Correlation between orthopaedic and radiographic examination findings and arthroscopic ligament fibre damage in dogs with cruciate ligament rupture.

OBJECTIVE: The objective is to study the correlations between physical examination and stifle radiography findings and severity of arthroscopic cranial cruciate ligament (CrCL) fibre damage in dogs with cruciate rupture (CR). DESIGN: Design Prospective clinical study. METHODS: Twenty-nine client-owned dogs with CR underwent physical examination, stifle radiography and arthroscopy, and the findings were recorded. Initial examination was repeated after sedation and after general anaesthesia. The Spearman rank correlations of examination variables with diagnostic imaging were examined. RESULTS: Overall, cranial tibial translation assessed by the tibial compression test in extension showed correlation with arthroscopic CrCL fibre damage (P < 0.05). Correlations between severity of cranial drawer laxity and arthroscopic CrCL fibre damage were not significant. Under general anaesthesia, stifle laxity tests were positively correlated with lameness severity grade (SR ≥ 0.41, P < 0.05). Meniscal damage was correlated with pain on the internal rotation of the tibia (SR = 0.42, P < 0.05) and severity of radiographic osteophytosis (SR = 0.53, P = 0.01). CONCLUSION: Detection and estimation of severity of cranial tibial translation enable the diagnosis of CR and also the inference of the severity of CrCL fibre rupture, particularly with the tibial compression test in extension. Severity of joint laxity is best assessed under general anaesthesia. Such knowledge should reduce the risk of misdiagnosis and may enhance early diagnosis and treatment of dogs with CR over time.

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation.

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

Salubrious effects of dexrazoxane against teniposide-induced DNA damage and programmed cell death in murine marrow cells.

The intention of the present study was to answer the question whether the catalytic topoisomerase-II inhibitor, dexrazoxane, can be used as a modulator of teniposide-induced DNA damage and programmed cell death (apoptosis) in the bone marrow cells in vivo. The alkaline single cell gel electrophoresis, scoring of chromosomal aberrations, micronuclei and mitotic activity were undertaken in the current study as markers of DNA damage. Apoptosis was analysed by the occurrence of a hypodiploid DNA peak and caspase-3 activity. Oxidative stress marker such as intracellular reactive oxygen species production, lipid peroxidation, reduced and oxidised glutathione were assessed in bone marrow as a possible mechanism underlying this amelioration. Dexrazoxane was neither genotoxic nor apoptogenic in mice at the tested dose. Moreover, for the first time, it has been shown that dexrazoxane affords significant protection against teniposide-induced DNA damage and apoptosis in the bone marrow cells in vivo and effectively suppresses the apoptotic signalling triggered by teniposide. Teniposide induced marked biochemical alterations characteristic of oxidative stress including accumulation of intracellular reactive oxygen species, enhanced lipid peroxidation, accumulation of oxidised glutathione and reduction in the reduced glutathione level. Prior administration of dexrazoxane ahead of teniposide challenge ameliorated these biochemical alterations. It is thus concluded that pretreatment with dexrazoxane attenuates teniposide-induced oxidative stress and subsequent DNA damage and apoptosis in bone marrow cells. Based on our data presented, strategies can be developed to decrease the teniposide-induced DNA damage in normal cells using dexrazoxane. Therefore, dexrazoxane can be a good candidate to decrease the deleterious effects of teniposide in the bone marrow cells of cancer patients treated with teniposide.

Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection.

Dendritic cell (DC) expansion is regulated by the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L). DCs are critical to the control of tumor growth and metastasis, and there is a positive correlation between intratumoral DC infiltration and clinical outcome. In this report, we first demonstrate that single intravenous (i.v.) injections of adenovirus (Adv)-Flt3L significantly increased splenic dendritic, B, T and natural killer (NK) cell numbers in both normal and mammary tumor-bearing mice. In contrast, the numbers of DCs and T cells infiltrating the tumors were not increased. Consistent with the minimal effect on immune cell infiltration, i.v. Adv-Flt3L injections had no therapeutic activity against orthotopic mammary tumors. In addition, we noted tumor and Adv-Flt3L expansion of Gr1(+)CD11b(+) immature myeloid suppressor cells (IMSCs), which may inhibit the therapeutic efficacy of Adv-Flt3L-expanded DCs.

Flt3 ligand bioactivity and pharmacology in neoplasia.

Fms-like tyrosine kinase 3 ligand (Flt3L) has multiple effects on the hematopoietic and immune systems. Further, preclinical studies have suggested potential therapeutic activity against cancer. Flt3L is a potent hematopoietic cytokine, capable of stimulating the expansion and differentiation of hematopoietic progenitor and stem cells. Administration of Flt3L mobilizes hematopoietic cells from the bone marrow (BM) into the blood, lymphoid organs, and parenchymal tissues. This mobilization activity, especially effective in combination with granulocyte colony stimulating factor (G-CSF), has stimulated studies of Flt3L in hematopoietic stem cell (HSC) transplantation. In addition to its effects on hematopoietic stem and progenitor cells, Flt3L has been shown to increase the frequency and number of dendritic cells (DCs) within the circulatory system and solid organs. DC expansion by Flt3L has been the focus of preclinical and clinical studies on antigen (Ag) specific T-cell mediated immunity. The mechanism for the augmentation of T-cell mediated immunity has yet to be completely identified, although Flt3L's ability to expand DCs in lymphoid and non-lymphoid tissues is involved. This expansion occurs primarily with DCs, which secrete interleukin (IL) 12. Consistent with the expansion of this DC population, treatment with Flt3L enhances T-cell mitogenesis and preferentially induces type 1 T-cell responses. However, the DCs resulting from Flt3L administration are immature, leading in some studies to the induction of tolerance. This review focuses on the effects of Flt3L on DCs and other effector populations, and on its potential activity as a therapeutic agent for cancer, alone and in combination with vaccines.

Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties.

Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C. Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection. Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII. A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g. proteolysis, phosphorylation, or adenylylation. Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases. Both enzymes are composed of 12 subunits, each approximately 51,000 daltons. However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II). Cd(II) activates EII more than EI, and only EI shows activity with Co(II). For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2. Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases. Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp. EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent. The divalent metal ion that is present can affect both specificity for analogs and substrate Km values. Kinetic binding plots (v versus [S]) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed. ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp. enzymes. For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.

Glutamine synthetase of Bacillus stearothermophilus. Regulation, site interactions, and functional information.

The action of various feedback modifiers on Bacillus stearothermophilus glutamine synthetase has been investigated by initial velocity kinetics, using the Mn2+-stimulated biosynthetic assay at 55 degrees C. The most potent inhibitors, used singly, are AMP, L-glutamine, and L-alanine. Other modifiers of significance include glycine, CTP, L-histidine, glucosamine 6-phosphate, and GDP. Marked synergism of action is observed for AMP in the presence of L-glutamine, L-histidine, ADP, or glucosamine 6-phosphate (glucosamine-6-P), and for CTP with ADP or GDP. Inhibition by saturating levels of many modifiers is either less than 100%, or is not overcome by elevated substrate levels, or both. This argues for modifier binding sites separate from substrate sites, notably in the cases of AMP, L-glutamine, glycine, L-alanine, glucosamine-6-P, and CTP. Glycine and L-alanine are Vmax inhibitors, whereas L-glutamine, glucosamine-6-P, GDP, and CTP alter the binding of L-glutamate. ADP and L-histidine apparently can compete directly with MnATP, but AMP alters Mn-ATP binding from a separate site. The action of several modifiers requires or is enhanced by bound substrates. Considerable antagonistic interaction is observed in experiments with modifier pairs, but the most potent inhibitors show synergistic or cumulative (independent) interactions. One may interpret antagonistic effects as due to (a) overlapping modifier domains, or (b) separate but antagonistically interacting sites. Either interpretation leads to a scheme for modifier-substrate and modifier-modifier site interactions in which the thermophilic enzyme must maintain and stabilize a great deal of complex functional information under extreme environmental conditions.