Influence of horse demographics, country of training and race distance on the rating of Thoroughbreds.

The aim of the research was to assess how age, sex, sire, country of foaling, country of training and race distance influenced the international racing and performance of Thoroughbreds. The research was based on performance ratings of 6216 horses assigned by the International Federation of Racing Authorities between 2004 and 2022. The most common sex was stallion (58.54 %) and more than half of the population consisted of 3- and 4-year-old horses (54.68 %). The majority of the horses had the USA as their country of foaling (25.92 %) and also as their country of training (24.87 %). The sire with the largest number of offspring in the International Federation of Horseracing Authorities (IFHA) databases was Galileo (IRE) (193 horses). Four of the 10 most frequently represented sires belonged to the Sadler's Wells (USA) paternal line. The analysis of the statistics in the database as a whole established a significant (p<0.001) influence of all observed factors. Stallions achieved a significantly higher rating (117.85) compared to geldings (117.17) and mares (117.13). The horses originating in Ireland achieved a statistically higher rating (117.99) than horses from Argentina, Australia, Brazil, New Zealand, a group of other countries designated "Others" and South Africa. Statistically conclusive differences were found between horses trained in Ireland (118.80) and all other countries except Great Britain and France. Five of the 10 sires with the best offspring rating belong to the Mr. Prospector (USA) paternal line.

Systemic administration of CpG oligodeoxynucleotide and levamisole as adjuvants for gene-gun-delivered antitumor DNA vaccines.

DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8(+) T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

DNA vaccination against bcr-abl-positive cells in mice.

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

Combined chemo- and immunotherapy of tumors induced in mice by bcr-abl-transformed cells.

For our experiments we selected two oncogenic, bcr-abl-transformed mouse cell lines, viz. B210 and 12B1. Both cell types are capable of inducing leukemia-like disease in syngeneic BALB/c mice after intravenous inoculation. 12B1 cells can moreover form solid tumors after subcutaneous injection. Since immunotherapy would expectedly be most effective in animals in which the tumor mass had been reduced by other therapeutic means, we attempted to develop a combined therapeutic system for suppressing tumor growth. In the present study, mice inoculated with the aggressive 12B1 cells were treated with imatinib mesylate (IM), mouse interferon alpha (IFNalpha) and cyclophosphamide (Cy) in combination with genetically modified tumor cells engineered to produce various cytokines. These cell vaccines had been derived from B210 cells. Therapy with IM or IFNalpha alone or cell immunotherapy alone resulted in partial suppression of tumor growth. Of the different therapeutic regimens tested, a combination of repeated doses of IM, IFNalpha and cell vaccines with one relatively high dose of Cy (200 mg/kg) was the most effective, resulting in tumor-free survival of a large portion of mice. The spleens, livers and bone marrows of the successfully treated animals were tested for the presence of bcr-abl-positive cells by means of RT-PCR technique. Results were negative, this suggesting that the animals had been cleared of residual disease.

Efficacy of reovirus therapy combined with cyclophosphamide and gene-modified cell vaccines on tumors induced in mice by HPV16-transformed cells.

Oncolytic virotherapy is a novel approach to cancer treatment. In the present study we tested the ability of reovirus type 3, strain Dearing, to suppress the growth of tumors induced in mice by HPV16-transformed TC-1 cells. In vitro, these cells are highly susceptible to the virus. In repeated in vivo tests the intratumoral inoculation of the virus resulted in only a minor slow-down of tumor growth, never in a complete cure. The effect of the treatment was not enhanced by the simultaneous administration of non-oncogenic, genetically modified TC-1 cells expressing either IL-2, IL-12 or GM-CSF, and, in fact, the oncolytic effect of the virus was even less expressed in some instances. When cyclophosphamide was used in combination with the viral treatment, a synergistic effect resulting in tumor suppression was observed. In most instances the tumor regression was transitory, however, and was followed by tumor progression. The outcome of these experiments was dependent on the timing of the two treatments.

Chemotherapy and immunotherapy of tumours induced by gene-modified HPV16-transformed cells.

HPV16 E6/E7 transformed mouse kidney cells designated MK16/1/IIIABC (MK16) were modified by the insertion of a suicide gene, viz. the thymidine-kinase gene of herpes simplex virus (HSV TK). Tumour induction by these cells, designated N2A, was suppressed by ganciclovir (GCV). The growth of already established tumours was partially inhibited by GCV. This effect was markedly potentiated by a single dose of cyclophosphamide (Cy). Ganciclovir- or GCV+ Cy-cured mice were not protected against challenge with MK16 cells. N2A tumour growth was suppressed by simultaneous administration of MK16-derived, non-oncogenic B9 and 181 cells, which express either mouse GM-CSF or mouse IL2, respectively, in addition to HSV TK. The animals treated were protected against challenge with MK16 cells. Animals with already established N2A tumours were treated with GCV and/or repeated doses of B9 or 181 cells. Ganciclovir treatment alone and immunotherapy alone resulted in partial suppression of tumour growth but not in tumour cure. On the other hand, combined chemo- and immunotherapy resulted in tumour rejection by nearly all animals. Similar results were obtained if the immunotherapy with homologous gene-modified cells was substituted by treatment with anti-CD4 antibody. The animals cured of tumours with GCV combined with cell-based vaccine therapy but not those cured by GCV and anti-CD4 antibody treatment were found resistant to challenge with MK16 cells. The present results suggest that combined specific and non-specific chemo- and immunotherapy of tumours induced by appropriately gene-modified cells might provide a special advantage in the treatment of established tumours.

Immunization with live HPV-16-transformed mouse cells expressing the herpes simplex thymidine kinase and either GM-CSF or IL-2.

From mouse (C57BL/6) HPV-16 transformed cells denoted MK16/1/IIIABC (MK16) a cellular thymidine kinase deficient (cTK-) cell line was isolated. These cTK- cells were transduced by bicistronic recombinant adeno-associated viruses (rAAV) carrying the herpes simplex virus thymidine kinase gene and the gene for either the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) or mouse interleukin-2 (IL-2). Transduced cells were highly sensitive to minute amounts of ganciclovir (GCV) and synthesized moderate amounts of the respective cytokines. A number of cell clones were tested for the cytokine production. The two best producer cell lines, the GM-CSF-producing cells denoted B9 and the IL-2-producing cells denoted 181, were selected for further experiments. Neither B9 nor 181 cells were tumorigenic in syngeneic animals. As inducers of antitumour immunity against challenge with MK16 cells, B9 cells proved superior to the 181 cells. GCV treatment did not markedly influence the level of immunity induced.