Characterisation of Canine and Feline Breast Tumours, Their Metastases, and Corresponding Primary Cell Lines Using LA-REIMS and DESI-MS Imaging.

Breast cancer, a complex disease with a significant prevalence to form metastases, necessitates novel therapeutic strategies to improve treatment outcomes. Here, we present the results of a comparative molecular study of primary breast tumours, their metastases, and the corresponding primary cell lines using Desorption Electrospray Ionisation (DESI) and Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) imaging. Our results show that ambient ionisation mass spectrometry technology is suitable for rapid characterisation of samples, providing a lipid- and metabolite-rich spectrum within seconds. Our study demonstrates that the lipidomic fingerprint of the primary tumour is not significantly distinguishable from that of its metastasis, in parallel with the similarity observed between their respective primary cell lines. While significant differences were observed between tumours and the corresponding cell lines, distinct lipidomic signatures and several phospholipids such as PA(36:2), PE(36:1), and PE(P-38:4)/PE(O-38:5) for LA-REIMS imaging and PE(P-38:4)/PE(O-38:5), PS(36:1), and PI(38:4) for DESI-MSI were identified in both tumours and cells. We show that the tumours' characteristics can be found in the corresponding primary cell lines, offering a promising avenue for assessing tumour responsiveness to therapeutic interventions. A comparative analysis by DESI-MSI and LA-REIMS imaging revealed complementary information, demonstrating the utility of LA-REIMS in the molecular imaging of cancer.

Foetal development of endocrine organs in dog.

The canine adenohypophysis starts to be identifiable from 25 day of pregnancy. ACTH-immunoreactive cells migrate until day 38 after which the number of ACTH-producing cells increases but their distribution does not change. The STH- and LH-producing cells first appear on day 38 of pregnancy. The primordium of the adrenal glands appears as a slender structure on day 27 and forms the definitive cortical structure on day 35. The histological pattern of the foetal adrenal cortex differs from the post-natal structure in so far as the three cortical zones (definitive zone, transitional zone and foetal zone) extend from the outside towards the inside of gland. The mass of foetal and neonatal adrenals is more than 10 times larger than the adult adrenals relative to body weight. The cortisol concentration in the amnion is slightly lower than in the allantois but the foetal serum cortisol concentration is significantly higher than in the maternal and foetal fluid compartments. The thyroxine concentrations in the allantois and amnion fluids exceed the foetal serum concentrations except in the ninth week of pregnancy, but thyroxine levels in foetal fluids and serum are below the physiological levels of adult animals. The exocrine and endocrine functions of the pancreas develop and act in parallel. Pancreatic cells are first detected at 30 days when the branched structure is clearly detectable immunohistochemically, and at that time, insulin-positive ß-cells and α-cells are visible as well. The foetal serum glucose concentration exceeds the healthy adult range, but the glucose concentration in the allantois and amnion fluid remains below the physiological blood glucose concentration of mature dogs. The insulin concentration in the allantois fluid greatly exceeds the foetal serum and amnion insulin concentrations.

Evaluation of the performance of a human D-dimer test in dogs with neoplasia.

The goal of this study was to evaluate the suitability of a commercially available D-dimer assay as a diagnostic tool for testing dogs. This assay is an immunoturbidimetric diagnostic test, capable of determining the D-dimer levels in human plasma by using 2B9 monoclonal antibody. Plasma samples of clinically healthy (n = 20) and tumour-bearing (n = 50) dogs were measured. The tumours were grouped on the basis of histological type and aggressiveness, and then the measured D-dimer concentrations of these groups were compared to those of the control group. The differences were analysed statistically. For benign tumours, we did not find alterations in the D-dimer levels. However, in the case of malignant tumours (lymphoma, sarcoma, and carcinoma) and in the presence of metastases, significantly elevated D-dimer levels were measured. The assay proved to be suitable for measuring the D-dimer levels in plasma samples of dogs. The calculated reference range for dogs was confirmed to be between 0.06 and 0.69 µg/mL fibrinogen equivalent unit.

The environmental obesogen bisphenol A increases macrophage self-renewal.

Self-renewal of macrophages is important for the healthy development and replenishment of tissue-resident macrophage pools. How this mechanism is controlled by endocrine signals is still largely unexplored. Here, we show that the endocrine disruptor bisphenol A (BPA) increases macrophage self-renewal. This effect was associated with phosphorylation of extracellular signal-regulated kinase (ERK) and a slight increase in the expression of liver X receptor alpha (LXRα). We found that LXRα inhibition induced, while LXRα activation impeded, macrophage self-renewal. LXRα signaling hence may protect from excessive macrophage expansion. Self-renewing macrophages, however, had negligible LXRα expression when compared with quiescent macrophages. Accordingly, tissue-resident macrophage pools, which are dominated by quiescent macrophages, were rich in LXRα-expressing macrophages. Overall, we show that BPA increases macrophage self-renewal and that this effect, at least in part, can be inhibited by increasing LXRα expression. Since BPA is accumulated in the adipose tissue, it has the potential to increase self-renewal of adipose tissue macrophages, leading to a condition that might negatively impact adipose tissue health.

A pilot study of total and allergen-specific IgE serum levels during anestrous, estrous and pregnancy in healthy female dogs.

BACKGROUND: Allergen-specific IgE serology is used for the determination of sensitization status in dogs with atopic dermatitis; the influence of the female reproductive cycle on the results of such methods has not been studied in dogs. OBJECTIVES: To compare the total and allergen-specific IgE of healthy bitches during anestrous, estrous and pregnancy. ANIMALS: Eight privately owned, healthy bitches. METHODS: Total and allergen-specific IgE levels were determined in eight bitches at three different time-points of their reproductive cycle: anestrous, estrous and pregnancy. RESULTS: Total IgE was significantly decreased (median: 74%) in female dogs during pregnancy when compared to anestrous. In 14 of 216 (6%), allergen-specific IgE test results were variably positive and negative at different stages of the reproductive cycle. This variation, however, was not related to changes in total serum IgE levels. CONCLUSIONS: Total IgE serum levels are reduced during pregnancy in female dogs. However, results of one allergen-specific IgE test did not appear to be markedly altered by the reproductive cycle in healthy bitches.

Adipose tissue macrophages in non-rodent mammals: a comparative study.

The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.

Effect of ELISA kit manufacturing process and incubation time on progesterone concentration measured in dog serum for ovulation diagnosis - Short communication.

Twenty-two serum samples of healthy bitches were tested with the frozen and lyophilised version of the same ELISA kit (Quanticheck, Faculty of Veterinary Science, Budapest, Hungary). Samples were chosen on the basis of their progesterone (P4) concentrations, which were between 1.00 and 20.00 ng/mL. As it is well known, this range has the highest clinical relevance in ovulation diagnosis. Both types of microplates were read at 15-min intervals from the 15th until the 90th minute (min) of incubation, and the results were compared with those of frozen plates at 60 min of incubation as 100 percent. Lyophilised microplates gave on average 18 percent higher results than the frozen version at equal incubation times. The highest difference between lyophilised and frozen samples was observed at 45 and 60 min of incubation. Ninety-four percent of the reaction in the frozen microplate occurred in the first 15 min, and during the subsequent 30 min the reaction seemingly stopped. After the 45th min of incubation, this 94 percent increased to 108 percent in the subsequent 30 min, which remained the final approximate result at the end of the 90 min of incubation. In contrast to the frozen microplate, the measured concentration increased continuously in the lyophilised version and reached the highest level at the 60th min. The results of the lyophilised microplate reached the same level at 30 min of incubation as those of the frozen version at 60 min. In conclusion, a mechanical increase or decrease of the incubation time does not generate a linear change in the test results. This study demonstrated that the results of a series of samples collected from the same bitch cannot be compared if they are measured with different laboratory methods or different ELISA kits.

Body fat distribution and metabolic consequences - Examination opportunities in dogs.

The relationship between metabolic disorders and the distribution of fat in different body regions is not clearly understood in humans. The aim of this study was to develop a suitable method for assessing the regional distribution of fat deposits and their metabolic effects in dogs. Twenty-five dogs were subjected to computed tomographic (CT) imaging and blood sampling in order to characterise their metabolic status. The different fat areas were measured on a cross-sectional scan, and the animals' metabolic status was evaluated by measuring fasting glucose, insulin and leptin levels. The volume of visceral adipose tissue is the main determinant of leptin levels. The correlation of visceral fat volume and leptin concentration was found to be independent of insulin levels or the degree of insulin resistance. There was a positive correlation between the visceral to subcutaneous fat volume ratio and serum insulin concentration, and a similar trend was observed in the relationship of fat ratio and insulin resistance. The distribution of body fat essentially influences the metabolic parameters in dogs, but the effects of adiposity differ between humans and dogs. The findings can facilitate a possible extrapolation of results from animal studies to humans with regard to the metabolic consequences of different obesity types.

[SPECT radiopharmaceuticals -- novelties and new possibilities]. / SPECT-radiofarmakonok -- újdonságok és új lehetoségek.

Actual state of affairs and future perspectives of SPECT radiopharmaceuticals regarding local and international data were summarized. Beyond conventional gamma-emitting radioisotopes, localization studies with beta emitting therapeutic radiopharmaceuticals hold increasing importance. Extension of hybrid (SPECT/CT) equipments has modified conventional scintigraphic and SPECT methods as well but more important changes come into the world through novel ligands for specific diagnoses and therapy.

An in situ depot for the sustained release of a TLR7/8 agonist in combination with a TGFß inhibitor promotes anti-tumor immune responses.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-ß inhibitor in preclinical tumor models in female mice.

Breast milk alkylglycerols sustain beige adipocytes through adipose tissue macrophages.

Prevalence of obesity among infants and children below 5 years of age is rising dramatically, and early childhood obesity is a forerunner of obesity and obesity-associated diseases in adulthood. Childhood obesity is hence one of the most serious public health challenges today. Here, we have identified a mother-to-child lipid signaling that protects from obesity. We have found that breast milk-specific lipid species, so-called alkylglycerol-type (AKG-type) ether lipids, which are absent from infant formula and adult-type diets, maintain beige adipose tissue (BeAT) in the infant and impede the transformation of BeAT into lipid-storing white adipose tissue (WAT). Breast milk AKGs are metabolized by adipose tissue macrophages (ATMs) to platelet-activating factor (PAF), which ultimately activates IL-6/STAT3 signaling in adipocytes and triggers BeAT development in the infant. Accordingly, lack of AKG intake in infancy leads to a premature loss of BeAT and increases fat accumulation. AKG signaling is specific for infants and is inactivated in adulthood. However, in obese adipose tissue, ATMs regain their ability to metabolize AKGs, which reduces obesity. In summary, AKGs are specific lipid signals of breast milk that are essential for healthy adipose tissue development.

Increased Serum and Urinary Oxytocin Concentrations after Nasal Administration in Beagle Dogs.

In recent years more and more studies have revealed the effect of extraneous oxytocin on the social behavior of dogs. The distribution of administered oxytocin in different physiologically relevant compartments is important because this knowledge forms the basis for the timing of behavior tests after the administration. Most behavioral studies rely on the non-invasive intranasal application of oxytocin. The aim of this study was to determine the time course of intranasal administered oxytocin secretion into blood and urine and also establish a connection between intranasal received oxytocin and urinary cortisol in dogs. In our experiment, four dogs received three puffs, 12 IU intranasal oxytocin treatment, two dogs received three puffs intranasal placebo treatment. Blood and urine samples were collected immediately prior to the administration then regularly during 4 h. After nasal oxytocin application, the serum oxytocin concentration increased, reached a maximum 15 min after the treatment and then rapidly returned to baseline levels 45 min later. The peak urinary oxytocin concentration occurred between 45 and 60 min after administration and returned to baseline levels slowly. We found considerable differences among individuals in the secretion of oxytocin in both the serum and the urinary oxytocin concentration measurements. Our results confirm that intranasally administered oxytocin passes into the blood stream. The time course of intranasally administered oxytocin secretion is similar to the time course of intravenously administered oxytocin secretion, and the peak values are also similar in both the serum and the urinary oxytocin concentration measurements, although there are large individual differences.

Effects on Sperms' Quality of Selegiline in Aged Rats.

BACKGROUND: Selegiline is used to treat Parkinsonian patients. Other indications of its use have recently been discovered. OBJECTIVE: Scouting special and beneficial side effects of selegiline treatment. METHOD: Two-year old male Wistar rats were daily treated with 0.25 mg/kg of selegiline s.c. (subcutaneous injection). The rats were sacrificed following a four-weeks' treatment. RESULTS: Mass of testes, number of sperms, progressive motility of sperms, and their viability definitely increased. CONCLUSION: Selegiline can successfully be used to stop/counterbalance certain symptoms of aging.

Effect of deslorelin on testicular function, serum dihydrotestosterone and oestradiol concentrations during and after suppression of sexual activity in tom cats.

Objectives The aim of the study was to evaluate the efficacy of a 4.7 mg deslorelin implant in tom cats. Methods Nine mature male cats were included in the deslorelin group and five cats in the control group. Before the study started, all cats were confirmed to have distinct sexually dimorphic behaviour. Blood samples were taken on the implantation day, at day 7 and at day 15, then monthly, in order to measure serum dihydrotestosterone (DHT) and 17beta(ß)-oestradiol concentrations. The deslorelin group (n = 9) was divided into two subgroups: five cats (cats 1-5) were neutered in the postimplantation period during suppression of sexually dimorphic behaviour, and four cats (cats 6-9) were neutered after re-expression of sexually dimorphic behaviour. The control group cats (n = 5) were castrated without administration of the implant. Results Sexually dimorphic behaviours ceased within a mean ± SD of 13-58 days (23.30 ± 14.17) after implantation. DHT concentration decreased within 30 days. The mean duration of suppression was 26.5 ± 7.42 months and reactivation coincided with increased DHT values reaching preimplantation concentrations within 1 month. 17ß-oestradiol concentrations significantly correlated with DHT concentrations ( P <0.01). For cats castrated during suppression of sexual behaviour, the length of the long axes of the nuclei of Leydig cells, the diameter of seminiferous tubules and the height of the epithelium of the seminiferous tubules did not change until 3-6 months after implantation, whereas at 12 and 32 months the measured values were even lower than in the control group. For cats castrasted after reactivation, the length of long axes of the nuclei of Leydig cells and the diameter of seminiferous tubules approached the values of the control group between 4 and 6 months after reactivation. Conclusions and relevance A deslorelin implant (4.7 mg) suppresses sexually dimorphic behaviour in tom cats without any side effects and with full reversibility; however, duration of suppression is highly individual.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Thyroxin and progesterone concentrations in pregnant, nonpregnant bitches, and bitches during abortion.

Serum progesterone and thyroxin concentrations were measured weekly until 61 to 62 days after ovulation in 24 pregnant bitches and in the control group of nine nonpregnant bitches in the luteal phase. Fourteen of the 24 dogs had a normal pregnancy and parturition. Ten of the 24 dogs showed mucinous or colored vaginal discharge, decreased appetite, or lethargy. These initial signs of abortion or fetal resorption were noted during the fourth week of pregnancy, and the process occurred over the next 2 weeks. Progesterone and thyroxin concentrations were measured by quantitative ELISAs validated to dog serum. The serum progesterone concentrations of the group going through abortions differed significantly from the third week until the end of the eighth week. The mean serum thyroxin concentrations of healthy pregnant and nonpregnant groups significantly exceeded the reference range (20-45 nmol/L). The serum thyroxin concentrations in the abortion group were between 16.15 ± 3.17 and 40.78 ± 8.97 nmol/L. The values in this group were significantly different from the other two groups at the third week of the luteal phase. Clinical signs of abortion or fetus resorption manifested in midpregnancy. The clinical signs of abortion coincided in each case with a low serum progesterone concentration (<10 ng/mL). This phenomenon indicated, in contrast with other studies, that the decrease of serum progesterone below 10 ng/mL at the fourth week of pregnancy may signal impending abortion. In the second half of pregnancy, the thyroid gland was not able to respond adequately to the elevated requirement in thyroid hormone, although in other periods of the ovarian cycle, there were no clinical signs of hypothyroidism.

Temperature increase induced by modulated electrohyperthermia (oncothermia®) in the anesthetized pig liver.

AIM OF STUDY: Is to show the intrahepatic temperature development in anesthetized pig. MATERIALS AND METHODS: Temperature development in the liver of anesthetized pig is measured to study the thermal effects of capacitive coupled energy transfer. The treatment was made by modulated electrohyperthermia (mEHT, trade name: oncothermia ®), controlled by a fluoroptical temperature sensing positioned by the ultrasound-guided process. Various fits of coupling were studied. RESULTS: The intrahepatic temperature at the end of the treatment ranged 40.5-44.8°C, while the skin temperature ranged 36.8-41.8°C depending on the coupling arrangement. CONCLUSION: mEHT is a feasible method to deliver deep heat to the liver of an anesthetized pig.

Evaluation of ¹77Lu-EDTMP in Dogs with Spontaneous Tumor Involving Bone: Pharmacokinetics, Dosimetry and Therapeutic Efficacy.

177Lu-EDTMP is currently being investigated as a potential agent for providing palliative care to the patients suffering from bone pain due to metastatic skeletal carcinoma. The present article describes the evaluation of 177Lu-EDTMP complex in four different canine patients with different types of primary and metastatic skeletal lesions with respect to its pharmacokinetic properties, dosimetry and therapeutic efficacy. The dogs were treated with a dose of ~44.4 MBq (1.2 mCi) per kg body weight of 177Lu-EDTMP, synthesized in-house with high radiochemical purity (98.8 ± 0.4 %) and excellent in vitro stability. The radiopharmaceutical showed favourable pharmacokinetic properties, such as, preferential accumulation at skeletal lesion sites and fast clearance from blood and other non-target organs through urinary route. The administered dose of the radiopharmacutical showed excellent therapeutic efficacy in case of a dog suffering from skeletal metastasis originating from primary tumor elsewhere. On the other hand, two of the remaining three patients with primary bone cancer showed stable disease intially with palliative effect. The fourth patient having metal implant induced osteosarcoma with severe limb oedema did not show any response to the treatment.