Novel chemotherapeutic agent FX-9 activates NF-κB signaling and induces G1 phase arrest by activating CDKN1A in a human prostate cancer cell line.

BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.

Combination of the PI3K inhibitor Idelalisib with the conventional cytostatics cytarabine and dexamethasone leads to changes in pathway activation that induce anti-proliferative effects in B lymphoblastic leukaemia cell lines.

BACKGROUND: The introduction of combined conventional cytostatics and pathway-specific inhibitors has opened new treatment options for several cancer types including hematologic neoplasia such as leukaemias. As the detailed understanding of the combination-induced molecular effects is often lacking, the identification of combination-induced molecular mechanisms bears significant value for the further development of interventional approaches. METHODS: Combined application of conventional cytostatic agents (cytarabine and dexamethasone) with the PI3K-inhibitor Idelalisib was analysed on cell-biologic parameters in two acute pro-B lymphoblastic leukaemia (B-ALL) cell lines. In particular, for comparative characterisation of the molecular signatures induced by the combined and mono application, whole transcriptome sequencing was performed. Emphasis was placed on pathways and genes exclusively regulated by drug combinations. RESULTS: Idelalisib + cytostatics combinations changed pathway activation for, e.g., "Retinoblastoma in cancer", "TGF-b signalling", "Cell cycle" and "DNA-damage response" to a greater extent than the two cytostatics alone. Analyses of the top-20 regulated genes revealed that both combinations induce characteristic gene expression changes. CONCLUSION: A specific set of genes was exclusively deregulated by the drug combinations, matching the combination-specific anti-proliferative cell-biologic effects. The addition of Idelalisib suggests minor synergistic effects which are rather to be classified as additive.

Claudin-1, -3, -4 and -7 gene expression analyses in canine prostate carcinoma and mammary tissue derived cell lines.

Claudins (CLDNs) are transmembrane proteins localised in the cell membrane of epithelial cells composing a structural and functional component of the tight junction protein complexes. In canine tumors deregulations of the CLDN expression patterns were described immunohistochemically. Targeting of claudin proteins has further been evaluated to establish novel therapeutic approaches by directed claudin binding. Precondition for the development of claudin targeting approaches in canine cells is the possibility to characterise claudin expression specifically and the availability of claudin positive cell lines. Herein PCR/qPCR assays were established allowing a rapid qualitative and quantitative characterisation of CLDN-1, -3, -4 and -7 gene expression in canine cell lines and tissues. Further commercially available antibodies were used to verify CLDN gene expression on protein level by Western blots. The developed assays were used to analyse six canine cell lines derived from mammary and prostate tissue for their CLDN-1, -3, -4 and -7 expressions. The canine cell line DT08/40 (prostate transitional cell carcinoma) was used for the establishment of specific CLDNs -1, -3, -4 and -7PCR/qPCR. The designed assays were verified by amplicon cloning and sequencing. Gene expressions were verified on protein level by Western blot. Additionally further cell lines were analysed for their CLDN-1, -3, -4 and -7 expression on mRNA and protein level (mammary derived cell lines: MTH53A (non-neoplastic), ZMTH3 (adenoma), MTH52C (carcinoma); prostate derived cell lines: DT08/46 and CT1258 (both adenocarcinoma).The screened cell lines showed expression for the CLDNs as follows: DT08/46 and DT08/40: CLDN-1, -3, -4 and -7 positive; CT1258: CLDN-1, -3, -4 and -7 negative; ZMTH3 and MTH52C: CLDN-1 and -7 positive, CLDN-3 and -4 negative; MTH53A: CLDN-1, -3 and -4 negative, CLDN-7 positive. Western blot analyses reflect the detected CLDN-1, -3, -4 and -7 expressions in the analysed cell lines. The established CLDN-1, -3, -4 and -7 PCR/qPCR assays allow a qualitative and quantitative characterisation of canine CLDN gene expression. Characterisation of CLDN expression in six canine cell lines led to the identification of two canine prostate tissue derived CLDN expressing cell lines. These cell lines serve as candidates for further research on CLDN-based functional and therapeutic approaches.

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BACKGROUND: Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. RESULTS: In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. CONCLUSION: Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection.

Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.

Cytogenetic analysis of CpG-oligonucleotide DSP30 plus Interleukin-2-Stimulated canine B-Cell lymphoma cells reveals the loss of one X Chromosome as the sole abnormality.

Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.

Two new cases of polysomy 13 in canine prostate cancer.

Besides man, the dog is the only known mammalian species that spontaneously develops carcinomas of the prostate with considerable frequency. For this reason, the dog is considered to be the only useful animal model for spontaneously occurring prostate malignancies in man. Cytogenetic investigations of human prostate cancers have revealed the frequent occurrence of trisomies 7, 8, and 17. Chromosome analyses of canine prostate carcinomas are rare. In this report we present 2 cases of canine prostate cancer showing a clonal polysomy 13 along with complex karyotype changes. Along with a previous report demonstrating polysomy 13 as the only karyotype deviation in a canine prostate cancer the present report supports the hypothesis that in canine prostate cancer, polysomy 13 is a recurrent cytogenetic aberration linked to the development of the disease. As human chromosomes (HSA) 8q and 4q and the canine chromosome (CFA) 13 share high homology, these results suggest that a conserved area on these chromosomes is involved in tumorigenesis in both species.

Suitability of ultrasound-guided fine-needle aspiration biopsy for transcriptome sequencing of the canine prostate.

Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated intra vitam on 43 dogs. Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using phenol-chloroform extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-Seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.

Histopathological Terminology Standards for the Reporting of Prostatic Epithelial Lesions in Dogs.

The terminology applied to canine prostatic epithelial lesions, especially carcinomas, is currently not standardized and this hampers the ability of pathologists to study the biological and clinical significance of these lesions. The aim of this review is to present the essential histomorphological diagnostic attributes of a wide spectrum of prostatic epithelial lesions in dogs. In addition to the traditionally recognized prostatic hyperplasia, hormonal atrophy, prostatitis, squamous metaplasia, adenocarcinoma and transitional cell (urothelial) carcinoma, new entities are described and discussed in order to provide veterinary pathologists with a basic atlas of common histological lesions of the canine prostate that is comprehensive and easy to use.

Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

High Mobility Group Box 1-Protein expression in canine haematopoietic cells and influence on canine peripheral blood mononuclear cell proliferative activity.

High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in mammalian tissues. HMGB1 demonstrates cytokine function and induces inflammation when actively released by haematopoietic cells or passively released during cell necrosis. This study aimed at the determination of HMGB1 expression in different cell types and at the evaluation of the role of HMGB1 in PBMC proliferation. Therefore we investigated the HMGB1 mRNA expression level in different canine haematopoietic cell types and the influence of exogenous rhHMGB1 on canine PBMC proliferation. Differentiated haematopoietic blood cells showed lower relative HMGB1 expression levels compared to CD34+ haematopoietic stem cells. Relative HMGB1 expression seemed also to decrease during differentiation of CD34+ stem cells into dendritic cells. Furthermore, peripheral blood CD14+ monocytes and granulocytes showed a lower relative HMGB1 expression in comparison to CD3+ T-lymphocytes. When exogenous rhHMGB1 at low concentrations was added to single PBMC cultures an increase of proliferation was obvious. However, in higher concentrations HMGB1 lost its stimulative effect. In conclusion, HMGB1 is broadly expressed in canine haematopoietic cells with highest levels in haematopoietic stem cells. HMGB1 induced directly PBMC proliferation.

Decitabine demonstrates antileukemic activity in B cell precursor acute lymphoblastic leukemia with MLL rearrangements.

BACKGROUND: Promotor hypermethylation of CpG islands is common in B cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed lineage leukemia (MLL) gene rearrangements. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) reduce DNA hypermethylation by incorporation into DNA and were successfully introduced into the clinic for the treatment of myeloid neoplasias. METHODS: Here, we investigated whether HMA induce comparable biological effects in MLL-positive BCP-ALL. Further, efficacy of HMA and concomitant application of cytostatic drugs (cytarabine and doxorubicin) were evaluated on established SEM and RS4;11 cell lines. In addition, promising approaches were studied on BCP-ALL cell line- and patient-derived xenograft models. RESULTS: In general, DEC effects were stronger compared to AZA on MLL-positive BCP-ALL cells. DEC significantly reduced proliferation by induction of cell cycle arrest in G0/G1 phase and apoptosis. Most sensitive to HMA were SEM cells which are characterized by a fast cell doubling time. The combination of low-dose HMA and conventional cytostatic agents revealed a heterogeneous response pattern. The strongest antiproliferative effects were observed when ALL cells were simultaneously exposed to HMA and cytostatic drugs. Most potent synergistic effects of HMA were induced with cytarabine. Finally, the therapeutic potential of DEC was evaluated on BCP-ALL xenograft models. DEC significantly delayed leukemic proliferation in xenograft models as demonstrated longitudinally by non-invasive bioluminescence as well as 18F-FDG-PET/CT imaging. Unexpectedly, in vivo concomitant application of DEC and cytarabine did not enhance the antiproliferative effect compared to DEC monotherapy. CONCLUSIONS: Our data reveal that DEC is active in MLL-positive BCP-ALL and warrant clinical evaluation.

Quantitative expression analysis in peripheral blood of patients with chronic myeloid leukaemia: correlation between HMGA2 expression and white blood cell count.

The architectural transcription factor HMGA2 is highly expressed during embryogenesis but scarcely detectable in non-dividing adult cells. Previously, HMGA2 re-expression was detected in blood from CML patients by conventional RT-PCR, while blood samples from healthy volunteers were HMGA2 negative. Using the sensitive method of real-time quantitative RT-PCR, herein HMGA2 expression was detectable not only in peripheral blood from leukaemia patients but also in blood from healthy donors. Statistical analysis revealed a highly significant correlation between white blood cell count and HMGA2 transcript levels. The results indicate that up-regulation of HMGA2 expression is correlated to the undifferentiated phenotype of leukaemic cells accumulating during progression of chronic phase to blast crisis.

Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release.

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-16,624pg/ml), IFNγ (4-80U/ml), IL-4 (0-5069pg/ml), IL-10 (49-1862pg/ml), and IL-17 (4-1244U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors - age being the dominant one - should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements.

Molecular characterization of the canine HMGB1.

Due to the close similarities of numerous canine diseases to their human counterparts, the dog could join the mouse as the species of choice to unravel the genetic background of complex diseases as e.g. cancer and metabolic diseases. Accordingly, the role of the dog as a model for therapeutic approaches is strongly increasing. However, prerequisite for such studies is the characterization of the corresponding canine genes. Recently, the human high mobility group protein B1 (HMGB1) has attracted considerable interest of oncologists because of what is called its "double life". Besides its function as an architectural transcription factor HMGB1 can also be secreted by certain cells and then acts as a ligand for the receptor for advanced glycation end products (RAGE). The binding of HMGB1 to RAGE can activate key cell signaling pathways, such as p38(MAPK), JNK, and p42/p44(MAPK) emphasizing the important role of HMGB1 in inflammation and tumor metastasis. These results make HMGB1 a very interesting target for therapeutic studies done in model organisms like the dog. In this study we characterized the molecular structure of the canine HMGB1 gene on genomic and cDNA levels, its predicted protein, the gene locus and a basic expression pattern.

A 3.4-kbp transcript of ZNF331 is solely expressed in follicular thyroid adenomas.

Translocations involving chromosomal region 19q13 are a frequent finding in follicular adenomas of the thyroid and might represent the most frequent type of structural aberration in human epithelial tumors. By positional cloning, a putative candidate gene, ZNF331 (formerly RITA) located close to the breakpoint was identified. Recently, aberrant expression of ZNF331 has been described in two cell lines of follicular thyroid adenomas with aberrations in 19q13 indicating an involvement of ZNF331 in tumorigenesis. Nevertheless, knowledge about structure and expression of ZNF331 is limited. We performed RACE-PCR and genomic sequence analyses to gain a deeper insight into its molecular structure. To elucidate ZNF331 expression patterns we performed Northern blot analyses on various normal tissues as well as on thyroid carcinoma and adenoma cell lines. Herein, unique expression of a 3.4-kbp transcript is described in thyroid adenoma cell lines with 19q13 aberrations, which was not detected either in normal tissues or in thyroid carcinoma cell lines.

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.

REASONS FOR PERFORMING STUDY: CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. OBJECTIVES: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. METHODS: Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). RESULTS: Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes. CONCLUSIONS: The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. POTENTIAL RELEVANCE: The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.

Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.

Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1ß, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1ß antibodies detected equine IL-1ß in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines.

Chromosome analyses in dogs.

Cytogenetics is the study of normal and abnormal chromosomes. Every species is characterized by a given number of chromosomes that can be recognized by their specific shape. The chromosomes are arranged according to standard classification schemes for the respective species. While pre- and postnatal chromosome analyses investigate the constitutional karyotype, tumor cytogenetics is focused on the detection of clonal acquired, tumor-associated chromosome aberrations. Cytogenetic investigations in dogs are of great value especially for breeders dealing with fertility problems within their pedigrees, for veterinarians and last but not least for the dog owners. Dogs and humans share a variety of genetic diseases, including cancer. Thus, the dog has become an increasingly important model for genetic diseases. However, cytogenetic analyses of canine cells are complicated by the complex karyotype of the dog. Only just 15 years ago, a standard classification scheme for the complete canine karyotype was established. For chromosome analyses of canine cells the same steps of chromosome preparation are used as in human cytogenetics. There are few reports about cytogenetic changes in non-neoplastic cells, involving predominantly the sex chromosomes. Cytogenetic analyses of different entities of canine tumors revealed that, comparable to human tumors, tumors of the dog are often characterized by clonal chromosome aberrations, which might be used as diagnostic and prognostic markers. The integration of modern techniques (molecular genetic approaches, adaptive computer programs) will facilitate and complete conventional cytogenetic studies. However, conventional cytogenetics is still non-replaceable.

Relevance of chromosome 13 aberrations in canine tumours.

For human tumours there are many reports documenting the correlation between chromosome aberrations and tumour entities. Due to the complex canine karyotypic pattern (78 chromosomes), cytogenetic studies of tumours of the dog are rare. However, the reports in the literature show, that canine chromosome 13 (CFA13) is predominantly involved in chromosomal changes. Interestingly, CFA 13 shows high homology to regions on the human chromosomes 4 (HSA 4) and 8 (HSA 8), which harbour the proto-oncogenes c-KIT and c-MYC . Both of these genes are involved in the development and progression of some human and canine tumour diseases.