ADAMTS-5 deficient mice do not develop mechanical allodynia associated with osteoarthritis following medial meniscal destabilization.

OBJECTIVE: To characterize pain-related behavior during the course of knee osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM) in wild type (WT) and in ADAMTS-5 null mice. METHODS: DMM surgery was performed in the right knee of CD-1 mice. At regular intervals up to 8 weeks after surgery, mice were assessed for the following parameters: mechanical allodynia (via withdrawal thresholds to von Frey filaments applied to the plantar surface of both hind paws or to the tail), thermal hyperalgesia, locomotor activity and gait analysis. In addition, mechanical allodynia was tested in C57BL/6 WT or ADAMTS-5 null mice following DMM surgery. RESULTS: In CD-1 mice, a robust and progressive decrease in withdrawal threshold was observed in both hind paws after DMM but not sham surgery. Allodynia was apparent as early as 14 days postoperatively. Both sexes developed OA changes after surgery with concurrent mechanical allodynia. No other pain-related behavioral changes were detected up to 8 weeks post-surgery. In C57BL/6 mice, a genetic background in which only males develop OA changes after DMM, males but not females developed allodynia in the ipsilateral hind paw. In contrast, C57BL/6 ADAMTS-5 null mice did not develop OA changes or mechanical allodynia up to 8 weeks post-surgery. CONCLUSION: Joint pathology following DMM surgery in mice is associated with progressive mechanical allodynia. ADAMTS-5 null mice are resistant to DMM-induced OA-like lesions and to the associated mechanical allodynia.

Effect of a short-term fast on intestinal disaccharidase activity and villus morphology of piglets suckling insulin-like growth factor-I transgenic sows.

The objectives of this study were to use transgenic sows that overexpress IGF-I in milk to investigate the effect of a short-term fast on piglet intestinal morphology and disaccharidase activity and to determine how milk-borne IGF-I influences the response to fasting. After farrowing, litters were normalized to 10 piglets. On d 6, piglets (n = 30) suckling IGF-I transgenic (TG) sows and piglets (n = 30) suckling nontransgenic sows (control) were assigned randomly to three treatments: fed piglets (0 h), which remained with the sow until euthanized on d 7, or fasted piglets, which were removed from the sow at either 6 or 12 h before euthanasia on d 7. Serum IGF-I and IGFBP, intestinal weight and length, jejunal protein and DNA content, disaccharidase activity, and villus morphology were measured. Fasting for 12 h resulted in a negative weight change between d 6 and 7 (quadratic response to fasting; P < 0.001). Piglets suckling TG sows tended to have greater intestinal length (P = 0.068), but no effect of IGF-I overexpression was noted for intestinal weight. Fasting, however, resulted in linear (P < 0.001) and quadratic (P = 0.002) decreases in intestinal weight. Serum IGF-I did not differ between control and TG sows, but decreased linearly (P = 0.003) with fasting. Serum IGFBP-4 decreased (linear and quadratic; P < or = 0.02) with fasting, whereas IGFBP-1 increased quadratically (P < 0.001) with fasting. Jejunal villus height, width, and crypt depth were all increased with fasting (linear and quadratic; P < 0.04). Disaccharidase activity was not affected by fed state; however, piglets suckling TG sows had greater jejunal lactase-phlorhizin hydrolase (P < 0.01) and sucrase-isomaltase (P = 0.02) activities than control piglets. In summary, intestinal weight, villus morphology, serum IGF-I, serum IGFBP-1 and -4, and piglet BW change were altered (P < or = 0.02) in response to fasting. Thus, the duration of food deprivation before euthanization should be considered when designing experiments to assess intestinal development or the IGF axis, as the magnitude of differences between the fed and fasted state may exceed those expected as a result of experimental treatment.

Three-dimensional microimaging (MRmicroI and microCT), finite element modeling, and rapid prototyping provide unique insights into bone architecture in osteoporosis.

With the proportion of elderly people increasing in many countries, osteoporosis has become a growing public health problem, with rising medical, social, and economic consequences. It is well recognized that a combination of low bone mass and the deterioration of the trabecular architecture underlies osteoporotic fractures. A comprehensive understanding of the relationships between bone mass, the three-dimensional (3D) architecture of bone and bone function is fundamental to the study of new and existing therapies for osteoporosis. Detailed analysis of 3D trabecular architecture, using high-resolution digital imaging techniques such as magnetic resonance microimaging (MRmicroI), micro-computed tomography (microCT), and direct image analysis, has become feasible only recently. Rapid prototyping technology is used to replicate the complex trabecular architecture on a macroscopic scale for visual or biomechanical analysis. Further, a complete set of 3D image data provides a basis for finite element modeling (FEM) to predict mechanical properties. The goal of this paper is to describe how we can integrate three-dimensional microimaging and image analysis techniques for quantitation of trabecular bone architecture, FEM for virtual biomechanics, and rapid prototyping for enhanced visualization. The integration of these techniques provide us with an unique ability to investigate the role of bone architecture in osteoporotic fractures and to support the development of new therapies.

Bone anabolic therapy with selective prostaglandin analogs.

Prostaglandin E(2) has been shown to increase bone mass in animals and humans but it also has considerable dose limiting systemic side effects. The molecular description of multiple seven transmembrane domain G protein coupled prostanoid receptors offered the opportunity to probe the skeletal effects of specific receptors using selective agonists. Bone effects have been reported with many of the prostanoid receptors, with most interest focused on the anabolic effects of EP2, EP4, and FP receptors. Current data suggests activity at the EP2 receptor stimulates formation, activity at the EP4 receptor stimulates resorption (and possibly formation), and activity at the FP receptor produces new trabeculae. However, caution must be exercised in extending the effects of prostanoids in isolated systems to systemic skeletal effects, since tissue level effects are the cumulative result of bone formation and bone resorption. Furthermore, species differences in receptor sequence and density confound extrapolation of effects from one model to another model. While these molecular targets increase our insight into how the skeleton can be affected pharmacologically, they still do not answer questions about the role of naturally occurring prostaglandins in skeletal health. This manuscript will review some of the recent advances in knowledge of the bone anabolic effects of selective prostanoid ligands.

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma.

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1x10 mm) afforded rapid isocratic elution of fluprostenol (t(R)=40 s) but still provided a relatively large k' value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml(-1) fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.

Preclinical development of agents for the treatment of osteoporosis.

Because of the high cost and long time frame of clinical testing, animal models play a crucial role in the identification and selection of agents for the treatment of osteoporosis. The use of animal models early in a program focuses on the establishment of efficacy, while animal models used later in a program to examine bone safety. More specifically, animal models are used to gain information on the skeletal mechanism of action, to examine multiple skeletal sites (axial and appendicular), and to examine the effects of higher doses than will be used in humans. Animal models also predict the usefulness of surrogate markers in clinical trials, such as formation and resorption markers, as well as bone density. The hazard of using surrogate markers for fracture prevention is highlighted by high dose fluoride administration, which can increase bone density (considered a strong predictor of fracture protection) while not protecting against fractures. Estrogen-deficient models are most commonly used to mimic the postmenopausal bone loss in women; these models are characterized by increased bone turnover and a negative bone balance. The timing of the administration of the new therapy in animal models can help determine whether the agent will be more effective in the prevention of osteoporosis or in the treatment of established osteoporosis. New methods for the measurement of bone mass or volume are less invasive, require shorter acquisition time, and have enhanced resolution, resulting in increased knowledge concerning architectural changes and specific sites of bone deposition. Finally, the measurement of biomechanical strength of bones from animal models can be used to predict protective effects on fracture rates in clinical trials. When used in combination with other methods, animal models can greatly increase our understanding of the pathophysiology of osteoporosis and can expedite the development of new therapies.

Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI.

Despite the compelling need mandated by the prevalence and morbidity of degenerative cartilage diseases, it is extremely difficult to study disease progression and therapeutic efficacy, either in vitro or in vivo (clinically). This is partly because no techniques have been available for nondestructively visualizing the distribution of functionally important macromolecules in living cartilage. Here we describe and validate a technique to image the glycosaminoglycan concentration ([GAG]) of human cartilage nondestructively by magnetic resonance imaging (MRI). The technique is based on the premise that the negatively charged contrast agent gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)2-) will distribute in cartilage in inverse relation to the negatively charged GAG concentration. Nuclear magnetic resonance spectroscopy studies of cartilage explants demonstrated that there was an approximately linear relationship between T1 (in the presence of Gd(DTPA)2-) and [GAG] over a large range of [GAG]. Furthermore, there was a strong agreement between the [GAG] calculated from [Gd(DTPA)2-] and the actual [GAG] determined from the validated methods of calculations from [Na+] and the biochemical DMMB assay. Spatial distributions of GAG were easily observed in T1-weighted and T1-calculated MRI studies of intact human joints, with good histological correlation. Furthermore, in vivo clinical images of T1 in the presence of Gd(DTPA)2- (i.e., GAG distribution) correlated well with the validated ex vivo results after total knee replacement surgery, showing that it is feasible to monitor GAG distribution in vivo. This approach gives us the opportunity to image directly the concentration of GAG, a major and critically important macromolecule in human cartilage.

Effect of prostaglandin and bisphosphonate on cancellous bone volume and structure in the ovariectomized rat studied by quantitative three-dimensional nuclear magnetic resonance microscopy.

The purpose of this work was to evaluate the potential of nuclear magnetic resonance microscopy (NMRM) in conjunction with a processing technique to monitor the effect of preventive agents in an ovariectomized (OVX) rat. Twenty-five female Sprague-Dawley rats were OVX at 6 months of age (except for the intact control group), allowed to lose bone for 60 days, and then treated for 60 days. During treatment, animals were administered vehicle, prostaglandin E2 (PGE2; 6 mg/kg), or alendronate (3 microg/kg) subcutaneously once a day. Subsequently, tibiae were harvested and the marrow removed. NMRM was carried out at 9.4 T, with the specimens immersed in 1.2 mM diethylenetriaminepentaacetic acid-gadolinium salt (Gd-DTPA) aqueous solution. A three-dimensional (3D) partial flip-angle pulse sequence was used, providing a 1283 array of (46 microm)3 isotropic voxels. Fifty of the 128 axial images in the 3D data set comprising approximately 2.4 mm volume distal to the growth plate were processed from each specimen using a probability-based method for determining bone volume fraction (BVF), tubularity, contiguity, as well as the mean trabecular plate thickness and separation. PGE2 and alendronate altered BVF consistently at all tibial regions. The effect of alendronate was to keep BVF about midway between intact and OVX, whereas PGE2 returned BVF to intact levels. The other parameters showed similar responses to treatment. The strongest discriminator was trabecular BVF, which could obviously differentiate the groups. The study establishes NMRM as a nondestructive histomorphometric method for the quantitative evaluation of drug response in a rat ovariectomy model.

Parathyroid hormone enhances fracture healing. A preliminary report.

This investigation tested the hypothesis that daily parenterally administered parathyroid hormone (1-34) improves fracture healing. Twenty, 3-month-old, male Sprague Dawley rats weighing approximately 400 g each, underwent the production of closed, unilateral mid-diaphyseal femoral fractures. Animals were divided into two groups of 10; the animals received either a daily subcutaneous injection of delivery vehicle (0.9% saline) or 80 micrograms/kg parathyroid hormone. On Day 21 after fracture the animals were euthanized, the femurs were removed and subjected to biomechanical testing, bone densitometry (dual energy x-ray absorptiometry, peripheral quantitative computed tomography), and histologic examination. Treatment with parathyroid hormone resulted in statistically significant increases in callus area and strength. Histologic examination of the calluses showed an increase in the amount of new bone formed. No differences were observed in the weights of the animals or the sizes of the bones. Values obtained using dual energy x-ray absorptiometry and peripheral quantitative computed tomography indicate an increase in density in the parathyroid hormone treated fractures consistent with the histologic appearance and the findings of increased strength, although these bone density changes did not achieve statistical significance. These results suggest that parenterally administered parathyroid hormone (1-34) may enhance or accelerate normal fracture healing and support the concept that this hormone be tested clinically as a systemic treatment for fractures that are slow to heal.

FeLV envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons.

In humans and animals, retroviruses have been implicated in nervous system disease. Our objective was to characterize the neurotoxicity of a peptide sequence derived from an animal retrovirus, the feline leukemia virus (FeLV). Using a peptide sequence from the subtype FeLV-C envelope protein variable region 5 (VR5), cytotoxicity was demonstrated in studies that evaluated neuronal survival, neurite outgrowth, and alterations in intracellular calcium ion concentration. The FeLV subtype isolate FeLV-CSarma possesses an envelope protein VR5 amino acid sequence that varies by four amino acids from the VR5 amino acid sequence of subtype FeLV-AGlasgow. The polypeptide representing the VR5 of FeLV-CSarma (FeLV-CVR5) is significantly more neurotoxic than the polypeptide sequence representing the VR5 of FeLV-AGlasgow (FeLV-AVR5). FeLV-CVR5 (> or = 3 microM) exposure resulted in significant dose-dependent neurotoxicity. Antibodies to FeLV-CVR5 blocked this effect. Neurite outgrowth was significantly reduced at all tested concentrations (3-12 microM) of FeLV-CVR5, with a 92% reduction in neurite length at 12 microM. FeLV-AVR5 was significantly less neurotoxic with respect to neurite outgrowth than was FeLV-CVR5. The significant reduction in neurotoxicity for FeLV-AVR5 illustrates the importance of the 4-amino-acid difference between it and FeLV-CVR5. Alterations in intracellular calcium ion concentration were associated with this neurotoxicity.

Validation and application of an immunoradiometric assay for the determination of human parathyroid hormone fragment 1-34 in dog plasma following subcutaneous and intravenous administration.

A method for the measurement of human parathyroid hormone fragment 1-34 (PTH1-34) in dog plasma was developed by modification of a commercially available immunodiometric assay (IRMA) designed for the determination of rat PTH1-34 in serum. Major modifications were made to the assay in order to circumvent significant problems encountered during the validation of the IRMA. PTH1-34 was found to be highly unstable in both rat serum and dog serum and plasma at room temperature, in contrast to literature reports. The addition of a protease inhibitor cocktail to serum or plasma samples was necessary to prevent in-vitro proteolytic degradation of human PTH1-34 prior to analysis. Additionally, plasma was chosen over serum as the sample matrix to expedite the separation of samples from cells, minimizing proteolytic degradation prior to the addition of cocktail. Finally, the reported 100% cross-reactivity between rat and human PTH1-34 was found to be only 65%; therefore, a human PTH1-34 standard was substituted for the rat standard. These modifications allowed the accurate measurement of human PTH1-34 in plasma obtained from dogs dosed intravenously and subcutaneously with human PTH1-34 using a commercially available kit.

Cytopathic feline leukemia viruses cause apoptosis in hemolymphatic cells.

Certain isolates of the oncoretrovirus feline leukemia virus (FeLV) are strongly cytopathic for hemolymphatic cells. A major cytopathicity determinant is encoded by the SU envelope glucoprotein gp70. Isolates with subgroup C SU gp70 genes specifically induce apoptosis in hemolymphatic cells but not fibroblasts. In vitro exposure of feline T-cells to FeLV-C leads first to productive viral replication, next to virus-induced cell agglutination, and lastly to apogenesis. This in vitro phenomenon may explain the severe progressive thymic atrophy and erythroid aplasia which follow viremic FeLV-C infection in vivo. Inappropriate apoptosis induction has also been hypothesized to explain the severe thymico-lymphoid atrophy and progressive immune deterioration associated with isolates of FeLV containing variant envelope genes. The influence of envelope hypervariability (variable regions 1 [Vr1] and 5 [Vr5] on virus tropism, viremia induction, neutralizing antibody development and cytopathicity is discussed. Certain potentially cytopathic elements in FeLV SU gp70 Vr5 may derive from replication-defective, poorly expressed, endogenous FeLVs. Other more highly conserved regions in FeLV TM envelope p15E may also influence apoptosis induction.

Acute feline leukemia virus infection causes altered energy balance and growth inhibition in weanling cats.

Naturally occurring retroviral infections cause progressive weight loss, immune suppression, invasion by opportunistic organisms, and eventual death. Feline leukemia virus (FeLV) inhibited growth and decreased energy intake in seven experimentally infected weanling cats compared with age- and sex-matched controls. Remarkably, changes in energy intake, energy expenditure, and weight gain occurred in the acute phase of infection prior to the systemic/productive bone marrow phase of FeLV infection. In other words, growth inhibition developed before FeLV infection was clinically detectable with use of standard enzyme-linked immunosorbent assay or fixed-cell immunofluorescence assays of circulating neutrophils and platelets. Acutely infected, previremic cats consumed 25% less energy [Day 4 postinoculation to Day 16 postinoculation (p < 0.05)] and expended 20% less energy [Day 8 postinoculation to Day 18 postinoculation (p < 0.05)] compared with control cats. Growth stunting of inoculated cats began by Day 11 postinoculation (p < 0.05) and was not corrected during the remaining 4 months of the study. Thus, experimental FeLV infection causes perturbations of metabolism and energy balance resulting in permanent growth impairment. Secondly, detrimental metabolic effects begin in the acute phase of retroviral infection, prior to the clinically detectable phase of FeLV infection.

Increased insensible water loss in feline retrovirus-infected cats.

Feline retroviruses such as feline leukemia virus (FeLV) adversely affect the regulation of many vital host systems such as the immune response, erythropoiesis, and nutrient metabolism. In this paper, we describe the disruption of an additional homeostatic mechanism-evaporative water loss-by FeLV. Viremic cats had greater evaporative water losses (24.0 +/- 1.4 gm water/kg per day) at low relative humidity levels (19% to 25% relative humidity) when compared to age- and sex-matched control cats (19.7 +/- 1.4 gm of water/kg per day [P < 0.05]). At relative humidity levels greater than 50%, viremic and control cats had similar evaporative water losses. Viremia also resulted in an elevation in the average body temperature (39.1 +/- 0.5 degrees C) compared to control cats (38.4 +/- 0.3 degrees C) (P < 0.001). However, the energy expenditure of viremic cats (17.14 +/- 1.60 kJ/kg/h) was not significantly different from the energy expenditure of control cats (17.02 +/- 2.22 kJ/kg/h). The elevated body temperature of viremic cats likely causes a greater increase in evaporative water loss at low relative humidity levels and suggests further study of water balance in retroviral infection is warranted.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes.

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.