Neural Pathways of Craniofacial Muscle Pain: Implications for Novel Treatments.

Craniofacial muscle pain is highly prevalent in temporomandibular disorders but is difficult to treat. Enhanced understanding of neurobiology unique to craniofacial muscle pain should lead to the development of novel mechanism-based treatments. Herein, we review recent studies to summarize neural pathways of craniofacial muscle pain. Nociceptive afferents in craniofacial muscles are predominantly peptidergic afferents enriched with TRPV1. Signals from peripheral glutamate receptors converge onto TRPV1, leading to mechanical hyperalgesia. Further studies are needed to clarify whether hyperalgesic priming in nonpeptidergic afferents or repeated acid injections also affect craniofacial muscle pain. Within trigeminal ganglia, afferents innervating craniofacial muscles interact with surrounding satellite glia, which enhances the sensitivity of the inflamed neurons as well as nearby uninjured afferents, resulting in hyperalgesia and ectopic pain originating from adjacent orofacial tissues. Craniofacial muscle afferents project to a wide area within the trigeminal nucleus complex, and central sensitization of medullary dorsal horn neurons is a critical factor in muscle hyperalgesia related to ectopic pain and emotional stress. Second-order neurons project rostrally to pathways associated with affective pain, such as parabrachial nucleus and medial thalamic nucleus, as well as sensory-discriminative pain, such as ventral posteromedial thalamic nuclei. Abnormal endogenous pain modulation can also contribute to chronic muscle pain. Descending serotonergic circuits from the rostral ventromedial medulla facilitate activation of second-order neurons in the trigeminal nucleus complex, which leads to the maintenance of mechanical hyperalgesia of inflamed masseter muscle. Patients with temporomandibular disorders exhibit altered brain networks in widespread cortical and subcortical regions. Recent development of methods for neural circuit manipulation allows silencing of specific hyperactive neural circuits. Chemogenetic silencing of TRPV1-expressing afferents or rostral ventromedial medulla neurons attenuates hyperalgesia during masseter inflammation. It is likely, therefore, that further delineation of neural circuits mediating craniofacial muscle hyperalgesia potentially enhances treatment of chronic muscle pain conditions.

The role of TRPA1 in muscle pain and mechanical hypersensitivity under inflammatory conditions in rats.

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is expressed in muscle afferents and direct activation of these receptors induces acute mechanical hypersensitivity. However, the functional role of TRPA1 under pathological muscle pain conditions and mechanisms by which TRPA1 mediate muscle pain and hyperalgesia are not clearly understood. Two rodent behavioral models validated to assess craniofacial muscle pain conditions were used to study ATP- and N-Methyl-D-aspartate (NMDA)-induced acute mechanical hypersensitivity and complete Freund's adjuvant (CFA)-induced persistent mechanical hypersensitivity. The rat grimace scale (RGS) was utilized to assess inflammation-induced spontaneous muscle pain. Behavioral pharmacology experiments were performed to assess the effects of AP18, a selective TRPA1 antagonist under these conditions. TRPA1 expression levels in trigeminal ganglia (TG) were examined before and after CFA treatment in the rat masseter muscle. Pre-treatment of the muscle with AP18 dose-dependently blocked the development of acute mechanical hypersensitivity induced by NMDA and α,ß-methylene adenosine triphosphate (αßmeATP), a specific agonist for NMDA and P2X3 receptor, respectively. CFA-induced mechanical hypersensitivity and spontaneous muscle pain responses were significantly reversed by post-treatment of the muscle with AP18 when CFA effects were most prominent. CFA-induced myositis was accompanied by significant up-regulation of TRPA1 expression in TG. Our findings showed that TRPA1 in muscle afferents plays an important role in the development of acute mechanical hypersensitivity and in the maintenance of persistent muscle pain and hypersensitivity. Our data suggested that TRPA1 may serve as a downstream target of pro-nociceptive ion channels, such as P2X3 and NMDA receptors in masseter afferents, and that increased TRPA1 expression under inflammatory conditions may contribute to the maintenance of persistent muscle pain and mechanical hyperalgesia. Mechanistic studies elucidating transcriptional or post-translational regulation of TRPA1 expression under pathological pain conditions should provide important basic information to further advance the treatment of craniofacial muscle pain conditions.

The role of TRPM2 in hydrogen peroxide-induced expression of inflammatory cytokine and chemokine in rat trigeminal ganglia.

Trigeminal ganglia (TG) contain neuronal cell bodies surrounded by satellite glial cells. Although peripheral injury is well known to induce changes in gene expression within sensory ganglia, detailed mechanisms whereby peripheral injury leads to gene expression within sensory ganglia are not completely understood. Reactive oxygen species (ROS) are an important modulator of hyperalgesia, but the role of ROS generated within sensory ganglia is unclear. Since ROS are known to affect transcription processes, ROS generated within sensory ganglia could directly influence gene expression and induce cellular changes at the soma level. In this study, we hypothesized that peripheral inflammation leads to cytokine and chemokine production and ROS generation within TG and that transient receptor potential melastatin (TRPM2), a well known oxidative sensor, contributes to ROS-induced gene regulation within TG. The masseter injection of complete Freund's adjuvant (CFA) resulted in a significantly elevated level of ROS within TG of the inflamed side with a concurrent increase in cytokine expression in TG. Treatment of TG cultures with H2O2 significantly up-regulated mRNA and protein levels of cytokine/chemokine such as interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 2 (CXCL2). TRPM2 was expressed in both neurons and non-neuronal cells in TG, and pretreatment of TG cultures with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of TRPM2, or siRNA against TRPM2 attenuated H2O2-induced up-regulation of IL-6 and CXCL2. These results suggested that activation of TRPM2 could play an important role in the modulation of cytokine/chemokine expression within TG under oxidative stress and that such changes may contribute to amplification of nociceptive signals leading to pathological pain conditions.

Ablation of galectin-3 induces p27(KIP1)-dependent premature senescence without oncogenic stress.

Premature senescence induced by oncogenic stimuli or tumor suppressor activation plays opposing roles in tumorigenesis. Here, we propose that galectin-3, a ß-galactoside-binding lectin, regulates premature senescence without oncogenic stress. We detected premature senescence, decreased Skp2, and increased p27(KIP1) expression in galectin-3 knockout MEFs and galectin-3-depleted gastric cancer cells. Interestingly, galectin-3 depletion did not affect other senescence inducers such as p14(ARF), p16(INK4A), and p21(WAF1/CIP1), suggesting that galectin-3-regulated senescence is p27(KIP1) dependent. We demonstrate that galectin-3 depletion decreases retinoblastoma protein (Rb) phosphorylation (Ser780, Ser807/811), cyclin D1 and CDK4 expression, and E2F1 transcriptional activation. Galectin-3 directly interacts with the cyclin D1/CDK4 complex and promotes hyperphosphorylation of Rb. It also blocks the inhibition of E2F1 transcription, thereby increasing the expression of Skp2 and reducing the stability of p27(KIP1) to promote the proliferation of gastric cancer cells. Xenograft mice with galectin-3-depleted gastric cancer cells display tumor growth retardation that is reversed by Skp2 overexpression. Increased expression of galectin-3 is also associated with the advanced TNM (tumor, lymph node, metastasis) system, clinicopathological stage, and lymph node metastases. The probability of survival was significantly decreased in gastric cancer patients with galectin-3(high) p27(KIP1-low)cells. Taken together, our results show that galectin-3 may accelerate gastric tumorigenesis by inhibiting premature senescence.

Sex differences in µ-opioid receptor expression in trigeminal ganglia under a myositis condition in rats.

BACKGROUND: Peripheral opioid receptor expression is up-regulated under inflammatory conditions, which leads to the increased efficacy of peripherally administered opioids. Sex differences in the effects of inflammation, cytokines and gonadal hormones on µ-opioid receptor (MOR) expression in trigeminal ganglia (TG) are not well understood. METHODS: MOR mRNA and protein levels in TG from male and female Sprague Dawley rats following complete Freund's adjuvant (CFA)-induced muscle inflammation were assessed. Cytokine-induced changes in MOR mRNA expression from TG cultures prepared from intact and gonadectomized male and female, and gonadectomized male rats with testosterone replacement were examined. Behavioural experiments were then performed to examine the efficacy of a peripherally administered MOR agonist in male, female and gonadectomized male rats under a myositis condition. RESULTS: CFA and cytokine treatments induced significant up-regulation of MOR expression in TG from male, but not from female, rats. The cytokine-induced up-regulation of MOR mRNA expression was prevented in TG from orchidectomized (GDX) male rats, which was restored with testosterone replacement. Peripherally administered DAMGO, a specific MOR agonist, significantly attenuated CFA-induced masseter mechanical hypersensitivity only in intact male rats. CONCLUSIONS: Collectively, these data indicate that testosterone plays a key role in the regulation of MOR in TG under inflammatory conditions, and that sex differences in the anti-hyperalgesic effects of peripherally administered opioids are, in part, mediated by peripheral opioid receptor expression levels.

Peripheral G protein-coupled inwardly rectifying potassium channels are involved in δ-opioid receptor-mediated anti-hyperalgesia in rat masseter muscle.

BACKGROUND: Although the efficacy of peripherally administered opioid has been demonstrated in preclinical and clinical studies, the underlying mechanisms of its anti-hyperalgesic effects are poorly understood. G protein-coupled inwardly rectifying potassium (GIRK) channels are linked to opioid receptors in the brain. However, the role of peripheral GIRK channels in analgesia induced by peripherally administered opioid, especially in trigeminal system, is not clear. METHODS: Expression of GIRK subunits in rat trigeminal ganglia (TG) was examined with reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. Chemical profiles of GIRK-expressing neurons in TG were further characterized. Behavioural and Fos experiments were performed to examine the functional involvement of GIRK channels in δ-opioid receptor (DOR)-mediated anti-hyperalgesia under an acute myositis condition. RESULTS: TG expressed mRNA and proteins for GIRK1 and GIRK2 subunits. Majority of GIRK1- and GIRK2-expressing neurons were non-peptidergic afferents. Inhibition of peripheral GIRK using Tertiapin-Q (TPQ) attenuated antinociceptive effects of peripherally administered DOR agonist, [D-Pen(2), D-Pen(6) ]-enkephalin (DPDPE), on mechanical hypersensitivity in masseter muscle. Furthermore, TPQ attenuated the suppressive effects of peripheral DPDPE on neuronal activation in the subnucleus caudalis of the trigeminal nucleus (Vc) following masseteric injection of capsaicin. CONCLUSIONS: Our data indicate that peripheral DOR agonist-induced suppression of mechanical hypersensitivity in the masseter muscle involves the activity of peripheral GIRK channels. These results could provide a rationale for developing a novel therapeutic approach using peripheral GIRK channel openers to mimic or supplement the effects of peripheral opioid agonist.

The role of androgen receptor in transcriptional modulation of cannabinoid receptor type 1 gene in rat trigeminal ganglia.

We have previously shown that anti-hyperalgesic effects of cannabinoid agonists under inflammatory condition are much greater in male than female, and that inflammatory cytokines upregulate cannabinoid receptor type 1 (CB1) expression in male, but not female, trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we investigated the mechanisms underlying the testosterone-mediated regulation of peripheral CB1 expression. We hypothesized that testosterone upregulates CB1 through transcriptional modulation by androgen receptor (AR). Interleukin-1 beta (IL-1ß), a pro-inflammatory cytokine, upregulated CB1 mRNA expression in TG of male rats. The cytokine-induced upregulation was prevented by the pretreatment with flutamide, a specific antagonist for AR, but not by ICI 182,780, a specific antagonist for estrogen receptor, suggesting that the effects of testosterone are not mediated by estradiol, a testosterone metabolite. The expression levels of AR and IL-1ß receptors were comparable between male and female TG, suggesting that the male specific IL-1ß effects on CB1 upregulation occurs downstream to these receptors. The chromatin immunoprecipitation assay showed AR binding to the CB1 promoter in the rat TG. Furthermore, luciferase reporter assay revealed that AR activated the CB1 gene in response to testosterone or dihydrotestosterone treatment. These experiments provided compelling evidence that testosterone regulates CB1 gene transcription in TG through AR following cytokine stimulation. These results should provide mechanistic bases for understanding cytokine-hormone-neuron interactions in peripheral cannabinoid systems, and have important clinical implications for pain patients in whom testosterone level is naturally low, gradually declining or pharmacologically compromised.

P2X3 and TRPV1 functionally interact and mediate sensitization of trigeminal sensory neurons.

Musculoskeletal pain conditions, particularly those associated with temporomandibular disorders (TMD) affect a large percentage of the population. Identifying mechanisms underlying hyperalgesia could contribute to the development of new treatment strategies for the management of TMD and other muscle pain conditions. In this study, we provide evidence of functional interactions between two ligand-gated channels, P2X3 and transient receptor potential V1 (TRPV1), in trigeminal sensory neurons, and propose that the interactions serve as an underlying mechanism for the development of mechanical hyperalgesia. Mechanical sensitivity of the masseter muscle was assessed in lightly anesthetized rats via an electronic anesthesiometer (Ro et al., 2009). Direct intramuscular injection of a selective P2X3 agonist, alpha,beta-methylene adenosine triphosphate (αßmeATP), induced a dose- and time-dependent hyperalgesia. Mechanical sensitivity in the contralateral muscle was unaffected suggesting local P2X3 mediate hyperalgesia. Anesthetizing the overlying skin had no effect on αßmeATP-induced hyperalgesia confirming the contribution of P2X3 from the muscle. Importantly, the αßmeATP-induced hyperalgesia was prevented by pretreatment of the muscle with a TRPV1 antagonist, AMG9810. P2X3 was co-expressed with TRPV1 in the masseter muscle afferents confirming the possibility for intracellular interactions. Additionally, in a subpopulation of P2Xv/TRPV1 positive neurons, capsaicin-induced Ca(2+) transients were significantly amplified following P2X3 activation. Finally, activation of P2X3 induced phosphorylation of serine, but not threonine, residues in TRPV1 in trigeminal ganglia cultures. Significant phosphorylation was observed at 15 min, the time point at which behavioral hyperalgesia was prominent. Previously, activation of either P2X3 or TRPV1 had been independently implicated in the development of mechanical hyperalgesia. Our data propose P2X3 and TRPV1 interact in a facilitatory manner, which could contribute to the peripheral sensitization known to underlie masseter hyperalgesia.

Activation of peripheral delta-opioid receptors leads to anti-hyperalgesic responses in the masseter muscle of male and female rats.

In this project, we examined peripheral δ-opioid receptor (DOR)-mediated anti-hyperalgesic responses in the context of an acute orofacial muscle pain condition in both male and female rats. We also investigated whether the ATP-sensitive K+ channel (KATP), a downstream target of OR signaling, contributes to DOR-mediated anti-hyperalgesic responses. Local pretreatment of the masseter with a DOR agonist, [D-Pen², D-Pen6]-enkephalin (DPDPE), dose-dependently attenuated capsaicin-induced mechanical hypersensitivity in both male and female rats. However, there were sex differences in the potency of local DPDPE in that a 10-fold higher dose of DPDPE was required in female rats to produce the level of anti-hyperalgesia achieved in male rats. The sex differences in the DPDPE effect may not be fully explained by DOR expression level since there was no significant sex difference in DOR mRNA levels in trigeminal ganglia (TG). Finally, pretreatment of the masseter with the KATP antagonist, glibenclamide, significantly blocked the effects of DPDPE in male rats suggesting that the peripheral DOR effect is mediated by the KATP. These studies revealed novel information about sex differences with regards to peripherally localized DOR-mediated anti-hyperalgesia under an orofacial muscle pain condition.

Lipopolysaccharide-induced pulpitis up-regulates TRPV1 in trigeminal ganglia.

Tooth pain often accompanies pulpitis. Accumulation of lipopolysaccharides (LPS), a product of Gram-negative bacteria, is associated with painful clinical symptoms. However, the mechanisms underlying LPS-induced tooth pain are not clearly understood. TRPV1 is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and hyperalgesia under inflammation or injury. Although TRPV1 is expressed in pulpal afferents, it is not known whether the application of LPS to teeth modulates TRPV1 in trigeminal nociceptors. By assessing the levels of protein and transcript of TRPV1 in mouse trigeminal ganglia, we demonstrate that dentinal application of LPS increases the expression of TRPV1. Our results suggest that the up-regulation of TRPV1 in trigeminal nociceptors following bacterial infection could contribute to hyperalgesia under pulpitis conditions.

Sex differences in the contribution of ATP-sensitive K+ channels in trigeminal ganglia under an acute muscle pain condition.

In this study, we examined whether functional subunits of the ATP-dependent K+ channel (KATP) are expressed in trigeminal ganglia (TG), which contains sensory neurons that innervate oral and facial structures. We also investigated whether direct activation of the KATP effectively attenuates mechanical hypersensitivity in the context of an acute orofacial muscle pain condition. The KATP expression in TG and behavioral studies were conducted in age matched male and female Sprague-Dawley rats. RT-PCR experiments showed that the mRNAs for the inwardly rectifying pore-forming subunits, Kir6.1 and Kir6.2, as well as the regulatory sulfonylurea subunits, SUR1 and SUR2, were reliably detected in TG. Subsequent western blot analysis confirmed that proteins for all four subunits are expressed in TG, and showed that Kir6.2 is expressed at a significantly higher level in male TG compared to that of female rats. This observation was confirmed by the immunohistochemical demonstration of higher percentages of Kir6 positive masseter afferents in female rats. Masseteric injection of capsaicin evokes a time dependent increase in masseter sensitivity to noxious mechanical stimulation. A specific KATP agonist, pinacidil, dose-dependently attenuated the capsaicin-induced mechanical hypersensitivity in male rats. The dose of pinacidil (20 µg) that completely blocked the capsaicin responses in male rats was ineffective in female rats regardless of their estrus phases. Only at the highest dose (300 µg) we used, pinacidil was partially effective in female rats. Similarly, another KATP agonist, diazoxide which targets different KATP subunits also showed sex specific responses in attenuating capsaicin-induced masseter hypersensitivity. These data suggested that sex differences in functional KATP expression in TG may underlie sex specific responses to KATP agonists. The present study provided novel information on sex differences in KATP expression in TG and its contribution under an orofacial muscle pain condition.

Effects of 7,8-dihydro-8-oxo-deoxyguanosine on antigen challenge in ovalbumin-sensitized mice may be mediated by suppression of Rac.

BACKGROUND AND PURPOSE: Earlier we reported that 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), an oxidatively modified guanine nucleoside, exerted anti-inflammatory activity through inactivation of the GTP binding protein, Rac. In the present study, the effects of 8-oxo-dG were investigated on responses to antigen challenge in sensitized mice, as Rac is also involved at several steps of the immune process including antigen-induced release of mediators from mast cells. EXPERIMENTAL APPROACH: Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG during the challenge. Effects of 8-oxo-dG were assessed by measuring lung function, cells and cytokines in broncho-alveolar lavage fluid (BALF) and serum levels of antigen-specific IgE. Rac activity in BALF cells was also measured. KEY RESULTS: 8-oxo-dG inhibited the increased airway resistance and decreased lung compliance of sensitized and challenged mice to the levels of non-sensitized control mice and lowered the increased leukocytes particularly, eosinophils, in BALF. Furthermore, 8-oxo-dG suppressed allergy-associated immune responses, such as raised anti- ovalbumin IgE antibody in serum, increased expression of CD40 and CD40 ligand in lung, increased interleukin-4, -5, -13, interferon-gamma and tumour necrosis factor-alpha in BALF and mRNA levels of these cytokines in BALF cells, dose-dependently. The corresponding purine, 8-oxo-guanine, showed no effects in the same experiments. Finally, 8-oxo-dG, but not 8-oxo-guanine, inhibited the increased Rac activity in sensitized and challenged mice. CONCLUSION AND IMPLICATIONS: 8-Oxo-dG had anti-allergic actions that might be mediated by Rac inactivation. This compound merits further evaluation of its therapeutic potential in allergic asthma.

Role of peripheral mu-opioid receptors in inflammatory orofacial muscle pain.

The aims of this project were to investigate whether inflammation in the orofacial muscle alters mu opioid receptor (MOR) mRNA and protein expressions in trigeminal ganglia (TG), and to assess the contribution of peripheral MORs under acute and inflammatory muscle pain conditions. mRNA and protein levels for MOR were quantified by reverse-transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, from the TG of naïve rats, and compared with those from the rats treated with complete Freund's adjuvant (CFA) in the masseter. TG was found to express mRNA and protein for MOR, and CFA significantly up-regulated both MOR mRNA and protein by 3 days following the inflammation. The MOR protein up-regulation persisted to day 7 and returned to the baseline level by day 14. We then investigated whether peripheral application of a MOR agonist, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin acetate salt (DAMGO), attenuates masseter nociception induced by masseteric infusion of hypertonic saline (HS) in lightly anesthetized rats. DAMGO (1, 5, 10 microg) or vehicle was administered directly into the masseter 5-10 min prior to the HS infusion. The DAMGO effects were assessed on mean peak counts (MPC) and overall magnitude as calculated by the area under the curve (AUC) of the HS-evoked behavioral responses. Under this condition, only the highest dose of DAMGO (10 microg) significantly reduced MPC, which was prevented when H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective MOR antagonist, was co-administered. DAMGO pre-treatment in the contralateral masseter did not attenuate MPC. The same doses of DAMGO administered into CFA-inflamed rats, however, produced a greater attenuation of both MPC and AUC of HS-evoked nocifensive responses. These results demonstrated that activation of peripheral MOR provides greater anti-nociception in inflamed muscle, and that the enhanced MOR effect can be partly explained by significant up-regulation of MOR expression in TG.

Peripheral metabotropic glutamate receptor 5 mediates mechanical hypersensitivity in craniofacial muscle via protein kinase C dependent mechanisms.

We previously demonstrated that peripherally located N-methyl-D-aspartic acid (NMDA) receptors contribute to acute muscle nociception and the development of chronic muscular hyperalgesia. In the present study, we investigated the potential role of peripheral group I metabotropic glutamate receptors (mGluRs 1/5) in the development of muscular hypersensitivity to mechanical stimulation, and attempted to elucidate intracellular signaling mechanisms associated with the mGluR activation in male Sprague-Dawley rats. First, our Western blot analyses revealed that mGluR 5 protein, but not mGluR 1 protein, is reliably detected in trigeminal ganglia and the masseter nerve. Subsequent behavioral studies demonstrated that the group I mGluR agonist, R,S-3,5-dihydroxyphenylglycol (DHPG), significantly decreased the mechanical threshold to noxious stimulation of the masseter, and that the DHPG-induced mechanical hypersensitivity can be effectively prevented by pretreatment of the masseter with 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGluR 5 antagonist, but not by 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), a selective mGluR 1 antagonist. Moreover, the DHPG-induced mechanical hypersensitivity was significantly blocked by inhibiting either the alpha or epsilon isoform of protein kinase C (PKC). Collectively, these data provide evidence that peripherally located mGluR 5 may play an important role in the development of masseter hypersensitivity, and that PKC activation is required for the modulatory effect of peripheral mGluR 5 in the craniofacial muscle tissue. Thus, selective targeting of peripheral mGluR 5 and PKCalpha, as well as PKCepsilon, might serve as an effective therapeutic strategy in the management of chronic muscle pain conditions, such as temporomandibular disorders.

Assessing abnormal gene promoter methylation in paraffin-embedded sputum from patients with NSCLC.

Aberrant methylation of CpG islands is an important pathway for regulation of gene expression. Recent data suggest that epigenetic abnormalities may occur very early in lung carcinogenesis. We studied the promoters of the four genes, HOX A9, p16(INK4a) (p16), MAGE A1 and MAGE B2 by methylation-specific PCR in matched normal tissue, tumour, and cytological negative sputum samples obtained from 22 patients with non-small cell lung cancer (NSCLC). We further report methylation abnormalities in sputum samples from 56 smokers with differential cytology readouts (negative, inflammatory changes, suspicious, and cancer). Our method was successfully performed on formalin-fixed and paraffin-embedded (FFPE) samples, and was fit to study only few cells obtained by a convenient non-invasive sputum collection and handling. The promoters of MAGE A1 and MAGE B2 had abnormal methylation patterns in, respectively, 50% and 41% of the cytologically negative sputum samples from NSCLC patients, whereas methylation abnormalities of p16 was observed in 27% of negative sputum samples. Interestingly, 95.5% (21 of 22) of the cytologically negative sputum samples from NSCLC patients had abnormal methylation in at least one of the four genes indicating a high sensitivity of this marker system. Moreover, a higher frequency of methylation abnormalities was observed in sputum samples from smokers with high cytological grade compared to low cytological grade. We conclude that the identification of abnormal gene methylation of a limited number of markers in FFPE sputum samples is feasible and may be investigated as a potential system for population-based screening of early stages of lung cancer.

Signal pathway of cytokines produced by reactive oxygen species generated from phorbol myristate acetate-stimulated HMC-1 cells.

The relationship of cytokine production and reactive oxygen species (ROS) generated in mast cells has not been reported yet. This study aimed to examine the signal pathway in the production of cytokines [interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha)] by ROS generated from phorbol myristate acetate (PMA)-stimulated human mast cell line-1 cells (HMC-1). HMC-1 cells were stimulated with 25 ng/ml of PMA. The ROS generation and production of cytokines (IL-8 and TNF-alpha) were measured by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay method, respectively. Phosphorylation of mitogen-activated protein kinase family (extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase) was detected by the Western blotting method. The expression of cytokine's mRNA was measured by reverse transcriptase--polymerase chain reaction, and the DNA-binding activity of the transcription factors [nuclear factor-kappaB (NF-kappaB) and activator protein-1] was detected by electrophoretic mobility shift assay. PMA-stimulated HMC-1 cells immediately generated ROS, and the generated ROS was inhibited by 1,3-dimethyl-2-thiourea (DMTU), but partially inhibited by catalase or N-acetyl-L-cysteine. The production of cytokines in PMA-stimulated HMC-1 cells reached the maximum at 3-5 h and was inhibited by DMTU and specific kinase inhibitor for p38, SB203580. DMTU and SB203580 also inhibited the expressed cytokine's mRNA level and the increased DNA-binding activity of transcription factors, NF-kappaB in PMA-stimulated HMC-1 cells. These data suggest that intracellular ROS generated from PMA-stimulated HMC-1 cells contributes to the production of inflammatory cytokines via p38 kinase/NF-kappaB.

Adenomatoid tumor of the adrenal gland: a clinicopathologic study of 3 cases.

Adenomatoid tumors are relatively uncommon benign neoplasms of mesothelial origin, usually occurring in the male and female genital tracts. Rare extragenital adenomatoid tumors have been identified in the adrenal glands, heart, mesentery, pleura, and lymph nodes. In the adrenal gland, adenomatoid tumors may pose a diagnostic challenge. The differential diagnosis includes adrenocortical carcinoma and metastatic carcinoma, especially signet ring cell carcinoma. Because of its glandular pattern, an adenomatoid tumor may be confused with an adenocarcinoma. We present 3 cases of adrenal adenomatoid tumors, including one with a concurrent large hemorrhagic vascular adrenal cyst. The adenomatoid tumors were unilateral, appeared solid and white, and varied from 1.7 to 4.2 cm in diameter. They occurred in 3 male patients aged 33, 33, and 46 years. One patient presented with abdominal pain due to the presence of a concurrent large adrenal cyst. The tumor was an incidental radiological finding in another case and was discovered during the course of a workup for hypertension in the third case. The light microscopic appearances were consistent with those of typical adenomatoid tumors. Immunohistochemical stains for calretinin and cytokeratin 5/6 were positive, confirming the tumors' mesothelial origin. Ultrastructural studies performed in 2 cases revealed microvilli and desmosomes. Follow-up showed no evidence of recurrence or metastasis. In our experience, the key to the diagnosis of this rare benign tumor is to consider adenomatoid tumor in the differential diagnosis of any glandular tumor occurring in the adrenal gland.