Quercetin and quercetin glycosides are widely consumed flavonoids found in many fruits and vegetables. These compounds have a wide range of potential health benefits, and understanding the bioavailability of flavonoids from foods is becoming increasingly important.This study combined an in vitro digestion, a lactase treatment and the Caco-2 cell model to examine quercetin and quercetin glucoside uptake from shallot and apple homogenates.p < 0.05), but had no significant effect on quercetin-3-glucoside recovery (p > 0.05). Digestion increased the Caco-2 cell uptake of shallot quercetin-4'-glucoside by 2-fold when compared to the non-digested shallot. Despite the loss of quercetin from the digested shallot, the bioavailability of quercetin aglycone to the Caco-2 cells was the same in both the digested and non-digested shallot. Treatment with lactase increased quercetin recovery from the shallot digestate nearly 10-fold and decreased quercetin-4'-glucoside recovery by more than 100-fold (p < 0.05), but had no effect on quercetin recovery from apple digestates. Lactase treatment also increased shallot quercetin bioavailability to the Caco-2 cells approximately 14-fold, and decreased shallot quercetin-4'-glucoside bioavailability 23-fold (p < 0.05). These Caco-2 cells had lactase activity similar to that expressed by a lactose intolerant human.The in vitro digestion alone significantly decreased quercetin aglycone recovery from the shallot digestate (The increase in quercetin uptake following treatment with lactase suggests that dietary supplementation with lactase may increase quercetin bioavailability in lactose intolerant humans. Combining the digestion, the lactase treatment and the Caco-2 cell culture model may provide a reliable in vitro model for examining flavonoid glucoside bioavailability from foods. Cardiovascular disease and cancer are the two most common causes of death in the United States and most industrialized nations. A diet high in fruits and vegetables has been correlated with a reduced risk for both cancer and heart disease ,2. It isA phytochemical of particular interest is quercetin, a strong antioxidant that is widely consumed in many fruits and vegetables. Quercetin has potential protective effects against both cancer and heart disease. Briefly, quercetin has been found to down regulate expression of mutant p53 in breast cancer cells, arrest human leukemic T-cells in G1, inhibit tyrosine kinase, and inhibit heat shock proteins . QuercetMore work is needed to understand the bioavailability of quercetin and other flavonoids from foods. The Caco-2 cell culture model is a well-established in vitro technique, extensively used to study intestinal cell absorption of compounds such as pharmaceuticals and nutrients; it is an excellent in vitro tool to study bioavailability of specific compounds. We have previously used the Caco-2 cell culture model to examine the uptake of quercetin from apple and onion extracts . Using tin vitro digestion has been paired successfully with the Caco-2 cell culture model to study iron and carotenoid bioavailability [in vitro digestion with the Caco-2 cell culture model could be quite useful in more specifically analyzing quercetin bioavailability from foods. At this time there is little information available describing the effects of digestion on flavonoids from foods. Vallejo et al. [To further understand quercetin bioavailability, it is important to examine the effects of digestion on foods prior to intestinal uptake. An lability ,24. Use o et al.  found tho et al. .in vitro model would aid in evaluating bioavailability of phytochemicals from foods by offering a simple method to screen for factors that may affect intestinal absorption of quercetin and quercetin glucosides, such as the food matrix, food processing, digestion, and interactions with other foods. Human and animal models can be expensive and time consuming, while a cell culture model allows for rapid, inexpensive screenings. The Caco-2 model has the potential to be a good model to measure quercetin absorption, however there are some drawbacks. Caco-2 cells have been shown to express significantly less lactase phlorizin hydrolase (LPH) than the average human small intestine [A good ntestine . Since tntestine , this deCaco-2 cells used in our lab have expressed greater LPH activity (3 mU/mg protein) than other Caco-2 cells (0.3 mU/mg protein) , resultiThe objectives of this study were (1) to develop an optimized in vitro digestion method for examining quercetin and quercetin glucoside recovery from digestates using onions and apples; (2) to examine the effect of lactase on shallot digestates; and (3) to examine Caco-2 cellular uptake of quercetin and quercetin glucosides from digested and lactase treated shallot.Kluyveromyces lactis, activity of 3000 units/mL), quercetin, and quercetin-4'-glucoside were purchased from Sigma Chemical Company . Quercetin-3-glucoside was purchased from Indofine Chemical Company, Inc . Caco-2 cells were obtained from the American Type Culture Collection  and were cultured in Dulbecco's Modified Eagle Medium  supplemented with 5% fetal bovine serum , 10 mM HEPES, 50 units/mL penicillin, 50 μg/mL streptomycin, and 100 μg/mL gentamicin, and were maintained at 37°C in 5% CO2.Shallots and onions (Northern Yellow) were obtained from a local grocery store. Apples (Red Delicious and Cortland varieties) were obtained from the Cornell Orchards . Porcine pepsin, bile extract, pancreatin, lactase  using a Waring blender, and then homogenized using a Virtis 45 homogenizer. The total homogenates were aliquotted in 15 mL centrifuge tubes and stored at -20°C until use.3. Porcine bile extract and pancreatin were added to a final concentration of 1.1 and 0.175 mg/mL, respectively. The pH was increased to 6.5 by drop-wise addition of 1M NaHCO3, and the samples were incubated for 1 hour in a water bath at 37°C. Following digestion the pH was decreased to 2 by addition of HCl and the digestates were stored at -80°C for further analysis.For the digestion treatment, 2 g aliquots of the food sample were placed in a centrifuge tube with an equal amount of saline. The pH was decreased to 2.0 by drop-wise addition of 1M HCl, and porcine pepsin was added to a final concentration of 1.3 mg/mL. The digestate was incubated in a shaking water bath at 37°C for 30 minutes. The pH of the digestate was then increased to 5.8 with the drop-wise addition of 1M NaHCOTo examine and optimize the effects of digestion time and pH on the recovery of compounds from the digestate, the above parameters were varied. To examine the effects of pepsin digestion time, the pepsin digestates were incubated for 0, 30, 60 or 90 minutes and then incubated with the intestinal digestion enzymes for 60 minutes. To examine the effects of intestinal digestion, the samples were incubated with pepsin for 30 minutes, then incubated with pancreatin and bile for 0, 30, 60, or 90 minutes.The effects of 100 μM ascorbic acid and a nitrogen environment on quercetin and quercetin glucoside recovery from onion and apple digestates were examined. Following homogenization and prior to digestion, the food samples were mixed 1:1 with saline containing 200 μM ascorbic acid, leaving a final sample concentration of 100 μM ascorbic acid. During the digestion procedure described above, the samples were flushed constantly with nitrogen.The effect of pH of either 6.5 or 7.0 during intestinal digestion on quercetin and quercetin-3-glucoside recovery was compared. The effect of the storage pH was examined by comparing digested samples having either a final pH of 2.0 or 6.5. The effect of storage pH on 20 μM pure quercetin and 20 μM quercetin-3-glucoside was examined by comparing recoveries from samples stored at pH = 2.0, 3.5, 5.0 or 7.0. All samples were stored overnight at -80°C. Prior to HPLC analysis samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL acidified methanol (pH 2.0).Doses of lactase (0.5 units to 3000 units per gram sample) were applied to 1 gram shallot extract and incubated for 15 minutes at 37°C. The final pH was brought to 2.0 and the samples were stored at -80°C. The time kinetics were examined by incubating 1 gram shallot extract with 100 units of lactase for 0, 15, 30, 60, 90, 120, 240, and 720 minutes.The effect of both lactase and digestion were examined by digesting the samples with pepsin and pancreatin as described above, then incubating the samples with 100 units of lactase for 30 minutes at 37°C. Samples were stored overnight at -80°C.Prior to HPLC analysis, all digestate samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL methanol.5 cells per well in a collagen coated 6-well, flat bottom plate and incubated at 37°C in a 5% CO2 environment. Caco-2 cells were used between passages #10–25, and the cells reached confluence approximately 5 days post seeding. Culture media was changed three times a week. On day 14 post seeding, the DMEM was removed and the cells were rinsed three times with phosphate buffered saline (PBS).Caco-2 cells were seeded at a density of 5 × 10Shallot homogenates were digested as previously described and were placed directly on the 14 day old Caco-2 cells, or the samples were diluted 1:2 or 1:4 in HBSS . Cells were also incubated with non-digested shallot homogenates for comparison. For each treatment, two wells of cells were used for each sample, and each treatment was repeated in triplicate.2. The shallot treatment and HBSS was removed and the cells were rinsed three times with 20% methanol in PBS. Cells were scraped in acidified methanol (pH = 2.0) and the wells were rinsed three times with methanol. The scraped cells were sonicated for 15 minutes, centrifuged at 1600 g for 5 minutes, and the methanol supernatant was collected. The cells were rinsed three more times with methanol, the supernatants were collected and the methanol extracts were evaporated to dryness under nitrogen and reconstituted in 400 μl acidified methanol for HPLC analysis.To examine the effect of lactase on quercetin uptake from shallots, shallots were digested then incubated with lactase  for 20 minutes at 37°C. The digested shallot homogenates and the digested plus lactase treated shallot homogenates were diluted 1:2 in HBSS and placed on the cells. In all experiments, Caco-2 cells were incubated with treatment for 30 minutes at 37°C in 5% CO5 cells per well in a 6-well flat bottom plate. The cells were cultured in DMEM spiked with different doses of either lactose , or forskolin . Media was changed every two days, and cells were harvested and lactase activity was measured at 14 days post-seeding. Lactase activity of Caco-2 cells was measured using a method adapted from Dahlqvist [Caco-2 cells were seeded at a density of 5 × 10ahlqvist . Cells wahlqvist . The resQuercetin and quercetin-3-glucoside content of untreated homogenates, digestates, and Caco-2 cell extracts were determined using an RP-HPLC procedure with a Supelcosil LC-18-DB column . Waters 515 HPLC pumps  and a Waters 2487 dual wavelength absorbance detector  set at 370 nm were used for all HPLC analysis. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside were used as standards. For the analysis of quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside in the apple peel extracts, shallot extracts, and digestate extracts, the solvent system used was (A) acidified water  and (B) acetonitrile. The gradient method was the following: 0.0 min, flow rate = 1.4, (A) 90% and (B) 10%; 53 min, flow rate = 1.5, (A) 80% and (B) 20%; 58 min, flow rate = 1.7, (A) 65% and (B) 35%; 64 min, flow rate = 1.4, (A) 90% and (B) 10%. Twenty μL injections were made for each sample. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside concentrations in the apple peel extracts, shallot extracts, and in the digestates were extrapolated from the pure quercetin and quercetin-3-glucoside standard curves.All data were reported as means ± SD for three replicates of each treatment. An analysis of variance (ANOVA) was used to compare results between treatment groups, and pairwise multiple comparisons were performed using Fisher's LSD with an individual error rate of 0.05. The statistical analysis was completed using Minitab Release 12 software .The total pepsin digestion time and pancreatin/bile digestion time had little to no effect on recovery of both quercetin and quercetin-3-glucoside from apple and onion homogenates when treated for up to 60 minutes (data not shown). After 90 minutes of pepsin digestion and 90 minutes of pancreatic digestion, quercetin and quercetin 3-glucoside in both the apple and the onion decreased slightly. Based on these results we chose to use a 30-minute pepsin digestion and a 60-minute pancreatin/bile digestion. In past studies, quercetin from onion and quercetin from quercetin-4-glucoside supplements reached the plasma in less than an hour following consumption by human volunteers . TherefoThe presence of ascorbic acid and nitrogen had no effect on quercetin or quercetin-3-glucoside recoveries from the digestates (data not shown). Quercetin and quercetin-3-glucoside recoveries from digested samples treated with ascorbic acid, nitrogen or both ascorbic acid and nitrogen were not different from the recoveries from the untreated digested samples.p > 0.05). However, following overnight storage at -80°C, the samples stored at pH 2.0 had significantly greater quercetin and quercetin-3-glucoside recoveries than samples stored at pH 6.5 and 7.0 (p < 0.05). Pure quercetin and quercetin-3-glucoside were also more stable at lower storage pH following digestion . Quercetin-3-glucoside recoveries from the digested apple, onion, and pure compound were 87.7, 89.5, and 86.4%, respectively. Quercetin recovery was significantly reduced in digested pure quercetin and digested onion samples, when compared to non-digested samples (p < 0.05). Quercetin recovery was lower than quercetin-3-glucoside recovery and tended to vary more, depending on the food matrix: 52.5% and 74.3%, from the onion and pure compound, respectively. There was no significant difference in quercetin recovery between to the non-digested and digested apple homogenates. There was only trace or no quercetin in non-digested apple homogenates, so the appearance of any quercetin following digestion resulted in a net increase.Based on these results, optimal digestion conditions were decided to be: pepsin digestion at pH 2.0 for 30 minutes, pancreatin/bile digestion at pH 6.5 for 60 minutes, and a final storage at pH 2.0. Ascorbic acid and nitrogen treatments were not continued. Using these conditions, the effect of digestion on quercetin and quercetin-3-glucoside recoveries from apples, onions, and pure quercetin and quercetin-3-glucoside was examined . Caco-2 cells treated with shallot homogenate absorbed approximately 2.9 ± 0.65 nmol of quercetin-4-glucoside, and Caco-2 cells treated with digested shallot absorbed 5.4 ± 0.04 nmol. Quercetin aglycone recovery from the digested shallot extract was only 47% that of the non-digested homogenate . Caco-2 cells absorbed 2.8 ± 0.4 nmol and 2.7 ± 0.2 nmol quercetin from the non-digested and digested shallot homogenates, respectively. Absorption of both quercetin-4-glucoside and quercetin from digested shallot followed a dose response. The Caco-2 cells absorbed quercetin 4'-glucoside and quercetin incrementally less from the digested samples that were diluted 1:2 or 1:4 in HBSS.Quercetin-4'-glucoside and quercetin were absorbed by Caco-2 cells following treatment with both digested shallot and non-digested shallot homogenates Figure . Quercete Figure  insert, Addition of lactose and forskolin, a specific inducer of lactase , to Cacop < 0.05; Figure p > 0.05).Treatment with 100 units lactase/g sample had a significant effect on both quercetin and quercetin-3-glucoside recoveries from the shallot . These cells had approximate lactase activity of between 2–4 mU/mg protein, consistent with what we previously reported . LactoseSince lactase activity could not be increased in the Caco-2 cells, we combined a lactase treatment with digestion to provide an intestinal uptake model that is more comparable to a lactose tolerant human, or a lactose intolerant human ingesting a lactase digestive aid to help improve lactose digestion. Not only did lactase increase the amount of quercetin in digested shallot homogenates, but it also increased the amount of quercetin taken up by the Caco-2 cells from the digested shallot extracts. This suggests that a lactase containing digestive aid may increase absorption of quercetin from onions in lactose intolerant humans. Combining the Caco-2 cell model with an in vitro digestion, simulating stomach and small intestinal digestion, and a lactase digestion may provide a more useful model to examine and screen for bioavailability of flavonoid glucosides from common foods Figure .Following an in vitro stomach and small intestinal digestion, the recovery of quercetin and quercetin-3-glucoside is optimized at a storage pH at or below 3.5. Storage at pH above 3.5 results in loss of most of the compounds. The in vitro digestion increased the uptake of shallot quercetin-4'-glucoside by the Caco-2 cells. Quercetin uptake by the Caco-2 cells was similar between digested and non-digested samples, despite the fact that approximately 50% of shallot quercetin is lost during digestion. Treating shallot digestates with lactase increased the recovery of quercetin aglycone 10-fold and decreased the recovery of quercetin-4'-glucoside. Lactase treatment increased the bioavailability of quercetin aglycone 14-fold and decreased the bioavailability of quercetin-4'-glucoside to the Caco-2 cells. Combining pepsin, pancreatin/bile, and lactase digestions with the Caco-2 cell culture monolayer may results in a useful model for studying flavonoid bioavailability from foods.Many advances have been made in understanding flavonoid bioavailability, however many questions still remain unanswered. Food processing, interactions with other compounds as well as with other foods are all factors that may affect bioavailability of flavonoids. A simple and inexpensive screening model would be beneficial as a first step in examining many of these factors. The Caco-2 cell model has the potential to be a good model for analyzing intestinal uptake of quercetin, quercetin glucoside and other flavonoids. While the Caco-2 cell model will never be an exact replica of a human small intestine, it could provide a valuable tool for initial screenings of large quantities of samples relatively quickly and inexpensively. In this study, it was found that a simple digestive aid such as lactase might increase quercetin bioavailability. This may be of importance to lactose intolerant people. Such significant trends may be examined in more detail using human or animal models.


Mutations of the BRAF gene are the most common genetic alteration in melanoma. Moreover, BRAF mutations are already present in benign nevi. Being overexpressed and mutated, B-Raf is a potential target for the immune system and as this mutation seems to be an early event, a humoral immune response against this antigen might serve as a diagnostic tool for detection of high risk patients.372 sera of 148 stage IV melanoma patients and 119 sera of non-melanoma patients were screened for B-Raf, B-Raf V599E and C-Raf specific antibodies by an ELISA assay. Sera were screened for specific total Ig and for IgG. Serum titers were compared with a two tailed Mann-Whitney U test. Sera with titers of 1:300 or higher were termed positive and groups were compared with a two tailed Fisher's exact test.B-Raf specific antibodies recognizing both B-Raf and B-Raf V599E were detected in 8.9% of the sera of melanoma patients and in 2,5% of the control group. Raf specific IgG was detected in some patients at very low levels. B-Raf specific antibody responses did not correlate with clinical parameters but in some cases, B-Raf antibodies emerged during disease progression.These findings imply that B-Raf is immunogenic in melanoma patients and that it might serve as a potential target for immunotherapy. However, B-Raf specific antibodies emerge at rather late stages of melanoma progression and are present only with a low frequency indicating that spontaneous B-Raf specific antibodies are not an early marker for melanoma, but rather may serve as a therapeutic target. Cutaneous malignant melanoma is responsible for 1% of all malignant tumors with a rising incidence in the Caucasian population . InvasivOn the molecular level, melanoma is associated with several genetic changes, including mutations or transcriptional variations in tumor suppressors like p53, CDKN2A/p16, CDKN1A/p21 or in oncogenes like N-Ras . Recentl+ T-cell responses in melanoma patients [+ T-cells in melanoma patients reactive against an HLA B27 restricted B-Raf V599E epitope encompassing the mutation identified using computer assisted algorithms [+ or CD8+ T-cell responses is not suitable for screening larger numbers of patients to estimate responder frequencies.Promising results with new approaches for melanoma therapy have been obtained with active immunizations against tumor associated antigens (TAA) like MAGE-3 , MART-1 patients . Moreovegorithms . HoweverTo determine the frequency of B-Raf specific responses in melanoma patients and to evaluate whether B-Raf specific immune responses could serve as a melanoma marker we examined the Raf specific humoral response using an ELISA assay with purified recombinant B-Raf, B-Raf V599E or C-Raf protein.Patient sera were obtained from frozen stocks and were collected over a period of 5 years. All patients gave informed consent to use their sera for scientific analysis. Control sera were obtained from patients of the dermatology department without signs of melanoma. Control patients were fully anonymized and no further information is available.Recombinant wild type B-Raf, B-Raf V599E and C-Raf proteins were expressed in Sf9 cells and purified as GST fusions  or His-tagged proteins (B-Raf) as described . Purity 405) in a TECAN Spectra Thermo microplate reader.372 sera of melanoma patients were analyzed for B-Raf V599E specific response, 271 sera were analyzed for B-Raf wt and C-Raf specific responses and 119 sera of non melanoma control patients of the dermatology department were used and analyzed for B-Raf V599E, B-Raf wt and C-Raf specific responses. For determination of serum levels of total Ig or IgG, an ELISA assay was developed. 1 μg/ml of purified B-Raf, B-Raf V599E or C-Raf protein in 100 μl coating-buffer or buffer alone as background control  was coated on NUNC 96-Well MaxiSorp plates at 4°C overnight. Plates were washed twice with washing buffer  in PBS) and blocked with 1% BSA (Sigma) in PBS. After washing twice, serial dilutions of human sera, starting at 1:100, in 100 μl conjugate buffer  were incubated for 1.5 hours at 37°C. After four wash steps, alkaline phosphatase coupled goat anti human Ig  or goat anti human IgG (Dianova) diluted 1:2000 in 100 μl conjugate buffer was added. After 1 h at 37°C and two washing steps, 50 μl of pNPP (Sigma) substrate in buffer was added. The reaction was incubated at room temperature and stopped after 20 min by 50 μl 1 M NaOH. Optical density was read at a wavelength of 405 nm . Each concentration was determined in duplicate. In addition, specificity was further confirmed using an unrelated antigen, prostate specific antigen , at concentrations of 1 μg/ml for ELISA analysis. Two patients exhibited non-specific serum responses and were excluded from further analysis.For competition ELISA performed with some positive sera, a similar protocol was engaged, using a fixed dilution of the sera and B-Raf or B-Raf V599E antigen for coating. Competition was performed by adding different concentrations of B-Raf or B-Raf V599E  to the human sera at the corresponding incubation step.405 value exceeding the Cut Off 0.275, corresponding to the mean + 1.5 SD of the background values of positive sera at serum dilutions of 1:50. Raf specific titers were compared to the background values using a two tailed Mann-Whitney U test with a 95% confidence interval. The amount of positive sera within the two groups were defined as sera exceeding a given titer; the final definition of positive sera were sera with titers of 1:300 or higher. Frequencies of positive sera were compared using two sided Fisher's exact test with 95% confidence interval.For the analysis of the ELISA data, the titer of the sera was defined as the last serial dilution with an OD405 and titers were attributed as described using a cut off OD405 of 0.275 (fig. 372 sera of 148 melanoma patients and sera of 119 control patients were screened for Raf specific antibody responses by an ELISA assay using a secondary antibody directed against IgG, IgA and IgM. Serial dilutions of the sera were analysed for their OD275 fig.  and 1B. 275 fig. . Specifi275 fig.  and WEST275 fig. , but mos275 fig. . In all 275 fig.  showed nComparison between Raf specific antibody responses in sera from melanoma patients and control patients reveals apparent differences figure . Raf speAnother interesting point to note is the small, although not significant, number of control patients with detectable levels of Raf specific antibodies. At least two out of 119 control patients showed a very strong B-Raf specific response in the same range as we have observed for positive melanoma patients figure . Due to For different extracellular tumor antigens, including gangliosides, in melanoma, positive associations between the induction of IgM and IgG antibodies and survival time have been reported  and the + or CD8+ T-cells. This question still remains to be clarified as up to now only advanced stage patients have been examined. It is obvious that at this stage the occurrence of antibodies is not correlated with prognosis and that it is very unlikely that the antibody response itself will have an influence on the disease. However, as it is rather unlikely that the frequency of a B-Raf specific T-cell response exceeds the frequency of antigen responses, our data can help to set the upper limit for the expected frequency of B-Raf or B-Raf V599E specific T-cells as 8.5% which allows the rational design of search strategies especially for Raf specific CD8+ T-cells.As oncogenic mutation of B-Raf and even transformation by other oncogenic events is frequently accompanied by Raf overexpression, the induction of an antibody response is not necessarily due to the presence of the mutation. Even though a polyclonal IgM response is very unlikely to be specific for a point mutation, this notion would explain the lack of specificity for the mutational epitope. However, it is also possible that a cellular immune response prior to the induction of Raf specific antibodies has occurred. Such a T-cell response directed against Raf or other tumor antigens, would also result in the lysis of tumor cells and the liberation of intracellular antigens. It is important to mention that the lysis leading to the formation of antibodies against intracellular antigens can be caused by T-cells other than the already observed B-Raf V599E specific CD4Even though the detection of B-Raf specific humoral and T-cell responses suggest that B-Raf/B-Raf V599E is immunogenic and that a response could be induced in at least a part of the patients, this notion does not allow conclusions on its suitability as a target for immunotherapy per se. In theory, an optimal tumor antigen is exclusively expressed in the tumor tissue and essential for tumor cell growth and survival to avoid the emergence of escape mutants or antigenic loss, whereas the immunological attack of the tumor cell is independent of the function of the antigen . To date+ and our earlier demonstration of B-Raf V599E specific CD8+ T-cell responses this study makes the screening for novel B-Raf V599E MHC class I epitopes for vaccination approaches a promising task.Taken together we have demonstrated that B-Raf/B-Raf V599E specific antibodies are detectable in 8.9% of advanced stage melanoma patients and B-Raf V599E might therefore be a valuable target for immune therapy. Combined with the independently described B-Raf V599E specific CD4None declared.JF and JCB designed the study and performed the data and statistical analysis. JF set up and supervised the ELISA and wrote the report. JCB set up and supervised the sample collection and was involved in writing the report. TP and VH were performing the ELISA assays, CSV was responsible for serum collection, transport and maintenance. EBB and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Supplementary figure with WESTERN Blot analysis of positive sera confirming specificity.Click here for file


In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated . Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation. The establishment of successful pregnancy requires a profound reorganization of uterine tissues. Rapid growth and differentiation of the endometrial stroma is the earliest and most striking event in pregnancy. Differentiation of stromal cells leads to the formation of unique cells, termed decidual cells, which differ greatly from the original stromal cells . The dec2α can induce different biological actions at the beginning and at the end of pregnancy. PGF2α, Prostaglandin E2 (PGE2), and 6-keto-PGF1α are produced by the pregnant uterus -dCTP , and a mixture of primers specific to each gene present in the array. All probes used had 5 to 10 × 106 cpm and the difference between control and experimental probes in each assay was less than 10%. cDNA were hybridized to an Atlas Rat 1.2 Array (#7854-1) nylon membrane . Hybridization and post-hybridization washes were performed according to the manufacturer's protocol. The signal was scanned with a phosphorImager  after 48 h of exposure. Control and experimental RNA were always processed in parallel.Total RNA was isolated from primary decidual cells (PDC) by using a RNAII isolation kit , following the manufacture's instructions. RNA isolated from wells treated with either PGFSpot intensities from scanned membranes were analyzed using the AtlasImage 1.5 software . Grids were orientated manually and adjusted to ensure optimal spot recognition using AtlasImage's fine tuning tools, discarding spots with dust or locally high background. The software makes the analysis for background correction and normalization versus housekeeping genes. It also calculate the ratio and indicates which genes has a ratio > 2 (up regulated), or ratio < 0.5 (down regulated) or 0.5 < ratio < 2 . Gene expression data were normalized using the Sum method included in the Atlas Image software. Data points where the expression was not greater than two standard deviations were discarded. For the final analysis, data points were the averages from triplicates and any non-reproducible data were discarded. The relative RNA expression with differences between control and treatment being higher or equal to 2 and lower than 0.5 were considered significantly different .2α .From the total genes available in the rat 1.2 membrane arrays, only 20% of the genes were detected in rat primary decidual cells. Sixty genes were significantly down regulated, whereas only six genes were up regulated by PGF2α in primary decidual cells are genes involved in the regulation of the ECM. Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase – all of which were significantly up regulated by PGF2α , casein kinase I, extracellular signal-regulated kinase (ERK-1), male germ cell-associated kinase (MAK), and Wee tyrosine kinase. PGF2α also significantly inhibited Crk-associated kinase (CAS), calcium calmodulin kinase II (CAMKII) and IV (CAMKIV), phospholipase C1, and protein phosphatases such as phosphatase 2A and protein tyrosine phosphatase 1B , guanine nucleotide-binding protein α stimulating (GN-BP α stimul.), Rab 11A and guanine nucleotide-binding protein α12 subunit (GN-BP α12) gene expression were down regulated by PGF2α  (Table The 5-hydroxytryptamine receptor 2A (HTR2A), adenosine A2A and A2B receptors (ADORA2A and ADORA2B respectively), guanine nucleotide-binding protein G α3 subunit (GN-BPG α2α Table . The mRNll Table  and leuk1) Table .2α in the uterus need to be under tight control to avoid interference with the establishment and progression of pregnancy. Pathophysiological elevations in PGF2α lead to excessive uterine contractions. Therefore, PGF2α production must be avoided to maintain a quiescent uterus [2α can target the decidua, an endocrine tissue whose integrity is fundamental for the success of implantation and for the progression of pregnancy. Our results demonstrate that elevated PGF2α down regulate the expression of decidual genes related to the proteasome protein component system, those involved in trafficking and signal transduction and genes associated with G-proteins, growth factors, chemokines and cytokines. However, interestingly, the main effect of PGF2α is related to the turnover and degradation of the ECM which provides mechanical strength to the tissue. Thus, in addition to inducing uterine contractions, PGF2α can have a noxious impact on the success of implantation and progression of pregnancy, at least in part by deregulating the turnover of the ECM in the decidua.Levels of PGFt uterus . In this2α is directly or indirectly related with the turnover and degradation of the ECM. Genes such as gelatinase A (MMP-2), TIMP2 and 3 [The largest group of genes down regulated by PGFP2 and 3 ,15, cathP2 and 3 , carboxyP2 and 3 . TGF-β iP2 and 3 . TGF-β cP2 and 3 -21.Endothelin-1 is a peptide characterized as a potent endothelial cell-derived vasoconstrictor. It is synthesized as an inactive precursor (preproendothelin) and processed to a mature active form (endothelin) by zinc metalloproteinases. The active form, endothelin-1, promotes synthesis of collagen types I and II by fibroblasts, affects the ECM remodelling and promotes the proliferation of mesangial cells . Endothe2α. This protein belongs to a family of intracellular proteins that binds membrane phospholipids in a calcium-dependent manner. Thus, it can also inhibit phospholipase A2 (PLA2) [Annexin IV  was differentially down regulated by PGF2 (PLA2) . ANX4 ca2 (PLA2) . It has 2 (PLA2) ,25.1, h2 and acidic calponin, and can be identified by the individual C-terminal tail sequences. The c-terminal sequences regulate actin association and the cytoskeleton [Calponin (CaP) has three genetic isoforms, hskeleton ,27. It iskeleton . On the skeleton .1 antiproteinase, plasma proteinase-Iα inhibitor, basic fibroblast growth receptor factor 1, phosphorylase B and proteasome component C9 were the only genes up regulated by PGF2α in PDC. Pro-stromelysin is a 53 kDa peptide and a pro-MMP3 precursor and is directly related with the ECM regulation [2α was BMP4, a gene which appears to be involved in specific stages of embryo development and is [Pro-stromelysin, αgulation . Plasma gulation , an impogulation . Growth gulation . bFGRFs gulation -35. Anott and is .2α. Proteosomal component systems are the main non-lysosomal proteolytic structures of the cells that participate in the elimination of abnormal proteins, short half-life proteins, and proteins controlling cell cycle [2α.Proteasome component system proteins, such as proteasome Iota, proteasome component C2, proteasome subunit RC-7 I, 26 S-proteasome and proteasome component C3 were down regulated by PGFll cycle . During ll cycle . Kinase ll cycle . On the ll cycle . Decorinll cycle , which i2α and is another gene related to the ECM. Active CAS can modulate changes in cell motility and gene expression by the various MAP kinase cascades, and modify the organization of the actin cytoskeleton [2α was SGK, which is a transcriptionally-regulated serine/threonine protein kinase with 45–55% homology to the catalytic domain of Akt/PKB protein kinase A [Crk-associated substrate (CAS) was down-regulated by PGFskeleton . Anotherkinase A . Skg is kinase A . Trafickkinase A .2α. The first and most important point of control of PP2A is at the transcriptional level. The increase of phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins in anchorage-dependent cells, but much lesser regulated in anchorage-independent cells [Protein phosphatase type 2A (PP2A) was down regulated by PGFnt cells .It is well known that members of the proinflammatory cytokine family can induce MMP expression in numerous tissues . Also, cin vivo on day 12 of pseudopregnancy (when the decidua undergoes regression) with that of the PGF2α treatment in rat PDC in vitro (data not shown). Interestingly, we found a 49% coincidence in the genes that were down regulated in both experimental situations. This coincidence suggests a relationship between the physiological regression and reorganization of the decidual tissue that occurs on day 12 of pseudopregnancy, with the pattern of gene expression in rat PDC after PGF2α treatment. On the other hand, many of the genes whose expression was affected by PGF2α in the rat PDC, are expressed in the decidua during decidualization and implantation. This suggests a possible connection between the action of PGF2α and the physiology of the decidual tissue. Moreover, the ECM is an important component of the decidualization and implantation process. Any modification in its turnover or degradation could affect the formation of the decidua, blastocyst invasion, and the timing of decidual regression and reorganization. It is known that the expression of MMPs/TIMPs plays an important role in the control of implantation. If PGF2α silences or decreases the expression of genes related with the systems aforementioned, it also could affect tissue remodelling by directly modifying the proteases involved in this process, or indirectly by affecting the ECM turnover and degradation, important in the accumulation of a spongy mass of tissue around each embryo during decidualization [2α's effects on the expression of genes involved in ECM turnover are indirect. Alterations of genes involved in different signalling pathways such as ERK-1, CaMCKII, PLCδ1 and G-protein subunits may impact the expression of a great number of transcripts.We have also compared the pattern of gene expression in rat decidual tissue lization . Most pr2α have a severe impact on the expression of numerous genes associated with the turnover of the ECM in the rat decidua. Future investigation should corroborate the differentially regulated genes, at the level of the message, protein, and in some cases, such as for the metalloproteinases, at the level of activity of the proteins. These data will contribute to the design of future studies on a cluster of gene candidates as targets of PGF2α action in this endocrine tissue.In summary, our results show, for the first time, that pathophysiological concentrations of PGF1 = phospholipase C delta 1; BMP 4 = bone morphogenetic factor 4; ADPRF 5 = ADP ribosylation factor 5; bFGFRF1 = basic fibroblast growth factor receptor F1; HTR2A = 5-hydroxytryptamine receptor 2A; ADORA2A = adenosine receptor A2A; ADORA2B = adenosine receptor A2B; GN-BPG α3 = guanine nucleotide-binding protein G α3; GN-BP α stimul. = guanine nucleotide-binding protein α stimulating; GN-BPG α12 sub. = guanine nucleotide-binding protein α12 subunitTIMP2 = tissue inhibitor metalloproteinases 2; TIMP3 = tissue inhibitor metalloproteinases 3; PAI 1 = plasminogen activator inhibitor 1; uPA = urokinase type; tPA = tissue type palsminogen activator; TGFβ = transforming growth factor β; SGK = serum glucocorticoid kinase; ERK-1 = extracellular signal-regulated kinase-1; MAK = male germ cell-associated kinase; CAS = crk-associated kinase kinase; CamKII = calcium calmodulin kinase II; CamKIV = calcium calmodulin kinase IV; PLC δEC and SFG carried out the decidualization, and primary decidual cell culture. EC isolated the RNA isolation, performed the cDNA array assay, the analysis of the results, and drafted the manuscript. GG conceived the study and edited the manuscript. All authors read and approved the final manuscript.


It is the certain fate of all antibacterials to be fought off eventually by the pathogens they target. We need new ways to defeat disease, and we will need them forever Streptococcus pneumoniae strains in the United States will resist both penicillin and erythromycin, according to a recent prediction from the Harvard School of Public Health. The forecast, based on mathematical modeling, was published in the spring of 2003. It's too early to tell whether that prediction is precisely on track, according to the senior author on that paper, Marc Lipsitch. But no one doubts that multidrug resistance in this common bug—responsible for diseases that range from sinus trouble and ear infections to meningitis and pneumonia—is speeding up.By next summer, more than 40% of It is the certain fate of all antibacterials to be fought off eventually by the pathogens they target. The fact that the process is accelerating has been alarming public health officials for some time, especially in the United States. We need new ways to defeat disease, and we will need them forever.Staphylococcus, Streptococcus, and Enterococcus.Antibiotics have traditionally been plucked from nature's battleground. For billions of years, tiny organisms have engaged in an arms race, hurling toxic molecules at each other in the struggle to prosper. Nearly all of today's antibiotics are versions of weapons long wielded by microbes and fungi. Chemical synthesis of entirely human-created antibiotics has so far yielded only fluoroquinolones, a group of broad-spectrum antibiotics that includes Cipro, which became famously scarce during the 2001 anthrax scare, and linezolid (trade-named Zyvox), which is effective against some resistant strains of Escherichia coli from transcribing RNA.The usual way to find a new antibiotic has been laborious screening of immense libraries of compounds, natural and otherwise. Some argue that screening chemical libraries is approaching a deadend. There may be diminishing returns from screening, but it's not quite dead yet: in October, researchers at the University of Wisconsin at Madison reported a new class of bacterial RNA polymerase inhibitors with antibiotic potential. They were found by screening for molecules that prevent Christopher T. Walsh of Harvard Medical School says screening's problem may be simply that libraries aren't good enough. Marine organisms have not been studied well, he points out, and 90% of organisms in the biosphere can't be cultured in standard ways. He says, “We're missing 90% of them every time we go and look in nature.”Walsh is doing his bit to create new libraries. He and his colleagues have recently employed combinatorial biosynthesis to learn how to use part of the machinery for assembling cyclic peptide antibiotics to control their architecture. The result was a small library of natural product analogs, some of which have improved antibiotic activity against common bacterial pathogens. “There are dozens of such enzymatic domains that in principle one could clone, express, and test with other substrates. I view that as the kind of thing we should do,” he says. For example, Walsh suggests, it is a reasonable approach to second-generation improvement of daptomycin, the antibiotic most recently approved for sale in the United States.Walsh collaborates with Chaitan Khosla of Stanford University on finding ways to make existing antibiotics better. They are studying biosynthesis of rifamycin, an antibiotic that is increasingly less effective against its prime target, tuberculosis (TB) see . “In the“Basically, what we do is to try and figure out new ways to hijack the biosynthesis of antibiotics in nature so as to modify their structures with the goal of improving them,” Khosla explains. He works with an important class of natural antibiotics called polyketides that have generated dozens of drugs, including erythromycin.PLoS Biology). Among them are the anthracyclines, a group of anticancer drugs and antibacterials that includes tetracycline. In this issue of PloS Biology, Khosla and his colleagues report that they can make selective positional modifications in existing anthracycline antibiotics by starting in a different way with a different starting molecule. The molecule came from a natural anthracycline antibiotic, an estrogen receptor antagonist called R1128. R1128 is made via two modules of enzymes that work sequentially; the first module starts the process, and the second completes it. This division of labor permitted the researchers to tack the first R1128 module onto two other enzyme systems, thus engineering completely new anthracyclines. Some were more active in two types of assays than the natural parent molecule. “One setting was an assay on an estrogen-sensitive cancer cell line. Another setting was an assay to probe activity of an enzyme that's of particular interest in Type 2 diabetes, called glucose-6-phosphate translocase.” The work also revealed fundamental mechanistic features of the polyketide synthases, Khosla says.Polyketides are secondary metabolites (which give their producers a competitive advantage in their environment) produced mostly by bacteria and fungi and made by a complex and structurally diverse family of enzymes called polyketide synthases , the United States Department of Defense's (DOD) central research and development organization, and once the researchers realized they wanted to develop drugs against three agents that have been considered bioterrorism threats — The most radical approach to new antibiotics may be the resurrection of an old idea: bacteriophage therapy see . Late inPhage were the model organisms of choice for genetics research in the 1930s and 1940s, but became less fashionable as research tools when investigators moved on to eukaryotes. A few held on, like Ry Young of Texas A&M University, who has made phage-induced cell lysis his life's work. “The cell is basically genetically dead as soon as the phage goes in there, but it will keep living as sort of an infected zombie for as long as the phage wants it to, with virus particles accumulating inside the cell,” he explains. “Only when the phage is ready and has decided that it's the right time will it pull the trigger. And the cell blows up.” The freed phage then spew forth to infect new cells.Antibiotic resistance has led to new interest in phage therapy by several small biotech companies. Young continues basic research at Texas A&M, but has also joined one of them, GangaGen, providing bacteriophage expertise to its labs.Phage do kill pathogenic bacteria effectively, and they do it without penetrating human cells, which they can't even recognize. So what is keeping phage therapy out of the clinic? Problems that some doubt can be overcome.Because bacteria develop resistance to phage rapidly, phage therapy companies will need to direct cocktails against a single pathogen, according to Vincent Fischetti at The Rockefeller University. Phage are also antigenic, and the antibodies they stimulate will neutralize their effects during subsequent treatment, he says. But the chief problem appears to be regulatory—regulatory in the political, rather than the genetic, sense. When bacteriophage package their DNA, they occasionally include varying amounts of their hosts' DNA, too. This miscellany, Fischetti points out, is likely to make the Food and Drug Administration unhappy. “Phage normally are very fragile, their tails break, so lot-to-lot homogeneity could be a problem too,” he adds. “So even though it will work, I think they'll have an uphill battle.” Phage may well enter agricultural or veterinary use, he predicts, but are probably not going to be available to patients in the United States any time soon.Strep pyogenes, pneumococci, Strep pneumoniae, Bacillus anthracis, Enterococcus faecalis, and group B Strep. The beauty of these enzymes is that they are targeted killing. You only kill the organism you intend to kill, without destroying or affecting the surrounding organisms that are necessary for health.”Fischetti chose a different approach to phage therapy. It does not rely on phage themselves, but on enzymes that phage produce to smash their way out of their host bacteria so they can infect new hosts. He and his colleagues employ these enzymes externally to kill bacteria. He reports, “We now have enzymes that will kill Pneumococcus, Staphylococcus, and group A Strep on contact with mucous membranes. The strategy might prevent bacterial infections from spreading in close quarters like hospitals, nursing homes, and daycare centers. Fischetti says, “Clinical trials would tell us how often we had to treat, but more important, we'd have a reagent that could treat people who walk out the door of the hospital to eliminate or reduce the transmission of resistant organisms into the community. We don't have that capability right now.”The enzymes can be loaded into a nasal spray that wipes out pathogens such as Bacillus anthracis spores, preventing them from germinating and seething through the bloodstream, producing deadly toxins. An IV drip would be started after exposure to the spores. The method, Fischetti reports, is already successful in mice; clinical trials will determine how long treatment must be continued, perhaps a week or so. They have also eliminated septicemia from pneumocci with the same intravenous method.Fischetti and his colleagues have moved on to using the enzymes systemically to wipe out Up to now the enzymes must make contact with bacteria to kill, but Fischetti is hoping that a new generation of engineered enzymes will be able to kill pathogens inside cells too. A second disadvantage is that they are effective only against gram-positive bacteria, although that group includes many vicious pathogens.But phage enzymes seem to offer one very big advantage: resistance to them has yet to develop. Fischetti says, “We've tried very hard to identify resistant bacteria, but so far we haven't found resistant organisms in all three of the enzymes we're working with. It appears to be a very rare event, much rarer than resistance to antibiotics.” Fischetti cautions against expecting that gladsome state to last forever, but he points out that even if widespread resistance takes the same 40 or 50 years that antibiotics required to become significantly resistant, phage enzymes could buy researchers decades for inventing other approaches.There is no shortage of ideas for unearthing new antibiotic candidates. Why are they so slow to enter medical practice? The bottleneck, researchers agree, lies in the development process of turning them into effective therapies. Several researchers blame the big pharmaceutical companies that got so big by leading the way to new drugs for battling infectious disease, but in recent years have dropped out. Fischetti complains, “These are the big companies that have the money to develop antiinfectives, but they leave it to small biotech companies, and it's not going to happen as rapidly as it should. I think it's really unconscionable for these big companies to drop the ball because it's not going to be a billion-dollar market for them and that's what they're looking for.”Half a billion at least, says Francis Tally, a big pharmaceuticals veteran who is now chief scientific officer at Cubist Pharmaceuticals, a biotech company located in Lexington, Massachusetts. According to Tally, Cubist produced daptomycin, approved in September 2003, by licensing it from Eli Lilly, which shelved the new compound after concluding its potential market was only $250 million.But, Tally argues, the size of the market is not the only barrier to new antibiotics. Combinatorial chemistry and the genomics revolution have simply not delivered on their early promise. “The pipeline is very dry,” he says. “There's been a real lag at the basic research level.”“Antibiotic discovery is hard,” Shapiro says. “It's a huge long process to get a decent antibiotic.” Walsh agrees. “It's easier to find inhibitors of particular enzymes for particular processes—and a very long road to convert that into something for development.”In the meantime, there is a rising clamor to slow down the rate at which bacteria develop resistance. Doctors are exhorted to cut back on prescribing antibiotics and decline to prescribe for viral diseases, which antibiotics can't combat, even when their patients badger them.But even if antibiotic consumption slowed, we will still need new antibiotics. “I always say it's not a matter of if, it's only a matter of when,” says Walsh. “There will always be a need for new antibiotics because the clock starts ticking on the useful lifetime of any antibiotic once you start to use it. That cannot be argued.”


Ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid (PA) formation by phospholipase D (PLD). The present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. Evidence is presented that the formation of PA and ceramide may be reciprocally linked during ethanol exposure.3H]-serine, ethanol caused a dose-dependent, biphasic increase of the [3H]-ceramide/ [3H]-sphingomyelin ratio after 1 and 18 hours of incubation. As primary alcohols such as ethanol and 1-butanol were shown to inhibit the phospholipase D (PLD)-mediated formation of PA, a mitogenic lipid messenger, we tested their effects on ceramide formation. In astrocytes prelabeled with [3H]-serine, ethanol and 1-butanol, in contrast to t-butanol, significantly increased the formation of [3H]-ceramide. Moreover, exogenous PA, added to transiently permeabilized astrocytes, suppressed ethanol-induced [3H]-ceramide formation. Vice versa, addition of C2-ceramide to astrocytes inhibited PLD activity induced by serum or phorbol ester.In cultured rat cortical astrocytes, ethanol  induced nuclear fragmentation and DNA laddering indicative of apoptosis. Concomitantly, in cells prelabeled with [We propose that the formation of ceramide in ethanol-exposed astrocytes is secondary to the disruption of phospholipase D signaling. Ethanol reduces the PA:ceramide ratio in fetal astrocytes, a mechanism which likely participates in ethanol-induced glial apoptosis during brain development. The proliferation of astrocytes is stimulated by polypeptide growth factors such as PDGF, EGF, bFGF and IGF-1 acting on cellular signaling pathways which involve tyrosine kinases, protein kinase C, and the Ras-Raf-MAP kinase pathway . AstroglThe molecular target of ethanol's antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated . Our groIn our previous work, we have found that astroglial PLD is activated by mitogenic factors including fetal calf serum (FCS), PDGF, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and PA formation in a parallel manner. 1-butanol reduced PA formation and DNA synthesis with the same potency while t-butanol was inactive for both effects . More relyso-PA, a potent mitogen in many cell types -ceramide was measured after labeling sphingomyelin with [3H]serine. Under basal conditions, the ratio of [3H]-ceramide to [3H]-sphingomyelin ("C/S ratio") was approximately 1:30. This ratio was not significantly changed during serum withdrawal  but did not significantly change the total labeling of the large pool of [3H]-sphingomyelin by [3H]-serine (data not shown). As the total incorporation of [3H]-serine into [3H]-sphingomyelin was somewhat variable between individual preparations, we used the C/S ratio to calculate ethanol-induced changes. As shown in Figs. Formation of H-ceramidal Figs. . The incThe rapid and transient phase of ceramide formation occurred within 15 min and reached a maximum at 1 hour after addition of ethanol . A second increase gradually developed after 4 hours and reached a maximum at 18 hours of ethanol exposure . At this later time point, staurosporine (1 μM) caused an increase of the C/S ratio to 7.97 ± 1.78 % .To investigate whether ceramide formation is secondary to a disruption of PLD signaling, we used the isomeric alcohols, 1-butanol and t-butanol. 1-Butanol – but not t-butanol – is a substrate of PLD for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of PA. The correlations between butanol exposures and disruption of PLD signaling  in astrocytes were documented in detail in our previous work . In the 3H]-ceramide levels (data not shown). Basal ceramide levels were also unchanged if exogenous PA was added to the astrocytes in the absence (data not shown) or presence of SL-O  of streptolysin-O (SL-O) to introduce the membrane-impermeable PA into the astroglial cytosol . In prelL-O Fig. . HoweverL-O Fig. . At the L-O Fig. . At the L-O Fig. .2-ceramide (50 μM) slightly but non-significantly reduced basal PLD activity by 30%  84 and 93 % and (for PDB stimulation) 53 and 64 %, respectively.As the previous experiments indicated an inhibitory effect of the PLD pathway on ceramide formation, the following experiment tested a possible effect of ceramide on PLD activity measured by the transphosphatidylation assay. In these experiments, the membrane-permeable C30% Fig. . However30% Fig.  or by PD30% Fig. , C2-ceraWe have previously documented that ethanol suppresses signaling through the mitogenic phospholipase D (PLD) pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes ,17,23. T2-ceramide , and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study -Serine and [3H]-glycerol were from Biotrend . Ceramide (from bovine brain), C2-ceramide (N-acetyl-D-erythro-sphingosine), and staurosporine were from Alexis . Hoechst 33258, L-α-phosphatidic acid  and 4β-phorbol-12β,13α-dibutyrate (PDB) were from Sigma ; most other chemicals and TLC plates were obtained from Merck  or Roth  at the highest purity available. Fetal calf serum (South America) was from Invitrogen, cell culture materials were from Sarstedt . Phosphatidylethanol (PEth) standard was synthesized as described -ceramide, we used one-dimensional TLC . For the determination of [3H]-sphingomyelin, we used two-dimensional TLC (TLC plates Merck 1.05721); solvent I was chloroform/methanol/25% aqueous ammonia (13:7:1), solvent II was chloroform/ methanol/water/acetic acid (30:30:2:5). 3H-lipids were visualized by using iodine vapor, spots were scraped and suspended in scintillation cocktail (Lumasafe Plus), and radioactivity was measured by liquid scintillation counting (Packard 1600 CA). Formation of [3H]-ceramide was calculated as percentage of radioactivity found in [3H]-sphingomyelin.Phospholipids were labeled by addition of -ceramide was determined as described above. Ethanol, if present, was present throughout the incubation period. Using 144 ng/ml of streptolysin-O, this procedure yielded transient permeabilization of > 80% of astrocytes followed by repair of the pore in > 80% of permeabilized cells. The procedure was previously found to allow the entry of approx. 106 molecules of PA per cell -glycerol (1 μCi/ml) for 24 h in order to label phospholipids. More than 60% of the phospholipid label was associated with phosphatidylcholine (not illustrated). Subsequently, the cells were washed and re-exposed to medium containing ethanol (2 %) and PDB (1.0 μM) or FCS  as stimulators. PDB and C2-ceramide, when used, were dissolved in DMSO (end concentration of DMSO < 0.1 %). After 30 min of reaction time, the cells were washed in cold phosphate-buffered saline and extracted as described above for ceramide determinations. After addition of phosphatidylethanol (PEth) and PA standards, aliquots of the lipid phase were separated by two-dimensional TLC (TLC plate Merck 1.05721) using chloroform/methanol/25 % aqueous ammonia (13:7:1) for the first run and the upper phase of ethylacetate/isooctane/ acetic acid/water (13:2:3:10) for the second run. Individual phospholipids were stained by iodine, and the spots corresponding to PEth, PA and phosphatidylcholine (PC) were isolated and counted for radioactivity in a scintillation counter. To determine PLD activity, formation of [3H]- PEth was calculated as percentage of [3H]-PC.Phospholipase D activity was determined using the transphosphatidylation assay . For thiData are shown as means ± SEM of N experiments whereby N refers to the number of different astroglial preparations from different animals. Results were obtained from two replicate dishes which were pooled to represent a single experiment. Statistical calculations were performed by GraphPad InStat 3.0 program package, using analysis of variance (ANOVA) of paired or unpaired data as indicated in text and figure legends.Cer, ceramide; FAS, fetal alcohol syndrome; FCS, fetal calf serum; PA, phosphatidic acid; PC, phosphatidylcholine; PDB, 4β-phorbol-12β,13α-dibutyrate; PEth, phosphatidylethanol; PKC, protein kinase C; PLD, phospholipase D; SL-O, streptolysin-O; SM, sphingomyelin.B.S. carried out the cell culture experiments and phospholipid measurements and participated in the experiments concerning apoptotic markers. S.J. contributed substantially to the apoptotic marker experiments. K.L. participated in the design of the study and the final draft of the manuscript. J.K. conceived and supervised the study, performed the statistical analyses and drafted the manuscript.


Medical education is increasingly being conducted in community-based teaching sites at diverse locations, making it difficult to provide a consistent curriculum. We conducted a randomized trial to assess whether students who viewed digital lectures would perform as well on a measure of cognitive knowledge as students who viewed live lectures. Students' perceptions of the digital lecture format and their opinion as whether a digital lecture format could serve as an adequate replacement for live lectures was also assessed.Students were randomized to either attend a lecture series at our main campus or view digital versions of the same lectures at community-based teaching sites. Both groups completed the same examination based on the lectures, and the group viewing the digital lectures completed a feedback form on the digital format.There were no differences in performance as measured by means or average rank. Despite technical problems, the students who viewed the digital lectures overwhelmingly felt the digital lectures could replace live lectures.This study provides preliminary evidence digital lectures can be a viable alternative to live lectures as a means of delivering didactic presentations in a community-based setting. Medical education is increasingly being conducted in community-based teaching sites outside of the traditional academic medical setting , At the Our institution has a community integrated structure where medical students spend the clinical portion of their training in one of six community campuses spread throughout the State of Michigan. Although this structure has many advantages, it is difficult to provide a consistent educational experience for the students. To help address this challenge, we implemented an all-day lecture series held at one of the community campuses two weeks before the end of the internal medicine clerkship.The students and faculty presenters from other campuses traveled from their home campus to the campus hosting the lecture series. End-of-clerkship feedback from the students has indicated the lecture series is both valuable and well received. Traveling to the host community, however, was inconvenient and time consuming for both students and faculty presenters. In addition, it is not practical for students at our rural medicine campus to attend due to the distance (approximately 400 miles) from the other communities.There is evidence that delivering the audio from a lecture in combination with the presenter's slides can be an effective means of delivering lectures at remote sites, and may even be as effective as traditional lectures,4. We saStudents taking the third-year required internal medicine clerkship during the 2003–2004 academic year at our institution were offered the opportunity to participate in the study. Those agreeing to participate were randomized into to one of two arms of the study. The random assignment of students to the two arms of the study was done within each community to control for the potential of community differences. The control group traveled to the host community campus and attended the live lectures with their colleagues who chose not to participate in the study. The experimental group stayed at their home campus on the same day and completed a parallel set of CD-ROM-based multimedia modules made from digital recordings of the previous year's lectures. They completed these digital lectures in computer laboratories in either the community campus office or within one of the teaching hospitals.The series included six lectures covering asthma, coronary artery disease (CAD), acute renal failure, liver disease, thyroid disease, and antibiotic pharmacology. The Clerkship Education Committee chose these topics based on their perceived importance and consistency with the Society for General Internal Medicine/Clerkship Directors of Internal Medicine (SGIM/CDIM) Curriculum Guide.Between the 2002–2003 academic year when the lectures were taped and the 2003–2004 academic year when the study was conducted, the clerkship faculty decided to revise the lecture series to be more case-based though the topics were kept the same. Two of the lectures, CAD and renal failure, were not modified and kept as consistent as possible with the previous year in order to conduct the study. Though there might have been minor inconsistencies between the digital and live versions of these two lectures, the same faculty member presented each lecture during both the 2002–2003 academic year when the lectures were taped and the 2003–2004 academic year when presented live. The two lecturers also attempted to keep the live lectures as consistent as possible with the digital lectures and used the same slides that they had used the previous year. While the format of the other four lectures changed, the material covered and instructional objectives remained consistent.At the end of the live lecture series, students were asked to complete a short examination that included four to five questions based on each of the six lectures. These questions were written by the presenters of the lectures and designed to assess student mastery of the lectures' key objectives. The students were informed that the purpose of the examination was to provide them with feedback on the mastery of the material and the presenters with feedback on the effectiveness of the lectures, and would not impact on their clerkship grade. After the students completed the exam, they were given a copy that included the correct answers and a short explanation for the correct answer. The exam forms contained no student identifiers, but students in the control group were asked to indicate on the examination form that they had agreed to participate in the study so they could be differentiated from the students who had chosen not to participate in the study.Students in the experimental arm of the study completed the same examination in their home community after they had completed the digital lectures. They were also asked to complete a short feedback form asking whether they had any technical problems using the modules, to rate their agreement with what the researchers felt to be three potential advantages and three potential disadvantages of the modules, and whether they felt the modules could serve as a suitable replacement for live lectures.The specific questions are listed below.Advantages of the Modules• Convenience of viewing the presentations when you choose.• Avoiding having to travel to another community for an all day lecture series.• Ability to keep copies of these presentations for use in the future.Disadvantages of these modules• Inability to ask questions of the presenter• Lack of group interaction/discussion of a topic• Just not like being in the room with the presenter. They included digitized video and audio from the taped presentation inserted as a window in the PowerPoint® slides from the presentation. As students displayed each of the slides, they were able to observe the presenter in the multimedia window discussing the slide that was being viewed.The CD-ROM modules were created using a technique developed by the first author. A manual outlining how to develop these modules is available from Nine of the items on the exam focused on the material in the CAD and acute renal failure lectures, where the lecturers presented the lectures in the same format as they had used in the previous year when the lectures were taped. The remaining 20 examination items covered material in the other four lectures. Group differences were tested for statistical significance by both an independent sample t-test for means and a Mann-Whitney test for ranks. A Levene's test for equality of variance between the two groups was also performed. These analyses were conducted separately for the subset of items covering the material in the CAD/acute renal failure lectures and the other four lectures. A power analysis was conducted to assess the magnitude of the difference between the groups that would likely be detectable given the number of students participating in the study. The coefficient alpha reliability of the exam was also estimated. The data were presented descriptively using means, standard deviations and mean ranks within the control and experimental groups. All analyses were conducted using the Statistical Package for the Social Sciences version 11. Approval for the project was obtained from the University Committee for Research Involving Human Subjects within our institution.A total of 96 students completed the internal medicine clerkship during the 2003–2004 academic year. As described below 56 of the students were eligible to participate in the study. A total of 29 students or 52% of the eligible students agreed to participate in the study. Complete data were available for 12 students who attended the live lectures and 17 students who completed the digital lectures.During the first rotation, there were some technical problems in a demonstration of the digital lectures. The net result was that very few students chose to participate during that rotation. During the second and third rotations, approximately two-thirds of the students agreed to participate. There were also 20 students who were ineligible to participate. These included six students from the rural medicine program at Marquette who do not participate in the live lecture series due to the distance from the other communities. Additionally, some of the communities conducted a fourth rotation of the internal medicine clerkship due to space limitations in the three regular rotations and a live version of the lecture series was not given for the students in the fourth rotation. These 20 students all completed the CD-ROM modules but because they could not be randomized between the live and digital formats, they were not able to participate in the study.Differences in the sample sizes for the two groups were due to some of the students in the live lecture group failing to mark that they were participating the study. During the second clerkship rotation, the proctor inadvertently failed to remind the students participating in the study to mark this information on their examinations when the exams were handed out.A power analysis indicated that with the number of subjects in the study, it would be possible to detect differences of nine tenths of a standard deviation with a power of 80%, p < 0.05 for a one-tailed t-test.Table The Levene's test for equality of variance between the control and experimental groups was statistically significant (p = 0.03) for the CAD and renal failure items. The variation among the scores of students who observed the CD-ROMs was almost twice as large as for students who observed the live lectures. There was no statistically significant difference among the groups for the variance of the items from the other four lectures.The coefficient alpha reliability for the items covering CAD/renal failure and the items covering the other four lectures were 0.33 and 0.66 for the 9 and 20 item scales respectively and was 0.70 for the combined 29-item exam.The 17 students who completed the digital lectures also completed a short feedback form on their experiences and impressions of the digital lecture format. These data are presented in Table There were no statistically significant differences found between students who viewed the live and CD-ROM based lectures. The observed mean scores in the two groups were in fact almost identical. Unfortunately, the small sample size limits the power of the study and confidence in which we can assert that digital lectures can be as effective as live lectures in increasing students' knowledge. The study does suggest that it is unlikely that there are large differences in the performance of students who view CD-ROM based lectures as opposed to live lectures and adds to the growing body of literature concerning the effectiveness of technology for implementing distance learning in medical education.There was a statistically significant difference in variances among the two groups for the items covering the CAD/renal failure lectures. The standard deviation in the scores was twice as large for the students who completed the digital modules. The differences in the dispersion are also evident in the range of values in each group. It is not clear why there was more variation in the scores among the students who completed the digital modules. It may be related to the technical problems encountered by many of the students in accessing the modules, though one would expect this would have resulting in extending the lower tail of the distribution but not the upper tail. It may have also in part reflected the impact of discussions that occurred during the live lectures that may have reduced the variability among the students in their responses to the examination.Despite the fact that almost all of the students experienced some technical difficulties using the modules, they all agreed and most strongly agreed the modules could serve as an adequate replacement for live lectures. They were particularly appreciative of not having to travel to another community to attend didactic presentations and having the flexibility of viewing the modules at their convenience. Of the three potential disadvantages of the format that were listed on the feedback form, they felt their inability to ask a question of the presenter was the most important. In the future we are considering using a web-based bulletin board system as a means of allowing students to ask questions of the presenter.The number of students with technical difficulties viewing the modules was surprising. We had tested the modules on a variety of different computers with very few problems. In a few cases, the CD-ROMs we distributed apparently had not been copied correctly. Additionally, we switched the video formats from MPEG to Windows media files. We assumed there would be less compatibility problems with the Windows media files given that this is a format developed by Microsoft. Unfortunately, we later found out the Windows media files require software that was not shipped with earlier versions of Windows. We expect this was a significant cause of the technical problems the students experienced.We are now using a commercial software package which greatly simplifies the process of creating the digital lectures and allows them to be distributed over the Web as well as via CD-ROM requires no special software minimizing the compatibility issues. Students who completed the fourth rotation of the clerkship and did not participate in the study were provided with the new version of the modules. Only one of the 12 students indicated they had technical problems accessing the lectures off the CD-ROM disks on which the modules were distributed and that student was able to access the modules via the Web. Such combined Web and CD-ROM distance learning formats have been shown to be effective in a number of educational settings ,7.There were several important limitations in the study. First is the very small sample size which limited the power of the study for detecting differences in the performance of the students completing the live and CD-ROM based lectures. It also increased the likelihood the two groups of students were not equivalent. Since there were no student identifiers on the exams, it was not possible to compare the characteristics of the two groups. The outcome measure was a locally developed test. While the items were written by the presenters and based on the major objectives in their lectures, there was no assessment of validity other than content validity. It is also not clear the extent the findings of this study can be generalized to other digital lecture formats.Although the data collected in this study were limited, it provides some evidence that digital lectures are both well received by students and can provide a satisfactory substitute for live lectures from a performance standpoint.The author(s) declare that they have no competing interests.DJS designed the study, developed the feedback instrument on for the digital modules conducted the statistical analyses and wrote the first draft of the paper. GSF, HSF and KK developed and gave presentations, wrote questions for the knowledge examination and edited and helped revise the manuscript.The pre-publication history for this paper can be accessed here:


Releases of hazardous materials can cause substantial morbidity and mortality. To reduce and prevent the public health consequences (victims or evacuations) from uncontrolled or illegally released hazardous substances, a more comprehensive analysis is needed to determine risk factors for hazardous materials incidents.Hazardous Substances Emergency Events Surveillance (HSEES) data from 1996 through 2001 were analyzed using bivariate and multiple logistic regression. Fixed-facility and transportation-related events were analyzed separately.For fixed-facility events, 2,327 (8%) resulted in at least one victim and 2,844 (10%) involved ordered evacuations. For transportation-related events, 759 (8%) resulted in at least one victim, and 405 (4%) caused evacuation orders. Fire and/or explosion were the strongest risk factors for events involving either victims or evacuations. Stratified analysis of fixed-facility events involving victims showed a strong association for acid releases in the agriculture, forestry, and fisheries industry. Chlorine releases in fixed-facility events resulted in victims and evacuations in more industry categories than any other substance.Outreach efforts should focus on preventing and preparing for fires and explosions, acid releases in the agricultural industry, and chlorine releases in fixed facilities. Recent high-profile hazardous materials incidents, such as an explosion at a pharmaceutical supply plant in Kinston, North Carolina  and an eA more comprehensive analysis is needed to determine which risk factors are associated with releases of hazardous substances that result in victims or evacuations. The Hazardous Substances Emergency Events Surveillance (HSEES) system, maintained by the Agency for Toxic Substances and Disease Registry (ATSDR), is an active, multistate surveillance system designed to collect and analyze information about acute releases of hazardous substances. Data elements captured in HSEES include time, date, and day of the week when the event occurred; event type (fixed-facility or transportation-related event); geographic information; factors contributing to the release; type of chemicals released; information about injured persons (victims); industry responsible for the event; and information about decontaminations and orders to evacuate. Data from HSEES were analyzed to identify potential risk factors associated with chemical releases involving victims or evacuations during 1996–2001.HSEES events are defined as sudden, uncontrolled, or illegal releases of at least one hazardous substance that had to be removed, cleaned up, or neutralized according to federal, state, or local law. A substance is considered hazardous if it might reasonably be expected to cause adverse human health outcomes. Threatened releases are also included in HSEES, if the amount threatened to be released would have required removal, cleanup, or neutralization under federal, state, or local law and the threat led to an action to protect the public health . Events involving only petroleum are excluded from HSEES.The purpose of HSEES is to reduce morbidity and mortality associated with acute releases of hazardous substances. The goals of the system are to: 1) describe the distribution and characteristics of hazardous substances emergencies, 2) describe morbidity and mortality of employees, emergency responders, and the general public resulting from hazardous substance releases, 3) identify risk factors for morbidity and mortality, and 4) identify strategies that might reduce future morbidity and mortality resulting from the release of hazardous substances.The pilot phase of the surveillance system was from January 1, 1990 through December 31, 1992. Data for 1996–2001, the most recent period for which complete data are available, were analyzed. Thirteen states participated in HSEES during the entire period analyzed: Alabama, Colorado, Iowa, Minnesota, Missouri, Mississippi, New York, North Carolina, Oregon, Rhode Island, Texas, Washington, and Wisconsin. An additional four states participated during portions of that period: Louisiana (2001), New Hampshire (1996), New Jersey (2000–2001), and Utah (2000–2001).State health department personnel used a variety of sources  to collect information about the hazardous events. Before January 1, 2000, data were entered into a computerized data-entry system designed by ATSDR and were transmitted quarterly to ATSDR for quality control checks and analysis. Beginning on January 1, 2000, data were entered into a web-based application that enabled ATSDR to instantly access the data.A standardized data-collection instrument was used to obtain information on each event. ATSDR provided the states with a training manual to ensure uniformity. A victim is defined as a person experiencing at least one documented adverse health effect  that probably resulted from the event and occurred within 24 hours after the release. The HSEES system does not identify the immediate cause of the adverse health effect other than the event itself. Official evacuations were recorded in the system if they were ordered by on-scene coordinators such as a fire or police chief, a member of a HazMat team, or some other type of official. Reasons for the evacuation are not captured.Industry codes for the type of industry responsible for each HSEES event were assigned according to the 1990 Industrial Classification System of the United States Census Bureau . The indData were analyzed using SAS  logisticFor each chemical reported, up to two entries for type of release could be selected from six choices . To describe an event, release type was assigned in a hierarchic arrangement  according to all release types reported for an event. When an event had multiple release types, the highest rank was used to assign the release type. For example, an event that had a chemical released as both an air emission and a fire  would be classified as fire. Threatened releases were analyzed only for events with evacuations. The area of evacuation recorded if the evacuation zone was downwind/downstream of the release, a circular area, the building(s) or affected part of the building(s), both a circular area and downwind/downstream, or if no criteria were used.For fixed-facility events, industry category and factor contributing to the release  were also included. Although up to two factors could have been selected for each event, we analyzed only the first factor selected, because only 13% of events had a second factor selected and specific combinations of factors were too sparse to provide meaningful analysis.We examined the likelihood of an event resulting in a victim or evacuation for specific substance and industry categories separately. Substance categories were coded as yes/no; the referent group was all other substances. Similarly, industry categories were coded as yes/no; the referent group was all other industries. Because most transportation-related events were coded into the transportation industry (79%), the association between industry type and an event with a victim (or evacuation) was examined only for fixed-facility events. Mode of transportation, categorized as ground , rail, water, air, and other, was also included in the analysis for transportation-related events.To further specify risk factors, multivariate and stratified analyses were performed to examine whether certain combinations of variables significant as univariate predictors were associated with a greater likelihood of an event with a victim or evacuation. A multiple logistic regression model was constructed to examine the potential interactions among time of day, weekday, and season. For fixed-facility events, analysis stratified by industry category examined the association between specific substances and an event with a victim or evacuation within industries.During 1996–2001, participating state health departments reported 39,766 events to the HSEES system. The analysis of risk factors for events with at least one victim excluded 568 events that involved only a threatened release, and thus focused on 39,198 events  fixed-facility events and 9,224 (24%) transportation-related events). At least one victim was reported for 2,327 (8%) of the fixed-facility events with the number of victims per event ranging from 1 to 259 (mean = 4 and median = 1). Similarly, at least one victim was reported for 759 (8%) of the transportation events with the number of victims per event ranging from 1 to 65 (mean = 2 and median = 1).The analysis of risk factors for evacuations focused on 39,622 events  fixed-facility events and 9,432 (24%) transportation-related events) because information about whether an evacuation was ordered was missing for 144 events. Evacuations were ordered for 2,961 (10%) fixed-facility events with the number of persons evacuated per event ranging from 0 (10 events) to 11,000 (mean = 112 and median = 20). For transportation events, 453 (5%) events involved an ordered evacuation; the number of persons evacuated per event ranged from 0 (3 events) to 8,700 (mean = 163 and median = 20). For fixed-facility events, the area of evacuation was primarily the building(s) or affected part of the building(s) , while for transportation events, the evacuation zone was primarily a circular area .Emergency chemical releases at fixed facilities involving victims were more likely to occur on weekdays than weekends  . Releases of chlorine  were most likely to result in fixed-facility events with victims compared with releases from all other chemical categories while releases of multiple substances from different categories  were most likely to result in transportation events with victims.Type of industry was examined as a potential risk factor for victims in fixed-facility events only. Industries classified as personal services were most likely to result in events with victims than were releases not involving these industries   Table . FurtherFixed-facility events with evacuations were most likely to occur in areas described as commercial compared with the referent group "other." Area type was not associated with ordered evacuations for transportation-related events. The release type fire and explosion was most likely to result in fixed-facility events with evacuations compared with spills . Releases of chlorine were most likely to result in events with evacuations compared with releases from all other chemical categories for both fixed-facility and transportation-related events .We examined type of industry as a potential risk factor for evacuations in fixed-facility events only. Industries classified as professional services were most likely to result in events with evacuations compared with releases not involving these industries  , day of week (weekday), and season (March through May) (p = 0.06); no interaction among temporal variables was indicated for evacuations.The likelihood of an event with at least one victim or evacuation in fixed facilities was examined for substances within industry category Table . The strAlmost 40,000 chemical release events were reported to HSEES during the 6-year period included in this analysis. Approximately 8% of the events resulted in almost 12,000 victims, and approximately 9% involved evacuation of more than 325,000 people. Because the public health consequences of chemical releases can be serious and affect large numbers of people, identifying the risk factors more likely to be associated with hazardous substance releases that result in victims or evacuations is important to target appropriate prevention activities.For fixed-facility events, fire and explosion was the strongest single risk factor for events involving either victims or evacuations. Illegal dumping or deliberate damage also was strongly associated with fixed-facility events resulting in victims. For transportation-related events, explosion was the strongest single risk factor for events involving victims, and fire and explosion was the strongest for events involving evacuations. The observed associations between time of day (0600–2359) and weekday occurrence are likely to be influenced by production intensity, however information on production intensity is not available in HSEES to examine this relationship further. When temporal variables were modeled together, fixed-facility events with victims were more likely to occur on weekday mornings in the spring, which may coincide with the planting season. These results are consistent with a previous analysis that showed that acute hazardous substance releases with victims were more likely during the planting season in Midwestern states .In the stratified analysis of substances within industry category, the strongest association was for acid releases in the agriculture, forestry, and fisheries industry for fixed-facility events involving victims. Previous analyses have shown that acute chemical releases are common in the agricultural industry, nitric acid and sulfuric acid are frequently released in agriculture events, and releases of acids result in significantly higher proportions of releases involving victims ,8,9.Chlorine releases in fixed facilities resulted in victims and evacuations in more industry categories than any other substance. This is consistent with an analysis that found chlorine releases had the greatest significant risk of having events with victims and were almost five times more likely than nonchlorine events to involve evacuations . This isThe HSEES system collected data in only 17 states during 1996–2001. Each state has different reporting requirements for the amount of hazardous substances released that has to be removed, cleaned up, or neutralized; and reporting of events to participating state health departments is not mandatory. Therefore, the total number of events, events with victims, and events with evacuations may be underestimated. However, HSEES is the only federal hazardous substances release database designed specifically to assess and record the public health consequences of hazardous substances emergency events.The HSEES system captures more events than other federal reporting systems, such as the United States Environmental Protection Agency's (EPA) Risk Management Program (RMP) . EPA's RThese results indicate that prevention activities should focus on ways to reduce fire and/or explosion hazards in facilities that store, use, or manufacture hazardous substances such as distributing material safety data sheets to employees, keeping work areas dust free, and storing chemicals away from ignition sources. Previous analyses of HSEES data have shown that human error and equipment failure are the most frequent causes of acute chemical releases ,16-18; rSeveral state health departments participating in HSEES already have developed strategies aimed at reducing releases and injuries associated with chlorine. These activities include distribution of fact sheets to county emergency management agencies, fire departments, other first responders, and industries; and presentations to municipal water directors, engineers, and hotel/motel swimming pool owners and operators.The predictors of public health consequences associated with acute chemical releases presented in this paper are broad categories of characteristics available in the national data. Future analyses of HSEES data may explore industry-specific root causes of events and risk of events with victims or evacuations for specific industries.The results of this analysis should help guide prevention activities aimed at reducing emergency chemical releases and their associated victims and evacuations. Attention should focus on preventing fires and explosions, releases from illegal dumping and deliberate damage, and acid releases in the agricultural industry. Efforts should continue to educate industry, first responders and other users about the potential hazards of chlorine.ATSDR, Agency for Toxic Substances and Disease RegistryCI, confidence intervalEPA, Environmental Protection AgencyHSEES, Hazardous Substances Emergency Events SurveillancePOR, prevalence odds ratioRMP, Risk Management ProgramThe author(s) declare that they have no competing interests.PZR led the writing of the manuscript and assisted with the statistical analysis. WAW led the statistical analysis and assisted with the writing of the manuscript. WEK conceived of the manuscript and participated in its writing and interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: