IL-6 restores dendritic cell maturation inhibited by tumor-derived TGF-ß through interfering Smad 2/3 nuclear translocation.

We previously found, in a canine transferable tumor model, that high concentration of IL-6 produced by tumor-infiltrating lymphocytes effectively restores the MHC expression of the tumor cells and T-cell activation inhibited by tumor-derived TGF-ß. This tumor also significantly suppresses monocyte-derived dendritic cells (DCs) differentiation and the functions of differentiated DCs with unknown mechanisms. In this study, we have demonstrated that a strong reaction of IL-6 was present to neutralize TGF-ß-down-regulated surface marker expression on DCs (MHC II, CD1a, CD40, CD80, CD83, CD86), TGF-ß-hampered DC functions and DC-associated T-cell activation. Western blotting and confocal microscopy results indicated that the presence of IL-6 markedly decreased the nuclear concentration of a TGF-ß signaling transducer, Smad 2/3. In addition, while Smad 7 is a potent molecule inhibiting Smad 2/3 nuclear translocation, no significant increase in Smad 7 gene expression upon addition of IL-6 in TGF-ß-pretreated DCs was detected, which suggested that the blockage of Smad 2/3 nuclear translocation by IL-6 did not occur through a Smad 7-inhibitory mechanism. In conclusion, IL-6 inhibited TGF-ß signaling and concomitantly antagonized the suppression activities of TGF-ß on DC maturation and activity. This study enables further understandings of host/cancer interactions an also provide hints facilitating improvements of DC-based cancer immunotherapy.

Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor.

BACKGROUND: Chemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-ß derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-ß in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT). RESULTS: We have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1ß and TGF-ß had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression. CONCLUSION: CXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.

Utilization of IκB-EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-κB Inhibitor Activity.

NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB-EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.

Electroporation-mediated IL-12 gene therapy in a transplantable canine cancer model.

Interleukin-12 (IL-12) is effective in treating many types of rodent tumors, but has been unsuccessful in most human clinical trials, suggesting that animal models of more clinical relevance are required for evaluating human cancer immunotherapy. Herein, we report on the effectiveness of gene therapy with plasmid encoding human IL-12 (pIL-12) through in vivo electroporation in the treatment of beagles with a canine tumor, the canine transmissible venereal tumor (CTVT). The optimal electroporation conditions for gene transfer into CTVTs were tested by luciferase activity and determined to be a voltage of 200 V and duration of 50 msec, with the number of shocks set at 10 pulses, and the use of an electrode with 2 needles. Under these conditions, intratumoral administration of as little as 0.1 mg pIL-12 followed by electroporation significantly inhibited the growth of well-established tumors and eventually led to complete tumor regression. Furthermore, local pIL-12 treatment also induced a strong systemic effect that prevented new tumor growth and cured established tumors at distant locations. Intratumoral administration of pIL-12 greatly elevated the IL-12 level in the tumor masses, but produced only a trace amount in the serum. A high level of IFN-gamma mRNA was also detected in the treated tumor masses. pIL-12 gene therapy attracted significantly more lymphocytes infiltrating the tumors, including CD4(+) and CD8(+) T cells, and the surface expression of MHC I and MHC II molecules on CTVT cells was greatly increased after pIL-12 therapy. This treatment also induced apoptosis of the tumor cells as detected by Annexin V. More importantly, delivery of pIL-12 with intratumoral electroporation did not result in any detectable toxicity in the dogs. We conclude that intratumoral electroporation of the pIL-12 gene could cause profound immunologic host responses and efficiently treat CTVT in beagle dogs. The results also indicate that CTVT is an excellent large animal cancer model for testing immunogene therapies mediated by electroporation.

CD5-low expression lymphocytes in canine peripheral blood show characteristics of natural killer cells.

NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5-low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 +/- 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8alpha (77.7%), CD8beta (53%), alpha/beta TCR (83%), and CD11/18 (80%), but the expression of gamma/delta TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5lo cells were larger than CD5-high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL-2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL-2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5hi cells. In addition, IL-2-stimulated, CD5lo-depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.

Synergistic anti-tumor effect of combination radio- and immunotherapy by electro-gene therapy plus intra-tumor injection of dendritic cells.

Interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) facilitate the maturation and functioning of injected DC. We developed a method of in situ electroporation using IL-2 and GM-CSF genes (EGT/cytokines), followed by intra-tumoral (i.t.) immature DC to determine the immune response at the tumor site using prostate-specific antigen-transfected CT26 cells. Three cycles of EGT/cytokines and i.t. DC inhibited tumor growth most effectively, but not superior to EGT/cytokines alone. However, the role of i.t. DC became significant when radiation was given after immunotherapy, which may have clinical implications on achieving better local control and prevention of systemic relapse.

Cytokine profiles of canine monocyte-derived dendritic cells as a function of lipopolysaccharide- or tumor necrosis factor-alpha-induced maturation.

In response to exogenous as well as endogenous signals, dendritic cells (DC) undergo programmed maturation to become efficient, antigen-presenting cells and mediate innate and adaptive immune responses. Very little is known, however, about the differential maturation responses of canine DC to endogenous and exogenous stimuli, especially the concomitant events related to the specific expression of cytokine genes. Canine monocyte-derived immature DC (iDC) were treated with an exogenous signal, bacterial lipopolysaccharide (LPS), or an endogenous signal, tumor necrosis factor-alpha (TNF-alpha), to generate mature DC (mDC). The mDC generated from either stimuli were characterized by significant increases in the expression of surface molecules, including CD11c, MHC class II, CD80, CD83, and CD86. Using real-time reverse transcriptase polymerase chain reactions, the cytokine expression profiles generated by these two stimuli were studied. Compared with the iDC, the LPS-stimulated mDC exhibited a significantly increased expression of IL-1 beta, IL-10, IL-12p40, IL-13, and TNF-alpha. Using the mixed lymphocyte reaction and cytokine intracellular staining, it was shown that the array of cytokines from LPS-generated mDC contributed to T cell priming and T helper cell type 1 (Th1) polarization. TNF-alpha-generated mDC increased the expression of a distinctly different panel of cytokines, namely IL-2, IL-4, IL-12p40, IL-13, TNF-alpha, TGF-beta, IFN-gamma, and MCP-2, and shifted naïve T cell differentiation to T helper cell type 2 (Th2) polarization. IL-13 expression was dramatically increased in canine TNF-alpha-generated mDC, which does not occur in other mammalian species, including humans. Because IL-13 is functionally similar to IL-4, IL-13 may contribute to the observed Th2 polarization. Thus, canine DC maturing from different stimuli release different cytokine profiles that in turn promote different immune responses and activate innate and adaptive immune responses.

Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes.

A panel of 380 commercially available monoclonal antibodies (mAbs) against human CD molecules from various sources was tested during the 8th Human Leukocyte Differentiation Antigen Workshop (HLDA8) for cross-reactivity on canine peripheral blood leukocytes by flow cytometry. In addition, all mAbs were used to label a 50:50 mixture of platelets and erythrocytes of the same dogs. This testing resulted in 51 cross-reacting mAbs. mAbs with specificity for CD9, CD29, CD42a, CD61, and CD41/CD61 showed cross-reactivity with canine platelets in a non-polymorphic and one mAb with the erythrocyte antigen CD235a in a polymorphic reaction pattern. Canine leukocyte-reactive mAbs included those with specificity for CD11a, CD11b, CD14, CD18, CD21, CD22, CD47, CD49d, CD49e, CD56, CD62L, CD91, CD94, and CD172a. In addition, several mAbs resulted in a staining pattern of canine cells which suggest that the canine epitope equivalents have an alternate expression pattern from that expected for humans (CD1a, CD35, CD44, CD45, CD75s, CD81). In summary, this study confirmed the reactivity of previously described cross-reactive mAbs with canine cells and resulted in the characterization of mAbs recognizing so far undetectable canine CD molecules.

Characterization of canine monocyte-derived dendritic cells with phenotypic and functional differentiation.

For therapeutic purposes, large numbers of dendritic cells (DCs) are essential. In this study, we used 2% autologous canine plasma, granulocyte/macrophage colony-stimulating factor (GM-CSF), fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin 4 (IL-4) in generating monocyte-derived DCs from peripheral blood mononuclear cells of dogs. The plasma enriched the population of CD14-positive monocytes by greatly enhancing the efficiency of monocyte adherence, the proportion of adherent cells increasing from 6.6% with 10% fetal bovine serum to 15.3% with 2% autologous canine plasma. Culturing the adherent monocytes for 6 d with human GM-CSF, canine IL-4, and human Flt3L significantly increased the yield of DCs, more than 90% of which were CD14-negative. Because, in the presence of lipopolysaccharide (LPS), monocytes that were CD14-positive expressed tumor necrosis factor ac much more than DCs with low levels of CD14, it is important to decrease the numbers of CD14-positive cells in generating monocyte-derived DCs. With flow cytometry and real-time reverse-transcriptase-mediated polymerase chain reaction assays, we found that in canine immature DCs (iDCs) the expression of DLA class II molecules, CD1a, CD11c, CD40, and CD86 was high and the expression of CD80, CD83, and CD14 either low or negative. During maturation (stimulated by LPS), the expression of CDla, CD40, CD83, and CD80 was upregulated. However, the expression of DLA class II molecules, CD11c, and CD86 was not increased in mature DCs. Incubating the iDCs with LPS decreased antigen uptake and increased the cells' immunostimulatory capacity (assessed by the allogeneic mixed-lymphocyte reaction), indicating that LPS accelerates the functional maturation of DCs. This protocol may facilitate the use of DCs in cellular immunotherapy.

Canine transmissible venereal tumor cell depletion of B lymphocytes: molecule(s) specifically toxic for B cells.

Canine transmissible venereal tumor (CTVT) is an excellent model for investigating the interaction between host immunity and tumor growth. Although CTVT is an allograft, initially the host immune system is unable to destroy the tumor cells, and the tumor grows progressively for about 4-6 months (P phase). After a short stable phase, the tumor undergoes regression (R phase). In this study, CTVT inoculation significantly reduced the proportion of B lymphocytes among all peripheral blood lymphocytes (PBL), but the proportion of B lymphocytes returned to normal after complete removal of CTVT. Following CTVT inoculation, immunoglobulin concentrations decreased gradually, coincident with B lymphocyte decline. Furthermore, CTVT secreted a soluble, heat- and protease K-sensitive cytotoxic molecule(s) that destroyed peripheral blood B lymphocytes (PBBL) but spared other types of immune cells regardless of whether mitogens, such as IL-2 or Con A, were present. The decrease in the proportion and viability of PBBL was caused by a cytotoxic molecule(s) that induced apoptosis. The molecular weight of the CTVT-derived cytotoxic molecule(s) was 30-100kDa. Human, domestic cat, horse and mouse B cells were also sensitive to the substance.

Effect of tumor infiltrating lymphocytes on the expression of MHC molecules in canine transmissible venereal tumor cells.

Canine transmissible venereal tumor (CTVT) can be allo-transplanted across major histocompatibility complex barriers. The expression of MHC molecules is usually low in the progression (P) stage and then greatly increases during tumor regression (R). We investigated the effects of tumor infiltrating lymphocytes (TIL) on the expression of MHC molecules of CTVT cells. Isolated, viable CTVT cells were inoculated at each of 12 sites (1 x 10(8) CTVT cells per site) on the back of six, mixed-breed dogs. Tumor masses were collected every 2-3 weeks and prepared for histopathologic, immunocytochemistry, flow cytometry and immunoblotting studies. The level of MHC expression on tumor cells from different stages of growth was measured. Initially, expression of MHC I and II molecules in P phase CTVT was low. Twelve weeks post-inoculation (PI), expression increased dramatically and it continued to increase during R phase. Tumor growth slowed after 12 weeks PI and tumors entered R phase around 17 weeks PI. We hypothesize that CTVT evades host immunosurveillance and grows progressively for 12 weeks, when it becomes vulnerable and subject to the host's anti-tumor immune responses. We further demonstrated that R phase, but not P phase, TIL were closely associated with the over-expression of MHC I and II molecules by CTVT cells. The number and proportion of TIL were higher in R phase tumors. Supernatants, from R phase co-cultures (CTVT+TIL) and TIL only, promoted MHC I and II expression on P phase CTVT cells. After culturing alone for 1 month, expression of MHC classes I and II molecules in R phase CTVT cells decreased to the level of P phase CTVT cells. However, the above-mentioned supernatants restored their expression of MHC I and II molecules. In contrast, supernatants from P phase TIL or CTVT cells increased expression slightly or had no effect. Therefore, TIL, not CTVT cells, produce the effective substance (s) to promote the expression of MHC molecules by the tumor cells. Heat treated supernatant was unable to promote the expression of MHC I and II molecules by CTVT cells. In conclusion, TIL isolated from R phase CTVT secreted a heat-sensitive, soluble substance(s) that triggered over-expression of MHC I and II after 12 weeks PI. This caused the tumor to enter R phase and helped stop CTVT growth. Our findings will facilitate the understanding and further investigation of the mechanisms that initiate host immune surveillance against tumors.

Identification of canine transmissible venereal tumor cells using in situ polymerase chain reaction and the stable sequence of the long interspersed nuclear element.

Canine transmissible venereal tumor (CTVT) is a unique tumor that can be transplanted across the major histocompatibility complex (MHC) barrier by viable tumor cells. In dogs, CTVT grows progressively for a few months and then usually regresses spontaneously. A long interspersed nuclear element (LINE) insertion is found specifically and constantly in the 5' end of the CTVT cell c-myc gene, outside the first exon. The rearranged LINE-c-myc gene sequence has been used with polymerase chain reaction (PCR) to diagnose CTVT. However, in CTVT cells, the total length of the inserted LINE gene is not constant. In this experiment, variation in the inserted LINE gene was studied to determine which parts of the LINE sequence can be used as primers to identify CTVT cells with in situ PCR (IS PCR). The LINE gene was inserted between the TATA boxes in the promoter region of c-myc. In CTVT cells, deletions of different lengths are frequent in this gene. However, the 550-bp segment at the 5' end of the LINE-c-myc gene was stable. Thus, primers were designed to cover the stable 0.55-kb segment from the 5' end outside the first exon of the c-myc gene to the 5' end of LINE gene stable segment. With these primers and IS PCR, individual CTVT cells in formalin-fixed tissue sections and CTVT cultures were identified. Cells from other canine tumors were negative for this gene. In addition, the CTVT-specific, 0.55-kb segment was not found in any spindle-shaped cells from progressive or regressive phase CTVT. The IS PCR technique also did not detect any positive spindle-shaped cells in CTVT cell cultures. Thus, fibroblastic terminal differentiation is less likely to be a mechanism for spontaneous regression of CTVT cells.

Echocardiographic features of pigs with spontaneous hypertrophic cardiomyopathy.

Ultrasonography is one of the most common, noninvasive techniques used for cardiovascular diagnosis because it provides reliable information and enhances patient safety. Two-dimensional (2-D) and M-mode echocardiography is conducted to assess the severity and distribution of myocardial hypertrophy. Hypertrophic cardiomyopathy (HCM) is a primary myocardial disease that has variable manifestations because interactions between the many facets of systolic and diastolic dysfunction of the heart are complex. The objective of the study reported here was to characterized clinical HCM in pigs. A commercial Vingmed (CFM-800) 3.25 MHz transducer was used to perform 2-D and M-mode echocardiography. Experimental pigs (about 100 kg in body weight) were anesthetized and positioned in left lateral recumbency. Echocardiographic images (2-D) were acquired in parasternal short-axis and long-axis views. The 2-D images provided M-mode under direct anatomic visualization. The pigs were sacrificed for pathologic study after echocardiographic examination. In typical HCM cases (n = 8), the interventricular septum thickness increased, the left ventricular (LV) end-systolic and end-diastolic dimensions decreased, and the left atrial dimensions and the indexes of systolic function, such as ejection fraction and velocity of fiber shortening, increased. The LV outflow tract narrowed, particularly when gross upper septal hypertrophy was evident. Moreover, systolic cranial motion (SCM) of the septal leaflet of the mitral valve was observed. Doppler evidence of mitral regurgitation often was associated with SCM. The echocardiographic findings from pigs with HCM resembled those from humans. Thus, porcine HCM may serve as a spontaneous animal model for the study of HCM in humans.

Serum acute phase proteins and swine health status.

The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%). On farm A, the percentage of pigs with 1 or more lesions was 88.2%. For culled pigs from farm A, the mean serum concentrations of HP and CRP were 2.23 +/- 0.14 mg/mL and 252.93 +/- 11.62 microg/mL, which were significantly higher than concentrations in clinically normal pigs (1.42 +/- 0.02 mg/mL and 84.88 +/- 2.61 microg/mL, respectively, P < 0.01). Moreover, among clinically normal farm A pigs, the mean HP concentration in pigs with lesions (1.43 +/- 0.02 mg/mL) was significantly higher than in pigs without lesions (1.32 +/- 0.07 mg/mL) (P < 0.05). However, the mean serum CRP concentrations in these animals were not significantly different. On farm B, the percentage of pigs with one or more lesions was 50.0%. Interestingly, the mean serum HP concentration in clinically normal pigs with lesions was significantly lower in farm B pigs (1.23 +/- 0.07 mg/mL) than in the farm A pigs (1.43 +/- 0.02 mg/mL; P < 0.01). However, serum CRP concentrations in farm A and B pigs were not significantly different. Serum HP concentration, which is a better indicator of inflammatory reactions in pig herds than serum CRP concentration, provides an important marker for swine health status.

Serum C-reactive protein in dairy herds.

The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd.

Serum haptoglobin concentration in cattle.

To obtain a basal concentration of serum Haptoglobin (Hp) in cattle in Taiwan, Hp concentrations were measured from serum samples collected from 10 healthy heifers, every week for one year. The values were also compared with those collected from 15 cows diagnosed with postpartum metritis. The heifers were successfully impregnated by artificial insemination six months after the tests. Hp concentrations were also measured in the serum collected from 11 other cows within 3 weeks after parturition. The Hp assay developed in this study gave a good correlation (r=0.893)with Western blotting. The Hp concentration of 454 serum samples from the 10 heifers had a mean value of 83.6 +/- 34.1 mg/l, and there was no significant difference among individual heifers. The basal value of Hp in heifers was calculated as less than 73.6 mg/l. No significant difference in Hp concentration was observed among the 10 heifers during cold and warm seasons (19.8 +/- 2.2 degrees C vs 27.3 +/- 1.4 degrees C), or before and after pregnancy. The mean serum Hp concentration from cows suffering from postpartum reproductive disorders was 1133.5 +/- 627.1 mg/l, which was significantly greater than the serum of healthy heifers and postpartum cows (104.6 +/- 61.0 mg/l) (P<0.05). These results demonstrate that Hp concentration may be a useful indicator for cows with postpartum reproductive disorders.

Gene-expression profiling to identify genes related to spontaneous tumor regression in a canine cancer model.

Microarray transcriptome study in cancer has been commonly used to investigate tumorigenic mechanisms. The unique growth pattern of spontaneous regression (SR) after progressive (P) growth in canine transmissible venereal tumor (CTVT) provides a valuable cancer model to study the genome-wide differences in samples between the two stages of growth. In this study, Affymetrix analysis was performed based on the canine genome to compare the gene expression profiles of CTVT P- and SR-phase tumors. A total of 459 (278 up-regulated and 181 down-regulated) genes were identified as being differentially-expressed during the SR phase by the 2-fold method. Further analysis of these genes revealed that the expression of three genes associated with IL-6 production -TIMD-4, GPNMB and PLTP - was significantly higher in SR-phase tumors than in P-phase tumors; these results were also confirmed by real time RT-PCR in tumor tissues of beagles. In addition, we found that Th17-related genes were over-expressed in the SR phase, suggesting autoimmune responses involvement in tumor regression. Although the interaction between CTVT and host immunity were partially investigated in previous studies, our results enable us to gain new insight into the genes and possible mechanisms involved in tumor regression and reveal potentially useful targets for cancer therapy.

Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid.

Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity.

Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model.

Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.

Canine CXCL7 and its functional expression in dendritic cells undergoing maturation.

Many cells, including leucocytes and stromal cells, express CXCL7, a member of the CXC chemokine family, also known as platelet basic protein. CXCL7 is a potent chemoattractant and activator of neutrophil function. Dendritic cells (DCs) play a pivotal role in antigen processing and presentation. Very little information is available on the ability of DCs to recruit neutrophils by producing chemokines. In this work, we have cloned canine CXCL7. Based on the predicted gene sequence and using the 3'RACE technique, the full-length gene was amplified from LPS-treated canine peripheral blood mononuclear cells. The cloned cDNA sequence consisted of 357 nucleotides and encoded a 118 amino acid protein, including a 38 amino acid signal peptide. The use of CXCL7-containing supernatants from CXCL7-transfected BALB/3T3 in the neutrophil migration assay confirmed that canine CXCL7 had chemoattractive activity for neutrophils. We then used canine monocyte-derived DCs to generate CXCL7 for the rest of the experiment. Expression of CXCL7 by DCs treated with LPS, IL-1beta, IL-6, TGF-beta, TNF-alpha, or IFN-gamma was compared using real-time RT-PCR and Western blotting. When treated with IL-1beta, IL-6, TNF-alpha, or TGF-beta, canine DCs expressed significantly higher levels of CXCL7 mRNA and protein than when treated with IFN-gamma or LPS. It is concluded that dog DCs express high levels of the neutrophil chemotactic factor CXCL7 when stimulated by proinflammatory cytokines, including IL-1beta, IL-6, TNF-alpha, or TGF-beta, and may play an important role in modulating inflammatory responses.