Role of ADAMTS-12 in Protecting Against Inflammatory Arthritis in Mice By Interacting With and Inactivating Proinflammatory Connective Tissue Growth Factor.

OBJECTIVE: It has been reported that ADAMTS-12 is a susceptibility gene for rheumatoid arthritis (RA) development, and its level is significantly increased in RA patients. In addition, ADAMTS-12 is reported to be required for inflammation in otherwise healthy subjects. This study was undertaken to determine the role of ADAMTS-12 and the underlying mechanisms in the pathogenesis of inflammatory arthritis. METHODS: The collagen-induced arthritis (CIA) model was established in ADAMTS-12-deficient mice and their control littermates to determine the role of ADAMTS-12 in vivo. Micro-computed tomography scanning was used to demonstrate the destruction of the ankle joint; histologic analysis illustrated synovitis, pannus formation, and bone and cartilage destruction; enzyme-linked immunosorbent assay was performed to measure serum levels of inflammatory cytokines; and protein-protein interaction assays were performed to detect the interactions of ADAMTS-12 and its various deletion mutants with connective tissue growth factor (CTGF). RESULTS: Deficiency of ADAMTS-12 led to accelerated inflammatory arthritis in the CIA mouse model. Loss of ADAMTS-12 caused enhanced osteoclastogenesis. In vitro and in vivo protein-protein interaction assays demonstrated that ADAMTS-12 bound and processed CTGF, a previously unrecognized substrate of ADAMTS-12. In addition, deletion of ADAMTS-12 enhanced, while overexpression of ADMATS-12 reduced, CTGF-mediated inflammation. Furthermore, ADAMTS-12 regulation of inflammation was largely lost in CTGF-deficient macrophages. Importantly, blocking of CTGF attenuated elevated inflammatory arthritis seen in the ADAMTS-12-deficient CIA mouse model. CONCLUSION: This study provides evidence that ADAMTS-12 is a critical regulator of inflammatory arthritis and that this is mediated, at least in part, through control of CTGF turnover.

High-Performance Liquid Chromatography (HPLC) Quantification of Liposome-Delivered Doxorubicin in Arthritic Joints of Collagen-Induced Arthritis Rats.

BACKGROUND Neoangiogenesis occurring in inflamed articular synovium in early rheumatoid arthritis (RA) is characterized by enhanced vascular permeability that allows nanoparticle agents, including liposomes, to deliver encapsulated drugs to arthritic joints and subsequently improve therapeutic efficacy and reduce adverse effects. However, the targeting distribution of liposomes in arthritic joints during RA has not been quantitatively demonstrated. We performed this study to evaluate the targeting distribution of PEGylated doxorubicin liposomes in the arthritic joints of collagen-induced arthritis (CIA) rats by high-performance liquid chromatography (HPLC). MATERIAL AND METHODS Two doxorubicin formulations were administered to CIA rats via tail intravenous injection at a single dose (50 mg/m²). CIA rats were sacrificed and the tissues of the inflamed ankle joints were collected. The content of doxorubicin in the arthritic joints was analyzed by a validated and reproducible HPLC method. A two-way ANOVA for 2×5 factorial design was used for statistical analysis. RESULTS The developed HPLC method was sensitive, precise, and reproducible. The method was successfully applied to quantify doxorubicin content in arthritic tissues. At each time point (6, 12, 24, 48, and 72 h), doxorubicin content in the arthritic joints of the doxorubicin liposome group (DOX-LIP group) was higher than in the free doxorubicin group (DOX group) (P<0.05). In the DOX-LIP group, doxorubicin levels in the arthritic joints increased gradually and significantly in the interval of 6-72 h post-administration. CONCLUSIONS PEGylated doxorubicin liposomes were targeted to, accumulated, and retained in the arthritic joints of CIA rats. The present study indicates that liposome encapsulation increases the therapeutic efficacy of antirheumatic drugs, presenting a promising therapeutic strategy for RA.

Capsaicin-sensitive sensory nerves exert complex regulatory functions in the serum-transfer mouse model of autoimmune arthritis.

OBJECTIVE: The K/BxN serum-transfer arthritis is a widely-used translational mouse model of rheumatoid arthritis, in which the immunological components have thoroughly been investigated. In contrast, little is known about the role of sensory neural factors and the complexity of neuro-immune interactions. Therefore, we analyzed the involvement of capsaicin-sensitive peptidergic sensory nerves in autoantibody-induced arthritis with integrative methodology. METHODS: Arthritogenic K/BxN or control serum was injected to non-pretreated mice or resiniferatoxin (RTX)-pretreated animals where capsaicin-sensitive nerves were inactivated. Edema, touch sensitivity, noxious heat threshold, joint function, body weight and clinical arthritis severity scores were determined repeatedly throughout two weeks. Micro-CT and in vivo optical imaging to determine matrix-metalloproteinase (MMP) and neutrophil-derived myeloperoxidase (MPO) activities, semiquantitative histopathological scoring and radioimmunoassay to measure somatostatin in the joint homogenates were also performed. RESULTS: In RTX-pretreated mice, the autoantibody-induced joint swelling, arthritis severity score, MMP and MPO activities, as well as histopathological alterations were significantly greater compared to non-pretreated animals. Self-control quantification of the bone mass revealed decreased values in intact female mice, but significantly greater arthritis-induced pathological bone formation after RTX-pretreatment. In contrast, mechanical hyperalgesia from day 10 was smaller after inactivating capsaicin-sensitive afferents. Although thermal hyperalgesia did not develop, noxious heat threshold was significantly higher following RTX pretreatment. Somatostatin-like immunoreactivity elevated in the tibiotarsal joints in non-pretreated, which was significantly less in RTX-pretreated mice. CONCLUSIONS: Although capsaicin-sensitive sensory nerves mediate mechanical hyperalgesia in the later phase of autoantibody-induced chronic arthritis, they play important anti-inflammatory roles at least partially through somatostatin release.

Role of tachykinin 1 and 4 gene-derived neuropeptides and the neurokinin 1 receptor in adjuvant-induced chronic arthritis of the mouse.

OBJECTIVE: Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse. METHODS: Complete Freund's Adjuvant was injected intraplantarly and into the tail of Tac1(-/-), Tac4(-/-), Tacr1(-/-) (NK1 receptor deficient) and Tac1(-/-/)Tac4(-/-) mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1ß concentrations of the tibiotarsal joints were performed. RESULTS: Mechanical hyperalgesia was significantly reduced from day 11 in Tac4(-/-) and Tacr1(-/-) animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1ß concentration in the joint homogenates were significantly smaller in Tac4(-/-) and Tac1(-/-/)Tac4(-/-) mice. CONCLUSIONS: Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1ß production is suggested.

Extra domain B fibronectin as a target for near-infrared fluorescence imaging of rheumatoid arthritis affected joints in vivo.

Abstract We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fibronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fluorescence imaging. The resulting AP39-TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fluorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

Disrupted bone metabolism in contaminant-exposed white storks (Ciconia ciconia) in southwestern Spain.

In 1998, the Aznalcóllar mine tailings dyke in southwestern Spain broke, flooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colonies of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects not seen before the spill, prompting this study. Nestlings with deformed legs had significantly lower plasma phosphorous (P) and higher Ca:P ratios than non-deformed cohorts in the first two years, but in the third year, when more, younger birds were studied, plasma P ranged from much higher to much lower in the affected colony compared with reference birds. Coefficients of variation for phosphorous were 19% and 60%, in reference and contaminated colonies, respectively. Storks from the contaminated colony were unable to control P levels and Ca:P ratios within the narrow limits necessary for normal bone development.

Response of broilers to feeding low-calcium and phosphorus diets plus phytase under different environmental conditions: body weight and tibiotarsus mineralization.

Three experiments on Ross broiler chickens were conducted in 3 locations: cages (Experiment 1), floor pens (Experiment 2), and commercial farms (Experiment 3). The effect of low-total P (TP) wheat-soybean based diets plus microbial phytase (Natuphos) was evaluated. Four experimental starter and finisher diets were used in a 2-phase feeding program, as follows: control diet (SC until 21 d, FC from 22 to 42 d); 2 diets (SL400 and SL600 until 21 d, FL400 and FL600 from 22 to 42 d) with low TP (0.61% for starter and 0.54% for finisher), including 400 and 600 U/kg of phytase, respectively; and a very low-TP (0.52% for starter and 0.44% for finisher) diet (SVL600 until 21 d, FVL600 from 22 to 42 d) with 600 U/kg of phytase. In Experiment 1 (broilers in cages had movement limitation and no access to litter), no differences in BW, tibiotarsus mineralization, or mineral metabolism were observed among diets. In Experiment 2 (broilers in floor pens had movement limitation and access to litter), at 21 d of age, the lowest tibiotarsus ash percentage and BW were shown by birds fed the SVL600 diet. At 42 d of age, broilers fed the FC diet were the lightest. For the rest of the parameters of tibiotarsus mineralization and mineral metabolism measured in Experiment 2, no differences were shown. In Experiment 3 (broilers in commercial farms had access to litter without movement limitation), the BW of broilers fed the SC diet was the highest at 21 d of age. At 42 d of age, the broilers fed FL400 and FL600 diets were the heaviest. At the end of Experiment 3, broilers fed the FC diet had the highest dry litter Ca and P, whereas broilers fed the FVL600 diet had the lowest values. In conclusion, the very low-TP wheat-soybean based diet supplemented with 600 U/kg of phytase was sufficient to optimize all the parameters measured in Experiment 1 but not in Experiments 2 and 3. Therefore, when evaluating Ca and P in phytase-supplemented diets for broilers, it is necessary to bear in mind the environmental conditions of experimentation.

Sequential expression of type IV collagen networks: testis as a model and relevance to spermatogenesis.

The six alpha chains of type IV collagen are organized into three networks: alpha1/alpha2, alpha3/alpha4/alpha5, and alpha1/alpha2/alpha5/alpha6. A shift from the alpha1/alpha2 to the alpha3/alpha4/alpha5 network occurs in the developing glomerular basement membrane, but how the alpha1/alpha2/alpha5/alpha6 network fits into this sequence is less clear, because the three networks do not colocalize. Here, we studied the seminiferous tubule basement membrane of normal canine testis where all three networks do colocalize: the alpha1/alpha2 network is expressed from birth, the alpha1/alpha2/alpha5/alpha6 network by 5-6 weeks of age, and the alpha3/alpha4/alpha5 network by 2 months of age. A canine model of Alport syndrome allowed study of the absence of alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks in testis. In Alport dogs, the seminiferous tubule basement membrane was thinner than in controls. Spermatogenesis began at the same time as with normal dogs; however, the number of mature sperm was significantly reduced in Alport dogs. Thus, it would appear that alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks are not essential for onset of spermatogenesis, but long-term function may be compromised by the loss of one or both networks. This situation is analogous to the glomerular basement membrane in Alport syndrome. In conclusion, testis can serve as a model system to study the sequence of type IV collagen network expression.

Tarsus determination in Drosophila melanogaster.

Arista versus tarsus determination is well investigated in Drosophila, yet it remains unresolved whether Antennapedia (ANTP) cell autonomously or noncell autonomously determines tarsus identity and whether Sex combs reduced (SCR) is the HOX protein required for normal tarsus determination. Three observations rule out a cell autonomous role for ANTP in tarsus determination. (i) Clonal ectopic overexpression of ANTP did not repress the expression of the arista determining protein Homothorax (HTH) in early 3rd stadium antennal imaginal discs. (ii) Clonal ectopic expression of ANTP did not transform the arista to a tarsus. (iii) Ectopic overexpression of ANTP, Labial (LAB), Deformed (DFD), SCR, Ultrabithorax (UBX), Abdominal-A (ABD-A), or Abdominal-B (ABD-B), using the dppGAL4 driver, resulted in arista-to-tarsus transformations, and repressed HTH/Extradenticle (EXD) activity noncell autonomously in early 3rd stadium antennal imaginal discs. SCR may not be the HOX protein required for normal tarsus determination, because co-ectopic expression of Proboscipedia (PB) inhibited the arista-to-tarsus transformations induced by ectopic expression of DFD, SCR, ANTP, UBX, ABD-A, and ABD-B. The proposal that SCR is the HOX protein required for normal tarsus determination is dependent on SCR being the sole target of PB suppression, which is not the case. Therefore, the possibility exists that normal tarsus determination is HOX independent.

Scintigraphic evaluation of the distal tarsal region in horses with distal tarsal pain.

Distal tarsal pain is a common reason for hind limb lameness, but diagnosis cannot always be made on radiographic examination. Scintigraphy may allow detection of subtle changes undetected by other diagnostic methods. We hypothesized that (1) distal tarsal pain would be associated with a loss of the expected pattern of radiopharmaceutical uptake (RU) detected in normal horses, (2) distal tarsal RU would be greater in limbs with tarsal pain than without pain, (3) RU in painful tarsi with radiographic evidence of osteoarthritis (OA) would be greater than in distal tarsal pain with no radiographic evidence of OA. The study aimed to describe radiopharmaceutical distribution in the distal tarsal region of horses with distal tarsal pain, and to compare this with the contralateral limb and results from horses without tarsal pain. Retrospective evaluation of scintigraphic images of the distal tarsal region was performed for 52 horses with distal tarsal pain: 15 with no radiographic evidence of OA (Group 1) and 37 with radiographic evidence (Group 2). The images were assessed using vertical and horizontal profile analysis across the distal tarsal region and regions of interest comparisons between the distal tarsal region and tibia within each horse (RU ratio). Painful limbs in unilaterally lame horses from Groups 1 and 2 had a significantly greater RU ratio than the respective contralateral limbs, and were significantly greater than the RU ratio in normal horses. On plantar images, mean region of interest counts were greater in the lame than the contralateral limb in Group 2 but not in Group 1. Although there was a positive correlation between lame and contralateral limb RU ratio in group 1, this was lost in group 2 horses. In lame limbs, the normal vertical activity profile was lost in 85% of group 1 and all of group 2, and the normal horizontal activity profile was lost in all of group 1 and 96% of group 2. There was a significant effect of lameness, but not of group on sites of peak activity on all profiles. The results of this study indicate that distal tarsal pain is associated with loss of the expected pattern of RU detected in normal horses. The findings also suggest that distal tarsal RU in lame limbs is greater than in limbs without pain, and that painful limbs with radiographic evidence of OA have a greater RU than painful limbs without radiographic evidence of OA.

Effects of continuous intra-articular infusion of gentamicin on synovial membrane and articular cartilage in the tarsocrural joint of horses.

OBJECTIVE: To determine the effects of a continuous intra-articular infusion of gentamicin on the synovial membrane and articular cartilage in the tarsocrural joint of horses. ANIMALS: 6 healthy adult horses. PROCEDURE: A balloon infusion system attached to a catheter placed in the plantarolateral pouch of both tarsocrural joints in each horse was used for continuous gentamicin solution (GM) or balanced electrolyte solution (BES) delivery for 5 days. Cartilage and synovial membrane specimens were collected on day 5 from 3 horses and on day 14 from the remaining 3 horses. Both infused joints from each horse were assessed, using gross evaluation and histologic scoring systems. RESULTS: Significant differences in the histologic scores of synovial membrane specimens between the GM- and BES-treated joints at either 5 or 14 days were not observed. Safranin-O-fast green staining scores were similar between cartilage specimens from GM- and BES-treated joints. Although the synovial membrane histologic scores and safranin-O-fast green staining scores improved from day 5 to 14, the changes in scores were not significant. Loss of synovial intimal cells from villi was found more commonly in sections of synovial membrane from GM-treated joints, compared with BES-treated joints. CONCLUSIONS AND CLINICAL RELEVANCE: Continuous infusion of GM into the tarsocrural joint of horses does not have significant effects on histologic scores of articular cartilage or synovial membrane, compared with those infused with BES. Continuous infusion of GM into the tarsocrural joint of horses for 5 days is an acceptable method for the treatment of septic arthritis.

Diffusion of mepivacaine between adjacent synovial structures in the horse. Part 2: tarsus and stifle.

This paper tests the hypothesis that the local analgesic agent mepivacaine diffuses between adjacent equine synovial structures in the hindlimb and with greater frequency than latex, gelatine dye or contrast media. We report the incidence of diffusion of mepivacaine between the tarsometatarsal, centrodistal and tarsocrural joints, and the 3 synovial compartments of the stifle in 33 fresh equine cadavers. The tarsometatarsal joint and one synovial compartment of the stifle in the left limb and the centrodistal joint and a different synovial compartment of the stifle in the right limbs were injected with mepivacaine. Following flexion and extension of the limb, synovial fluid was aspirated from the noninjected centrodistal and tarsometatarsal joints and the tarsocrural joints of the hock and the noninjected compartments of the stifle. Concentrations of mepivacaine in these samples were assayed using an enzyme linked immunosorbent assay. For samples obtained by dilution of synovial fluid the concentration of mepivacaine was determined by comparing the concentration of urea in the diluted synovial fluid and the concentrations of the serum urea. Mepivacaine was detected in 25/25 (100%) adjacent tarsometatarsal and centrodistal joints after diffusion in both directions, in 23/25 (92%) of tarsocrural joints after diffusion from tarsometatarsal joints and in 22/25 (88%) tarsocrural joints after diffusion from centrodistal joints in the hocks. Diffusion from the femoropatellar to medial and lateral femorotibial joints and between the medial and lateral femorotibial joints in both directions were 20/20 (100%). Diffusion from the lateral femorotibial to the femoropatellar joint was 18/20 (90%) and from the medial femorotibial to femoropatellar joints 17/20 (85%). Mepivacaine was detected at concentrations >0.3 mg/l in a proportion of samples ranging from 15/25 (60%) in the tarsocrural joint following tarsometatarsal joint injection to 18/20 (90%) in the lateral femorotibial joint after femoropatellar joint injection. At mepivacaine concentrations >100 mg/l, detection ranged from 3/20 (15%) in the lateral femorotibial joint from the medial femorotibial joint to 19/25 (76%) in the centrodistal joint from the tarsometatarsal joint. At mepivacaine concentrations >300 mg/l, detection ranged from 1/25 (4%) in the tarsocrural joint from the tarsometatarsal joint to 16/25 (64%) in the from centrodistal joint the tarsometatarsal joint. The results show greater diffusion of mepivacaine between these adjacent synovial structures than assumed from previous anatomical, latex injection and contrast arthrographic studies. Therefore, commonly performed intrasynovial local analgesic techniques in the hindlimb of the horse are not as specific as first thought.

Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis.

OBJECTIVE: To examine the expression of the novel CX3C chemokine fractalkine (Fkn) and its receptor (CX3CR1) in rheumatoid arthritis (RA) and rat adjuvant-induced arthritis (AIA), a model of RA. METHODS: Immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and chemotaxis assays were used. RESULTS: In rat AIA, synovial tissue (ST) macrophages, fibroblasts, endothelial cells, and dendritic cells were Fkn immunopositive, whereas lymphocytes did not significantly express Fkn. Significant staining for CX3CR1 was found in ST macrophages, fibroblasts, and dendritic cells, whereas only a small percentage of endothelial cells stained for CX3CR1 in rat AIA. We immunolocalized Fkn to RA ST macrophages, fibroblasts, endothelial cells, and dendritic cells. We also found intense ST macrophage and dendritic cell staining for CX3CR1 in RA ST. Flow cytometry analysis of RA synovial fluid (SF) and peripheral blood revealed a greater percentage of monocytes expressing Fkn and CX3CR1 compared with T cells. By ELISA, we found significantly elevated soluble Fkn (sFkn) levels in RA SF compared with SF from patients with osteoarthritis or other forms of arthritis. By RT-PCR, we found enhanced expression of Fkn and CX3CR1 mRNA on day 18 in rat AIA, a time of pronounced inflammation in the rat joint. Soluble Fkn-depleted RA SF showed significantly decreased chemotactic activity for monocytes compared with sham-depleted RA SF. CONCLUSION: These results indicate that Fkn and its receptor are both expressed in RA and in rat AIA, and that sFkn is up-regulated in RA SF. Furthermore, our data suggest a new role for Fkn in monocyte chemotaxis in the inflamed RA joint.

Iontophoresis of dexamethosone-phosphate into the equine tibiotarsal joint.

In human rehabilitation medicine, dexamethasone-phosphate is theoretically iontophoresed to localized subcutaneous tissue where conversion to dexamethasone occurs. This delivery system has recently been introduced into veterinary medicine for the same purpose. However, the pharmacokinetic justification for parenteral delivery of this prodrug remains undocumented. Utilizing iontophoretic methods that are relevant to both human and veterinary clinical practice, the present investigation compared injection and iontophoresis of dexamethasone-phosphate into the equine tibiotarsal joint, also known as the tarsocrual joint. The tibiotarsal joints of seven horses were injected with 4 mL of 6 mg/mL dexamethasone-phosphate. With a similar drug concentration and over the same application site, six different horses underwent simultaneous cathodic iontophoresis (4 mA, 40 min) or passive application (0 mA, 40 min) on contralateral limbs. Following all applications, tibiotarsal joint synovium was collected. Local venous blood samples were also collected from the iontophoretic and passive application sites for analysis of plasma drug concentrations. Because of the potential for conversion of dexamethasone-phosphate to dexamethasone, an extraction and analysis protocol was developed for both chemicals. The technique demonstrated a linear range of detection (0.39-12 microg/mL) and a capability for measuring both chemicals in plasma and synovium. Conversion of dexamethasone-phosphate to dexamethasone occurred during synovial incubation (37 degrees C) and following freeze-thaw cycles. In contrast to the measurable synovial concentrations of dexamethasone-phosphate (2.3 +/- 0.96 mg/mL) and dexamethasone (0.27 +/- 0.07 mg/mL) following injection, neither drug was detected in the synovium or the local venous blood following iontophoretic or passive applications. In conclusion, these results do not confirm iontophoretic or passive delivery of measurable dexamethasone-phosphate into the tibiotarsal joint using current clinical methods.

Cytokine mRNA in the joints and draining lymph nodes of rats with adjuvant arthritis and effects of cyclosporin A.

TNF-alpha and IL-1beta promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF-alpha and IFN-gamma mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1beta and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1beta and TNF-alpha throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-alpha, IL-1beta, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF-alpha levels were reached on d20, while IL-1beta peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN-gamma mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1beta or TNF-alpha was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN-gamma mRNA in synovium being analogous to human rheumatoid arthritis.

Lead concentration in the bones of the feral pigeons (Columba livia): sources of variation relating to body condition and death.

This paper reports on lead concentrations in the tarsometatarsi of 84 individuals of adult feral pigeons Columba livia found dead or experimentally captured in Bratislava, Slovakia. The interrelationships between lead concentrations and time of death, place of death, body measurements, sex, condition, and plumage phenotype were investigated. Size and shape of pigeons was not correlated with bone lead contamination. Sex and plumage color and pattern were slightly associated with variation in bone lead levels, females and melanic (urban) phenotypes tending to have higher bone lead concentrations than males and blue-bar (wild) individuals. Birds with antibodies to chlamydiae did not contain significantly higher lead concentrations in the tarsometatarsi than birds without antibodies. Concentration of lead in tarsi was significantly higher in birds dying in winter, compared to birds dying at the end of summer. Chronic lead poisoning probably causes mortality in pigeons in winter. The natural stressor, cold weather, has the capability of exacerbating the effects of lead poisoning, and the mortality is due to lead exposure coupled with cold stress.

Regulation of growth region cartilage proliferation and differentiation by perichondrium.

Endochondral bone formation in vertebrates requires precise coordination between proliferation and differentiation of the participating chondrocytes. We examined the role of perichondrium in this process using an organ culture system of chicken embryonic tibiotarsi. A monoclonal antibody against chicken collagen type X, specifically expressed by hypertrophic chondrocytes, was utilized to monitor the terminal differentiation of chondrocytes. Proliferation of chondrocytes was examined by a BrdU-labeling procedure. The absence of perichondrium is correlated with an extended zone of cartilage expressing collagen type X, suggesting that the perichondrium regulates chondrocyte hypertrophy in a negative manner. Removal of perichondrium, in addition, resulted in an extended zone of chondrocytes incorporating BrdU, indicating that the perichondrium also negatively regulates the proliferation of chondrocytes. Partial removal of perichondrium from one side of the tibiotarsus led to expansion of both the collagen type X-positive domain and the BrdU-positive zone at the site of removal but not where the perichondrium remained intact. This suggests that both types of regulation by the perichondrium are local effects. Furthermore, addition of bovine parathyroid hormone (PTH) to perichondrium-free cultures reversed the expansion of the collagen type X-positive domain but not that of the proliferative zone. This suggests that the regulation of differentiation is dependent upon the PTH/PTHrP receptor and that the regulation of proliferation is likely independent of it. Taken together, these results are consistent with a model where perichondrium regulates both the exit of chondrocytes from the cell cycle, and their subsequent differentiation.

Degenerative joint disease in poultry--differences in composition and morphology of articular cartilage are associated with strain susceptibility.

The morphology and basic biochemical composition of articular cartilage from two strains of fowl were examined. Broiler breeder fowl are considered susceptible to degenerative joint disease (DJD); histological examination of one-year-old broiler breeders showed in some samples, articular cartilage thinning, fibrillation and chondrocyte cluster formation, features considered typical of DJD. Examination of similar samples from laying strain fowl showed only minor age-related changes such as some slight cartilage thinning and very mild fibrillation. The articular cartilage from the broiler breeder birds was significantly more hydrated with a higher uronic acid content than that of the laying strain birds. In addition, unloaded articular surfaces such as the proximal humerus had significantly higher amounts of uronic acid than the loaded cartilage surfaces of the proximal tarsometatarsus and the distal tibiotarsus; this suggested that the joint loading may have a role in any biochemical differences found between joints and between strains of fowl. These findings concur with other reports in mammals that showed increased hydration and uronic acid in association with early DJD and in models of osteoarthritis (OA). Thus, despite some differences between avian and mammalian articular cartilage, studies on avian DJD may give insights into mammalian disease.

Relationships between the bone pathologies, ash and mineral content of long bones in 35-day-old broiler chickens.

Histological examinations and estimations of the contents of ash, phosphorus and calcium were used to investigate the femora and tibiotarsi from lame and normal 35-day-old broilers from Holland, Northern Ireland and Scotland. The prevalence of different pathologies varied with the source of the broilers and there were correlations between histological and bone ash values. The most common condition causing lameness was bacterial infection within the physis and cartilaginous epiphysis (bacterial chondronecrosis) of the proximal tibiotarsus, and there was a possible link between rickets attributable to a relative phosphorus deficiency and this condition. There were wide variations between birds in the cortical bone quality as assessed histologically and by estimates of the bone ash content and phosphorus to calcium ratios. Theses variations may be related to different probabilities of bone fracture.

Projections of ankle joint afferents to the spinal cord and brainstem of the chicken (Gallus g. domesticus).

The projections of the ankle joint capsule afferents were studied by transganglionic transport of horseradish peroxidase injected directly into the ankle joint. The number and size of the labelled dorsal root ganglion cells were measured from synsacral nerves 2-9. In the dorsal root ganglia, all sizes of sensory neurones were labelled, and the largest number of labelled cells was in ganglia 5-7. The extensive sympathetic innervation of the ankle joint was identified by the large number of cell bodies labelled in the sympathetic ganglia of the paravertebral chain. Labelled afferent fibres projected to the spinal cord from the 2nd to the 8th synsacral nerves, with the rostral projection mainly via Lissauer's tract and the dorsal funiculus. Terminal labelling in the dorsal horn was identified in laminae I-III and VI, with a slight projection to V. Two areas of dense labelling, which did not correspond with the largest number of labelled dorsal root ganglion cells, were identified. A rostral area with the highest density of label was observed at the level of synsacral nerves 3-4 and a second slightly less dense area between synsacral nerves 7-8. In the caudal medulla, diffuse terminal labelling was observed in the nucleus gracilis et cuneatus, nucleus of the tractus solitarius, and the nucleus cuneatus externus. These results are discussed in a comparative context to identify similarities and differences between different primary afferent projections in birds and mammals and to highlight the possible functional significance of the avian articular afferent projection.