On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils.

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.

Tumor bone diseases: molecular mechanisms and opportunities for novel treatments.

A variety of cancers are associated with bone. Primary tumors can arise in bone, common cancers, such as those of breast and prostate origin, metastasize to bone, and multiple myeloma neoplastic disease affects bone profoundly. The cellular and molecular mechanisms underlying these pathological processes are increasingly being understood. The interaction of tumor cells with bone cells, osteoblasts and osteoclasts, and with the bone local environment is a new promising direction in research, which should help to develop new therapies. In this article we will relate the newest developments in the molecular research to the pathology of the tumor bone disease. Potential new targets for drugs, aimed specifically at tumor bone diseases, will be highlighted. Furthermore, we will describe the existing compounds that are either used in treatment or have a potential as therapeutic agents, such as bisphosphonates, Src inhibitors, and selective estrogen receptor modulators.

A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation of its major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo.

The tyrosine kinase Src has been implicated in the process of osteoclast-mediated bone resorption. Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitro, CGP77675 inhibited phosphorylation of peptide substrates and autophosphorylation of purified Src (concentration producing half-maximal inhibition [IC50] values 5-20 and 40 nmol/L, respectively). The compound was selective toward other protein kinases: the Src IC50 value was lower than those for Cdc2 (>500-fold), epidermal growth factor (EGF) receptor (7.5-fold), and vascular endothelial growth factor receptor (>50-fold), and for v-Abl (15-fold) and focal adhesion kinase (Fak) (>25-fold). The Src kinase family members Lck and Yes were inhibited with IC50 values 20-fold higher than or equal to Src. To measure the inhibition of cellular Src activity, we identified the major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin. CGP77675 potently inhibited tyrosine phosphorylation of the Src substrates Fak and paxillin, but had much less effect on Src (IC50 values 0.3, 0.5, and 5.7 micromol/L). The phosphorylation of Src in IC8.1 cells reflected phosphorylation of the negative regulatory tyrosine 527 (Y527); thus, the inhibitor was selective against the Y527 C-terminal Src kinase Csk. In osteoblastic MC3T3-E1 cells, CGP77675 inhibited signaling induced by PDGF at the receptor level, but not signaling by EGF, basic fibroblast growth factor, insulin-like growth factor-1, and phorbol 12-myristate 13-acetate. The effect of CGP77675 on bone resorption was evaluated in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 micromol/L. CGP77675 dose-dependently reduced the hypercalcemia induced in mice by interleukin-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated with elevated bone loss.

A rat model of bone cancer pain.

This study describes the first known model of bone cancer pain in the rat. Sprague-Dawley rats receiving intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) syngeneic MRMT-1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10-14 after inoculation of 3 x 10(3) MRMT-1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate-resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat-killed MRMT-1 cells. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) MRMT-1 cells, heat-killed cells or vehicle did not show changes in body weight and core temperature over 19-20 days. The general activity of animals after injection with live or heat-killed MRMT-1 cells was higher than that of the control group, however, the activity of the MRMT-1 treated group declined during the progress of the disease. Rats receiving intra-tibial injections of MRMT-1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12-14 or 10-12 following injection of 3 x 10(3) or 3 x 10(4) cells, respectively. These symptoms were not observed in rats receiving heat-killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti-nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1-3mg/kg, subcutanously (s.c.)) produced a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra-tibial injections of 3 x 10(3) MRMT-1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10-30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3 x 10(3) MRMT-1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT-1 mammary tumour cell line provides a valid pre-clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX-2 inhibitor, celebrex, has no influence on the pain-related behavioural changes in this model.

The bisphosphonate zoledronate prevents vertebral bone loss in mature estrogen-deficient rats as assessed by micro-computed tomography.

The effect of long-term treatment with the bisphosphonate zoledronate on vertebral bone architecture was investigated in estrogen-deficient mature rats. 4-month-old rats were ovariectomized and development of cancellous osteopenia was assessed after 1 year. The change of bone architectural parameters was determined with a microtomographic instrument of high resolution. After 1 year of estrogen-deficiency, animals lost 55% of vertebral trabecular bone in comparison to sham operated control animals. Trabecular number (Tb.N) and trabecular thickness (Tb.Th) were significantly reduced in ovariectomized animals, whereas trabecular separation (Tb.Sp), bone surface to volume fraction (BS/BV) and trabecular bone pattern factor (TBPf) were significantly increased, indicating a loss of architectural integrity throughout the vertebral body. 3 groups of animals were treated subcutaneously with zoledronate for 1 year with 0.3, 1.5 and 7.5 microgram/kg/week to inhibit osteoclastic bone degradation. Administration started immediately after ovariectomy and treatment dose-dependently prevented the architectural bone deterioration and completely suppressed the effects of estrogen deficiency at the higher doses. The results show that microtomographic determination of static morphometric parameters can be used to quantitate the effects of drugs on vertebral bone architecture in small laboratory animals and that zoledronate is highly effective in this rat model.

Effect of imidazole on prostaglandin and thromboxane accumulation in urate arthritis.

High concentrations of prostaglandins E2 and F2alpha and much lower concentrations of thromboxane B2 occurs in joint washes of chicken 1--3 h after intra-articular injection of urate crystals. Pretreatment with 200 mg/kg imidazole i.v. reduced the concentration of prostaglandins and of thromboxane B2 in the joint washes significantly. Simultaneously, leucocyte invasion was delayed and development of oedema was inhibited. The results suggest that in urate crystal arthritis the effect on prostaglandin and thromboxane accumulation at the site of inflammation contributes to the anti-inflammatory activity of imidazole.

Leukotrienes reduce nociceptive responses to bradykinin.

The biotransformation of arachidonic acid leads to two important groups of inflammatory mediators, the leukotrienes and the prostaglandins. Hyperalgesic effects have been demonstrated for prostaglandins in a variety of animal models but the effects of leukotrienes on inflammatory pain are less well documented. Using the isolated rabbit ear model of algesia we have shown that perfusion of the ear with the leukotrienes B4, C4 and D4 (10(-8)-10(-7) M) causes a reversible, dose- and time-dependent reduction of the reflex fall in systemic blood pressure and the "head flick" response induced by injection of bradykinin (400 ng), without affecting similar responses induced by the neurotransmitter acetylcholine. The antagonistic effect of leukotrienes on the algesic action of bradykinin could be reversed by the leukotriene antagonist FPL55712 (2 micrograms/ml). This result implies that the leukotrienes may have a desensitizing effect on the nociceptor during an inflammatory response in contrast to the pain threshold-lowering action of the E- and I-type prostaglandins.

Neutralization of interleukin-1 beta activity in vivo with a monoclonal antibody alleviates collagen-induced arthritis in DBA/1 mice and prevents the associated acute-phase response.

Interleukin-1 (IL-1) has been implicated in the development and progression of a variety of acute and chronic inflammatory diseases. Due to its pro-inflammatory and tissue-degrading activities, IL-1 is regarded as a major mediator of chronic inflammatory joint diseases, including rheumatoid arthritis in man, adjuvant arthritis in rats and collagen-induced arthritis in mice. However, conclusive experimental evidence for the crucial role of IL-1 in the development of joint destruction has not been presented as yet. In the present study, we investigated the effect of a neutralizing monoclonal mouse antibody against mouse IL-1 beta (IgG1 isotype) on the development and progression of collagen-induced arthritis in DBA/1 mice. The antibody was injected intraperitoneally 3 times a week, either from day 3 or from day 21 after primary immunization, to day 60. In the positive control group an arthritis incidence of 80% was observed after 60 days. The injection of a control antibody of the same isotype did not influence the incidence of arthritis, whereas injection of anti-IL-1 beta from day 21 reduced the arthritis incidence to about 30%. Injection of anti-IL-1 beta starting at day 3 totally prevented both the development of arthritis and the associated increase of the acute phase protein serum amyloid P (SAP). Anti-collagen antibody titers, which increased significantly after immunization, were not influenced by the injection of anti-IL-1 beta antibodies, in spite of the suppressive effect on arthritis development.(ABSTRACT TRUNCATED AT 250 WORDS)

A sulphonamido-indanone derivative CGP 28237 (ZK 34228), a novel non-steroidal anti-inflammatory agent without gastro-intestinal ulcerogenicity in rats.

CGP 28237 (5-methylsulphonylamino-6-phenoxy-1-indanone) belongs to a series of structurally novel indanones. The compound is a weak acid (pK = 6.98), but it does not contain a carboxylic group. CGP 28237 exhibits potent anti-inflammatory activity in developing and established adjuvant arthritis in rats (ED40 approximately 0.5 mg/kg p.o.) and good activity in carrageenin oedema (ED40 approximately 3 mg/kg p.o.). It inhibits yeast-induced fever in rats with ED50 values of 1, 2 and 10 mg/kg p.o. at 1, 3 and 5 hours after drug administration. The antinociceptive activity in mice (phenyl-p-benzoquinone writhing) and rats (acetic-acid writhing) is weak. CGP 28237 has been shown to be non-ulcerogenic in rats under acute and chronic test conditions: it does not cause mucosal lesions in the stomach at 2 X 400 mg/kg p.o., it does not enhance gastro-intestinal blood loss during 10 days' oral treatment with 400 mg/kg p.o., and it did not induce gastro-intestinal lesions in a 4-week toxicity study up to 1000 mg/kg p.o. Although CGP 28237 is not a cyclooxygenase inhibitor in bovine seminal vesicle microsomes, it inhibits prostaglandin synthesis in zymosan-stimulated murine macrophages (IC50 approximately 3 X 10(-6) mol/l) and protects rabbits against arachidonic acid-induced lung embolism with 10 mg/kg p.o. CGP 28237 may represent a novel anti-inflammatory drug with excellent gastro-intestinal tolerability.

[Similarities and differences in the therapy of drug-dependent and alcoholic patients: advantages and disadvantages of combined treatment]. / Gemeinsamkeiten und Unterschiede in der Therapie von Drogensüchtigen und Alkoholikern: Vor- und Nachteile der gemeinsamen Behandlung.

Alcoholics and (other) drug addicts show similar and different qualities. Differences affecting their "combined treatment" are based mainly on age and life style and much less on pharmacological nature of the drug concerned. Therapists and patients experience certain problems in "combined treatment" of addicts and alcoholics but also an extension of mental horizon-having to deal with such a heterogeneous population. Close, daily contact with older, more mature, experienced alcoholics offers young drug addicts an opportunity for a more rapid development of motivation, cooperation with treatment, and emotional maturation, than may be possible in a community formed by young addicts only. According to Wanke, in drug dependence the foundation of scientific understanding is even more insecure than in dependence on alcohol and medicaments. He quotes Lothar Schmidt's view that it is important to create a motivating framework for the patient (during his treatment) which determines motives for lifelong abstinence and maturation of personality. Possibly it is the close collaboration with older alcoholics which creates this motivating framework for certain young drug misusers.

Arthritis in MRL/LPR mice and in collagen II sensitized DBA-1 mice and their use in pharmacology.

For assessment of anti-arthritic drugs, we used mice of the autoimmune MRL/lpr strain and DBA-1 mice sensitized with collagen type II. Our studies showed that in these two mouse arthritis models, unlike the classical rat adjuvant arthritis, the nonsteroidal antiinflammatory compounds tested were either ineffective or minimally affected a chosen number of humoral parameters and the incidence and severity of arthritis. Interestingly, both arthritis models showed a distinct pharmacological pattern in response to the slow-acting anti-rheumatic drugs, whereas steroids and cyclophosphamide inhibited both arthritic processes. Therefore, these two mouse models are useful for studying the immunopathological events operating in chronic inflammation and could potentially serve the characterization of new antirheumatic drugs.

A family systems nursing approach to premenstrual syndrome.

The paper presents a family systems nursing approach to premenstrual syndrome. The interaction between premenstrual syndrome and family dynamics becomes the focus for the clinical nurse specialist and her team. A case example illustrates the use of the interview guidelines of hypothesizing, circularity and neutrality and various systemic-strategic interventions. through this process the couple's maladaptive patterns of interaction are interrupted. Symptom prescription, symptom sharing, symptom escalation, and positive connotation are among the creative interventions used to release premenstrual syndrome from its triangulated position in the marriage.