In search of a counter you can count on: relative efficacy of human visual and automated colony counting.

To evaluate comparative efficiency of traditional vs automated colony counting methods, cultures of Escherichia coli (ATCC 25945), Staphylococcus epidermidis (ATCC 12225), Streptococcus pyogenes (ATCC19615) and Streptococcus pneumoniae (ATCC49619) were prepared as pure cultures and mixed cultures at 0·5 McFarland standard and serial dilutions were performed. Plates were inoculated in triplicate with 50, 125, 250 and 500 colony forming units and counted by four researchers, visually and using each of the automated counters. Colony count and counting time were recorded. The pattern of efficiency for all bacterial species was similar: plates with low counts were accurate and quick to count for all methods, with an increase in time and a decrease in accuracy and precision as counts rose. Higher counts of single round colonies required less time and had greater precision with automated counters than human visual counting counts with no loss of accuracy; however, counts were reduced in accuracy and increased in time for species with less regular morphology or when plates had mixed species. Surprisingly, a free phone application was only slightly less precise and more time consuming than the high-end professional counter indicating that automation may be achievable at lower cost than expected. SIGNIFICANCE AND IMPACT OF THE STUDY: Colony quantification is essential in clinical and research settings as well as pedagogy at the college level. Human visual (HV) counting, the most common method, is time consuming and fraught with errors. The time, accuracy and precision of HV counting were compared to a high-end professional automated counter, an inexpensive phone application and a free phone application. Low cost benefits of increased speed and accuracy with automated counting are maximized when counting single round colonies; but much reduced if colonies have irregular morphology or demonstrate haemolysis.

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.

Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 1.

The synthesis and testing of potential multisubstrate inhibitors of tyrosine-specific protein kinases are described. One of the substrates, ATP, was mimicked by the known kinase inhibitor 5'-[4-(fluorosulfonyl)benzoyl]adenosine, which was covalently linked via the sulfonyl moiety to tyrosine mimics. The resulting multisubstrate inhibitors were tested for their ability to inhibit the transfer of phosphate from ATP to a protein acceptor by p60v-abl, the tyrosine kinase encoded by the transforming gene (v-abl) of the Abelson murine leukemia virus (A-MuLV). Although the series of inhibitors displayed moderately potent activity (IC50 values as low as 19 microM), the absence of large effects produced by modification of the tyrosine mimic suggests that they do not behave as multisubstrate inhibitors but bind primarily through the adenosine moiety common to all the inhibitors. This interpretation is strengthened by the finding that the inhibitors lack specificity, inhibiting a serine kinase at comparable concentrations.

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 2.

Tyrosine-specific protein kinases that transfer the terminal phosphate from ATP to protein acceptors are associated with certain transforming viruses and cell surface growth factor receptors. Here we describe the synthesis and testing of potential multisubstrate inhibitors of this class of enzymes. The inhibitors were prepared by covalent attachment of the terminal phosphate of ATP or its tetraphosphate analogue to tyrosine mimics. Testing against p60v-abl, the tyrosine kinase from the Abelson murine leukemia virus, showed that the series of inhibitors was moderately potent (IC50 values as low as 13 microM). However, structural modification of the tyrosine mimic, including replacement with a serine-like moiety, had little effect on potency. It is therefore concluded that the ATP moiety is largely responsible for binding and that the enzyme requires additional structural features for recognition of the tyrosine-containing substrate.

Antagonism of TGF-alpha receptor binding and TGF-alpha induced stimulation of cell proliferation by methyl pheophorbides.

Over 4,500 natural product extracts were screened for their abilities to inhibit binding of radiolabeled TGF-alpha to A431 cells; several plant extracts were identified as potential leads with IC50 values of less than 30 micrograms/mL. The active components of one extract were purified to homogeneity and identified as the porphyrin structures, methyl pheophorbides a and b. These compounds inhibited both TGF-alpha receptor binding and the TGF-alpha induced proliferation of NRK-49F cells in soft agar. To construct a structure-function relationship, a series of commercially available porphyrin derivatives was evaluated. The most potent compound, hematoporphyrin IX, inhibited TGF-alpha functions in a dose-dependent fashion with IC50 values slightly lower than the methyl pheophorbides. Further studies revealed that inhibition of TGF-alpha binding was light dependent and that inhibition did not involve direct competition of porphyrins for the TGF-alpha binding site. To determine the specificity of inhibition, the porphyrins were tested in a number of other receptor-ligand assays. TNF-alpha and beta-adrenoceptor bindings were unaffected, whereas IL-1 beta binding to EL-4 membranes and platelet-derived growth factor induced thymidine incorporation in NIH-3T3 cells were both antagonized by the most active porphyrins. Inhibition of TGF-beta binding to NRK-49F cells and TGF-beta-induced growth of AKR-2B cells was also observed. In summary, we report that methyl pheophorbides are naturally occurring, photodynamic antagonists of TGF-alpha, and although the inhibitory properties of these molecules were not confined to TGF-alpha alone, some level of receptor selectivity was observed.

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

Inhibition of TGF alpha-induced second messengers by anti-EGF receptor antibody-425.

Monoclonal antibody 425 binds to a protein epitope of the human EGF receptor and blocks EGF dependent functions such as EGF receptor phosphorylation and mitogenesis (1). We now show that MAb 425 blocks TGF alpha-induced second messenger signals, namely inositol 1,4,5 triphosphate and Ca2+ in two carcinoma cell lines, A 431 and SW-948. In this study we have further characterized the specificity of this antibody for inhibiting TGF alpha induced mitogenesis in MRC-5, a EGF-receptor expressing fibroblast cell line.

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function.

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.

Growth-inhibitory activity of interferon-beta against human colorectal carcinoma cell lines.

Recombinant human interferon beta (rIFN-beta) inhibited in a time- and dose-dependent manner the proliferation of 18/18 human colon carcinoma cell lines in monolayer culture and 8/9 lines in a soft agar assay but had no effect on 4 human fibroblast cell lines. Maximal inhibition of cell proliferation by rIFN-beta required repetitive treatment (every 2 days) with lymphokine (50 units/ml). Furthermore, the inhibitory activity of rIFN-beta was neutralized by polyclonal antibodies against natural IFN-beta. In contrast to rIFN-beta, rIFN-alpha was inactive against all colon cell lines tested, and rIFN-gamma, with the exception of HT-29 cells, was similarly ineffective. These data demonstrate that rIFN-beta is a potent growth inhibitor of colon carcinoma cells in vitro, and suggest that studies on its mechanism of action may lead to a better understanding of the regulation of colon tumor cell proliferation.

Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by the microphthalmia transcription factor family.

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix-loop-helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found-and TFE3 has been suggested-to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase.

We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).

Structure-function analysis of human transforming growth factor-alpha by site-directed mutagenesis.

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

Disease-modifying activity of SK&F 106615 in rat adjuvant-induced arthritis. Multiparameter analysis of disease magnetic resonance imaging and bone mineral density measurements.

OBJECTIVE: To evaluate the effect of SK&F 106615 on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced in Lewis rats on day 0, and the animals were treated either prophylactically (days 0-16 or days 0-23) or therapeutically (days 10-23) with SK&F 106615. Efficacy was determined by measurements of paw inflammation, bone mineral density (BMD) using dual x-ray absorptiometry, and magnetic resonance imaging (MRI). Joint integrity was also determined histologically, and serum interleukin-6 (IL-6) levels were measured as a marker of the antiinflammatory effects of the compound. RESULTS: Prophylactic treatment (days 0-16) of AIA rats with SK&F 106615 significantly inhibited paw volume at doses of 545 mg/kg/day given orally on 5 days each week. Extensive evaluation of joint integrity in rats treated with SK&F 106615 20 mg/kg/day orally for 23 days showed inhibition of paw volume, normalization of BMD, and significant improvement in disease by MRI and histologic assessment compared with the AIA controls. Elevated levels of serum IL-6 in AIA rats were reduced dramatically by SK&F 106615. Therapeutic treatment (days 10-23) resulted in similar protective effects measured by paw inflammation, BMD, and MRI. In the therapeutic protocol, serum IL-6 appeared to be a more sensitive marker of antiinflammatory activity than paw edema. CONCLUSION: Symptoms of AIA in rats are significantly reduced by prophylactic and therapeutic treatment with SK&F 106615. Of particular note, this compound appears to exert a protective effect on joint integrity and to have disease-modifying properties.

Cloning and characterization of a novel integrin beta3 subunit.

We have identified a novel integrin beta3 subunit, termed beta3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3'-untranslated region of the beta3C subunit differs from the previously reported beta3A (platelet) and beta3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The beta3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the beta3A gene. HEK 293 cells were stably co-transfected with alphaV and either beta3C (HEKbeta3C) or beta3A (HEKbeta3A). The viability of HEKbeta3C cells was lower than that of HEKbeta3A cells, and HEKbeta3C cells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both alphaVbeta3A and alphaVbeta3C isoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKbeta3A, HEKbeta3C cells failed to adhere to osteopontin, an alphaVbeta3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the beta3 integrin in cell adhesion and suggest a potential role for the beta3C integrin subunit in modulating cell-matrix interactions.

A novel human bone marrow stroma-derived cell line TF274 is highly osteogenic in vitro and in vivo.

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-beta), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and beta-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased 45Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OKa blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo.

SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridim idi n-4-yl)imidazole; IC(50) = 44 nM vs. p38 alpha], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3-30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC(50) of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis.

OBJECTIVE: To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS: SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION: Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

Idoxifene, a novel selective estrogen receptor modulator, is effective in a rat model of adjuvant-induced arthritis.

Idoxifene, a selective estrogen receptor modulator, was evaluated in male and female rats with adjuvant-induced arthritis (AA). AA was induced in Lewis rats with Mycobacterium butyricum in paraffin oil injected into the base of the tail, and the animals were treated with idoxifene prophylactically (days 0-21) or therapeutically (days 10-21). Efficacy was determined by measurements of paw inflammation, bone mineral content, and bone mineral density (BMD) with dual X-ray absorptiometry and by histological evaluation. Serum interleukin-6 levels were measured as a marker of the anti-inflammatory effects of the compound. Estrogen was included for comparison and was administered at 5 mg/kg, three times a week s.c. Prophylactic treatment of male AA rats with idoxifene at 10, 3, and 1 mg/kg and estrogen at 5 mg/kg significantly inhibited paw inflammation. There was improved joint integrity measured by BMD and reduced serum interleukin-6 levels in animals treated with 10 mg/kg/day idoxifene. Idoxifene and estrogen were as effective for AA in female Lewis rats as in male rats, significantly inhibiting paw inflammation and improving BMD. Histological evaluation of the tibiotarsal joints of female rats treated with 10 mg/kg showed protection of bone, cartilage, and soft tissue. Therapeutic treatment with either idoxifene or estrogen (starting on day 10 of disease) of male and female Lewis rats also was effective in reducing paw inflammation in these animals, although the effect was much less than that observed with the prophylactic dosing protocol.