Multiprobe Ribonuclease Protection Assay Research Articles

A Personal Perspective on My Scientific Career

A Personal Perspective on My Scientific Career

Homeopathic Medicines Do Not Alter Growth and Gene Expression in Prostate and Breast Cancer Cells In Vitro

Homeopathy is an alternative medical system practiced in all parts of the world. Although several theories are proposed to explain the mechanisms of action, none are scientifically verified. In this study, the authors investigate the effect of selected homeopathic remedies often used to treat prostate and breast cancer. The authors investigated the effect of the homeopathic medicines Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias, Phytolacca, and Carcinosin on prostate and breast cancer cell (DU-145, LNCaP, MAT-LyLu, MDA-MB-231) growth and on gene expression that regulates apoptosis, using MTT and multiprobe ribonuclease protection assay. None of the homeopathic remedies tested in different potencies produced significant inhibitory or growth-promoting activity in either prostate or breast cancer cells. Also, gene expression studies by ribonuclease protection assay produced no significant changes in mRNA levels of bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, or FasL after treatment with homeopathic medicines. The results demonstrate that the highly diluted homeopathic remedies used by homeopathic practitioners for cancer show no measurable effects on cell growth or gene expression in vitro using currently available methodologies.

Primary glia expressing the G93A-SOD1 mutation present a neuroinflammatory phenotype and provide a cellular system for studies of glial inflammation

Detailed study of glial inflammation has been hindered by lack of cell culture systems that spontaneously demonstrate the "neuroinflammatory phenotype". Mice expressing a glycine → alanine substitution in cytosolic Cu, Zn-superoxide dismutase (G93A-SOD1) associated with familial amyotrophic lateral sclerosis (ALS) demonstrate age-dependent neuroinflammation associated with broad-spectrum cytokine, eicosanoid and oxidant production. In order to more precisely study the cellular mechanisms underlying glial activation in the G93A-SOD1 mouse, primary astrocytes were cultured from 7 day mouse neonates. At this age, G93A-SOD1 mice demonstrated no in vivo hallmarks of neuroinflammation. Nonetheless astrocytes cultured from G93A-SOD1 (but not wild-type human SOD1-expressing) transgenic mouse pups demonstrated a significant elevation in either the basal or the tumor necrosis alpha (TNFα)-stimulated levels of proinflammatory eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4); inducible nitric oxide synthase (iNOS) and •NO (indexed by nitrite release into the culture medium); and protein carbonyl products. Specific cytokine- and TNFα death-receptor-associated components were similarly upregulated in cultured G93A-SOD1 cells as assessed by multiprobe ribonuclease protection assays (RPAs) for their mRNA transcripts. Thus, endogenous glial expression of G93A-SOD1 produces a metastable condition in which glia are more prone to enter an activated neuroinflammatory state associated with broad-spectrum increased production of paracrine-acting substances. These findings support a role for active glial involvement in ALS and may provide a useful cell culture tool for the study of glial inflammation.

Pseudomonas aeruginosa–Induced Inflammation in the Rat Extended-Wear Contact Lens Model

To examine the early host response to Pseudomonas aeruginosa challenge in the extended contact lens-wearing rat model. Lewis rats were fitted with extended-wear lotrafilcon A hydrogel lenses in the left eye, and the right eye served as the control. Bacterial challenge was initiated in the experimental eye by fitting a bacteria-soaked contact lens and by topical delivery of the bacteria. On first detection of corneal opacity, slitlamp examination, histopathologic examination, viable bacteria counts, enzyme-linked immunosorbent assay, myeloperoxidase, Langerhans cell detection, and multiprobe ribonuclease protection assays were used to evaluate the early corneal response. Analysis of bacterially challenged contact lens-wearing versus control rats showed Langerhans cells and polymorphonuclear neutrophils only in the experimentally challenged cornea. In addition, in the experimentally challenged cornea, ribonuclease protection and enzyme-linked immunosorbent analyses showed an upregulation of proinflammatory cytokines, including interleukins 1beta and 6, suggesting that with contact lens wear, these cytokines contribute to the early corneal response and, potentially, disease. The contact lens-wearing rat model allows a unique analysis of the early effects of bacterial challenge in extended-wear contact lenses in the absence of corneal scarring, used in most rodent models. The rat model should be valuable to delineate further the effects of contact lens wear, including the testing of additional contact lens-related complications.

Steroid hormone receptors and coregulators in endocrine‐resistant and estrogen‐independent breast cancer cells

Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ERbeta upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ERalpha levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy.

Evidence that T-Helper Type 2 Cell-Derived Cytokines and Eosinophils Contribute to Acute Rejection of Orthotopic Corneal Xenografts in Mice

Because guinea pig corneal xenografts are rejected acutely (within 16 days) in mouse eyes by a T-cell-dependent mechanism, the authors wished to determine the functional phenotype of CD4+ effector T cells. Orthotopic corneal xenotransplantation was performed from strain 13 guinea pigs to BALB/c mice. Grafted eyes were removed at specified times and examined histologically or subjected to cytokine and chemokine mRNA analysis using a multi-probe ribonuclease protection assay. Draining cervical lymph node cells were harvested at specified times and stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells. Supernatants were assayed by enzyme-linked immunosorbent assay for content of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, and IL-10 and cells were used for mRNA analysis. Rejected corneal xenografts were heavily infiltrated with polymorphonuclear leukocytes, the majority of which were eosinophils. These eyes contained mRNA for IL-4, IL-6, IL-10, IL-13, IL-15, and IFN-gamma. When stimulated with guinea pig spleen cells, T cells from draining cervical lymph nodes secreted primarily IL-4, IL-5, IL-10, and IFN-gamma. Eotaxin was overexpressed in eyes with rejected corneal xenografts. Acute rejection of corneal xenografts in mice is mediated by T cells that display a mixed T-helper (Th) type 2/Th1 phenotype and secrete eotaxin, an eosinophil chemoattractant. Eosinophil-dependent xenograft rejection bears similarities to immune elimination of parasites.

Topical application of culture supernatant from human amniotic epithelial cells suppresses inflammatory reactions in cornea

Human amniotic epithelial cells (HAEC) may be a source of soluble anti-inflammatory factors. The purpose of this study is to determine the effect of topically applied HAEC culture supernatant on corneal inflammatory reactions. HAEC were obtained from a placenta and cultured for 48 hr, and the supernatant was collected. The conditioned medium from HAEC contained small amounts of human interleukin-1 receptor antagonist (IL-1ra). Intrastromal sutures were placed in the cornea of BALB/c mice to induce corneal neovascularisation. Superficial cauterisation was applied to induce recruitment or activation of antigen presenting cells (APCs) in the cornea without neovascularisation. HAEC conditioned medium, placebo, or recombinant human IL-1ra was topically applied three times daily for 2 weeks. Suture-induced corneal neovascularisation was evaluated microscopically for 8 weeks. The cauterised corneas were harvested at 2 weeks, and the MHC class II + APCs were quantified by immunofluorescent staining and confocal microscopy. Inflammatory cytokine gene expression in the cauterised corneas was analyzed by a multiprobe ribonuclease protection assay. Conditioned medium from HAEC led to a profound suppression of corneal neovascularisation and fewer MHC class II + APCs in the epithelium. In contrast, human IL-1ra was only slightly effective in suppressing corneal inflammatory reactions. mRNA expression of murine IL-1ra and IL-1beta in the cauterised corneas was markedly suppressed after application of the conditioned medium. These results suggest that HAEC are a source of soluble anti-inflammatory factors and that conditioned medium from HAEC contains factors other than IL-1ra that suppress corneal inflammation.

Multi-probe RPA template sets to study RNA modulation and transcriptional control of BH3-only members of the Bcl-2 family

The Bcl-2 family of apoptotic regulators comprises both pro- and anti-apoptotic proteins. Pro-apoptotic BH3-only members of the Bcl-2 family are subject to stringent control in normal proliferating cells, but can be activated by a variety of mechanisms in response to diverse apoptotic signals. To facilitate the simultaneous identification of BH3-only targets that are regulated at the RNA level, we have designed and assembled multi-probe ribonuclease protection assay templates and demonstrated their ability to detect multiple BH3-only transcripts in a variety of human cell lines and primary tissues. Following this, we have determined the modulation of BH3-only transcripts to a variety of apoptotic stimuli, in selected primary and transformed cell lines. Early experiments, showing that a given cell type upregulates a unique subset of BH3-only RNAs in response to different death-inducing stimuli, indicate that the multi-probe BH3-only RPA template sets could serve as useful diagnostic and analytical tools in cancer research.

Regulation of nuclear receptor and cofactor expression in breast cancer cell lines.

The aim of this study was to compare the expression profile of nuclear receptors (NRs) and cofactors in different breast cancer cell lines as well as their regulation by estradiol, insulin and progestin R5020. Expression of NRs and cofactors were determined from MCF-7, T-47D and ZR-75-1 breast cancer cell lines. Multiprobe ribonuclease protection assay and real-time RT-PCR were used to quantitate mRNA levels of steroid receptors, vitamin D receptors (VDR) and retinoic acid receptors (RAR) and cofactors: amplified in breast cancer-1, cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, p300, nuclear receptor corepressor and silencing mediator of repressed transcription. Basal expression levels of NRs and cofactors varied depending on the cell line. Cell line-specific regulation of androgen receptor, estrogen receptor-alpha (ERalpha), RARalpha, RARgamma and VDR expression was observed after estradiol treatment. Likewise, differences in the regulation of ERalpha, RARalpha and VDR expression after R5020 treatment were observed. We did not observe significant regulation of cofactor expression after estradiol, insulin or progestin treatment in any cell line analyzed. The results showed that not only is the expression profile of the NRs and cofactors cell line specific but also the regulation of NR expression. Thus the determinants of the ligand action (receptor and cofactor expression) varied considerably among different cell clones of the breast cancer cells. This suggested a gradient of NR-ligand sensitivities in the hormone-dependent breast cancers, which produces an additional challenge in developing novel ligands for hormone replacement therapy and breast cancer treatment.

Effect of preexposure to ultrafine carbon black on respiratory syncytial virus infection in mice.

Epidemiological studies have indicated that exposure to elevated levels of particulate matter exacerbates several pulmonary diseases, including asthma, bronchitis, and viral infections. Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in infants and may lead to the development of asthma in childhood. To determine whether particle exposure modulates the immune response to RSV, eight-week-old female BALB/c mice received an intratracheal (i.t.) instillation of either 40 micro g ultrafine carbon black (CB) particles or vehicle. The following day, mice were i.t. instilled with either 106 pfu RSV or uninfected media. End points were examined 1, 2, 4, 7, and 10 days during RSV infection. Compared with RSV alone, tumor necrosis factor-alpha (TNF-alpha) protein was reduced in the bronchoalveolar lavage fluid (BALF) on days 1 and 2 of infection; there was also a reduction in BALF lymphocyte numbers on day 4, which correlated with reductions in both IFN-gamma-inducible protein (IP-10), lymphotactin, and IFN-gamma mRNAs in the lungs of RSV + CB mice. Multiprobe ribonuclease protection assays of RSV + CB lung tissue showed no changes in the RSV-associated chemokines regulated upon activation, normal T cell expressed and secreted (RANTES), eotaxin, monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP)-1 alpha or MIP-1 beta. Viral titers in RSV + CB mice were lower than RSV on days 2-4 of infection. By day 7 of infection, however, neutrophil numbers, proinflammatory cytokine mRNA expression, and protein levels of TNF-alpha and the Th2 cytokine interleukin (IL)-13 were increased in the lungs of RSV + CB mice, indicating an exacerbation of infection. These data indicate that preexposure to ultrafine particles induces an inflammatory milieu promoting allergic immune responses rather than IFNgamma production necessary for microbial defense.

Mechanisms of immunotherapeutic intervention by anti-CD154 (CD40L) antibody in high-risk corneal transplantation.

This study aimed to determine the effects of anti-CD154 on T cell cytokine profiles and ocular chemokine gene expression after high-risk corneal transplantation and to specifically determine if CD154 blockade is associated with a switch from a Th1 to a Th2 alloimmune response. Mice were used as recipients of syngeneic or multiple minor H or MHC antigen-mismatched corneal grafts. Recipient beds were neovascularized (high-risk). Hosts were randomized to receive either anti-CD154 antibody or control immunoglobulin (Ig) perioperatively. Two weeks after corneal transplantation, allospecific delayed-type hypersensitivity (DTH) was evaluated. Frequencies of interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in the hosts were measured by enzyme-linked immunospot (ELISPOT) assay. Ocular chemokine gene expression in anti-CD154-treated and control hamster Ig-treated groups was determined using a multiprobe ribonuclease protection assay (RPA). Leukocyte infiltration of corneal grafts was evaluated microscopically. Anti-CD154-treated mice did not exhibit allospecific DTH. The frequencies of Th1 cytokine-producing but not Th2 cytokine-producing T cells were significantly reduced in anti-CD154-treated hosts. Postoperative mRNA levels of RANTES and macrophage inflammatory protein-1beta (MIP-1beta) in anti-CD154-treated eyes were substantially suppressed compared with hamster Ig-treated controls. Leukocyte infiltration was profoundly suppressed in grafts of anti-CD154-treated hosts. These data demonstrate that blockade of the CD40-CD154 costimulatory pathway after corneal transplantation inhibits Th1-mediated responses but does not induce a switch to a Th2-specific response. In addition, anti-CD154 therapy suppresses ocular chemokine gene expression and leukocytic infiltration into allografts.

Simultaneous quantitation of equine cytokine mRNAs using a multi-probe ribonuclease protection assay

A rapid multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNA levels in activated equine monocyte-derived macrophages (EMDM) and equine peripheral blood mononuclear cells (EPBMC). Eleven template plasmids specific to 10 equine cytokine genes and the β-actin gene were generated from which radiolabeled anti-sense RNA probes were produced. The multi-probe RPA simultaneously quantitated mRNA levels of equine IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IFN-γ, TGF-1β and TNF-α in EPBMC and EMDM with coefficients of variation as low as 0.03–0.08 (3–8%) when normalized to β-actin expression. This sensitive and rapid assay provides a valuable tool for studies of equine immune responses.

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.

Cytokine Profiling for Chemical Sensitizers: Application of the Ribonuclease Protection Assay and Effect of Dose

Exposure to chemicals in domestic and occupational settings may contribute to increases in asthma and allergy. Airway hypersensitivity (AHS) is T helper-2 (Th2) cell associated, whereas contact hypersensitivity (CHS) is T helper-1 (Th1) cell associated. The distinct cytokine profiles produced by these cells may provide a means of distinguishing respiratory sensitizers from contact sensitizers. In this study, female BALB/c mice were exposed twice on the flanks and three times on the ears using the airway sensitizer trimellitic anhydride (TMA) or the contact sensitizer dinitrochlorobenzene (DNCB). At various times following exposure, total mRNA was extracted from draining lymph node cells and cytokine mRNA profiles analyzed using a multiprobe ribonuclease protection assay (RPA). The Th2 cytokines IL4, IL10, and IL13 were significantly increased in response to TMA compared to DNCB, with optimal detection occurring 14 days following initial exposure. To determine its effect, dose was varied in flank exposures, ear exposures, or both simultaneously. When dose was varied during flank exposures only, TMA induced higher levels of Th2 cytokines than DNCB at all doses tested. DNCB did not induce Th1 cytokines at any dose tested. Variation of TMA dose during both exposures similarly induced Th2 cytokines. Dose only appeared to be a factor when TMA concentration was varied during the ear exposures alone. Thus, these studies suggest that quantitative differences in Th2 responses between TMA and DNCB may be demonstrated over a wide range of doses and these differences may be detected by RPA following dermal exposure to these sensitizers.

Chemokine gene expression in iris-ciliary body during experimental autoimmune uveoretinitis

Purpose. To evaluate, through differential gene expression of chemokines in iris-ciliary body (I/CB), the extent to which leukocyte recruitment to the ocular anterior segment participates in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in mice. Methods. B10.A mice were immunized with 50 µg of inter-photoreceptor retinoid binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at 0, 11, 17, and 28 days and assayed for leukocyte content and protein levels. Enucleated eyes were subjected to histologic analysis. Chemokine gene expression in I/CB was determined at these same time points by a multiprobe ribonuclease protection assay (RPA) system. Results. Inflammation was detected in the anterior chamber (AC) at 11 days and leukocyte recruitment continued thereafter. Polymorphonuclear neutrophils (PMN) were the predominant cell type in the AC, whereas macrophages/ monocytes and lymphocytes were predominant in the retina/subretinal space at 17 days. Peak gene expression of macrophage inflammatory protein (MIP)-1a, MIP-1ß, and MIP-2 and interferon- ?-inducible protein of 10kd (IP-10) was detected in I/CB on day 11, whereas peak expression of RANTES and eotaxin was observed at 17 days. Conclusions. Early I/CB peak expression of mRNA for MIP-2 followed by PMN recruitment into the AC, suggests that PMN may play an important role in EAU pathogenesis.

Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen α1(I) and TIMP-1

Background/Aims : Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism. Methods : Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen α1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) β1 were determined by multi-probe ribonuclease protection assay. Results : After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen α1(I), TIMP-1 and TGF β1 ( P <0.01). Silymarin downregulated elevated procollagen α1(I), TIMP-1 and TGF β1 mRNA levels by 40–60% ( P <0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III. Conclusions : Silymarin suppresses expression of profibrogenic procollagen α1(I) and TIMP-1 most likely via downregulation of TGF β1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

Cytokine mRNA expression in mouse colon: IL-15 mRNA is overexpressed and is highly sensitive to a fibre-like dietary component (short-chain fructo-oligosaccharides) in an Apc gene manner

On the basis of studies using the Min mouse model of colon carcinogenesis, we have recently proposed that a fibre-like food (short-chain fructo-oligosaccharides, sc-FOS) fermented in the colon may stimulate a mechanism of cancer immunosurveillance. In the present paper, we have investigated the expression of cytokines as potential effector molecules. Interleukin (IL-)4, IL-5, IL-13, IL-15 and interferon (INF)-γ mRNAs were detected by a multi-probe ribonuclease protection assay in C57BL/6J and Min mouse colons. IL-15 mRNA expression was significantly amplified (P=0.01) by the sc-FOS-enriched diet in the colon of Min mice.

Proliferating Bile Duct Epithelial Cells Are a Major Source of Connective Tissue Growth Factor in Rat Biliary Fibrosis

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-beta1) and thus a potential target for antifibrotic treatment strategies. CTGF is up-regulated in disorders such as atherosclerosis, scleroderma, and fibrosis of kidneys and lungs. We investigated the temporospatial expression patterns of CTGF and TGF-beta1 mRNA in rat livers with acute fibrogenesis (after a single dose of CCl(4)) and with advanced fibrosis (6 weeks after complete bile duct occlusion). Multiprobe ribonuclease protection assay revealed increasing TGF-beta1 and CTGF mRNA levels 6 hours after injection of CCl(4), with peak levels after 72 hours. In biliary fibrosis TGF-beta1 and CTGF mRNA levels increased fourfold and sevenfold, respectively (P: < 0.001). In situ hybridization combined with cell-specific markers revealed CTGF transcripts in desmin-positive cells after a single dose of carbon tetrachloride, whereas no transcripts were found in normal livers. In biliary fibrosis, however, proliferating bile duct epithelial cells were the predominant source of CTGF mRNA. We conclude that in rat liver fibrogenesis CTGF is up-regulated in close association with TGF-beta1 and that, contrary to a previous report, not solely hepatic stellate cells but activated bile duct epithelial cells are the main source of this profibrogenic factor.

The determination of draining lymph node cell cytokine mRNA levels in BALB/c mice following dermal sodium lauryl sulfate, dinitrofluorobenzene, and toluene diisocyanate exposure.

Differential modulation has been demonstrated in interleukin-4 (IL-4), IL-10, and interferon gamma (IFN-gamma) mRNA and protein secretion patterns of cells isolated from the draining lymph nodes of mice following exposure to T cell and respiratory sensitizers. Using a multiprobe ribonuclease protection assay, the following investigation examined the mRNA expression patterns of multiple cytokines associated with respiratory sensitization for modulation following exposure to chemicals known primarily to induce irritation (sodium lauryl sulfate), respiratory sensitization (toluene diisocyanate), or T cell-mediated hypersensitivity (dinitrofluorobenzene) responses. On days 0 and +5 female BALB/c mice were exposed to either test article or vehicle on the shaven dorsal lumbar region; on days +10 through +12 the mice received test article on the dorsal aspect of each ear. On day +13 animals were euthanized, draining lymph nodes were excised, and mRNA was isolated immediately or following 24 or 48 h of culture in the presence or absence of concanavalin (Con) A. Differential expression of cytokine mRNA was most notable following 24 h incubation with Con A. Modulation of IL-4, -10, and IFN-gamma following chemical exposure was consistent with previous studies. In addition, IL-9, -13, and -15 were significantly elevated only following toluene diisocyanate exposure. Further investigations of these cytokines may provide additional insight into the mechanisms of chemically induced respiratory sensitization and provide endpoints for the detection of a chemical's ability to elicit IgE-mediated hypersensitivity responses.

Absence of Proinflammatory Cytokine Gene Expression in Nasal Biopsies from Wood Surface-coating Industry Workers

Symptoms of nasal, pharyngeal and ocular discomfort have been reported among workers in the wood surface-coating industry. Symptoms were reported more often by workers using ultraviolet radiation-curable acrylate coatings (UV coatings), which contain potential chemical sensitizers, than by those using acid-curing coatings. Furthermore, increased levels of eosinophil cationic protein (ECP) and albumin, but not tryptase, in nasal lavage from workers exposed to UV coatings have been observed. To further examine whether air contaminants present in the UV-coating industry are causing the observed increase in symptoms, the inflammatory process in the nasal mucosa of workers exposed to UV coatings was investigated. Clinical and biochemical endpoints were selected to distinguish between specific and non-specific hypersensitivity and to test the hypothesis that the symptoms were consistent with Type IV hypersensitivity. The nasal lavage and nasal biopsy were performed under local anesthetic at the workplace during working hours after a minimum of 2 h of work in both the exposed and control groups. Albumin and ECP, and the cytokines tumor necrosis factor- f (TNF- f ) and interleukin-8 (IL-8), were used as inflammatory markers. A multi-probe ribonuclease protection assay was used to attempt to detect cytokine variation in human nasal biopsies. The cytokine genes analyzed were TNF- f , GM-CSF, interferon- n , IL-2, IL-4 and IL-5. L32 and GAPDH were used as control genes for mRNA expression levels. Mucosal inflammation symptoms correlated with increased levels of albumin, but not with increased levels of ECP, secreted proinflammatory cytokines or cytokine gene mRNA expression. We conclude that the symptoms are non-specific and do not correlate with occupational exposure to UV coatings under the conditions of this investigation.