Angle-stable interlocking nailing in a canine critical-sized femoral defect model for bone regeneration studies: In pursuit of the principle of the 3R's.

Introduction: Critical-sized long bone defects represent a major therapeutic challenge and current treatment strategies are not without complication. Tissue engineering holds much promise for these debilitating injuries; however, these strategies often fail to successfully translate from rodent studies to the clinical setting. The dog represents a strong model for translational orthopedic studies, however such studies should be optimized in pursuit of the Principle of the 3R's of animal research (replace, reduce, refine). The objective of this study was to refine a canine critical-sized femoral defect model using an angle-stable interlocking nail (AS-ILN) and reduce total animal numbers by performing imaging, biomechanics, and histology on the same cohort of dogs. Methods: Six skeletally mature hounds underwent a 4 cm mid-diaphyseal femoral ostectomy followed by stabilization with an AS-ILN. Dogs were assigned to autograft (n = 3) or negative control (n = 3) treatment groups. At 6, 12, and 18 weeks, healing was quantified by ordinal radiographic scoring and quantified CT. After euthanasia, femurs from the autograft group were mechanically evaluated using an established torsional loading protocol. Femurs were subsequently assessed histologically. Results: Surgery was performed without complication and the AS-ILN provided appropriate fixation for the duration of the study. Dogs assigned to the autograft group achieved radiographic union by 12 weeks, whereas the negative control group experienced non-union. At 18 weeks, median bone and soft tissue callus volume were 9,001 mm3 (range: 4,939-10,061) for the autograft group and 3,469 mm3 (range: 3,085-3,854) for the negative control group. Median torsional stiffness for the operated, autograft treatment group was 0.19 Nm/° (range: 0.19-1.67) and torque at failure was 12.0 Nm (range: 1.7-14.0). Histologically, callus formation and associated endochondral ossification were identified in the autograft treatment group, whereas fibrovascular tissue occupied the critical-sized defect in negative controls. Conclusion: In a canine critical-sized defect model, the AS-ILN and described outcome measures allowed refinement and reduction consistent with the Principle of the 3R's of ethical animal research. This model is well-suited for future canine translational bone tissue engineering studies.

Minimally invasive application of a radial plate following placement of an ulnar rod in treating antebrachial fractures. Technique and case series.

OBJECTIVE: To describe a surgical technique for placement of a minimally invasive radial plate following application of an ulnar rod (MIPR) for treatment of antebrachial fractures. METHODS: Medical records (November 2005-June 2009) were searched to identify dogs with diaphyseal radius and ulna fractures stabilised by MIPR. Data retrieved included signalment, weight, limb affected, cause of injury, open versus closed fracture, number of fragments, implant size, number of screws used and cortices engaged, number of open screw holes, operative time, rod removal, complications and time to radiographic healing. To be included, dogs had to have evidence of radiographic healing during follow-up. RESULTS: Eight dogs with diaphyseal radius and ulna fractures treated with MIPR were included in the case series. All fractures were due to trauma and two fractures were open (grade 1). Rod loosening and osteomyelitis of the ulna occurred in one case which subsequently resolved with rod removal. Healing occurred in all cases with no implant failures. Median time to radiographic union was 10.5 weeks (mean ± SD = 17 ± 15 weeks; range 4-52 weeks). CLINICAL RELEVANCE: Use of MIPR constructs on diaphyseal fractures of the radius and ulna is an effective technique for managing these fractures using principles of biological osteosynthesis. An intramedullary rod in the ulna assists with fracture reduction and stabilisation and rod removal is recommended once fracture healing has occurred.

Meniscal injury following initial cranial cruciate ligament stabilization surgery in 26 dogs (29 stifles).

OBJECTIVE: To describe clinical signs, arthroscopic findings, and outcome in a group of dogs undergoing second look arthroscopy for the treatment of meniscal tears following original surgery to correct a CCL deficient stifle joint. METHODS: The medical records of 26 dogs from the Veterinary Teaching Hospital at Texas A&M University and the Veterinary Orthopedic Center (Round Rock, Texas) that had second look arthroscopy for lameness following an original surgical procedure were reviewed. Pre-operative clinical findings, 2nd look arthroscopic findings and owner assessed outcome were documented. RESULTS: Postliminary bucket handle tears of the medial meniscus were detected in 22 (75.9%) cases. Other postliminary meniscal injuries included frayed caudal horn tears of the medial meniscus 6 (20.7%), and longitudinal tears of the lateral meniscus 1 (3.4%). An audible or palpable click was present in 27.6% of cases. An improvement or resolution of lameness was reported in 96.5% of cases reported. In conclusion, tears of the medial meniscus are a significant cause of lameness in dogs subsequent to surgery for cranial crucial ligament ruptures. Increased lameness or acute onset of lameness after surgery for cranial crucial rupture is a consistent finding. In rare cases, a palpable or audible click will be appreciated. Arthroscopic evaluation and partial meniscectomy improve or resolve lameness in the majority of cases. CLINICAL SIGNIFICANCE: Sudden or increased lameness in dogs with historical CCL stabilization surgery should be evaluated and treated arthroscopically for postliminary meniscal injury if another cause for lameness can not be determined.

Soy saponins and the anticancer effects of soybeans and soy-based foods.

While the cancer protective effect of soy-based diets has been the subject of numerous studies, the constituents of soy that may give rise to this effect remain elusive. Recent publications describing anticancer activity of crude and purified soybean saponins have sparked a renewed interest in these compounds. In this review, I summarize the epidemiological studies concerning the cancer protective effects of soy and the efforts to elucidate the constituents responsible for this effect. The recent reports of the anticancer activity of soy saponins is placed in context with reports of promising anticancer activity of structurally related non-dietary saponins from other legumes. While recent studies have demonstrated a direct effect of soy saponins on cancer cells, alternative mechanisms of cancer prevention by these agents are also discussed. It is concluded that the soy saponins may represent promising leads both in terms of elucidating the soy constituents involved in the cancer protective effect of soy as well as in the discovery of anticancer agents with novel mechanisms of action.

Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry.

The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes.

Synthesis, DNA cleavage, and cytotoxicity of a series of bis(propargylic) sulfone crown ethers.

Compounds that couple molecular recognition of specific alkali metal ions with DNA damage may display selective cleavage of DNA under conditions of elevated alkali metal ion levels reported to exist in certain cancer cells. We have prepared a homologous series of compounds in which a DNA reactive moiety, a bis(propargylic) sulfone, is incorporated into an alkali metal ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ethers to bind Li(+), Na(+), and K(+) ions was determined. For the series of crown ethers, the association constants for Li(+) ions are generally low (< 2 x 10(4)M(-1)). Only two of the bis(propargylic) sulfone crown ethers associate with Na(+) or K(+) ions (K(a) 4-8 x 10(4)M(-1)), with little discrimination between Na(+) or K(+) ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of Li(+), Na(+), and K(+) ions was determined. The two crown ethers that bind Na(+) and K(+) display a modest increase in DNA cleavage efficiency in the presence of Na(+) or K(+) ions as compared to Li(+) ions. These two bis(propargylic) sulfone crown ethers are also more cytotoxic against a panel of human cancer cell lines when compared to a non-crown ether macrocyclic bis(propargylic) sulfone.

Draining tracts and nodules in dogs and cats.

Draining tracts and nodules in the dog and cat can present a diagnostic challenge to the veterinarian. A systematic approach and a complete list of differential diagnoses are needed to define the underlying disease, so that appropriate therapeutic management can be instituted and prognosis can be discussed with the owner. The purpose of this article is to review a complete list of differential diagnoses for draining tracts and nodules in the dog and cat, and discuss the appropriate diagnostic steps including cytology, biopsy and histopathology, culture and sensitivity, serology, and diagnostic imaging that are an important part of the work-up for draining tracts and nodules.

Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176.

The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA.

G-Quadruplex DNA as a target for drug design.

Telomeres are structures on the ends of chromosomes that are required for chromosomal stability. Telomeric DNA contains a single-stranded G-rich DNA overhang, which may adopt a G-quadruplex structure. Telomere shortening has been implicated in cellular senescence. Telomerase is an enzyme which synthesizes the G-rich strand of telomere DNA. Telomerase activity is highly correlated with cancer and may allow cancer cells to escape senescence. Based on these observations, telomerase has been proposed as a potential target for anticancer drug design. The targeting of telomerase is associated with potential problems, including the existence in some cancer cells of telomerase-independent mechanisms for telomere maintenance, and the long delay time between telomerase inhibition and effects on proliferation. One promising approach for inhibiting telomerase involves targeting the G-quadruplex DNA structures thought to be involved in telomere and telomerase function. Compounds that specifically bind G-quadruplex DNA may interact directly with telomeres, in addition to inhibiting telomerase, and produce more immediate antiproliferative effects. The diamidoanthraquinones, porphyrins, and perylene diimides have all been shown to bind G-quadruplex DNA and inhibit telomerase. Most of these compounds also bind double-stranded DNA and are cytotoxic at the concentrations required to inhibit telomerase; however, certain perylene diimides appear to be non-cytotoxic, G-quadruplex selective telomerase inhibitors. Biological characterization of such compounds may provide validation for the concept of the G-quadruplex as a target in drug design.

Deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours.

Medical records of 55 dogs with a diagnosis of cutaneous mast cell tumour were reviewed. Twenty-seven of the dogs were treated with surgery plus deionized water and the remaining 28 with surgery alone. A survival analysis was performed to determine whether deionized water, as an adjunct to surgery for cutaneous mast cell tumour, affected survival time or time to tumour recurrence. Dogs in which mast cell tumour recurred had a significantly shorter survival time compared with dogs with no recurrence (P = 0.05), regardless of the method of treatment. A significant negative association between tumour recurrence and method of treatment (P = 0.0097) and clinical stage (P = 0.0223) was observed. Dogs treated with surgery and deionized water had a significantly shorter time to recurrence of their mast cell tumour (P = 0.0113). Based on these results, deionized water does not appear to be beneficial in prolonging survival time or time to tumour recurrence for dogs with cutaneous mast cell tumours.

Caudal cervical intervertebral disk disease in the small dog: role of distraction and stabilization in ventral slot decompression.

The clinical outcomes in 112 dogs weighing less than 35 pounds that were presented with cervical intervertebral disk protrusions were retrospectively evaluated. Although the second to third cervical (C2 to C3) intervertebral space was the most common site (27%) of disk protrusion, 57% of disk protrusions presented were caudal to the fourth cervical (C4) vertebra. Dogs with cranial intervertebral disk protrusions, including the C2 to C3 and C3 to C4 intervertebral disk spaces, responded favorably to ventral slot decompression. By comparison, caudal intervertebral disk protrusions (within the C4 to the seventh cervical [C7] intervertebral disk spaces) responded less favorably to ventral slot decompression, demonstrating significantly more severe clinical effects in motor function, comfort, recovery, and long-term outcome following surgery. Significant improvement in clinical results was seen in caudal disk protrusions when additional surgical distraction and stabilization were provided following ventral slot decompression.

Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors.

One hundred twenty-six cutaneous mast cell tumors obtained by excisional biopsy from 106 dogs were evaluated using immunohistochemical staining for the presence of p53 protein. A standard avidin-biotin immunohistochemical protocol was used incorporating a polyclonal antibody of rabbit origin (CM-1) as the primary antibody. Histopathologic grading of tumors was performed on hemotoxylin and eosin-stained samples. There was a significant difference in the percentage of cells staining positive for p53 for the histopathologic grades (P = 0.0005). Grade III tumors had a significantly greater p53 content than did grade I or II tumors (P < 0.05). Clinical data obtained retrospectively was available for 54 dogs. Tumor recurred in 19 of 54 (35.2%) dogs. Twenty-nine dogs died by the end of the study; 9 of 29 (31.0%) died of mast cell tumor disease. Histopathologic grade showed a significant negative association with survival time. Both clinical stage and histopathologic grade showed a significant negative association with time to recurrence. The percentage of cells staining positive for p53 did not significantly improve the forward analysis. Immunohistochemical detection of p53 did not appear useful in characterizing the clinical association between cutaneous mast cell tumor cellular features and survival time or time to tumor recurrence in dogs.

Cervical spinal cord compression caused by cryptococcosis in a dog: successful treatment with surgery and fluconazole.

A six-year-old, male Doberman pinscher was presented for acute onset of upper motor neuron tetraparesis. An extradural compressive lesion compatible with intervertebral disk rupture at the sixth to seventh cervical (C6-C7) disk space was evident on myelography. A large, gelatinous mass of pure cryptococcal organisms causing spinal cord compression was identified upon exploratory surgery. Removal of the mass caused relief of clinical signs. No evidence of involvement of other organ systems was found; however, serum and cerebrospinal fluid titers were positive for cryptococcal infection. The dog was treated with fluconazole (5.5 mg/kg body weight, per os sid) until serum titers for cryptococcal infection were negative at seven months postsurgery. To the authors' knowledge, this is the only report of a dog with cryptococcosis treated successfully using fluconazole as a sole agent.

Design of new topoisomerase II inhibitors based upon a quinobenzoxazine self-assembly model.

A new class of pyridobenzophenoxazine compounds has been developed as topoisomerase II inhibitors for anticancer chemotherapy. These compounds were designed based on a proposed model of a quinobenzoxazine self-assembly complex on DNA. They showed excellent inhibitory effects on several tumor cell lines with nanomolar IC50 values. Their cytotoxic potency correlates with their ability to unwind DNA and inhibit topoisomerase II.

NMR-Based model of a telomerase-inhibiting compound bound to G-quadruplex DNA.

The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Musculoskeletal system. Joint and vertebral column diseases.

Owner complaints that refer to the musculoskeletal system are common in older dogs and cats. When the veterinarian is presented with these types of complaints, the differential lists include chronic intervertebral disk disease, diskospondylitis, degenerative joint disease, spondylosis with nerve root compression, joint/ ligament instability, and/or cancer. The diagnosis and management of some of these conditions is presented in detail with the general goal in mind that the older dog or cat is provided the best quality of life possible through good mobility along with being pain free.

Thermally induced DNA.RNA hybrid to G-quadruplex transitions: possible implications for telomere synthesis by telomerase.

Telomerase is a specialized reverse transcriptase that contains its own RNA template for synthesis of telomeric DNA [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337; Shippen-Lentz, D., & Blackburn, E. H. (1990) Science 247, 546-552]. The activity of this ribonucleoprotein enzyme has been associated with cancer cells [Kim et al. (1994) Science 266, 2011-2015] and is thus a potential target for anticancer chemotherapy. Telomeric DNA.RNA hybrids are important intermediates in telomerase function and form after extension of the growing telomere on the telomerase RNA template. Translocation is a critical step in telomerase function and consists of unwinding of the telomeric DNA.telomerase RNA hybrid followed by repositioning of the 3'-end of the extended telomere. A central question in telomerase function is how translocation of the extended telomere occurs in the absence of ATP or GTP. It has been hypothesized that unwinding of the telomeric hybrid may be facilitated by the formation of stable hairpins or G-quadruplexes by the telomere product (i.e., a hybrid to G-quadruplex transition) and that this may provide at least part of the driving force for translocation [Shippen-Lentz & Blackburn, 1990; Zahler et al. (1991) Nature 350, 718-720]. However, so far there has been no effort aimed at examining the possibility that a hybrid/G-quadruplex equilibrium can occur and to what extent this equilibrium depends on buffer and concentration conditions. Examination of these transitions may provide insight into telomerase function and may also provide clues for the development of anti-telomerase agents. Using a model system consisting of the DNA.RNA hybrid d(GGTTAGGGTTAG).r(cuaacccuaacc), we present evidence that a thermally induced transition of telomeric DNA.RNA hybrid to G-quadruplex can occur under certain conditions. These results provide support for the hypothesis that G-quadruplex formation by the telomere product may in fact regulate telomerase function at the translocation step (Zahler et al., 1991) and suggest an Achilles' heel for indirectly targeting telomerase. Thus, on the basis of the insight gained from the present studies and the results of Zahler et al. (1991), we propose that ligands that selectively bind or cleave G-quadruplex structures may modulate telomerase processivity.

Deep-frozen allogeneic cancellous bone grafts in 10 dogs: a case series.

Deep-frozen, aseptically collected and processed allogeneic cancellous bone was implanted in eight dogs during the surgical repair of diaphyseal long bone fractures and in two dogs during arthrodeses. A combined allogeneic and autogeneic cancellous bone graft was used in two fractures with a segmental bone loss of more than 5 cm. Bone union occurred in five fractures and in both arthrodeses. Failure of fixation occurred in two dogs with nonunion fractures and in a third dog with an open, infected fracture. Biopsies from the fracture sites were obtained from these dogs following failure of their fracture fixation. The cancellous bone graft appeared to be in the process of normal incorporation in each case. Failure of fixation was attributed to technical or case management errors or both, in each of the three fractures that failed to achieve bony union. Frozen allogeneic cancellous bone grafts were effectively incorporated when used in the primary repair of fractures and arthrodeses. Combined autogenous and allogeneic cancellous bone grafts may be particularly useful in the repair of fractures with large segmental diaphyseal bone defects. The use of allogeneic cancellous bone grafts in nonunion fractures requires further investigation before it can be recommended.

Hepatic aspiration and biopsy techniques.

Hepatic biopsy can be a key part of the diagnostic plan in dogs or cats with liver disease. There are a wide variety of techniques for the clinician to choose from based on the patient's condition, liver size, equipment available, experience of the operator, and financial considerations. Although major complications are rare, the clinician should not attempt a biopsy procedure without being prepared to diagnose and treat complications appropriately if they occur. Many techniques for hepatic biopsy are easy to learn and to perform and should be a part of small animal practice at every level.