divided into three groups. The mice in the first group were selected as the control group. The second group of animals was injected with 0.5 mg of anti-IL-10 after mating, while those in the third group were intraperitoneally injected with 0.5 mg of anti-TGFß. The animals in all groups were decapitated on the 13thday after mating and their blood samples were taken. The uteri were removed to determine pregnancy. The mice's uterine irrigation fluids were also obtained. We used the multiplex immunoassay technique to determine the cytokine concentrations in uterine fluid and blood serum of the mice. RESULTS: We observed no intergroup difference with respect to conception rates. A comparison of the cytokine concentrations in the uterine fluids of pregnant mice revealed higher TGFß concentrations (p<0.01) in the second group injected with the anti-IL-10 antibody compared with the other groups. There was no difference detected in pregnant animals with regards to both uterine fluid and blood serum concentrations of the other cytokines. CONCLUSION: Post-mating administration of anti-IL-10 and anti-TGFß antibodies in mice may not have any effect on conception rates.

What is the impact of SOCS3, IL-35 and IL17 in immune pathogenesis of recurrent pregnancy loss?

OBJECTIVE: To investigate the plasma levels of interleukin-4 (IL-4), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), IL-17, IL-35 and suppressor of cytokine signaling 3 (SOCS3) in the women with history of idiopathic recurrent pregnancy loss (RPL) and in the fertile controls. METHODS: This study was conducted with 60 idiopathic RPL cases and 40 age-matched fertile controls. Mid-follicular plasma levels of IL-17, IFN-gamma, TNF-alpha, TGF-beta, IL-6, IL-4, IL-10, SOCS3 and IL-35 were assayed by an enzyme linked immunosorbent assay. RESULTS: The mean age of RPL and control cases were 31.6 ± 0.6 and 32.1 ± 0.7 years, respectively. While plasma IL-35 and SOCS3 levels of RPL group were significantly lower than that of the control group; IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10, IL-17 and TGF-beta levels of RPL group were significantly higher than that of the control group. The comparison of cytokine ratios between RPL and control groups indicated significantly high TNF-alpha/IL-10, TNF-alpha/IL-4, IFN-gamma/IL-10, IFN-gamma/IL-6 and IFN-gamma/IL-4 ratios in the RPL group. IL-35/IL-17 ratio was significantly low in the RPL group compared to that in the control group. Overstimulation of TNF-alpha presented moderate influence on recurrent miscarriage risk. CONCLUSION: Decreased SOCS3 and IL-35 plasma levels and increased Th1/Th2 cytokine ratios in RPL cases pointed out the supression of anti-inflammatory process and this supression might play an important role in the pathogenesis of idiopathic RPL.

Absence of link between abortion and seropositivity of cystic hydatid disease in ewes and female goats in Turkey.

This study was conducted to test whether there is a statistically significant association between seropositivity to cystic echinococcosis and abortion in ewes and female goats from the Turkish provinces of Elazig (east Anatolia), Sanliurfa (south-east Anatolia) and Kayseri (Inner Anatolia) using the enzyme-linked immunosorbent assay (ELISA). A total of 20 of 133 sera (15.1%) from ewes and 5 of 101 sera (4.9%) from goats with a history of abortion gave seropositive results that were not significantly different (p>0.05) from these, 9.9% (10/101) were reported for ewes and 1.6% (2/122) for female goats without a history of abortion. Serological prevalence rates among those animals with a history of abortion were not significantly different from the control group. No positive association was established between seropositivity for cystic echinococcosis and abortion in ewes and female goats.

The effects of Kisspeptin antibodies on delayed estrus in rats.

In recent years, numerous studies have been conducted on the effects of Kisspeptin/GPR54 system on sexual cycle, which proved that this system regulated gonadotropin release through GnRH. This study aims to determine the effects of hyperimmune serum containing antibodies produced against Kisspeptin on the sexual cycle and GnRH receptors in rat pituitaries. To this end, five Wistar female rats were passively immunised using a hyperimmune serum obtained from two Wistar female rats against Kisspeptin 10. Another five rats were selected as the control group. Anti-Kisspeptin antibodies of the hyperimmunised rats in the serum were identified by ELISA. The sexual cycles of the rats were followed by the measurements of vaginal irrigations and the estradiol concentrations in their blood samples. The number of follicles and corpora lutea in their ovaries was determined through histopathological tests, and the GnRH receptors in their pituitaries were identified by immunohistochemistry. Consequently, strong seropositivity was detected in all passively immunised rats and the hyperimmune serum. However, no difference was found between the groups with regard to the number of estrous cycles observed, the interval between estrous periods, estradiol concentrations, the number of follicles and corpora lutea and immunohistochemical results.