The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours. Investigating these cell subpopulations may lead to more precise diagnosis of these malignancies.

Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Masitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action. The effect of masitinib on the proliferation of CMT-U27 and CMT-U309 cells was assessed by MTT assay and DNA fragmentation. Flow cytometric analysis was used to measure the effect of masitinib on apoptosis and the cell cycle. Additionally, vascular endothelial growth factor levels (VEGF) were measured, and the proliferation marker Ki-67 was visualised in immunocytochemical stainings in CMT cells. Treatment with masitinib inhibited the proliferation of CMT cells in a concentration-dependent manner. Maximal apoptotic activity and DNA fragmentation were observed at approximately IC₅₀ of masitinib in both cell lines. In addition, cell cycle distribution was altered and VEGF levels and Ki-67 proliferation indices were decreased in masitinib-treated cells in comparison with control cells. In this study, masitinib suppressed cell proliferation concomitantly via induction of apoptosis and cell cycle arrest by decreasing VEGF levels and the Ki-67 proliferation index in CMT-U27 and CMT-U309 cells in vitro, suggesting its potential as a therapeutic tool in the clinical setting of mammary cancer treatment in dogs.

Introduction: Canine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine. Material and Methods: A biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model. Results: Histopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR+, CD5.1+, CD14+, CD45+, CD83+, CD163+, and Ly-6G-Ly-6C+. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×108 did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells. Conclusion: The present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention.

Bovine leukaemia virus (BLV) is a Deltaretrovirus responsible for enzootic bovine leukosis, the most common neoplastic disease of cattle. It deregulates the immune system, favouring secondary infections and changes in the blood and lymphatic tissues. Blood homeostasis depends on functional haematopoietic stem cells (HSCs). Bone marrow is populated by these cells, which express CD34+ and CD35+ surface antigens and produce and release cytokines involved in the maintenance of haematopoiesis. The aim of the study was determination of the profile of cytokine production by CD34+ stem cells of cattle naturally infected with BLV.

Piperlongumine (PL) is a bioactive alkaloid and medicinal compound of piperamide isolated from the long pepper (Piper longum Linn). It has demonstrated bactericidal action against Mycobacterium tuberculosis (MTB), the cause of pulmonary tuberculosis; nevertheless, immunomodulatory activity had not been identified for it in MTB-triggered granulomatous inflammation. This study investigated if piperlongumine could inhibit such inflammation. Mycobacterium tuberculosis strain H37Rv was subjected to a broth microdilution assay. Piperlongumine at 5, 15, and 25 μg/mL, 0.2% dimethyl sulphoxide as control or 4 μM of dexamethasone were tested in vitro on MH-S murine alveolar macrophages. BALB/c mice were orally administered PL at 50, 100 and 150 mg/kg b.w. after trehalose-6,6-dimycolate (TDM) stimulation. Chemokine and cytokine concentrations were determined in lung supernatants. Flow cytometry and Western blot analysis were performed to determine phosphorylated spleen tyrosine kinase (Syk), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways. Piperlongumine inhibited inflammatory mediators and adherence of lymphocyte function-associated antigen 1 to MH-S cells following TDM activation. It also improved macrophage clearance of MTB. In TDM-stimulated MH-S cells, PL significantly influenced the macrophage inducible Ca²⁺-dependent lectin receptor (Mincle)-Syk-ERK signalling pathway. Oral dosing of PL effectively suppressed the development of pulmonary granulomas and inflammatory reactions in the TDM-elicited mouse granuloma model. PL as an inhibitor of MTB-triggered granulomatous inflammation may be an effective complementary treatment for mycobacterial infection.

Turkey histomonosis poses a serious threat to poultry production due to the ban on the use of effective drugs. The aim of the study was to evaluate the influence of a phytoncidal feed supplement on the course of histomonosis. The preparation was also analysed for immunomodulatory properties. Clinical observations and production monitoring were conducted in a flock of turkeys with histomonosis from their 11ᵗʰ to 56ᵗʰ weeks of life which were treated with the adiCoxSᴼᴸPF soluble supplement in a dose of 2.5 mL/L water. Later the preparation was used in a preventive dose (1 mL/L). The influence on the immune system was evaluated in broiler turkeys having been given adiCoxSᴼᴸPF for 3 days in doses of 1 or 3 mL/L. The T and B lymphocyte percentages in turkey blood and spleen tissue were analysed with flow cytometry. ELISA was implemented to evaluate antibody titres after Ornithobacterium rhinotracheale vaccination, and biochemical analyses were performed to evaluate the supplement’s safety. AdiCoxSᴼᴸPF was found effective in therapy and prevention of histomonosis. Additionally, adiCoxSᴼᴸPF stimulated both humoral and cell-mediated immune mechanisms, without impairing the functions of internal organs. The treated turkeys also yielded better production results (eggs/hen, fertility, and hatchability). AdiCoxSᴼᴸPF possesses immunomodulatory properties and it can be used successfully in the prevention and therapy of histomonosis in turkeys.

Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression. We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway. PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors. PEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

Introduction: The aim of the study was to evaluate the effect of administration of therapeutic doses of ceftiofur and tulathromycin on the circulating lymphocyte subpopulations in healthy pigs. Material and Methods: The study was conducted on thirty healthy 7- to 10-week-old pigs, assigned to three groups: the TUL group, injected with tulathromycin (n = 10); the CEF group, injected with ceftiofur (n = 10); and the C group, the control with no antibiotic administration (n = 10). Blood samples were collected before, during, and after treatment with antimicrobials. Lymphocyte subpopulations circulating in the blood were determined by immunostaining and flow cytometry analyses. Results: Following administration of a therapeutic dose of tulathromycin, there were no changes in the lymphocyte subpopulations circulating in blood. In contrast, administration of ceftiofur at the recommended dose decreased the absolute number of CD3+, CD21+, CD4+CD8-, CD4-CD8+, and double positive CD4CD8 cells. Conclusion: Results from the study indicate that ceftiofur possesses the ability to modulate the immune system in healthy pigs by decreasing lymphocyte subpopulations circulating in blood.

Changes in selected blood lymphocyte subpopulations in dogs administered with a soluble parasitic antigen (SPA) derived from a supernatant of 18S RNA-A and 18S RNA-B Babesia canis cell culture were investigated. The studies included 20 dogs divided into three groups: group I (n=8) - comprised of dogs receiving SPA twice, at 3 week intervals; group II (n=5) - nonvaccinated control dogs, and group III (n=7) - dogs vaccinated twice with a commercial B. canis vaccine. Cytometric analysis revealed that vaccination with SPA derived from B. canis culture had similar effects to the vaccination with a commercial vaccine. The vaccination lowered the percentage of T lymphocytes (CD3+), T helper cells (CD4+), cytotoxic/supressor T cells (CD8+), B lymphocytes (CD21+), and MHC II lymphocytes in the blood in comparison to non-vaccinated dogs. Statistical analysis of the results demonstrated that mean values of the tested parameters at each stage of the study were similar in groups I and III and significantly higher in group II. The lowered level of the lymphocyte subpopulations in groups I and III persisted during the whole period of the study. The results presented that SPA has immunosuppressive effect in the first period after being administrated.