Aleksandra Pawlak

Immunophenotypic characterization of canine malignant lymphoma: a retrospective study of cases diagnosed in Poland Lower Silesia, over the period 2011–2013

Lymphoma is the most frequently diagnosed cancer of the canine haematopoietic system. In this study, the flow cytometry and polymerase chain reaction (PCR) analysis were used to characterize a series of canine lymphomas in detail. The aim of this study was to determine the incidence of B- and T-cell high-grade lymphomas and their immunophenotypic characterization in Lower Silesia, Poland. The results show that the frequency of each type of lymphoma is 71% for B-cell and 17% for T-cell lymphomas. In two cases the PCR techniques confirmed the presence of simultaneous double gene rearrangements of the BCR and TCR receptors.

A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

[Reptile-associated salmonellosis as an important epidemiological problem].

of food poisoning. One of the reservoirs of Salmonella are reptiles, which are increasingly kept as pets. Most reptiles are asymptomatic carriers of Salmonella. These strains, isolated from reptiles, can cause serious infections, especially in infants, young children and people with immunodeficiencies. The disease called reptile-associated salmonellosis (RAS) may manifest with bloody diarrhea, meningitis, and arthritis, and consequently can cause bacteremia and sepsis. Among the strains described in the literature, Salmonella strains possessing the O48 antigen are an important group. Lipopolysaccharide (LPS) of Salmonella O48 contains sialic acid (NeuAc) in an O-specific-chain. LPS containing NeuAc exhibits antigenic similarity to antigens found in the human body, including blood serum, and therefore is correlated with the occurrence of the dangerous phenomenon of molecular mimicry. Bacteria containing NeuAc in their outer structures can evade the immunological response of the host, which significantly increases their virulence. Most data about RAS come from the USA, but in recent years cases from European countries are more frequent in the literature. Unfortunately, the occurrence of RAS in Poland has not been monitored so far. There is also no campaign to inform the public about the health risks connected with contact of people with reptiles.

Methotrexate induces high level of apoptosis in canine lymphoma/leukemia cell lines

INTRODUCTION Methotrexate is an antimetabolite used in the treatment of cancer and non-malignant diseases including rheumatoid arthritis, psoriasis and graft vs. host disease. Combination therapy with methotrexate was successful in the treatment of canine lymphoma, mammary tumor and invasive urinary bladder cancer. Lymphoma, the most common hematopoietic cancer in dogs, and leukemia are sensitive to chemotherapy, which is why methotrexate may be an important treatment option for these diseases. Although methotrexate is already used in veterinary oncology its effects on canine cancer cells has not been tested. The aim of the study was to evaluate for the first time methotrexate concentration-dependent cytotoxicity and its capability of inducing apoptosis in selected canine lymphoma/leukemia cell lines: CLBL-1, GL-1 and CL-1 as a first step before the in vitro development of new therapeutic options with the use of methotrexate. RESULTS Methotrexate exhibited concentration-dependent inhibitory effect on proliferation of all the examined cell lines with different degree of apoptosis induction. The most methotrexate sensitive cells belonged to CL-1 cell line derived from T cell neoplasia and previously characterized by high resistance to the majority of anticancer drugs used in the therapy of lymphoma/leukemia in dogs. Canine lymphoma and leukemia cell lines are sensitive to methotrexate, and this drug may be useful in effective treatment of canine neoplasms and especially of T-type leukemia/lymphoma.

In vitro drug sensitivity in canine lymphoma

Abstract Introduction: Due to the high heterogeneity of canine lymphoma, the aim of the present study was to test in vitro the chemosensitivity of canine high-grade primary lymphoma cells to various cytostatic drugs commonly used to treat dogs: 4-HO-cyclophosphamide, doxorubicin, dexamethasone, prednisolone, vincristine, etoposide, chlorambucil, lomustine, and cytosine arabinoside. Material and Methods: To determine the cell viability and drug ability to induce apoptosis two different tests were used: an MTT assay and annexin V/propidium iodide staining. Results: Both in vitro tests were found to be useful tools. Significant differences in the sensitivity, depending on the drug type, between B-, T- and mixed/null-type lymphoma cells were found for the majority of the tested drugs. B-type cells were the most sensitive in vitro, whereas T-type cells seemed to be the most resistant. Doxorubicin, chlorambucil, etoposide, and vincristine most strongly reduced the cell viability and induced apoptosis. Conclusion: In vitro assays, such as the MTT test and especially the annexin V/PI assay, may be useful tools for predicting a response to the treatment of high-grade lymphoma in dogs or improving the treatment outcomes in individual animals.

Antitumor Activity of Betulinic Acid and Betulin in Canine Cancer Cell Lines

Background/Aim: Betulinic acid (BA) and betulin (BT) exhibit a variety of pharmacological properties including anti-cancer, anti-inflammatory and anti-oxidant ones. Canine lymphoma and osteosarcoma have a high mortality rate and need more effective therapeutic approaches. In this study, the anti-proliferative and pro-apoptotic effects of BA and BT were investigated in canine T-cell lymphoma (CL-1), canine B-cell lymphoma (CLBL-1) and canine osteosarcoma (D-17) cell lines. Materials and Methods: The cultured cells were treated with several concentrations of BA or BT for 24, 48 and 72 h, and cell proliferation was assessed by the MTT assay. Cell apoptotic rate and cell cycle were analyzed using flow cytometry. Results: Anti-proliferative effect of BT and BA was concentration- and time-dependent. Moreover, BA and BT arrested cell cycle in S phase in CL-1 and D-17 cells, and in G0/G1 phase in CLBL-1 cells. Conclusion: Both compounds showed an antitumor activity, and the effects of BA were stronger than that of BT.

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL‐associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross‐reactivity of B5 and E11 with the human B‐cell line, Raji. By the ectopic expression of the hybrid murine/canine I‐E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B‐cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement‐dependent and antibody‐mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR‐targeted, preclinical trials involving CL.

Propentofylline, phosphodiesterase and adenosine reuptake inhibitor modulates lymphocyte subsets and lymphocyte activity after in-vivo administration in non-immunized and SRBC-immunized mice

The aim of the study was to investigate immunomodulatory effect of in‐vivo administered propentofylline on the subsets and activity of murine lymphocytes.

Experimental immunology The activity of phagocytic cells after in vivo administration of propentofylline in mice

Propentofylline, a phosphodiesterase (PDE) inhibitor, increases the intracellular level of cyclic nucleotides (cAMP and cGMP) and, in consequence, can change the activity of many cells, including immune cells. The studies were conducted on female Balb/c mice (8 weeks of age). Propentofylline was administered orally once or six times at 12 h intervals at a therapeutic dose of 3 mg/kg. The production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide (NO) by resident peritoneal murine macrophages stimulated in vitro with lipopolisaccharide (LPS) from Escherichia coli and the phagocytic activity of granulocytes and monocytes from peripheral blood were determined 12 h and 24 h after a single dose or after the last of six doses of propentofylline administration. A temporary decrease in TNF-α synthesis and an increase in IL-1β production were noted 12 h following a single administration of propentofylline. No effect on the TNF-α and IL-1β release was observed after six doses of the drug applied. Propentofylline, irrespective of the number of subsequent doses applied, did not change the synthesis and release of NO by peritoneal murine macrophages stimulated in vitro with LPS. No effect on the percentage of phagocytosing granulocytes and monocytes was observed after a single administration of propentofylline. However, an increase in the fluorescence intensity of the granulocytes was observed 12 h and 24 h after a single dose of the drug administered. Propentofylline administered six times did not change the fluorescence intensity of granulocytes and monocytes, but an increase in the percentage of phagocytosing monocytes was noted. The results obtained in the present study showed that propentofylline administered in vivo did not exhibit an immunosuppressive influence on the activity of phagocytic cells. The changes after propentofylline administration were short-lived and were balanced during the treatment.