Artemisia dracunculus L. Ethanolic Extract and an Isolated Component, DMC2, Ameliorate Inflammatory Signaling in Pancreatic ß-Cells via Inhibition of p38 MAPK.

Non-resolving pancreatic islet inflammation is widely viewed as a contributor to decreases in ß-cell mass and function that occur in both Type 1 and Type 2 diabetes. Therefore, strategies aimed at reducing or eliminating pathological inflammation would be useful to protect islet ß-cells. Herein, we described the use of 2',4'-dihydroxy-4-methoxydihydrochalcone (DMC2), a bioactive molecule isolated from an ethanolic extract of Artemisia dracunculus L., as a novel anti-inflammatory agent. The ethanolic extract, termed PMI 5011, reduced IL-1ß-mediated NF-κB activity. DMC2 retained this ability, indicating this compound as the likely source of anti-inflammatory activity within the overall PMI 5011 extract. We further examined NF-κB activity using promoter-luciferase reporter constructs, Western blots, mRNA abundance, and protein secretion. Specifically, we found that PMI 5011 and DMC2 each reduced the ability of IL-1ß to promote increases in the expression of the Ccl2 and Ccl20 genes. These genes encode proteins that promote immune cell recruitment and are secreted by ß-cells in response to IL-1ß. Phosphorylation of IκBα and the p65 subunit of NF-κB were not reduced by either PMI 5011 or DMC2; however, phosphorylation of p38 MAPK was blunted in the presence of DMC2. Finally, we observed that while PMI 5011 impaired glucose-stimulated insulin secretion, insulin output was preserved in the presence of DMC2. In conclusion, PMI 5011 and DMC2 reduced inflammation, but only DMC2 did so with the preservation of glucose-stimulated insulin secretion.

Mechanisms of Artemisia scoparia's Anti-Inflammatory Activity in Cultured Adipocytes, Macrophages, and Pancreatic ß-Cells.

OBJECTIVE: An ethanolic extract of Artemisia scoparia (SCO) improves adipose tissue function and reduces negative metabolic consequences of high-fat feeding. A. scoparia has a long history of medicinal use across Asia and has anti-inflammatory effects in various cell types and disease models. The objective of the current study was to investigate SCO's effects on inflammation in cells relevant to metabolic health. METHODS: Inflammatory responses were assayed in cultured adipocytes, macrophages, and insulinoma cells by quantitative polymerase chain reaction, immunoblotting, and NF-κB reporter assays. RESULTS: In tumor necrosis factor α-treated adipocytes, SCO mitigated ERK and NF-κB signaling as well as transcriptional responses but had no effect on fatty acid-binding protein 4 secretion. SCO also reduced levels of deleted in breast cancer 1 protein in adipocytes and inhibited inflammatory gene expression in stimulated macrophages. Finally, in pancreatic ß-cells, SCO decreased NF-κB-responsive promoter activity induced by IL-1ß treatment. CONCLUSIONS: SCO's ability to promote adipocyte development and function is thought to mediate its insulin-sensitizing actions in vivo. Our findings that SCO inhibits inflammatory responses through at least two distinct signaling pathways (ERK and NF-κB) in three cell types known to contribute to metabolic disease reveal that SCO may act more broadly than previously thought to improve metabolic health.

Response to adrenocorticotropic hormone or corticotrophin-releasing hormone and vasopressin in lactating cows fed an immunomodulatory supplement under thermoneutral or acute heat stress conditions.

Adrenal responsiveness was tested in nonpregnant, lactating Holstein dairy cows fed diets supplemented with OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ), an immune modulator, and in nonsupplemented control (CON) cows following bolus infusions of a combination of corticotropin-releasing hormone (CRH; 0.3 µg/kg of BW) and arginine vasopressin (VP; 1.0 µg/kg of BW) or ACTH (0.1 IU/kg of BW) in 2 environments: thermoneutral [TN; temperature-humidity index (THI) <60] for 24 h/d and heat stress (HS; THI >68 for 17 h/d). Cows (506) were initially fed OG (n = 254) or CON (n = 252) diets for 44 d before selection of a subgroup of cows (n = 12; 6 OG, 6 CON) for the study. The 2 subgroups were balanced for parity, milk yield, and days in milk. All cows were transported to and housed in 2 environmentally controlled rooms at the University of Arizona Agricultural Research Complex (Tucson). Cows were given 3 d to acclimate to the rooms and then underwent 12 d of TN conditions and then 8 d of HS conditions for a total of 24 d on experiment. Cows were infused with CRH-VP on d 9 of TN and on d 1 of HS and with ACTH on d 10 of TN and on d 2 of HS. Hormone infusions took place at 1000 h (0 h) on each infusion day. Blood samples, taken in 30-min intervals, were first collected at 0800 h (-2 h) and were drawn until 1800 h (8 h). Before infusion, serum progesterone was elevated in OG cows compared with CON cows. Infusion of releasing factors (CRH-VP or ACTH) caused increases in serum cortisol and progesterone, but cortisol release was greater in CON cows than in OG cows during HS, whereas progesterone did not differ between the 2 treatments. Serum ACTH increased following infusion of releasing factors, but this increase was greater following CRH-VP infusion than ACTH infusion. Serum bovine corticosteroid-binding globulin also increased following infusion of releasing factors in both treatment groups, but this increase was greater during HS in cows fed OG. The free cortisol index (FCI) increased following CRH-VP and ACTH and was higher in HS than in TN for both OG and CON cows. However, the FCI response was blunted in OG cows compared with CON cows during HS. Heat stress enhanced the adrenal response to releasing factors. Additionally, the adrenal cortisol and FCI response to releasing factors was reduced during acute heat stress in cows fed OG. Collectively, these data suggest that OG supplementation reduced the adrenal responsiveness to factors regulating cortisol secretion during acute HS.

Effects of niacin and betaine on bovine mammary and uterine cells exposed to thermal shock in vitro.

The objective of this study was to investigate the direct effects of feed supplements niacin and betaine on the heat shock responses of in vitro cultured cells derived from bovine mammary and uterine tissues. First, we determined the mRNA expression profiles of the niacin receptor (GPR109A) in bovine tissues (liver, skin, uterus, udder, and ovary) and in cells derived from bovine mammary epithelium (mammary alveolar cells, MAC-T; bovine mammary epithelial cells, BMEC) and endometrium (bovine endometrial cells, BEND). We found that GPR109A was distributed in all examined tissues and cells, and the highest expression was in cells from skin and udder. Second, we evaluated the effects of niacin treatment on the mRNA abundance of heat shock proteins 70 and 27 (HSP70 and HSP27) in MAC-T, BMEC, and BEND under thermoneutral conditions and heat stress, and whether these effects were associated with alterations in the mRNA expression of prostaglandin E2 synthesis-related genes, including cyclooxygenase 1 and 2 (COX-1 and COX-2) and microsomal prostaglandin E synthase 1 and 2 (mPGES-1 and mPGES-2). Quantitative PCR data indicated that niacin suppressed HSP70 mRNA expression in BMEC and both HSP70 and HSP27 in BEND under thermoneutral conditions. Only COX-2 expression was downregulated by niacin in BMEC; other prostaglandin E2 synthesis-related genes stayed unaltered in BMEC and BEND. The mRNA abundance of HSP70, COX-1, COX-2, and mPGES-1 were elevated in niacin-treated MAC-T. During heat stress, niacin increased mRNA levels of HSP70 and HSP27 in MAC-T and HSP27 in BEND, but decreased HSP70 in BMEC. Although mPGES-2 was stimulated by niacin in BEND, the mRNA expression of prostaglandin E2 synthesis-related genes were consistent with neither HSP70 nor HSP27 expression patterns in niacin-treated BMEC and MAC-T. These data suggest that the effects of niacin on heat shock protein expression and prostaglandin E2 synthesis were not well coupled in these cells. Finally, we tested the effects of betaine treatment on viability and apoptosis in BMEC. Compared with control cultures, viability was higher in betaine-treated cells at 8 h under thermoneutral conditions and at 16 h in heat stress, and apoptotic rates were lower at 8 h. Our data support a dual role for niacin in regulating heat shock protein expression in normal and heat-shocked cells derived from mammary and uterine tissues, and positive effects of betaine in regulating mammary cell viability during heat stress.

Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice.

Type 1 diabetes mellitus results from autoimmune-mediated destruction of pancreatic islet ß-cells, a process associated with inflammatory signals. We hypothesized that dietary supplementation with botanicals known to contain anti-inflammatory properties would prevent losses in functional ß-cell mass in nonobese diabetic (NOD) mice, a rodent model of autoimmune-mediated islet inflammation that spontaneously develops diabetes. Female NOD mice, a model of spontaneous autoimmune diabetes, were fed a diet supplemented with herbal extracts (1.916 g total botanical extracts per 1 kg of diet) over a 12-week period. The mice consumed isocaloric matched diets without (controls) and with polyherbal supplementation (PHS) ad libitum starting at a prediabetic stage (age 6 weeks) for 12 weeks. Control mice developed hyperglycemia (>180 mg/dL) within 16 weeks (n = 9). By contrast, mice receiving the PHS diet did not develop hyperglycemia by 18 weeks (n = 8). Insulin-positive cell mass within pancreatic islets was 31.9% greater in PHS mice relative to controls. We also detected a 26% decrease in CD3(+) lymphocytic infiltration in PHS mice relative to mice consuming a control diet. In vitro assays revealed reduced ß-cell expression of the chemokines CCL2 and CXCL10 after overnight PHS addition to the culture media. We conclude that dietary PHS delays initiation of autoimmune-mediated ß-cell destruction and subsequent onset of diabetes mellitus by diminishing islet inflammatory responses.

A dose-response evaluation of rumen-protected niacin in thermoneutral or heat-stressed lactating Holstein cows.

Twenty-four multiparous high-producing dairy cows (40.0±1.4kg/d) were used in a factorial design to evaluate effects of 2 environments [thermoneutral (TN) and heat stress (HS)] and a dose range of dietary rumen-protected niacin (RPN; 0, 4, 8, or 12g/d) on body temperature, sweating rate, feed intake, water intake, production parameters, and blood niacin concentrations. Temperature-humidity index values during TN never exceeded 68 (stress threshold), whereas temperature-humidity index values during HS were above 68 for 24h/d. The HS environment increased hair coat and skin, rectal, and vaginal temperatures; respiration rate; skin and hair coat evaporative heat loss; and water intake and decreased DMI (3.5kg/d), milk yield (4.1kg/d), 4% fat-corrected milk (2.7kg/d), and milk protein yield (181.7g/d). Sweating rate increased during HS (12.7g/m(2) per h) compared with TN, but this increase was only 10% of that reported in summer-acclimated cattle. Niacin supplementation did not affect sweating rate, dry-matter intake, or milk yield in either environment. Rumen-protected niacin increased plasma and milk niacin concentrations in a linear manner. Heat stress reduced niacin concentration in whole blood (7.86 vs. 6.89µg/mL) but not in milk. Reduced blood niacin concentration was partially corrected by dietary RPN. An interaction existed between dietary RPN and environment; dietary RPN linearly increased water intake in both environments, but the increase was greater during HS conditions. Increasing dietary RPN did not influence skin temperatures. During TN, supplementing 12g/d of RPN increased hair coat (unshaved skin; 30.3 vs. 31.3°C at 1600h) but not shaved skin (32.8 vs. 32.9°C at 1600h) temperature when compared with 0g/d at all time points, whereas the maximum temperature (18°C) of the room was lower than skin temperature. These data suggest that dietary RPN increased water intake during both TN and HS and hair coat temperature during TN; however, core body temperature was unaffected. Thus, encapsulated niacin did not improve thermotolerance of winter-acclimated lactating dairy cows exposed to moderate thermal stress in Arizona.

Fatal case of sorafenib-associated idiosyncratic hepatotoxicity in the adjuvant treatment of a patient with renal cell carcinoma.

BACKGROUND: Sorafenib is an orally available kinase inhibitor with activity at Raf, PDGFß and VEGF receptors that is licensed for the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Current evidence-based post-nephrectomy management of individuals with localized RCC consists of surveillance-based follow up. The SORCE trial is designed to investigate whether treatment with adjuvant sorafenib can reduce recurrence rates in this cohort. CASE PRESENTATION: Here we report an idiosyncratic reaction to sorafenib resulting in fatal hepatotoxicity and associated renal failure in a 62 year-old man treated with sorafenib within the SORCE trial. CONCLUSION: This is the first reported case of sorafenib exposure associated fatal toxicity in the adjuvant setting and highlights the unpredictable adverse effects of novel adjuvant therapies.

Zyflamend reduces the expression of androgen receptor in a model of castrate-resistant prostate cancer.

Prostate cancer is the most commonly diagnosed solid malignancy, and tumor cells eventually transform to castrate resistance through multiple pathways including activation of the androgen receptor via insulin-like growth factor receptor (IGF-1R) signaling involving phospho-AKT (pAKT). In this study, a mixture of herbal extracts, Zyflamend®, was used as a treatment in a model of castrate-resistant prostate cancer using CWR22Rv1 cells. Zyflamend reduced androgen receptor and IGF-1R expression along with a reduction of IGF-1-mediated proliferation of CWR22Rv1 cells. IGF-1 induced downstream AKT phosphorylation; however, the induction of pAKT was not associated with androgen receptor expression. Further, constitutively active form of AKT had no effect on nuclear expression of androgen receptor, indicating that upregulation of pAKT did not promote androgen receptor expression or nuclear translocation in castrate-resistant CWR22Rv1 cells. Conversely, Zyflamend reduced androgen receptor expression following IGF-1 stimulation and in cells overexpressing pAKT. These results demonstrated that Zyflamend inhibited IGF-1-stimulated cell growth, IGF-1R expression, and androgen receptor expression and its nuclear localization, but these effects were not dependent upon phosphatidylinositol 3-kinase/pAKT signaling. In conclusion, Zyflamend decreased cell proliferation and inhibited IGF-1R and androgen receptor expression in a phosphatidylinositol 3-kinase/pAKT independent manner.

Alterations of A549 lung cell gene expression in response to biochemical toxins.

Health risks associated with the inhalation of potentially toxic materials have been a topic of great public concern. In vitro cellular analyses can provide mechanistic information on the molecular-level responses of lung-derived cell lines to a variety of these hazards. This understanding may be used to develop methods to reduce the damage from such toxins or to detect early stages of their effects. Here we describe an evaluation of the alterations in gene expression of an immortalized lung cell line (A549, human type II epithelia) to a variety of inhalation health hazards including etoposide, gliotoxin, streptolysin O, methyl methansesulfonate (MMS), and Triton X-100. The A549 cells display a dose-response relationship to each toxin with initial responses including alterations in metabolic activity, increases in membrane permeability, and initiation of response genes. In general, membrane-damaging agents (streptolysin O and Triton X-100) induce production of new ion channel proteins, structural proteins, and metabolic enzymes. Gliotoxin impacted the metabolic machinery, but also altered ion channels. Etoposide and MMS caused alterations in the cell cycle, induced DNA repair enzymes, and initiated apoptotic pathways, but MMS also induced immune response cascades. The mechanism of cell response to each toxin is supported by physiological analyses that indicated a fairly slow initiation of cell response to all compounds tested, except for Triton, which caused rapid decline in cell function due to solubilization of the cell membrane. However, Triton does induce production of a number of cell membrane-associated proteins and so its effects at low concentrations are likely translated throughout the cell. Together these results indicate a broader array of cellular responses to each of the test toxins than have previously been reported.

Injury prevention training: a cluster randomised controlled trial assessing its effect on the knowledge, attitudes, and practices of midwives and health visitors.

OBJECTIVE: To evaluate the effectiveness of injury prevention training. DESIGN: Cluster randomised controlled trial. SETTING: Primary care facilities in the East Midlands area of the United Kingdom. SUBJECTS: Midwives and health visitors. INTERVENTION: Evidence based training session on the risks associated with baby walkers. MAIN OUTCOME MEASURES: The primary outcome measures were knowledge of baby walker use and walker related injury, attitudes towards walkers and towards walker education, and practices relating to walker health education. RESULTS: Trained midwives and health visitors had greater knowledge of the risks associated with baby walkers than untrained midwives and health visitors (difference between the means 0.22; 95% confidence interval (CI) 0.12 to 0.33). Trained health visitors had more negative attitudes to baby walkers (difference between the means 0.35; 95% CI 0.10 to 0.59) and more positive attitudes towards baby walker health education (difference between the means 0.31; 95% CI 0.00 to 0.62) than untrained health visitors. Midwives who had been trained were more likely to discuss baby walkers in the antenatal period than those who were not trained (odds ratio 9.92; 95% CI 2.02 to 48.83). CONCLUSIONS: Injury prevention training was associated with increased knowledge, more negative attitudes towards walkers, and more positive attitudes towards walker education. Trained midwives were more likely to give advice antenatally. Training did not impact on other practices. Larger trials are required to assess the impact of training on parental safety behaviours, the adoption of safety practices, and injury reduction.

Relaxation training for psychiatric inpatients.

In Exp. 1 the effects of progressive and imaginal relaxation training were examined for 51 psychiatric inpatients. Relaxation Inventory scores indicated significant changes in the direction of greater relaxation for each training procedure; there were no significant differences in responses to the two types of training. Significant relaxation effects were found for each of three training sessions, but the effects were not cumulative. Only one patient was withdrawn because reaction to training was overtly negative. Exp. 2 was an analysis of Exp. 1 data in combination with data from a prior study. Patients and college students responded much alike but students reached greater relaxation within sessions. Further experimentation on relaxation training with psychiatric inpatients appears justified.

Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: not all bleaching is the same.

Cell coloration changes from normal blue-green to yellow or yellow-green when the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient. We found that this bleaching process (chlorosis) in cells deprived of sulfur (S) was similar to that in cells deprived of nitrogen (N), but that cells deprived of phosphorus (P) bleached differently. Cells divided once after N deprivation, twice after S deprivation, and four times after P deprivation. Chlorophyll (Chl) accumulation stopped almost immediately upon N or S deprivation but continued for several hours after P deprivation. There was no net Chl degradation during N, S, or P deprivation, although cellular Chl content decreased because cell division continued after Chl accumulation ceased. Levels of the light-harvesting phycobiliproteins declined dramatically in a rapid response to N or S deprivation, reflecting an ordered breakdown of the phycobilisomes (PBS). In contrast, P-deprived cultures continued to accumulate PBS for several hours. Whole PBS were not extensively degraded in P-deprived cells, although the PBS contents of P-deprived cells declined because of continued cell division after PBS accumulation ceased. Levels of mRNAs encoding PBS polypeptides declined by 90 to 95% in N- or S-deprived cells and by 80 to 85% in P-deprived cells. These changes in both the synthesis and stability of PBS resulted in a 90% decline in the PC/Chl ratio of N- or S-deprived cells and a 40% decline in the PC/Chl ratio of P-deprived cells. Therefore, although bleaching appears to be a general response to nutrient deprivation, it is not the same under all nutrient-limited conditions and is probably composed of independently controlled subprocesses.

Dilator actions of arginine in human peripheral vasculature.

1. L-Arginine is the physiological precursor for the formation of endothelium-derived nitric oxide. The synthesis of nitric oxide is stereospecific: D-arginine is not a substrate for nitric oxide synthase. It is possible that the provision of excess L-arginine substrate might increase the vascular synthesis of nitric oxide. We have examined this possibility by studying the effects of local infusion of L- and D-arginine in the forearm resistance bed and the superficial dorsal hand veins of healthy subjects. 2. Drugs were either infused locally into a vein on the back of the hand and then the vein diameter was measured using a linear displacement technique, or into the brachial artery and then the forearm blood flow was measured by venous occlusion plethysmography. 3. In the superficial hand veins, L- and D-arginine free base and L- and D-arginine hydrochloride (all four preparations at a dose of 5 mumol/min) all caused a significant increase in venous diameter. The responses of the L- and D-enantiomers did not differ significantly from one another. 4. In the forearm resistance bed, L- and D-arginine free base and L- and D-arginine hydrochloride were without effect at doses of 10 and 40 mumol/min. However, at doses of 160 mumol/min all three preparations of arginine caused a significant increase in forearm blood flow compared with control values. The responses to the three preparations of arginine did not differ significantly from one another. 5. These results show that arginine in high dose is a vasodilator in both human resistance vessels and superficial veins in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

A therapeutic benefit from combining normobaric carbogen or oxygen with nicotinamide in fractionated X-ray treatments.

The ability of normobaric oxygen and carbogen (95% O2 + 5% CO2) combined with nicotinamide to enhance the radiosensitivity of two rodent adenocarcinomas and of mouse skin and kidneys, using a 10 fraction radiation schedule, was compared with the effect of radiation in air with and without the drug. Tumour response was assayed using local control and regrowth delay, and compared with acute skin reactions, decreased renal 51Cr-EDTA clearance and reduction in haematocrit. Nicotinamide increased the radiation sensitivity of CaNT tumours under all three different oxygen concentrations tested (21, 95 and 100% oxygen). The effect was statistically significant for oxygen and carbogen but not for air; the combination of nicotinamide with carbogen gave the greatest increase in tumour radiosensitivity. Relative to treatments in air without the drug, the enhancement ratios (ER) at the TCD50 level were 1.17, 1.65 and 1.83 for CaNT tumours irradiated in air, oxygen or carbogen and injected with nicotinamide 1 h before each fraction. The ER in CaRH tumours irradiated in carbogen plus the drug was 1.83, which was greater, but statistically not significantly different, to that seen with carbogen alone (ER = 1.68). In skin, relative to air without the drug, the increase in radiosensitivity by nicotinamide was greater in oxygen and carbogen than in air (1.29, 1.36 and 1.08, respectively). The ERs for both assays of renal damage were similar and lower than those in skin: less than or equal to 1.07, less than or equal to 1.13 and less than or equal to 1.16 for irradiations done in air, oxygen and carbogen plus nicotinamide, relative to air alone. A comparison of these results in the tumours and normal tissues showed that a significant therapeutic benefit was obtained with normobaric oxygen and carbogen combined with nicotinamide. This benefit is greater than observed with other radiosensitizers tested so far. Toxic side effects of the treatment are unlikely in a clinical situation, since prolonged administration of nicotinamide is well tolerated in man. The combination of normobaric carbogen with nicotinamide could be an effective method of enhancing tumour radiosensitivity in clinical radiotherapy where hypoxia limits the outcome of treatment.

Nitric oxide synthesised from L-arginine mediates endothelium dependent dilatation in human veins in vivo.

Endothelium derived relaxing factor (EDRF) has been identified as nitric oxide, synthesised from the amino acid L-arginine, a process which is inhibited by the L-arginine analogue NG-monomethyl L-arginine (L-NMMA). We have studied the effect of local infusions of L-NMMA on venous reactivity in healthy volunteers. Studies were performed using the veins on the back of the hand. The diameter of a single dorsal hand vein was measured in healthy subjects who had taken 600 mg of aspirin 30 min before the experiment. Changes in diameter were recorded during local infusions of noradrenaline, bradykinin, acetylcholine, glyceryl trinitrate, L- and D-arginine and its NG-monomethyl derivatives. L-NMMA (100 nmol.min-1) stereospecifically inhibited vasodilatation induced by acetylcholine and bradykinin (p less than 0.02) but not that induced by the endothelium independent vasodilator glyceryl trinitrate. L-NMMA (100 nmol.min-1) potentiated the venoconstrictor effect of a high dose of acetylcholine (100 nmol.min-1) without affecting the action of noradrenaline and without having a direct venoconstrictor effect in doses up to 10 mumol.min-1. These results show that the venous effects of certain vasodilators in man are mediated through the release of nitric oxide (EDRF) synthesised from L-arginine. They also highlight differences in basal and stimulated production of nitric oxide between arteries and veins.

Relaxation training side effects reported by seriously disturbed inpatients.

The extent to which seriously disturbed inpatients report side effects related to passive and progressive relaxation training was examined. Most reported few side effects; only 1 of 64 subjects was removed because of a negative reaction to training. There was no significant difference in side effects reported in response to the two training procedures. Comparisons were made with data from a survey of therapists who practice relaxation training.

Sodium cromoglycate and atropine block the fall in FEV1 but not the cough induced by hypotonic mist.

In a group of patients with mild asthma the inhalation of mist derived from ultrasonically nebulised distilled water caused an increase in cough and a fall in FEV1. Double blind administration for five minutes of sodium cromoglycate (from an original solution containing 30 mg/ml) or atropine (2 mg/ml) by inhalation from a Minineb nebuliser, 30 minutes before the mist challenge, caused a significant reduction in the fall in FEV1 (p less than 0.05), but not in cough, by comparison with the protection afforded by placebo (saline). In a second study the fall in FEV1 caused by the inhalation of distilled water was not significantly different from that seen in response to hypotonic sodium chloride (1.7 g/l, 58 mmol/l), but both produced a significantly greater fall than did a similar mist containing sodium cromoglycate at an original concentration of 10 mg/ml (58 mmol/l). The results show that both atropine and sodium cromoglycate can block the fall in FEV1 due to mist and that protection by sodium cromoglycate is immediate. These results suggest that sodium cromoglycate blocks the nervous reflexes concerned in the response to mist, probably in the afferent limb of the reflex.