Administration of DNA Encoding the Interleukin-27 Gene Augments Antitumour Responses through Non-adaptive Immunity.

DNA-mediated immunization of a tumour antigen is a possible immunotherapy for cancer, and interleukin (IL)-27 has diverse functions in adaptive immunity. In this study, we examined whether IL-27 DNA administration enhanced antitumour effects in mice vaccinated with DNA encoding a putative tumour antigen, ß-galactosidase (ß-gal). An intramuscular injection of cardiotoxin before DNA administration facilitated the exogenous gene expression. In mice received ß-gal and IL-27 DNA, growth of ß-gal-positive P815 tumours was retarded and survival of the mice was prolonged. Development of ß-gal-positive Colon 26 tumours was suppressed by vaccination of ß-gal DNA and further inhibited by additional IL-27 DNA administration or IL-12 family cytokines. Nevertheless, a population of ß-gal-specific CD8(+) T cells did not increase, and production of anti-ß-gal antibody was not enhanced by IL-27 DNA administration. Spleen cells from mice bearing IL-27-expressing Colon 26 tumours showed greater YAC-1-targeted cytotoxicity although CD3(-)/DX5(+) natural killer (NK) cell numbers remained unchanged. Recombinant IL-27 enhanced YAC-1-targeted cytotoxicity of IL-2-primed splenic NK cells and augmented a phosphorylation of signal transducer and activator of transcription 3 and an expression of perforin. These data collectively indicate that IL-27 DNA administration activates NK cells and augments vaccination effects of DNA encoding a tumour antigen through non-adaptive immune responses.

Marked depletion of the water-channel protein, AQP5, in the canine nictitating membrane glands might contribute to the development of KCS.

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Adenoviruses-mediated transduction of human oesophageal carcinoma cells with the interferon-λ genes produced anti-tumour effects.

BACKGROUND: Interferon-λs (IFN-λs) are novel cytokines with multiple functions, like IFN-α and -ß. We examined possible anti-tumour effects produced by adenoviruses bearing the IFN-λ1 or -λ2 gene (Ad/IFN-λ) with the type-35 fibre-knob structure. METHODS: Proliferation of oesophageal carcinoma cells transduced with Ad/IFN-λ and mechanisms of the inhibited growth were investigated. RESULTS: Transduction with Ad/IFN-λ upregulated the expression of the class I antigens of the major histocompatibility complexes and induced the growth suppression. Increased sub-G1 populations and the cleavage of caspase-3 and poly (ADP-ribose) polymerase were detected in IFN-λ-sensitive YES-2 and T.Tn cells. The cell death was accompanied by cytoplasmic cytochrome C and increased cleaved caspase-9 and Bax expression, suggesting mitochondria-mediated apoptosis. Adenovirus/IFN-λ-infected YES-2 cells subsequently reduced the tumourigenicity. Adenovirus/IFN-λ-infected fibroblasts, negative for the IFN-λ receptors, induced death of YES-2 or T.Tn cells that were co-cultured. Inoculation of YES-2 cells in nude mice, when mixed with the Ad/IFN-λ-infected fibroblasts, resulted in retardation of the tumour growth. The growth suppression was not linked with upregulated CD69 expression on natural killer cells or increased numbers of CD31-positive cells. CONCLUSION: Adenovirus/IFN-λ induced apoptosis, and fibroblast-mediated delivery of IFN-λs is a potential cancer treatment by inducing direct cell death of the target carcinoma.

Purification and partial primary sequence of a chemotactic protein for polymorphonuclear leukocytes derived from human lung giant cell carcinoma LU65C cells.

A chemotactic protein for polymorphonuclear leukocytes (lung carcinoma-derived chemotaxin [LUCT]) was purified from culture fluid of the human lung giant cell carcinoma LU65C cells to electrophoretically homogeneous form through five sequential purification steps: DEAE-Sepharose, CM-Sepharose, HPLC on carboxyl-methylated-polyvinylalcohol resin, hydrophobic, and reversed-phase. The molecular mass was determined as approximately 10 kD by SDS-PAGE and isoelectric point was 10.7. The chemotactic activity (ED50 0.75 x 10(-9) M) was sevenfold more potent than that of FMLP (5 X 10(-9) M) and comparable with that of C5a (10(-9) M). NH2-terminal amino acid sequence and amino acid composition of LUCT strongly suggest that it may be closely related to the putative protein encoded by the cDNA clone (3-10C) and almost identical with a part of sequence of the chemotactic factor derived from stimulated human leukocytes in the 6th to 32nd, but not the NH2-terminal 5 amino acids. These results indicate that the carcinoma cells produce LUCT without any added stimulant and suggest that the previously isolated chemotactic monokines may correspond to des(1-5) of LUCT in the NH2-terminal region.

Dose-dependent effect of romifidine on intraocular pressure in clinically healthy buffalo (Bubalus bubalis).

In the present study, changes in intraocular pressure (IOP) associated with romifidine sedation in buffalo were evaluated. Eighteen healthy adult, non-pregnant, buffalo without ocular abnormalities were used in a prospective randomized trial. Buffalo were allocated into three groups (six each). Buffalo in the treated groups received an intramuscular injection (IM) of romifidine at 40 or 50 µg/kg. The control group was administrated an equivalent volume of sterile saline (0.9% NaCl; 0.4 ml/100 kg). Baseline IOP (T0) values were obtained using applanation tonometry. Immediately afterwards, romifidine was administered and IOP values of both eyes were measured at 5, 15, 30, 45, 60, 90, 120, and 180 min post-administration. The pre-administration values (T0) of IOP for both the left and right eyes ranged from 30-36 (mean, 33 ± 1.5) mmHg and 30-35 (mean, 33.7 ± 1.4), respectively. IOP values decreased significantly after administration of both doses of romifidine compared with the placebo (P < 0.01). Compared with the control, the IOP decreased significantly in animals treated with both doses from 5-90 min post-administration in both eyes (P < 0.05). In the right eye, the lowest IOP value in the romifidine treated groups was observed at T30 (21.6 ± 1.0 and 23.3 ± 1.4 mmHg), respectively. In the left eye, the lowest IOP was observed at T60 (22.5 ± 3.0 and 23.3 ± 2.8 mmHg), respectively. In conclusion, romifidine could be recommended as an alternative analgesic in buffalo, especially for ocular affections associated with increased IOP. A dose of 40 µg/kg could be used at a low cost.

IL-27-mediated activation of natural killer cells and inflammation produced antitumour effects for human oesophageal carcinoma cells.

Interleukin IL-27, composed of p28 and EBV-induced gene 3 subunits, has diverse functions in enhancing cell-mediated immunity and silencing the immunity. We examined whether forced expression of the p28-linked EBI3 gene in human oesophageal carcinoma cells (Eca109) produced antitumour effects in a T cell-defective condition. Tumour growth of Eca109 cells expressing IL-27 (Eca109/IL-27) was retarded in nude mice compared with parental and vector DNA-transduced tumours and survival of the mice inoculated with Eca109/IL-27 cells was prolonged. Expression of the tumour necrosis factor-alpha, IL-1beta and IL-6 genes was up-regulated in Eca109/IL-27 tumour specimens while the tumours remained small in size but the increased transcription was subsequently down-regulated in enlarged tumours. Spleen cells from mice-bearing Eca109/IL-27 tumours produced interferon-gamma and showed YAC-1-targeted cytotoxic activities greater than those of mice inoculated with parental or vector DNA-transducer tumours. Numbers of DX5+CD69+ natural killer cells in spleen of mice-bearing Eca109/IL-27 tumours and those of CD31+ cells within Eca109/IL-27 tumours remained the same as found in mice-bearing parental or vector DNA-transduced tumours. These data collectively suggest that the IL-27-mediated antitumour effects produced in a mature T cell-defective condition were attributable to enhanced interferon-gamma production and natural killer activities.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

The effects of FasL on inflammation and tumor survival are dependent on its expression levels.

The apoptosis-inducing Fas ligand (FasL) is expressed in a variety of human cancers and has been implicated in tumor immune evasion. Paradoxically, ectopic expression of FasL in experimental tumors triggers a neutrophil-mediated inflammatory response and tumor rejection. To resolve these conflicting findings, we have established B16 melanoma and P29 Lewis lung carcinoma lines expressing different levels of FasL and examined their tumorigenicity in vivo. While tumors with a high level of FasL were rapidly rejected as previously reported, those expressing a low level of FasL were not rejected but grew faster than did FasL-negative parental cells. The growth enhancement of FasL(low) tumors was not observed in T-cell-deficient nude mice, suggesting that FasL expressed in tumors at low levels counteracted against T-cell-dependent antitumor responses. In support of this notion, FasL(low) tumors were found to grow faster than parental cells in mice that had acquired tumor-specific immunity. Furthermore, histological examinations revealed apoptosis of lymphocytes in tissue sections of FasL(low) tumors. These results collectively suggest that FasL on tumors is a double-edged sword: at high levels it triggers tumor rejection whereas at low levels it facilitates tumor growth possibly by suppressing antitumor immune responses.

Lateralization index but not contralateral suppression at adrenal vein sampling predicts improvement in blood pressure after adrenalectomy for primary aldosteronism.

Adrenal vein sampling (AVS) is essential in differentiating unilateral from bilateral sources of aldosterone excess in primary aldosteronism (PA). However, its ability to predict blood pressure (BP) improvement after adrenalectomy has not been well studied. This is a retrospective observational study of 119 patients who underwent AVS by sequential technique followed by adrenalectomy for PA at the Hospital of the University of Pennsylvania from 1997 to 2015. Median age was 52 years (interquartile range 44-59), 67% were male and median duration of hypertension was 10 (interquartile range 6-20) years. A total of 76% and 90% of patients experienced BP improvement at 0-6 months or at any time point after surgery, respectively. Lateralization index (LI) >8, but not the presence of contralateral suppression, was significantly associated with BP improvement after surgery by multivariate logistic regression analysis adjusted for potential confounders (odds ratio (95% confidence interval): 17.1 (1.7-171.6) and 6.39 (0.06-641.8), respectively). A prediction score was created by covariates that was significantly associated with BP improvement in logistic regression analysis (duration of hypertension, body mass index, preoperative systolic BP and number of antihypertensive medications). Receiver-operating characteristic curve analyses showed that the addition of LI >8 to the score increased its ability to predict BP improvement (area under the curve 0.73-0.80). In conclusion, LI is useful in predicting improvement in BP after adrenalectomy for PA. The results of this study suggest that patients with long-standing severe hypertension may still benefit from surgery if LI >8.

Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.

Induction of immunity in peripheral tissues combined with intracerebral transplantation of interleukin-2-producing cells eliminates established brain tumors.

Cytokine gene therapy for the induction of potent immune responses against central nervous system tumors has proven to have significant potential. However, this strategy needs improvement in the process of antigen presentation and/or insufficient recruitment of immunocompetent cells to achieve successful eradication of established brain tumors. We investigated the therapeutic potential of induced systemic immunity in peripheral tissues combined with interleukin-2 (IL-2) production in the vicinity of brain tumors to treat established brain tumors. Sequential magnetic resonance image monitoring showed that the combinatory therapy consisting of intracerebral (i.c.) transplantation of IL-2-producing rat gliosarcoma 9L (9L/IL-2) cells and s.c. vaccination using irradiated 9L or 9L/IL-2 cells could cure 9L-bearing rats, whereas either the i.c. injection of 9L/IL-2 cells or the s.c. vaccination produced little or marginal antitumor effects, respectively. Xenogeneic murine neuroblastoma cells secreting IL-2 could substitute for 9L/IL-2 cells, producing significant antitumor effects in the vaccinated rats. Tumor-specific cytotoxic activity was induced in the vaccinated rats but not fully in the rats treated only with i.c. injection of 9L/IL-2 cells. Immunohistochemical analysis revealed that a number of CD4(+) and CD8(+) T cells infiltrated into the brain tumors which were treated with the combinatory therapy. The level of cell infiltration was similar to that found in s.c. 9L/IL-2 tumors which were subsequently rejected. In contrast, the brain tumors treated with either i.c. transplantation of 9L/IL-2 cells or the s.c. vaccination showed only moderate infiltration of T cells. The combinatory strategy, i.c. grafting of IL-2-producing cells, and s.c. immunization of irradiated whole tumor cell vaccine, is, thus, effective for recruiting activated T cells into the brain tumor site and could be a potential therapy for brain tumors.

Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity.

To define the role of neuropeptides in lung cancer biology, we evaluated the effect of seven peptide classes on signal transduction and growth in human lung and breast cancer cell lines. Flow cytometric methods were used to quantitate the calcium response in individual cells produced by these peptides alone or in combination. The effects on growth were assessed by [3H]thymidine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agarose colony assays. All lung cancer cells demonstrated calcium responses to one or more peptides with classic small cell lines displaying the greatest responsiveness, followed by variant small cell lines and non-small cell lines. Breast cancer cell lines demonstrated little or no response. There was great variability in the magnitude of calcium response and pattern of response between lung cancer cell lines to individual neuropeptides. Bradykinin was the most potent peptide and produced responses in the highest fraction of lung cancer cell lines. Combinations of peptides produced greater intracellular calcium release than the single peptides, although in less than a quantitatively additive manner. Each peptide produced a refractory period which was peptide class specific. The growth stimulating effects of these neuropeptides were absent or small in magnitude and did not correlate with calcium signal transduction. These results imply that lung cancer cells display a wide sensitivity to neuropeptides but in a very heterogeneous manner. Knowledge of this heterogeneity should be incorporated into the design of antitumor strategies based on this autocrine pathway.

DNA polymerase kappa, implicated in spontaneous and DNA damage-induced mutagenesis, is overexpressed in lung cancer.

DNA polymerase kappa (Pol kappa) is a newly identified low-fidelity polymerase implicated in spontaneous and DNA damage-induced mutagenesis. As an initial study to investigate its possible involvement in tumorigenesis, we compared the expression level of Pol kappa in tumors and adjacent nontumorous tissues by Northern blot, semiquantitative RT-PCR, and Western blot analyses. In this study, paired tumor and normal specimens from 29 patients with stages I to IIIb non-small cell lung cancer (NSCLC), including 13 adenocarcinomas, 15 squamous cell cancers, and 1 adenosquamous carcinoma, were analyzed, among which different levels of tumor-associated Pol kappa overexpression were observed in 21 of 29 matched specimens. In addition, five matched specimens exhibited elevated Pol kappa expression in both tumor and control tissues, whereas only one nontumorous tissue expressed a higher level of Pol kappa than its tumor counterpart. The preferential up-regulation of Pol kappa expression in tumors was highly significant (P < 0.001). There was no apparent correlation of Pol kappa expression levels with tumor histology, grade, and stage or with smoking history. Southern blot analysis did not show amplification of the Pol kappa gene, indicating that the elevated Pol kappa expression is likely attributable to dysregulated transcription. Our data suggest that Pol kappa may contribute to lung tumor development by accelerating the accumulation of mutations.

Effects of neuropeptide analogues on calcium flux and proliferation in lung cancer cell lines.

Small cell lung cancers (SCLC) and some non-small cell lung cancers (NSCLC) have neuroendocrine features which include production of a variety of neuropeptides, cell surface expression of the receptors for these peptides, and autocrine stimulation by the peptides. Previous studies showed that some peptide antagonists and anti-peptide antibodies inhibited the growth of SCLC cell lines which expressed receptors for the specific peptide. We and others showed that the heterogeneity of peptide receptor expression and responsiveness was a major potential obstacle for developing therapeutic uses of peptide antagonists. In this manuscript we evaluated the effects of 11 peptide antagonists (3 bombesin-specific, 2 cholecystokinin-specific, 1 arginine vasopressin (AVP)-specific, and 5 substance P derivatives with broad specificity) on peptide-induced calcium mobilization and growth of SCLC and NSCLC cell lines. For each antagonist, we determined the dose-response effects, specificity of peptide antagonism, and biological stability in serum using Indo-1AM-based flow cytometric assays. We found that the three bombesin antagonists, S30, SC196, and L336,175, varied in potency from 10 nM to 10 microM, varied in serum stability from 6 h to more than 24 h, and had no effect on the calcium response elicited by other peptides. None of these compounds effectively inhibited the growth of SCLC cell lines in [3H]dThd and cell growth assays in vitro. Similarly, the three cholecystokinin and AVP antagonists were highly specific for cholecystokinin and AVP, respectively, had widely varying potency, but had little inhibitory effect on SCLC growth in vitro. In contrast, the five substance P derivatives inhibited the calcium response to bombesin, AVP, bradykinin, and fetal bovine serum. None of these five antagonists were as potent as the six specific antagonists described above, but they were more effective in inhibiting the growth of SCLC cell lines in vitro. These substance P derivatives inhibited the growth of peptide-sensitive SCLC cell lines more efficiently than their inhibition of peptide-insensitive NSCLC or breast cancer cell lines. Relatively high concentrations of these substance P derivatives were required to inhibit in vitro growth, even in the absence of added peptide. It is likely that more potent broad spectrum antagonists, toxins, or radiolabeled stable antagonists will need to be developed for maximal clinical development of this type of anti-growth factor therapy.

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Reactivity of mouse monoclonal antibody M2590 against B16 melanoma cells with chemically synthesized GM3 ganglioside.

A mouse (C57BL/6) monoclonal antibody M2590, which is established against syngeneic melanoma B16 cells, reacted with chemically synthesized GM3. NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1' ceramide (24:0/d 18:1), but not with its stereoisomer, NeuAc beta 2-3Gal beta 1-4Glc beta 1-1' ceramide (24:0/d 18:1).

Endovascular treatment for elderly patients with ruptured aneurysm.

We report our results of endovascular treatment for elderly patients with ruptured aneurysm and discuss the indication for treatment. One hundred and thirty four consecutive patients with ruptured aneurysm treated in our institute during the last 4 years were retrospectively evaluated. Fifty eight patients were included in group A (over 70 years old), and 76 patients in group B (under 69 years old). In both groups, the outcome was strongly related to the preoperative Hunt & Kosnik grade. However, significant risk factors (i.e. pneumonia, rupture of extracranial aneurysm) which make prognosis poor were more common in group A. Group A showed poor outcome in grade III patients, although there were no outcome differences between the two groups in patients of other grades. Endovascular treatment for elderly patients with ruptured aneurysms seemed to be useful. Their outcome was strongly related to their preoperative condition. General risk factors should be evaluated before treatment, especially in elderly patients. Patients with low Hunt & Kosnik grade seem to be most suitable for endovascular treatment. On the other hand, outcome of patients with poor preoperative grade was worse despite the less invasive nature of endovascular treatment. An improvement of outcome in grade III patients is desirable.

Histological and immunohistological analysis of degenerative changes in the cranial cruciate ligament in a canine model of excessive tibial plateau angle.

OBJECTIVE: To create a canine model of excessive tibial plateau angle (eTPA) and assess the chondroid metaplasia and extracellular matrix alteration in the cranial cruciate ligament. METHODS: Seven mature female Beagles were included. Cylindrical osteotomy was performed bilaterally in the proximal tibia. The TPA was increased to approximately 40° in the left tibia (eTPA stifle) and left unchanged in the right tibia (control stifle). Exercise stress was started at three months postoperatively, and at 12 months postoperatively the dogs were euthanatized and the cranial cruciate ligaments were collected. The specimens were subjected to haematoxylin and eosin staining to assess the ligamentocyte morphology and immunostaining to assess the type I (COLI), type II (COLII), and type III (COLIII) collagen, and the sry-type HMG box 9 (SOX9) staining. RESULTS: Macroscopic cranial cruciate ligament injury was absent in six dogs but present in the eTPA stifle of one dog, which was excluded from the analysis. The ligamentocyte density decreased and the percentage of round ligamentocytes increased in the eTPA stifles. The COLII, COLIII, and SOX9 staining increased significantly and COLI deposition decreased in the eTPA stifles compared to the control stifle. CLINICAL SIGNIFICANCE: The extracellular matrix changed, COLI deposition decreased, and COLIII and SOX9 staining increased in the cranial cruciate ligament of the eTPA stifles. SOX9 may contribute to COLII synthesis in the extracellular matrix of the cranial cruciate ligament in eTPA stifles, and eTPA may promote chondroid metaplasia and extracellular matrix alteration.

Expression of p53 synergistically augments caspases-mediated apoptosis induced by replication-competent adenoviruses in pancreatic carcinoma cells.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Recombinant human basic fibroblast growth factor accelerates fracture healing by enhancing callus remodeling in experimental dog tibial fracture.

Effect of recombinant human basic fibroblast growth factor (bFGF) on fracture healing was investigated using a tibial fracture in beagle dogs. Transverse fractures in the middle of the diaphyses were created in the right tibiae and bFGF was injected into the fracture sites at a single dose of 200 micrograms. The time course of changes in callus volume and morphology of the fracture sites were evaluated at weeks 2, 4, 8, 16, and 32 after treatment, and the fracture strength was analyzed at weeks 16 and 32. At week 2, a radiogram of the fracture site showed obvious membranous ossification in the group injected with bFGF. Basic FGF extended the callus area at week 4 and increased the bone mineral content (BMC) in the callus at week 8. bFGF also increased the osteoclast number in the periosteal callus at weeks 2 and 4. In the bFGF group, a maximal increase in the osteoclast index was found at week 4, and an identical increase was recognized in the control group at weeks 8 and 16. These findings strongly suggested that bFGF stimulated not only callus formation but osteoclastic callus resorption. BMC in the bFGF group was followed by a rapid decrease from week 8, while that in the control group was identical from week 4. Fracture strength of the bFGF group showed significant recovery by week 16, and recovery was still evident by week 32. We conclude that bFGF promotes the fracture healing in dogs by the stimulation of bone remodeling.