The three major forms of immune‐mediated inflammatory myopathy are dermatomyositis (DM), polymyositis (PM), and inclusion‐body myositis (IBM). They each have distinctive clinical and histopathologic ...features that allow the clinician to reach a specific diagnosis in most cases. Magnetic resonance imaging is sometimes helpful, particularly if the diagnosis of IBM is suspected but has not been formally evaluated. Myositis‐specific antibodies are not helpful diagnostically but may be of prognostic value; most antibodies have low sensitivity. Muscle biopsy is mandatory to confirm the diagnosis of an inflammatory myopathy and to allow unusual varieties such as eosinophilic, granulomatous, and parasitic myositis, and macrophagic myofasciitis, to be recognized. The treatment of the inflammatory myopathies remains largely empirical and relies upon the use of corticosteroids, immunosuppressive agents, and intravenous immunoglobulin, all of which have nonselective effects on the immune system. Further controlled clinical trials are required to evaluate the relative efficacy of the available therapeutic modalities particularly in combinations, and of newer immunosuppressive agents (mycophenolate mofetil and tacrolimus) and cytokine‐based therapies for the treatment of resistant cases of DM, PM, and IBM. Improved understanding of the molecular mechanisms of muscle injury in the inflammatory myopathies should lead to the development of more specific forms of immunotherapy for these conditions. Muscle Nerve 27:407–425, 2003
Many cancers express an array of chemokines which have the capacity to modulate the nature and function of intratumoural leukocyte infiltrates. In malignant mesothelioma (MM) neither the chemokine ...signalling networks nor their regulation have been investigated despite the prominence of leucocytic infiltrates in both clinical and experimental tumours. In this study, we examined constitutive and cytokine-regulated expression of CC and CXC chemokine genes in mesothelioma and mesothelial cell cultures derived from two different mouse strains (BALB/C and CBA/CaH). In mouse MM and mesothelial cells MCP-1/JE, GRO-α/KC and RANTES were expressed whereas MIP-1α and MIP-2 were infrequently expressed. Comparison of basal chemokine expression showed that GRO-α/KC mRNA was overexpressed in the malignant cells whereas MCP-1 gene expression and release was downregulated. Treatment of mesothelioma cells with IL-4, IFN-γ or TNF-α revealed that chemokine genes could be more responsive to cytokines in the malignant compared to their mesothelial cells. TNF-α was consistently the most potent positive regulator of both CC and CXC chemokine expression and MCP-1 release. The present study for the first time provides a mechanistic insight into the differential regulation of chemokine expression in malignant mesothelioma cells and has implications for mesothelial chemokine signalling in mouse models.
Abstract Susceptibility to sIBM is strongly associated with the HLA-DRB103 allele and the 8.1 MHC ancestral haplotype (HLA-A1, B8, DRB103) but little is known about the effects of allelic ...interactions at the DRB1 locus or disease-modifying effects of HLA alleles. HLA-A, B and DRB1 genotyping was performed in 80 Australian sIBM cases and the frequencies of different alleles and allele combinations were compared with those in a group of 190 healthy controls. Genotype–phenotype correlations were also investigated. Amongst carriers of the HLA-DRB103 allele, DRB103/01 heterozygotes were over-represented in the sIBM group ( p < 0.003) while. DRB103/04 heterozygotes were under-represented ( p < 0.008). The mean age-at-onset (AAO) was 6.5 years earlier in DRB103/01 heterozygotes who also had more severe quadriceps muscle weakness than the rest of the cohort. The findings indicate that interactions between the HLA-DRB103 allele and other alleles at the DRB1 locus can influence disease susceptibility and the clinical phenotype in sIBM.
PURPOSE OF REVIEWThe pathogenesis of sporadic inclusion body myositis is complex and the disease has a relentless course. Recent observations regarding possible mechanisms of disease may provide ...targets for therapy.
RECENT FINDINGSEvidence is strengthening that specific T-cell and B-cell responses are ongoing in skeletal muscle in sporadic inclusion body myositis and that cytokines and chemokines generated by an autoimmune response are likely to influence antigen presentation by intramuscular dendritic cells and muscle cells, expression of amyloid precursor protein and the endoplasmic reticulum stress response. Early β-amyloid expression and perhaps aberrant expression of protein processing enzymes, such as E3 ligases, seem to be involved in the myopathic process. NF-κB activation by endoplasmic reticulum stress and cytokine action further stimulates amyloid precursor protein production, exacerbates endoplasmic reticulum stress and increases myostatin content in muscle contributing to muscle atrophy.
SUMMARYUnderstanding the paradoxes in sporadic inclusion body myositis is important in determining rational therapy for the disease. Amyloid precursor protein is expressed in muscle in other inflammatory muscle diseases but the cellular distribution differs and inclusions do not form so that other metabolic defects seem to be important. Intramuscular immune cells influence muscle function and viability in inclusion body myositis but immunotherapy is ineffective. A useful target for therapy may be restoration of muscle regenerating capacity.
In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human ...airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo‐oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX‐2), both COX‐2 and iNOS may be co‐expressed in response to an inflammatory stimulus. Although regulation of the COX‐2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
The effect of endogenous and exogenous NO on the COX‐2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX‐2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml−1, interleukin‐1β (IL‐1β) 1 u ml−1 and lipopolysaccharide (LPS) 10 μg ml−1 induced nitrite formation which could be inhibited by the competitive NOS inhibitor NG‐nitro‐L‐arginine‐methyl‐ester (L‐NAME). IL‐1β alone (1–50 u ml−1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX‐2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml−1, IL‐1β 1 u ml−1 and LPS 10 μg ml−1 to induce both the iNOS and COX‐2 pathways, and IL‐1β 3 u ml−1 to induce COX‐2 without iNOS activity.
Cells treated with IFNγ 100 u ml−1, IL‐1β 1 u ml−1 and LPS 10 μg ml−1 for 48 h either alone, or with the addition of L‐NAME (0 to 10−2 M), demonstrated inhibition by L‐NAME of PGE2 (3.61±0.55 to 0.51±0.04 pg/104 cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/104 cells; P<0.001) production. Restoration of the PGE2 response (0.187±0.053 to 15.46±2.59 pg/104 cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L‐NAME 10−6 M, but with the addition of the NOS substrate L‐arginine (0 to 10−5 M).
Cells incubated with IL‐1β 3 u ml−1 for 6 h, either alone or with addition of the NO donor S‐nitroso‐acetyl‐penicillamine (SNAP) (0 to 10−4 M), demonstrated increased PGE2 formation (1.23±0.03 to 2.92±0.19 pg/104 cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL‐1β 3 u ml−1 for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10−3 M) also demonstrated an increased PGE2 response (2.56±0.21 to 4.53±0.64 pg/104 cells; P<0.05).
These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX‐2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.
Abstract Previous studies have differed as to whether APOE ε4 is a susceptibility factor for developing sporadic inclusion body myositis (sIBM), with a positive association being found only in an ...Australian cohort of cases. We have now re-examined this in a larger cohort of 57 sIBM cases and have also carried out a meta-analysis of all the published studies looking for evidence of a risk association or effect of APOE alleles on disease expression. Our findings argue against a specific role for any APOE alleles in conferring susceptibility to sIBM but have demonstrated a non-significant trend towards an earlier age-of-onset in patients with the ε2 allele.
Nitric oxide (NO) synthesized by airway epithelium may be important in the regulation of airway inflammation and reactivity. As such, the expression and localization of nitric oxide synthase (NOS) ...isoforms was assessed in human airway tissue obtained following thoracotomy, and in cultured human airway epithelial (BEAS-2B) cells. NOS expression was determined by reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot analysis, and localized by in situ hybridization and immunohistochemistry. No synthesis by cell cultures was detected by nitrite assay. Endothelial and neuronal constitutive NOS mRNAs were not detected in airways or cell cultures. Inducible NOS (iNOS) mRNA was detected in 5 of 6 airway specimens, and in situ hybridization demonstrated iNOS mRNA expression in columnar epithelial cells. This was confirmed by immunohistochemistry using an iNOS specific antibody. BEAS-2B cell cultures were stimulated with (I) combinations of tumor necrosis factor alpha (TNF alpha) (50-2,000 U/ml)/interferon gamma (IFN gamma) (20-500 U/ml)/lipopolysaccharide (LPS) (10 micrograms/ml) or (2) histamine (10(-3) M-10(-5) M). Cell cultures treated with TNF alpha/IFN gamma/LPS in combination expressed iNOS mRNA, and this was associated with increases in supernatant nitrite concentrations over 24 h; however, this response diminished with successive passage of cells. Histamine treatment did not result in iNOS mRNA expression or detectable NO synthesis. We conclude that iNOS in human airway tissue is localized to the airway epithelium. Cytokine/ LPS stimulation, but not histamine, results in iNOS mRNA expression and NO synthesis in BEAS-2B cells. BEAS-2B cells may not be a suitable model for the study of NO biology in airway epithelium.
Malignant mesothelioma (MM) is an aggressive and highly chemo-resistant tumour. In this study, we examined cisplatin-induced apoptosis in mouse models of this disease and investigated the role of ...constitutive and inducible expression of apoptosis related genes in this process. All of the four mouse MM cell lines examined expressed Bax, Bcl-xL, c-Myc, and caspase-3 but not Bcl-2. Cisplatin-induced apoptosis characterised by DNA fragmentation and cell death while caspase-3/7 was activated in 3 of 4 cell lines. Quantitation of basal gene expression showed significant differences but there was no correlation between single genes and cisplatin sensitivity. In the AC29 and AB1 models, both cisplatin and TNF-α downregulated Bcl-xL gene expression, indicating that this gene was a common transcriptional target in these cells. The findings of the present study provide insights into apoptotic mechanisms in mesothelioma cells and show similar patterns of gene expression to that reported in the human disease.
We have previously prepared two B7-1 transfectant clones (AC29 B7-6 and AC29 B7-7) from the AC29 murine mesothelioma (MM) cell line which displayed markedly different in vivo growth rates and ...susceptibility to cytotoxic T cell killing. Using suppression subtractive hybridisation (SSH), we searched for factors which may determine the biological distinction seen in these clones. We isolated 19 cDNA clones from two SSH generated libraries by screening using subtracted cDNA probes and characterised them using Northern hybridisation, sequencing, RT-PCR and real-time RT-PCR. The 19 cDNA clones comprised 16 different transcripts of which 15 were identified by homology to known genes and one was novel. Expression of a murine endogenous retroviral (mERV) transcript mERV-AC29 was found in the immunogenic AC29 B7-6 clone and parental AC29 but absent in AC29 B7-7. Real-time RT-PCR was used to confirm that galectin-1, the disintegrin/metalloproteinase MDC9 and ribonucleotide reductase M1 were overexpressed in AC29 B7-7. Our results show that SSH is a powerful method for the identification of genes expressed differentially between phenotypically different tumour cell lines or clones. Characterisation of the role of those identified here will provide useful information in understanding genes responsible for differential tumorigenicity.
Objectives
This qualitative study explored the barriers and enablers influencing Western Australian (WA) community pharmacists’ knowledge, confidence, willingness and practice in engaging older ...clients (>60 years) in alcohol‐related health discussions.
Methods
Two focus groups were conducted with a total of 14 community pharmacists who had previously completed a formative quantitative survey (n = 63), and indicated willingness to participate in a follow‐up focus group. Focus group questions, informed by the survey results, explored participants’ perceptions about barriers and enablers to delivering health information and advice about alcohol to older clients (60+ years). Shaw and colleagues’ theoretical framework was used to understand barriers and enablers in relation to role legitimacy, role adequacy and role support.
Key findings
Participants acknowledged that providing health information about alcohol to older clients is a legitimate part of a community pharmacist's role, and most were confident performing this role in situations perceived as core to their professional practice, such as while dispensing medicines. However, many participants identified limited knowledge, skills and confidence in assisting older clients who may have alcohol issues, beyond advising them on medication and alcohol use. Structural barriers such as time and financial barriers were also identified.
Conclusion
Routine professional practice including dispensing medicine and home medicine reviews may provide valuable opportunities to engage older clients in alcohol‐related discussions. However, limited knowledge concerning appropriate strategies to assist older clients reduce their alcohol consumption, coupled with limited skills and confidence among community pharmacists in raising sensitive alcohol‐related issues with clients, suggest the need for specific alcohol‐related training and support.
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