WITHDRAWN: Etidronate for treating and preventing postmenopausal osteoporosis.

BACKGROUND: Osteoporosis is a clinical syndrome of reduced bone mass and increased fracture susceptibility. There are now a number of options, including etidronate which can decrease the risk of fractures. OBJECTIVES: To systematically review the efficacy of etidronate on bone density, fractures and toxicity in postmenopausal women. SEARCH STRATEGY: We searched MEDLINE from 1966 to December 1998, examined citations of relevant articles, and the proceedings of international osteoporosis meetings. We contacted osteoporosis investigators to identify additional studies, primary authors, and pharmaceutical industry sources for unpublished data. SELECTION CRITERIA: We included thirteen trials (with 1010 participants) that randomized women to etidronate or an alternative (placebo or calcium and/or vitamin D) and measured bone density for at least one year. DATA COLLECTION AND ANALYSIS: For each trial, three independent reviewers assessed the methodological quality and abstracted data. MAIN RESULTS: The data suggested a reduction in vertebral fractures with a pooled relative risk of 0.60% (95% CI 0.41 to 0.88). There was no effect on non-vertebral fractures (pooled relative risk 1.00, (95% CI 0.68 to 1.42)). Etidronate, relative to control, increased bone density after three years of treatment in the lumbar spine by 4.27% (95% CI 2.66 to 5.88), in the femoral neck by 2.19% (95% CI 0.43, 3.95) and in the total body by 0.97% (95% CI 0.39, 1.55). Effects were larger at 4 years, though the number of patients followed was much smaller. AUTHORS' CONCLUSIONS: Etidronate increases bone density in the lumbar spine and femoral neck. The pooled estimates of fracture reduction with etidronate are consistent with a reduction in vertebral fractures, but no effect on non-vertebral fractures.

WITHDRAWN: Risedronate for the prevention and treatment of postmenopausal osteoporosis.

BACKGROUND: Postmenopausal osteoporosis results in an increased susceptibility to low-trauma fractures due to reduced bone volume and microarchitectural deterioration. Risedronate, a third generation bisphosphonate, has been shown in multiple clinical trials to reduce fracture risk and improve bone mineral density in postmenopausal women with osteoporosis. First and second generation bisphosphonates are known to have gastrointestinal side-effects and risedronate may be better tolerated. OBJECTIVES: To systematically review the efficacy of risedronate on bone density, and fracture reduction in postmenopausal women. SEARCH STRATEGY: The Cochrane Controlled Trials Registry Medline, and Current Contents were searched from 1990 - 2001. The electronic search was supplemented by handsearching four osteoporosis journals and their conference proceedings, as well as contacting content experts and industry sources for unpublished data. SELECTION CRITERIA: We included eight trials that randomised women to risedronate or an alternative (placebo or calcium and /or vitamin D) and measured bone mineral density for at least one year. DATA COLLECTION AND ANALYSIS: For each trial three independent reviewers assessed the methodological quality and abstracted data. Data was extracted for outcomes of fracture, bone mineral density and adverse events. The more conservative random effects model was used to pool data. The quality of trials was assessed according to the Jadad five-point scale. MAIN RESULTS: Both vertebral and non-vertebral fractures were statistically and clinically reduced with risedronate. Eleven out of one hundred women who received risedronate had a vertebral fracture compared to 17 out of one hundred of those who received calcium and vitamin D (pooled relative risk for vertebral fractures of 0.64 (95% CI 0.52 - 0.77). Three percent of participants who received risedronate had a non-vertebral fracture compared to 4.6% of those who received calcium and vitamin D (pooled relative risk for nonvertebral fractures of 0.73 (95% CI 0.61 - 0.87). The weighted mean difference for the percent change from baseline for bone mineral density with 5 mg daily for lumbar spine, femoral neck and trochanter was 4.54% (95%CI 4.12 - 4.97), p<0.01; 2.75% (95% CI 2.32 - 3.17), p<0.01; and 4.38% (95% CI 3.51 - 5.25), p<0.01 respectively. AUTHORS' CONCLUSIONS: There is good evidence for the efficacy of risedronate in the reduction of both vertebral and non-vertebral fractures. In addition, there is evidence from randomized trials that risedronate is able to achieve this without increasing risk for overall withdrawals due to adverse effects.

Acupuncture and electroacupuncture for the treatment of rheumatoid arthritis.

BACKGROUND: Acupuncture has been used by rehabilitation specialists as an adjunct therapy for the symptomatic treatment of rheumatoid arthritis (RA). Acupuncture is a traditional Chinese medicine where thin needles are inserted in specific documented points believed to represent concentration of body energies. In some cases a small electrical impulse is added to the needles. Once the needles are inserted in some of the appropriate points, endorphins, morphine-like substances, have been shown to be released in the patient's system, thus inducing local or generalised analgesia (pain relief). This review is an update of the original review published in July 2002. OBJECTIVES: To evaluate the effects of acupuncture or electroacupuncture on the objective and subjective measures of disease activity in patients with RA. SEARCH STRATEGY: A comprehensive search of MEDLINE, EMBASE, PEDro, Current Contents , Sports Discus and CINAHL, initially done in September 2001, was updated in May 2005. The Cochrane Field of Rehabilitation and Related Therapies and the Cochrane Musculoskeletal Review Group were also contacted for a search of their specialized registries. Handsearching was conducted on all retrieved papers and content experts were contacted to identify additional studies. SELECTION CRITERIA: Comparative controlled studies, such as randomized controlled trials and controlled clinical trials in patients with RA were eligible. Trials published in languages other than French and English were not analyzed. Abstracts were excluded unless further data could be obtained from the authors. DATA COLLECTION AND ANALYSIS: Two independent reviewers identified potential articles from the literature search and extracted data using pre-defined extraction forms. Consensus was reached on all the extracted data. Quality was assessed by two reviewers using a five point validated tool that measured the quality of randomization, double-blinding and description of withdrawals. MAIN RESULTS: After the updated searches were conducted, five further potential articles were identified; however, these did not meet the inclusion criteria. Two studies involving a total of 84 people were included. One study used acupuncture while the other used electroacupuncture. In the acupuncture study, no statistically significant difference was found between groups for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), visual analogue scale for patient's global assessment (VAS G), number of swollen joints and tender joints, general health questionnaire (GHQ), modified disease activity scale (DAS) or for the decrease in analgesic intake. Although not statistically significant, pain in the treatment group improved by 4 points on a 0-100mm visual analogue scale versus no improvement in the placebo group. In the second study, using electroacupuncture, a significant decrease in knee pain was reported in the experimental group, 24 hours post treatment, when compared to the placebo group (WMD: -2.0 with 95% CI -3.6,-4.0). A significant decrease was found also at four months post-treatment (WMD -0.2, 95% CI: -0.36, -0.04) AUTHORS' CONCLUSIONS: Although the results of the study on electroacupuncture show that electroacupuncture may be beneficial to reduce symptomatic knee pain in patients with RA 24 hours and 4 months post treatment, the reviewers concluded that the poor quality of the trial, including the small sample size preclude its recommendation. The reviewers further conclude that acupuncture has no effect on ESR, CRP, pain, patient's global assessment, number of swollen joints, number of tender joints, general health, disease activity and reduction of analgesics. These conclusions are limited by methodological considerations such as the type of acupuncture (acupuncture vs electroacupuncture), the site of intervention, the low number of clinical trials and the small sample size of the included studies.

Transcutaneous electrical nerve stimulation (TENS) for the treatment of rheumatoid arthritis in the hand.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic, inflammatory, system disease. It commonly affects the small peripheral joints (such as fingers and wrist). The main goals of intervention for RA are preventing joint deformity, preserving joint function, and reducing inflammation and pain. Transelectrical nerve stimulation (TENS) is a form of electrotherapy and is thought to produce analgesia according to the gate control theory. OBJECTIVES: To determine the efficacy and safety of TENS in the treatment of RA of the hand. The primary outcomes of interest were relief of grip pain and resting pain intensity, relief of joint tenderness, number of tender joints and patient assessment of disease. The secondary objective was to determine the most effective mode of TENS application in pain control. SEARCH STRATEGY: We searched for relevant studies, in English, in the Cochrane field of physical and related therapies, the Cochrane Controlled Trials Register, MEDLINE, EMBASE, HEALTHSTAR, Sports Discus, CINAHL, Current Contents, and the PEDro database, up to October 2002. SELECTION CRITERIA: Two independent reviewers selected the trials that met predetermined inclusion criteria. DATA COLLECTION AND ANALYSIS: Study results were extracted by two independent reviewers. Continuous outcomes were analyzed by weighted mean difference (WMD) using a fixed effects model. MAIN RESULTS: Three RCTs, involving 78 people, were included in this review. AL-TENS and C-TENS were compared to placebo and to each other. Administration of 15 minutes of AL-TENS a week, for 3 weeks, resulted in a significant decrease in rest pain (67% relative benefit, 45 points absolute benefit on 100 mm VAS scale) but not in grip pain compared to placebo. AL-TENS did result in a clinical beneficial improvement in muscle power scores with a relative difference of 55%, and an absolute benefit of 0.98, compared to placebo. No significant difference was found between one 20-minute treatment duration of C-TENS versus AL-TENS, or C-TENS versus placebo on decrease in mean scores for rest pain or grip pain, or on the number of tender joints. Results showed a statistically significant reduction in joint tenderness, but no clinical benefit from C-TENS over placebo in relief of joint tenderness. No statistically significant difference was shown between 15 days of treatment with C-TENS or AL-TENS in relief of joint pain, although there was a clinically important benefit of C-TENS over AL-TENS on patient assessment of change in disease (risk difference 21%, NNT 5). REVIEWER'S CONCLUSIONS: There are conflicting effects of TENS on pain outcomes in patients with RA. AL-TENS is beneficial for reducing pain intensity and improving muscle power scores over placebo while, conversely, C-TENS resulted in no clinical benefit on pain intensity compared with placebo. However C-TENS resulted in a clinical benefit on patient assessment of change in disease over AL-TENS. More well designed studies with a standardized protocol and adequate number of subjects are needed to fully conclude the effect of C-TENS and AL-TENS in the treatment of RA of the hand.

Acute inflammation and recovery in rats after intratracheal instillation of a 1-->3-beta-glucan (zymosan A).

Although endotoxin is a known potent stimulant of inflammatory responses, the magnitude of pulmonary response following exposure to various organic dusts does not always correlate with endotoxin content of the dusts alone. Other components, such as 1-->3-beta-glucans, derived from the inner cell wall of yeasts and fungi, have been implicated in organic dust toxic syndrome. However, animal studies report conflicting results concerning the inflammatory potency of 1-->3-beta-glucan. In this experiment, the pulmonary reaction of rats to 1-->3-beta-glucan (zymosan A) exposure was assessed. Male Sprague-Dawley rats were exposed via intratracheal instillation (IT) to zymosan A (dose range 0-5 mg/kg body weight). Rats were sacrificed 1-7 d postexposure and the following pulmonary responses were monitored: (1) breathing frequency, (2) differential cell counts of hronchoalveolar lavage (BAL) cells, (3) chemiluminescence (CL) as a measure of alveolar macrophage activation, (4) nitric oxide production by alveolar macrophages, (5) albumin levels, and (6) lactate dehydrogenase (LDH) activity in the first acellular lavage fluid. Upon challenge with zymosan A, rats exhibited a dose-dependent pulmonary response at 1 d post IT that was significantly higher than the control level at a dose of 1-2.5 mg/kg body weight for each of these pulmonary parameters. Post-IT enhancement of breathing frequencies and polymorphonuclear leukocytes (PMN) obtained by BAL both correlated very well with zymosan A concentration (r = .95 and .99, respectively). Elevation of albumin levels and LDH activity of the acellular BAL fluid also correlated (r = .80) with the dose of zymosan. The recovery from a single intratracheal administration of zymosan A (2.5 mg/kg body weight) was monitored over 7 d. PMN and CL showed significant recovery from d 1 level by 3 d postexposure. Breathing frequencies and nitric oxide production showed significant recovery from d 1 level by 4 d postexposure. A good correlation (r2= .8) between recovery of PMN in BAL, CL, or nitric oxide production and the days postexposure was observed.

Time course of pulmonary response of rats to inhalation of crystalline silica: histological results and biochemical indices of damage, lipidosis, and fibrosis.

Previous studies have determined that alpha-quartz (crystalline silica) can cause pulmonary inflammation, damage, and fibrosis. However, the temporal relationship between silica inhalation and pulmonary inflammation, damage, and fibrosis has not been fully examined. To address this gap in our knowledge of silica-induced pulmonary fibrosis, a chronic inhalation study using rats was designed. Specifically, rats were exposed to a silica aerosol (15 mg/m3 silica, 6 h/d, 5 d/wk, 116 d), and measurements of pulmonary inflammation, damage, and fibrosis were monitored throughout the study. We report (1) data demonstrating that the silica aerosol generation and exposure system produced a consistent silica aerosol of respirable size particles; (2) the time course of silica deposition in the lung; (3) calculations that demonstrate that the rats were not in pulmonary overload; (4) histopathological data demonstrating time-dependent enhancement of silica-induced alveolitis, epithelial hypertrophy and hyperplasia, alveolar lipoproteinosis, and pulmonary fibrosis in the absence of overload; and (5) biochemical data documenting the development of lipidosis, lung damage, and fibrosis.

Comparative pulmonary toxicity of 6 abrasive blasting agents.

Inhalation of silica dust is associated with pulmonary fibrosis. Therefore, substitute abrasive materials have been suggested for use in abrasive blasting operations. To date, toxicological evaluation of most substitute abrasives has been incomplete. Therefore, the objective of this study was to compare the pulmonary toxicity of a set of substitute abrasives (garnet, staurolite, coal slag, specular hematite, and treated sand) to that of blasting sand. Rats were exposed to blasting sand or an abrasive substitute by intratracheal instillation and pulmonary responses to exposure were monitored 4 weeks postexposure. Pulmonary damage was monitored as lactate dehydrogenase (LDH) in the acellular lavage fluid. Pulmonary inflammation was evaluated from the yield of polymorphonuclear leukocytes (PMN) obtained by bronchoalveolar lavage. The activity of alveolar macrophages was determined by measuring zymosan-stimulated chemiluminescence. Blasting sand caused lung damage and showed histologic evidence for inflammation and fibrosis. Garnet, staurolite, and treated sand exhibited toxicity and inflammation that were similar to blasting sand, while coal slag caused greater pulmonary damage and inflammation than blasting sand. In contrast, specular hematite did not significantly elevate LDH or PMN levels and did not stimulate macrophage activity 4 weeks postexposure.

Activation of mitogen-activated protein kinase p38 and extracellular signal-regulated kinase is involved in glass fiber-induced tumor necrosis factor-alpha production in macrophages.

In a previous study, we demonstrated that the length of glass fibers was a critical determinant of fiber potency in induction of tumor necrosis factor (TNF)-alpha and that activation of NF-kappaB was an important factor in this response. In the present study, we analyzed the role of mitogen-activated protein (MAP) kinases in the induction of TNF-alpha by glass fibers. Glass fibers induced phosphorylation of MAP kinases, p38, and ERK in primary rat alveolar macrophages, and this phosphorylation was associated with TNF-alpha gene expression. Long fibers were more potent than short fibers in activation of MAP kinases. Results from mechanistic analysis support that MAP kinases activate transcription factor c-Jun. The activated c-Jun acts on the TNF-alpha gene promoter through two binding sites, the cyclic AMP response element and the activator protein 1-binding site. These results suggest that in addition to the NF-kappaB pathway for TNF-alpha production, glass fibers are able to activate c-Jun through MAP kinase pathways that lead to induction of TNF-alpha expression.

Ovalbumin aeroallergen exposure-response in Brown Norway rats.

A major route of exposure to allergens is through the respiratory tract. Comparatively few animal studies have used aerosolized high-molecular-weight allergens for sensitization, and in these studies, proper characterization of the aeroallergen exposure was usually missing. The purpose of this study was to profile the exposure-response relationship in Brown Norway rats (BNR) to well-characterized ovalbumin (OVA) aerosols. Rats were exposed 30 min/wk x 6 wk to respirable OVA aerosols from <1 mg/m(3) to 64 mg/m(3) air. Ovalbumin-specific circulating immunoglobulin (Ig)E, IgG, and IgA were measured throughout the study period. Rats were sacrificed 1 day after the last exposure. Pulmonary tissue was processed for histopathological and histochemical analysis. Tracheas were isolated, perfused, and assessed for in vitro responsiveness to methacholine. Serum concentrations of OVA-specific antibodies increased with both exposure concentration and number of exposures. The number of BNR with measurable titers also increased with both dose and time. Pulmonary inflammatory changes were noted only in BNR exposed to higher OVA concentrations (15 and 64 mg/m(3) air). Increased tracheal reactivity to methacholine was not found in any of the sensitized BNR. In summary, sustained aeroallergen concentration-dependent changes in specific antibody responses and pulmonary inflammation have been demonstrated.

Acute inflammatory reaction in rats after intratracheal instillation of material collected from a nylon flocking plant.

Several cases of interstitial lung disease have been diagnosed among workers at a nylon flock plant, but the etiologic agent for the disease outbreak was unknown. The results of a medical survey and industrial hygiene study indicated that the dust present in the plant may be responsible. Thus, airborne dust collected at the plant was examined for its inflammatory potential in rat lungs. The endpoints measured were: (1) breathing rates, (2) differential cell counts of bronchoalveolar lavage cells, (3) alveolar macrophage (AM) chemiluminescence, (4) albumin concentration and matrix metalloprotease activities in the acellular fluid from the initial bronchoalveolar lavage, and (5) pulmonary histopathology. In the first study, rats received a single dose of the airborne dust sample (10 mg/kg body weight) by intratracheal (IT) instillation. At 1 d post-IT, all inflammatory endpoints were significantly increased versus controls, but by 29 d post-IT they did not differ significantly from controls. Histopathology demonstrated mild to moderate, multifocal, suppurative pneumonia, usually centered around bronchioles, at 1 d post-IT. At 29 d post-IT, pulmonary inflammation was minimal to mild and characterized by alveolar histocytosis usually restricted to the immediate area of retained bire-fringent fibers. In subsequent experiments, airborne dust was extracted with water and the dust (washed airborne dust) and water extract (soluble fraction) were separated by centrifugation for further study. Nylon tow dust was prepared in the laboratory by milling uncut nylon strands (called tow) that had not been treated with the finish or dyes that are commonly used in the flock plants. Rats were administered a single dose of a dust sample (10 mg/kg body weight) or the soluble fraction (1.3 ml/kg body weight) by IT administration and the same endpoints were measured at 1 d post-IT. The dust samples caused significant increases in all of the inflammatory endpoints; however, the soluble fraction was much less active. Histological analysis of the lungs 1 d post-IT confirmed lung inflammation was occurring and tended to center around bronchioles. The results suggest that: (1) nylon flocking generates particles of respirable size that can interact with AM in the lung and can be detected in the lung 29 d after exposure, (2) the dust samples examined cause an inflammatory response, (3) water-extractable agent(s) from airborne dust contribute only minimally to the inflammatory response, and (4) the acute inflammatory response to these dusts is substantial when compared to other pathologic occupational dusts previously examined in our laboratory.

Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process.

Acute lung injury induced by a commercial leather conditioner.

Following a formulation change, a leather conditioner was involved in a 1992 nationwide outbreak of respiratory illness. We investigated the composition and toxicity of the conditioner produced before (previous product) and after (new product) the disease outbreak. The new product induced tachypnea, pulmonary edema, pulmonary hemorrhage, and sporadic deaths in exposed guinea pigs and rats. Ultrastructurally, these changes were associate with direct pulmonary cytotoxicity characterized by necrosis of alveolar type I cells and alveolar septal interstitial edema. Chemical analyses suggested major alterations in the fluorohydrocarbon constituents in the new formulation of the leather conditioner. While these alterations could not be specifically identified, they appeared to include changes from fluoralkanes to fluoroalkenes, fluorophenyl, and/or fluoroalcohol compounds. Changes in solvent composition were consistent with traces of 2-butoxyethanol and isomers of dipropylene glycol methyl ether, and additional C10-C12 alkanes. In this study, we demonstrated the toxicity of the new product in laboratory animals. Some of the altered constituents of the new product have been identified and are potential candidates for additional investigations to identify specific etiologic agents.

Pulmonary reactions to organic dust exposures: development of an animal model.

Acute inhalation of organic dusts such as cotton, hay, silage, grain, animal confinement, or compost dust can result in illness characterized by fever, pulmonary inflammation, chest tightness, and airway obstruction. These agricultural materials are complex mixtures of plant, bacterial, and fungal products. Elucidation of the time course of disease onset, the mechanisms of disease progression, and the identity of etiologic agents is essential for effective prevention and treatment. Toward this end, animal models for acute organic dust-induced reactions have been developed and characterized. Information concerning the applicability of various animal models to humans and progress toward elucidation of causative agents and mechanisms of action is presented.

Nitric oxide synthase inhibitor and lipopolysaccharide effects on reactivity of guinea pig airways.

The in vivo and in vitro effects of nitric oxide (NO) synthase inhibitors and lipopolysaccharide (LPS) on reactivity of guinea pig airways were examined. In isolated, perfused tracheas from untreated animals, the NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME; 10(-4)M), NG-methyl-L-arginine (L-NMMA; 10(-4) M) and aminoguanidine (10(-4) M) had no effect or inhibited reactivity to extraluminally (EL) or intraluminally (IL) applied methacholine and histamine. L-NMMA (10(-4) M) did not appreciably contract resting or metacholine-contracted preparations (+/- 3 x 10(-4) M L-arginine) and L-arginine only weakly relaxed contracted tracheas (+/- L-NMMA). Sodium nitroprusside and S-nitroso-N-penicillamine elicited relaxant responses and were more potent extraluminally than intraluminally. Methylene blue (10(-5) M) antagonized relaxation to sodium nitroprusside. Incubation with Escherichia coli LPS (10 micrograms/ml; 30 min incubation) alone in the EL and IL baths depressed methacholine and histamine concentration-response curves. In the presence of LPS, L-NAME potentiated responses to intraluminally applied methacholine but did not affect responses to extraluminally added methacholine. Four days after i.p. injection of animals with LPS (4 mg/kg), L-NAME potentiated responses to IL methacholine, and L-arginine acquired greater relaxant activity. LPS injection increased sensitivity to intraluminally added but not extraluminally added isoproterenol. LPS given by i.p. injection or by inhalation did not affect basal specific airway resistance of conscious animals or reactivity to methacholine aerosol during a postexposure period of 6 to 72 h. NO seems to have little role in regulating reactivity of guinea pig airways to bronchoconstrictor agonists, except after in vitro or in vivo exposure to LPS. After LPS injection the in vitro changes suggestive of NO synthase induction are not associated with altered airway reactivity to inhaled methacholine.

Activity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis.

The suppression of focal growth of initiated mouse keratinocytes by co-culture with normal keratinocytes can be overcome by treatment of co-cultures with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This keratinocyte co-culture model was developed as an analog of initiated mouse epidermis to facilitate the study of tumor promotion in cell culture. A number of promoters of TPA-type [those with protein kinase C (PKC) as receptor] were compared to non-TPA-type promoters for activity in the keratinocyte co-culture model. The TPA-type promoters teleocidin and aplysiatoxin show comparable activity to that of TPA. Exposure of co-cultures to oleoyl-2-acetylglycerol, a diacylglycerol activator of PKC, also induces focal outgrowth of initiated cells, suggesting that PKC is likely to be involved in the mechanism of action of these compounds. However, the involvement of alternative pathways (not involving PKC directly) for clonal selection are evident since the non-TPA-type promoters okadaic acid, staurosporine and thapsigargin are also active in the assay. Thus, the keratinocyte co-culture model differs from fibroblast models of normal and neoplastic co-cultures in which only TPA-type promoters are active. In further contrast to certain fibroblast co-cultures, TPA does not inhibit cell-cell communication under conditions that suppress focus formation. Taken together with previous data, we conclude that the keratinocyte co-culture model may have broad application for detecting skin tumor promoters, and may be useful for dissecting the mechanism by which normal epidermal cells suppress the growth of initiated cells.

Activation of the rasHa gene in urethane-initiated papillomas induced by protocols with high and low frequencies of malignant conversion.

The number of papillomas that develop in mice initiated with a single exposure to urethane and promoted by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) is increased greater than 4-fold by pretreating the skin once with TPA 24 hr before administration of urethane. In contrast, the carcinoma incidence was increased only 2-fold by the TPA pretreatment. Individual papillomas developed from the two protocols, differing in the potential for conversion to malignancy, were compared for activation of the rasHa gene. An activated oncogene that transformed 3T3 cells was found in the DNA of four of five papillomas from urethane-initiated, TPA-promoted mice and in eight of eleven papillomas from similarly promoted mice exposed to TPA before urethane initiation. Southern analysis of DNA from tumors or 3T3 foci demonstrated that the rasHa gene was activated by an A----T transversion at the second base of codon 61 in all mutated alleles. Thus, tumors induced by the two protocols did not differ in rasHa activation.

Coculture of neoplastic and normal keratinocytes as a model to study tumor promotion.

In order to study skin tumor promotion, a cell culture model system analogous to initiated mouse epidermis was developed. Keratinocytes of the neoplastic cell line 308 display the initiated phenotype since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal keratinocytes results in the suppression of growth of colonies of 308 cells. This inhibition, which is calcium dependent, epidermal cell specific, and requires cell contact, can be overcome by exposure to various tumor promoters. Promoters and antipromoters of diverse structure and mechanism of action are recognized in this keratinocyte coculture model.

Development of an in vitro analogue of initiated mouse epidermis to study tumor promoters and antipromoters.

To facilitate the study of skin tumor promotion, a cell culture model system with characteristics analogous to initiated mouse epidermis was established. Cells of the keratinocyte cell line 308, derived from adult mouse skin initiated with 7,12-dimethylbenz[a]anthracene, display the initiated phenotype, since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal primary keratinocytes resulted in the inhibition of growth of colonies of 308 cells. Addition of fresh keratinocytes weekly was required to sustain the inhibition for 3-4 weeks. Inhibition of 308 cell colonies required culture medium with a calcium concentration of 1.2 mM; normal keratinocytes did not inhibit 308 cells in medium with 0.05 mM calcium. Growth of 308 cells was not inhibited by coculture with confluent fibroblasts or by 1.2 mM calcium medium conditioned by either keratinocytes or fibroblasts. During continuous exposure of the cocultures to tumor promoters, 308 cell colonies became apparent within 2-3 weeks. A limited number of promoters were tested in this model system and 12-O-tetradecanoylphorbol-13-acetate, 12-O-retinoylphorbol-13-acetate, mezerein, and benzoyl peroxide were all active. The number of colonies which developed during promoter exposure in cocultures showed a dose-response curve which differed from the dose-response curve for stimulation of growth of 308 colonies in the absence of normal keratinocytes. Simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and known inhibitors of skin tumor promotion, such as retinoic acid, fluocinolone acetonide, and bryostatin 1, blocked colony formation of 308 cells in cocultures but not in cultures with only 308 cells. In this model system, the actions of promoters and inhibitors both appear to be mediated by normal keratinocytes.

Levels of bacteria, fungi, and endotoxin in bulk and aerosolized corn silage.

Three samples of silage taken from the surface of a silo and from depths of 20 and 45 cm in the silo were studied for identification of the potential agents causing symptoms of organic dust toxic syndrome. The samples were examined by dilution plating before and after aerosolization in an acoustical dust generator. Aerosol samples were collected by liquid impinger and filter cassettes. The samples were examined for total aerobic bacteria, anaerobic bacteria, gram-negative bacteria, lactobacilli, listeriae, thermophilic actinomycetes, fungi, and endotoxin. Very high levels of total aerobic bacteria and fungi were found in the surface sample (up to 10(9) CFU/g in the bulk sample and up to 10(9) CFU/m3 after aerosolization), whereas the corresponding values from the deepest site were 100 to 50,000 times lower. Aspergillus fumigatus predominated among the fungi, whereas Bacillus and gram-negative organisms (Pseudomonas, Alcaligenes, Citrobacter, and Klebsiella species) prevailed among bacteria. Thermophilic actinomycetes occurred in numbers up to 10(7) CFU/g in the bulk samples, whereas anaerobic bacteria, lactobacilli, and listeriae were only few or absent. The concentration of endotoxin was high in the surface sample (up to 211.4 Endotoxin Units/mg) and about 200-fold lower in the sample from the deepest site. The results show that contact with dust from the surface of silage carries the risk of exposure to high concentrations of microorganisms, of which A. fumigatus and endotoxin-producing bacteria are the most probable disease agents.

Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra.

Stachybotrys atra is the etiologic agent of stachybotryotoxicosis, and this fungus and its trichothecene mycotoxins were recently implicated in an outbreak of unexplained illness in homes. S. atra was grown on sterile rice, autoclaved, dried, and then aerosolized by acoustic vibration. The distribution of particles (mass and number) was monitored on an aerodynamic particle sizer interfaced with a computer. Dust was collected on preweighed glass-fiber filters and extracted with 90% aqueous methanol. Extracts were tested for the ability to inhibit protein synthesis in rat alveolar macrophages, the ability to inhibit the proliferation of mouse thymocytes, and the presence of specific trichothecene mycotoxins. Virtually all of the particles were less than 15 micron in aerodynamic diameter, and the mass median diameter was 5 micron. Thus, most of the particles were respirable. Microscopic analysis of the generated dust revealed that ca. 85% of the dust particles were conidia of S. atra, another 6% were hyphal fragments, and the remainder of the particles were unidentifiable. Thus, greater than 90% of the particles were of fungal origin. The extracts strongly inhibited protein synthesis and thymocyte proliferation. Purified satratoxin H was also highly toxic in the same systems. Each of the individual filters contained satratoxin H (average, 9.5 ng/mg of dust). Satratoxin G and trichoverrols A and B were found in lesser amounts in some, but not all, of the filters. The limit of analysis is ca. 50 ng. These results establish that the conidia of S. atra contain trichothecene mycotoxins. In view of the potent toxicity of the trichothecenes, the inhalation of aerosols containing high concentrations of these conidia could be a potential hazard to health.