The etiology of ulcerative colitis (UC) is to be understood. The basic pathological feature of UC is intestinal chronic inflammation. Superantigen, such as Staphylococcus enterotoxin B (SEB), is reported to compromise intestinal barrier function by increasing epithelial permeability and initiate inflammation in the intestinal mucosa. Inasmuch as anatomic position of the sinus, chronic sinusitis-derived SEB may follow the secretion and to be swallowed down to the gastrointestinal tract and induce lesions to the intestinal mucosa.Sinus wash fluid  was collected from a group of patients with both chronic sinusitis (CS) and UC. A group of mice were sensitized to ovalbumin (OVA) in the presence of SWF. The sensitized mice were challenged with the specific antigen OVA. The inflammatory status of the colonic tissue was determined with histology, serology and electron microscopy. Using horseradish peroxidase (HRP) as a tracer, another group of mice was stimulated with SWF for 2 hours. The HRP activity was detected in the colonic tissue with enzymatic approaches and electron microscopy.2 tissue); (3) increased MPO activity (12.9 ± 3.2 U/g tissue) and inflammatory scores (1.8 ± 0.3); (4) mucosal surface ulcers; (5) edema in the lamina propria; (6) bacterial translocation and abscess formation in the subepithelial region.Epithelial hyperpermeability in colonic epithelium was induced by stimulating with SWF. The HRP activity in the colonic mucosa was almost 11 times more in the SWF treated group (3.2 ± 0.6 μg/g tissue) than the control group (0.3 ± 0.1 μg/g tissue). Mice were sensitized using a mixture of SWF and OVA (serum OVA-specific IgE was detected with a highest titer as 1:64). Challenge with OVA induced extensive inflammation in the colonic mucosa by showing (1) marked degranulation in mast cells  and eosinophils ; (2) inflammatory cell infiltration (MC = 145.2 ± 11.4; Eo = 215.8 ± 12.5; mononuclear cell = 258.4 ± 15.3/mmIntroducing Sinusitis-derived SEB-containing SWF to the gastrointestinal tract compromised colonic mucosal barrier function increasing epithelial permeability to luminal macromolecular protein in mice. The SWF facilitated colonic mucosal sensitization to luminal antigen. Multiple challenging the sensitized colonic mucosa with specific antigen OVA induced inflammation, induced a condition similar to human ulcerative colitis. Ulcerative colitis (UC) is a disease characterized by inflammation and ulcers in the mucosa of the large intestine with unknown etiology. The inflammation usually occurs in the rectum and lower part of the colon, but it may affect the entire colon. The inflammation speeds up colonic motility and causes diarrhea. Ulcers form in places where the inflammation has killed the cells lining the colon; the ulcers bleed and produce pus. Theories about what causes ulcerative colitis abound, but none have been proven. The most popular theory is that the body's immune system reacts to a virus or a bacterium causing ongoing inflammation in the intestinal mucosa; others include genetic predisposition, autoimmune disorders and impaired immune regulation -3.over the past 15 years, more than 2000 patients with chronic sinusitis (CS) including some patients with both CS and UC visited our clinic and were treated with different remedies, including medical treatment and functional sinus endoscopic surgery (FESS). Apart from improvement of chronic sinusitis, those patients with both CS and UC showed great improvement of UC as well (data not shown) that couldn't be explained with the specific treatment alone. Therefore, we postulated that there might be an association between CS and UC in these patients. Microbial infection is the most common cause of CS. The microbial products, such as lipopolysaccharide (LPS), staphylococcus aureus enterotoxin B (SEB) ,5 can beStaphylococcus aureus (S. aureus) [S. aureus enterotoxins [Staphylococcus enterotoxin B is an extracellular toxin produced by certain strains of  aureus) . Many carotoxins . In addirotoxins  and haverotoxins . SEB is rotoxins . SEB is rotoxins . SEB canrotoxins  that mayS. aureus, and a quantity of SEB was detected in the nasal cavity of the patients with allergic rhinitis [The nasal cavity and sinus are primary sites of colonization by rhinitis ,16. BaseSinus-wash fluids (SWF) were collected from 32 patients with both CS and UC . We diagnosed CS in the patients as an inflammation of the sinus mucosa with a persistent mucoid or mucopurulent nasal discharge for longer than 3 months that resisted antimicrobial therapy and antral irrigation. Diagnosis was made on the basis of clinical history, rhinoscopic findings, and computed tomographic scan of paranasal sinuses. CS was confirmed by computed tomographic examination that showed diffuse mucosal thickening in the ethmoid or/and maxillary sinuses bilaterally with scores higher than 12 by the Lund-Mackay staging system . Twenty-The diagnosis of UC was based on clinical history, colonoscopy and histology. The history included persistent bloody diarrhea, rectal urgency, or tenesmus. Examinations and sigmoidoscopy and biopsy were performed to confirm the presence of colitis and to exclude the presence of infectious etiologies.Every patient underwent maxillary sinus puncturing and washing. The SWF was collected prior to other procedures. Five ml saline was injected into the sinus cavities and re-collected and stored at -70°C for further use. SEB content in SWF was evaluated with ELISA (All the reagents used in this study were purchased from Sigma unless otherwise mentioned). None of the subjects had recent upper respiratory acute infections. This study was approved by the Ethical Committee of the First Hospital of Shanxi Medical University.For the purpose of verifying our hypothesis of an association between sinus pathology and colitis, an animal model of ulcerative colitis was developed. Mice were sensitized to a model food antigen, ovalbumin (OVA) with or without the presence of SEB-containing SWF. Animal experiments were approved by Animal Use and Care Committee at Shanxi Medical University. Male Balb/c mice (10 week old) were purchased from Beijing Animal Research Institute and maintained in the animal center at Shanxi Medical University. All mice were housed according to guidelines of the animal center. Water was available continuously through automatic ports, and a commercial mouse diet was provided ad libitum.2O2 (pH 7.6) for 30 min, and postfixed with 2% osmium tetroxide for 60 minutes, followed by staining en bloc with 4% uranyl acetate for 30 minutes. Tissue samples were dehydrated through a graded series of ethanol, cleared in propylene oxide, and embedded in Epoxy embedding medium. Thin sections were prepared and stained with 4% uranyl acetate for 5 min and subsequently with 2.5% lead citrate for 2 min and examined at 80 kV with a JEM 1200 electron microscope. HRP containing endosomes were photographed randomly for further analysis.A group of mice was introduced 0.2 ml SWF  via intragastric gavage under a light anesthetization. Mice were killed by cervical dislocation 2 hours later . Control groups were designed as: a naïve control group, treated with HRP 10 mg in 0.2 ml PBS via intragastric gavage; an inactivated-SWF control group, the SWF was pretreated with anti-SEB (100 μg in 0.2 ml) for 30 min, then introduced to the mice in gavage with HRP. Colon was removed; a piece of colon (3 cm) was opened; the contents were collected and dissolved in 1 ml PBS for HRP assay; the colon tissue was snap frozen and stored at -70°C for HRP assay; another piece of colon (2 × 2 × 4 mm) was fixed with 2.5% glutaraldehyde for 2 hours; then rinsed in sodium cacodylate buffer, incubated in 3,3'-diaminobenzidine tetrahydrochlorine and H2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4 (sodium ortho-vanadate), 1 μg/ml leupeptin) and homogenized on ice; the homogenates and colon content were centrifuged at 1,500 g for 20 minutes at 4°C respectively, the supernatants were collected for HRP assay. HRP amount in colon content and colon tissue was determined by assaying enzyme activity in the collected supernatant according to the previous report [2O2 and 0.025 ml of a solution of o-dianisidine di-HCl (10 mg/ml). The optical density was determined at 460 nm with a spectrophotometer.Frozen colon tissue was weighed and immersed into lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Nas report . BrieflyTen mice were used for each group. A group of mice was sensitized by means of intragastric gavage with 50 μg OVA in 0.2 ml alum-SWF . Another group was also treated with the same protocol, but the SWF in the adjuvant was pretreated with an anti-SEB antibody (100 μg in 0.2 ml). Control groups were designed as: mice were only exposed to OVA without the presence of SWF, or were only exposed to SWF without OVA; naïve controls were treated with 0.2 ml saline. Each mouse was injected with 50 ng pertussis toxin peritoneally.From the 14th day after sensitization, all mice were challenged with 50 mg OVA in 0.3 ml saline by means of intragastric gavage 3 times, 48 h apart. Diarrhea was determined by visually monitoring mice for up to 1 hour following intragastric challenge. Mice demonstrating profuse liquid stool were recorded as diarrhea-positive animals. Two days after the last challenge, mice were sacrificed by decapitation; blood samples were collected, serum was separated and stored at -70°C for further analysis. Colon was removed, one piece was fixed with 4% paraformaldehyde for histology; one piece was snap frozen for MPO analysis; one piece was fixed with Carnoy solution for mast cell count; one piece was fixed with 2% glutaraldehyde for electron microscopy.2O2 in 1 minute at 25°C.Myeloperoxidase (MPO) activity was determined according to the method of Bradley et al . Tissue 2 of mucosa. All sections were coded to avoid observer bias. The degree of inflammation on microscopic tissue sections was scored as follows: : 0, no leukocyte infiltration; 1, low level of leukocyte infiltration; 2, moderate level of leukocyte infiltration; 3, high vascular density and thickening of the colon wall; and 4, transmural leukocyte infiltration, loss of goblet cells, high vascular density, and thickening of the colon wall. Grading was done in a blinded manner [The morphology of the epithelium, villi, and subepithelial layer of colon mucosa were assessed, and the numbers of eosinophils and mononuclear cells were counted in 10 randomly selected fields  for each mouse (100/group). To determine mast cell numbers, tissues were fixed in Carnoy's fixative and paraffin sections were stained with 0.5% toluidine blue. Mast cells were counted in 10 fields for each mouse (100/group). Cell numbers were expressed per mmd manner .Colon tissue was processed with routine procedures and observed with an electron microscope. Activation of mast cell and eosinophil in colonic mucosa was determined by the phenomenon of degranulation .Passive cutaneous anaphylaxis (PCA) was employed to determine serum OVA-specific IgE level. The serum was diluted in phosphate-buffered saline (PBS) from 1:4 to 1:128. The mice were injected intradermally with 50 μl serum into each of 4 dorsal shaved skin sites. The sites were outlined with a water-insoluble red marker. Forty-eight hours later each mouse received an injection of 0.1 ml OVA (1 mg/ml) containing 4% Evans blue via the tail vein. The results were recorded thirty minutes after the challenge; the diameter of the blue spot on the injection sites larger than 6 mm was recorded as positive reaction.t test was used to compare the difference between groups. p < 0.05 was accepted as significant criteria.Data were expressed as mean ± SD; Student The content of SEB was significantly higher in the SWF of patients with both UC and CS  than in the nasal wash fluids  of healthy control subjects.Horseradish peroxisase activity was detected in colonic content in both control mice (group of HRP-only and group of anti-SEB treated SWF) and SWF treated mice Fig . The HRPPassive cutaneous anaphylaxis (PCA) is the gold standard method to measure allergen-specific IgE antibody levels in mouse models of allergy. The present study demonstrated that only the group treated with both SWF and OVA showed positive results with the highest titer 1:64. No specific IgE was detected in the serum of mice in control groups.Activation of mast cells and eosinophils was observed as degranulation of the granules in the cytoplasm. Piecemeal type degranulation was observed more often than the anaphylactic type in the mast cells in the colonic mucosa of the sensitized mice with EM. Crystal core degranulation and matrix degranulation were observed in eosinophils of the sensitized mice. Granules were categorized intact or degranulated for each mast cell or eosinophil. Each type granule was numerated with EM. After challenge with specific antigen OVA, degranulation was observed in both mast cells and eosinophils of the sensitized mice. The ratio of degranulation was much higher in the sensitized mice compared with controls Fig .Challenge with specific antigen OVA resulted in inflammatory cell infiltration in the lamina propria and subepithelial regions. The infiltrate consisted of mainly mast cells, eosinophils and mononuclear cells. Compared with control, the sensitized group showed significantly more inflammatory cell infiltration, most of them might be activated immune cells Fig . CapillaInflammatory status in the colonic mucosa was assessed by colonic tissue MPO measurement, inflammatory cell infiltration and mucosal surface condition observation. MPO is a critical parameter that indicates activation of neutrophils. We observed a significant increase in MPO and inflammatory score in colonic mucosa after challenge with OVA in the mice treated with SWF and OVA Fig .Electron microscopy revealed ultrapathology in the sensitized and OVA-challenged colonic mucosa that included: (i) epithelial cell destruction Fig ; (ii) baFollowing the protocol of challenge with specific antigen OVA, the sensitized mice developed diarrhea 15–30 minutes after each challenge that lasted up to 1 hour. The number of diarrhea episodes (3 to 7 times) increased in parallel with the number of OVA challenges. No diarrhea was noted in control mice although those mice also received the same number of OVA challenges. Diarrhea was also noted by direct observation of the colon and cecum; the liquid stool observed following OVA challenge-induced diarrhea in the sensitized mice contrasts with the solid pellets seen in the distal colon of other mice in control groups.Our knowledge about the etiology of ulcerative colitis is still limited. Although some theories about its origins have been advanced, such as genetic predisposition, autoimmune disorders, infection, and so on ,24, the S. aureus in sinuses is frequently encountered [Rhinosinuses are empty cavities lined with mucosa. The anatomic feature, only having a small ostium, makes them very easily to be blocked and subsequently infected. Infection with ountered ,5. Thus,There are many toxic substances in the secretions from chronic sinusitis. SEB is one that has been well characterized. The unique feature of SEB is that it can down regulate intestinal barrier function ,13, actiIntestinal epithelial cells form a barrier between the luminal contents and the subepithelial region. The barrier restricts substances to be absorbed. It only allows some small molecules such as water to pass it freely. Antigens are macromolecular proteins that are not allowed to be absorbed before being digested to small peptides or amino acids under normal physiological conditions. But in reality, intact antigens do pass the intestinal barrier to reach lamina propria to induce inappropriate immune reactions under certain circumstance. How antigens cross the intestinal epithelial barrier is still a mystery. By introducing both SWF and HRP to mouse gastrointestinal tract, intact HRP in the colonic tissue was increased nearly 11 times compared to control. The results implicate that superantigen SEB is one of the factors that facilitate antigens to be absorbed without destroying their antigenicity. Lu et al also reported that SEB significantly increased colonic mucosal permeability in a mouse study .We have begun to appreciate that food allergy plays a role in the inflammation of intestinal mucosa . The resIn chronic allergic diseases such as asthma, during continuous antigen exposure, eosinophils are primed by IL-5 and attracted by chemokines, infiltrating the local tissue . We alsoMarked mast cell hyperplasia in the colonic tissue was observed in this study. In general, mast cell numbers in tissues are relatively constant, even though mast cell hyperplasia is observed in both the inflammatory and in repair/remodeling stage of various inflammatory disorders . The funStriking epithelial damage of colonic mucosa of the sensitized mice after challenge with OVA was observed in this study. This phenomenon has been well-documented in airway allergy . AlthougIn summary, we reported a murine model of ulcerative colitis in this paper that was induced with sinusitis-derived SEB and OVA sensitization followed by repeat challenges with specific antigen OVA. The histopathology of the colonic mucosa included inflammatory cell infiltration, activation of mast cell/eosinophil, epithelial barrier damage, micro-ulcer formation on the surface of colonic mucosa, bacteria translocation and abscesses formation in the subepithelial region.UC, ulcerative colitis; CS, chronic sinusitis; FESS: functional endoscopic sinus surgery; OVA, ovalbumin; SEB, Staphylococcus enterotoxin B; MPO, myeloperoxidase; HRP, horseradish peroxidase.The author(s) declare that they have no competing interests.PCY was involved in study design, histology, EM observation, data analysis and manuscript preparation; CSW and AZY were involved in SWF collection and animal model.The pre-publication history for this paper can be accessed here:


CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology. Toll gene, the expression of one of it's relatives we are reporting here concerning human malignant tumors, originally was characterized for its role in specifying dorsoventral polarity of the Drosophila embryo[Toll are also present in plants, mammalian toll-like genes are products of an ancient evolutionary process beginning before the separation of animals and plants [Drosophila thus far nine toll-like genes were identified, ten different human toll-like genes are currently described. In contrast to Drosophila, the mechanisms taking place in mammalian embryogenesis concerning TLR are widely unknown. The discovery of immune function for Toll in Drosophila led to a new understanding of innate immunity mechanisms.The la embryo. Since hd plants . Within Human TLR recognize pathogen-derived products, also termed pathogen-associated molecular patterns (PAMP) . These aDifferent studies show an immunostimulatory capacity of bacterial components which can mediate anti-tumor activity. The first reported use of such a therapy for a nonbacterial disease took place 1890, evaluating the anti-tumor activity of living streptococci directly injected into tumor masses . ShimadaIn order to characterize possible interactions between malignant cells and CpG-ODN, we investigated whether TLR9 is present in malignant tumors. A variety of malignant solid tumors and cell lines were tested for TLR9 expression; in addition, we examined direct effects of CpG-ODN upon apoptosis and chemokine production of tumor cells.Samples of human tumors and tumor-free tissues were obtained from lobectomies because of lung cancer. Tumor-free tissues were taken at least 5 cm away from the tumor-border. The specimens were fixed and paraffin-embedded using the HOPE-technique . SectionFor increased comparability of the staining intensities in malignant and non malignant cells we additionally performed IHC on tumor-bearing and tumor free lung tissues which have been assembled on one slide by use of a mechanical tissue arrayer device .2 polystyrene flasks with Dulbecco's modified Eagle's medium DMEM (Sigma) with 10 % heat-inactivated fetal calf serum (PAA Laboratories), 100 μg/ml penicillin G, 100 μg/ml streptomycin and 2 mM L-glutamine (Sigma), maintained under 5 % CO2 by routine passage every 3 days. Cells were seeded in 35-mm dishes (Nunc).A549 cells and HeLa cells were grown in 25 cmFor IHC cells were cytocentrifuged and treated by the HOPE-technique , the celTotal RNA was extracted from lung tissues according to the manufacturer's recommendations . After destroying residual DNA with DNase (Invitrogen), cDNA was synthesized by reverse transcription . PCR wasHybridization, detection of signals and controls were carried out as previously described  ,22.Primary antibody  was applied in a dilution of 1/100 in PBS for 16 h at 4°C. Negative controls comprised omission of the primary antibody. Detection was performed by horseradish-peroxidase labeled streptavidine-biotin technique  .A549, HeLa, BEAS 2b, U937, and NCI-H727 cell lines were used. RT-PCR was performed like described above using TLR9 specific primers  spanning an amplicon of 260 bp. Mononuclear cells were included as positive control as well as RT-PCR targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  (not shown). PCR-products were separated along with a molecular weight marker  using 2 % agarose gels Fig. .A549-cells were seeded in 35-mm glass bottom dishes (MatTek Corp.) overnight. Cells were transfected with GFP-huTLR9 using Polyfect (Qiagen) according to the manufacturer's instructions or incubated in medium.Cells were washed in tris-buffered-saline, containing 0.2 % Tween 20 (TTBS), fixed with 4 % paraformaldehyde in phosphate-buffered-saline (PBS) for 10 min on ice, and permeabilized with 0.25 % Triton-X100 (Roche) in PBS for 10 min. Cells were washed with TTBS, blocked with 10 % bovine-serum-albumine (BSA) in TBS for 20 min, and incubated with primary antibody  or isotype  1:150 in TBS 10 % BSA for 30 min. Cells were washed with TTBS, incubated for 30 min with Alexa-568/goat-anti-MouseIgG1 (Molecular Probes Inc.) 1:500 in TBS containing 10 % BSA, and washed with TTBS. Counterstaining was achieved using TOTO-3 1:500 in TBS containing 10 % BSA. Cells were washed with TTBS, fixed again as above, mounted and analyzed using a confocal laser microscope. The GFP-TLR9 plasmid was kindly provided by Terje Espevik, Trondheim, Norway.For CpG-ODN stimulation the M362 sequence was used in a concentration of 1 μM; as control M383 was used as described by Hartmann et al.  (MWG-BioAnnexin-V FITC apoptosis kit I and PE-conjugated active caspase-3 apoptosis kit I were used according to the manufacturer's instructions (BD Pharmingen). TLR9 antibody and isotype control  were stained after fixation and permeabilization using Intraprep (Beckmann Coulter) according to the manufacturer's instructions. Flowcytometric data  collected from 10,000 cells are reported as percentages of positive cells (Becton Dickinson).Cell culture supernatant (50 μl per sample) was analyzed using the Bio-Plex system and a Luminex 100TM analyzer (BioRad) according to manufacturer's instructions.2 for 24 h, and either stimulated or not stimulated with 1 μM of CpG-ODN (M362 sequence). These blocks from adjacent locations of the same lung-tumors were fixed using the HOPE-technique and paraffin embedded. RT-PCR was carried out like described above using primers targeting human MCP-1  resulting in a 149 bp PCR-product and GAPDH  resulting in a 257 bp PCR-product. PCR products were separated on 2 % agarose gels along with a molecular weight marker (pBR322-Msp1) and the results displayed in figure Tissue blocks from lung cancer specimens (edge length approximately 0.5 cm) were cultivated in RPMI 1640 at 37°C and 5 % COin situ hybridization (ISH). The generation of TLR9-signals was achieved within 10 minutes, whereas unspecific signals were not detected in the control preparations. We found high signal intensities for TLR9 transcripts in the cytoplasm of tumor cells in the majority of lung cancer specimens. Immunohistochemistry (IHC) revealed strong TLR9 protein expression within tumor cells of tissues and cell lines. In contrast normal lung tissues sporadically showed weak expression of TLR9 mainly in cells revealing morphological characteristics of alveolar macrophages and alveolar epithelial cells as displayed in figure To investigate the expression of TLR9 in human lung tumors and lung tumor cell lines we used the recently described HOPE-fixation method. HOPE-fixed  specimenA cytoplasmic localization of TLR9 was confirmed by confocal microscopy fig. . This fiThe expression of TLR9 in tumor cells and cell lines rises up the question, whether this receptor is functional active in these cells. As shown in figure Here we investigated, whether CpG-ODN can modulate their own receptor. We found no differences in TLR9 expression with and without CpG-ODN stimulation. However, in the presence of TNF-α/CHX the expression of TLR9 was strongly reduced, whereas CpG-ODN stimulation counteracted this downregulation Fig.  and 3b.In order to obtain further information about the functional activity of TLR9 in tumors we studied cytokine release upon CpG-ODN stimulation. The measurement of cytokines from stimulated HeLa and A549 cells revealed a significantly enhanced release of monocyte chemoattractant protein-1 (MCP-1) after 24 h of stimulation in response to CpG-ODN or TNF-α Fig. . The proex vivo experiments. We conclude the expression of a functionally active TLR9 in human malignant tumors.By application of a novel fixation technique we specify for the first time the expression of TLR9 protein and mRNA in a selection of human non small cell lung cancer tissues as well as cell lines. Stimulation of the TLR-9 expressing cell lines A549 and HeLa with CpG-ODN showed a marked antiapoptotic effect. In addition, there was substantially enhanced release of MCP-1 from the cell lines upon CpG-ODN stimulation which was also shown in Spaetzle. Such a ligand could play a role for the activation of human TLR9. Whether the expression of TLR9 in human malignant cells takes advantage of TLR9-function in embryogenesis therefore remains unclear.The presence of molecules involved in ontogenesis e.g. the carcinoembryonic antigen (CEA) is frequently observed in malignant tumors suggesting a kind of "shift-back" towards earlier developmental stages . The sigOn the other hand TLR9 in malignant cells could have similar functions as in cells of the innate and adaptive immune system. In humans TLR9 has been described to be mainly expressed in B-lymphocytes, monocytes and plasmacytoid dendritic cells . In addiThe CpG-ODN sequence M362 used in our study is known to potently activate TLR9-expressing immune cells in humans including plasmacytoid dendritic cells and B cells as shown by Hartmann et al.  B cells These mechanisms are both reflected in the results we obtained in our study after CpG-ODN stimulation of malignant cells:in vivo showed anti-tumor effects. However, this anti-tumor effect appears to be effective indirectly and is related to enhanced NK cell activity. In a murine model of lymphoma the immunostimulatory effect of CpG-ODN was demonstrated to be responsible for the observed anti-tumor effects [et al. have shown that CpG-ODN in vivo induced rejection of neuroblastoma xenografts [in vivo requires further investigation.Firstly, stimulation of the A549 and HeLa cells with CpG-ODN showed an antiapoptotic effect. This was demonstrated for spontaneous as well as induced apoptosis with TNF-α and CHX after 24 h. Our observation is consistent with previous evidence in other cell lines. Yi et al. demonstrated antiapoptotic effects of CpG-ODN in a mouse B lymphoma cell line , and sim effects . Carpentnografts . In contnografts . To whatin vitro in this study could further enhance this scenario.Secondly, tumor cell lines (A549 and HeLa) stimulated with CpG-ODN showed strong secretion of the CC chemokine MCP-1. Furthermore a similar effect was observed in the investigated tumor tissues. Immunostimulatory properties together with anti-tumor activity of bacterial DNA were initially reported for a DNA fraction derived from mycobacteria by Tokunaga and coworkers . It is kRegarding TLR9 expression in nonmalignant lung tissue our data confirm the findings of low TLR9 expression in respiratory cells of Platz et al. , who havin vitro and ex vivo experiments, our data provide evidence for the second as well as the third possibility; the sum effect of these two counteracting mechanisms in an in vivo setting can not be estimated from these experiments and could even differ from tumor entity to tumor entity.Biological explanations for the TLR9 expression in malignant cells require further investigations. Three possibilities are conceivable: Either this could represent a bystander phenomenon, a side effect of a pathway functional to a different purpose. Secondly the upregulation of TLR9 could be beneficial to the tumor, promoting tumor cell survival. Thirdly, it even might help immune control strategies of the organisms an element of a pathway directing defense mechanisms against malignantly transforming cells. While the first possibility seems unlikely in the light of our findings of a functionality of the receptor in various In conclusion, we showed in a selection of samples that human malignant tumors express functionally active TLR9 and respond to CpG treatment with prolonged survival and chemokine release. This might influence the effects of CpG-ODN based anti-tumor therapies. Broad screening approaches will be worthwhile to further substantiate these initial results.While recent strategies in tumor-immunology mainly target a strengthening of the host-defense, we provide evidence that the malignant cells themselves can be regarded active players in the complex struggle between tumor and host. In any case CpG-ODN based anti-tumor therapies should be reconsidered in the light of our findings since CpG-ODN products are currently in Phase I/II clinical trials both as a monotherapy and as part of multi-drug regimens.DD carried out the flow cytometry and cytokine assays and was involved in the design and coordination of the study and drafting the manuscript. DA and AJU carried out the confocal microscopy, RT-PCR with cell lines and were involved in drafting the manuscript. JG was involved in immunohistochemistry of cell lines and the design of the study. DB conducted the surgical part of the study. EV conducted the pathological part of the study and was involved in the design of the study. KD and PZ conducted the clinical part of the study and were involved in the design and coordination of the study. TG performed the immunohistochemistry, in situ hybridization and RT-PCR with tissues and conceived of the study. All authors read and approved the final manuscript.


SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied.in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA).Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro, in which polymerase α-primase is one of its main targets.We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug. DNA replication in eukaryotic cells is a tightly regulated process . The regMuch of what is currently known about the mechanism of DNA replication in eukaryotic cells has come from studying SV40 and related viruses. SV40 virus can use the host replication machinery for its own DNA replication together with the virally encoded SV40 T-antigen. SV40 T-Ag is a multifunctional regulatory protein with numerous biochemical activities, and it has been classified as a member of superfamily III helicase and can unwind dsDNA and RNA . All othPsammaplysilla sponge dTTP (300 cpm/pmol), 0.6 μg of SV40 T-Ag, 0.23 μg of pUC-ori+, HeLa extracts, and psammaplin A as indicated. The reactions ran at 37°C for 2 hr, after which the acid-insoluble radioactivity was measured dTTP (500 cpm/pmol), 0.1 μg of poly (dA)4500: oligo (dT)25 (20:1), DNA pol α-primase, and psammaplin A as indicated. After incubation at 37°C for 30 min, acid-insoluble radioactivity was determined [DNA pol α-primase activities were assayed as described previously  with thetermined .Values are presented as mean ± SD. Data was initially analyzed by one-way analysis of variance (ANOVA) and comparison of groups was made using Turkey test (SPSS software).in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Psammaplin A is rather interesting owing to its two identical domains which are linked through a disulfide bridge. Macrophage cells are one of the key players in the early innate immune response, and they release inflammatory chemicals known as cytokines when they are activated. This sort of inflammation is not always a good thing, and overactive macrophage cells have been implicated in a number of human diseases, including arthritis and sepsis. When we studied the effect of psammaplin A on the viability of macrophage cell line RAW264.7, a reduced cell count was observed in the psammaplin A-treated cells and this decrease in the number of living cells also showed good dose-dependent  that is required for replication. The former mechanism is unlikely, because we have directly checked the effect of psammaplin A on DNA and didn't find any detectable damages to the template (data not shown). In order to find what proteins in DNA replication were affected by psammaplin A, we checked topo I activity at first, which plays key roles in DNA replication, transcription, and recombination by forming transient DNA single-strand breaks and acting as DNA strand transferase. In addition, the topoisomerase is now considered to be an important cancer chemotherapeutic target. The inhibitory effect of psammaplin A on the catalytic activity of topo I was shown in Fig Nicolaou and his colleagues reported that the DTT present in many enzyme assays could reduce the disulfide bond of psammaplin A to the corresponding free thiol . In thei50, which appeared as two distinct bands in the polyacrylamide gel. The ssDNA binding activity of RPA was inhibited by psammaplin A in a concentration-dependent manner, and 500 μM of psammaplin A totally inhibited the ssDNA-binding activity of RPA.In replication, RPA mediates unwinding of SV40 origin-containing DNA in the presence of SV40 T-Ag and topo I. It interacts with SV40 T-Ag and the DNA pol α-primase complex, which is necessary for the initiation of SV40 DNA replication ,9. Here,As described above, DNA pol α-primase complex is necessary for the initiation of SV40 DNA replication. To further investigate the inhibitory effect of psammaplin A in replication, we tested psammaplin A for inhibition of pol α-primase activity to see whether it's inhibitory effect on DNA replication correlate with pol α-primase activity. As shown in Fig in vitro system attempting to find out the target process and molecules of psammaplin A and have a glimpse on cell cycle regulation.Psammaplin A has exhibited inhibition on general bacterium, some actinomycetes and fungi, and it also has cytotoxicity toward several cancer cell lines. In our research, we found that psammaplin A deliver significant cytotoxic activity against macrophage cell line RAW264.7. Which process is mostly affected by psammaplin A in cell cycle? What is the mechanism of the inhibition? Enlightened by the inhibitory effects of psammaplin A on bacterial DNA synthesis, bacterial DNA gyrase and eukaryotic topo I, we investigated the effect of psammaplin A on DNA replication using SV40 replication in vitro. In order to clarify the inhibition mechanism, further work were carried out. In SV40 DNA replication, three factors, SV40 T-Ag, RPA, and pol α-primase complex, are essential for initiation process. In the presence of topo I, SV40 T-Ag will continue to unwind the DNA to form a highly unwound DNA [In our study, we found that psammaplin A inhibited SV40 DNA replication ound DNA . DNA synound DNA . We haveound DNA . Psammapin vitro DNA replication system includes a large number of proteins, while in topo I assay, RPA binding assay and pol α-primase assay, purified proteins were used. Many of the proteins in the crude extracts can affect each other by physical or functional interactions. For example, in the process of DNA replication initiation, RPA interacts with T-Ag and DNA pol α-primase, and it is believed that RPA can both stabilize the unwound DNA and stimulate DNA pol α. The universal protein-protein interactions in crude extracts make its working environment quite different from that of purified protein assay system. Second, even for the same protein, for example, pol α-primase, the concentration in the cell extract and in the purified pol α-primase assay is not comparable before any quantification of the replication proteins in the cell extract. Third, due to the active disulfide moiety in the structure of psammaplin A, it is possible that psammaplin A could interact with some particular cellular targets in the crude extract, which may lead to covalent modification of the biological targets and psammaplin A itself. Therefore, the free available concentration of psammaplin A in the crude extract might be different from that in purified protein assay system.It is puzzling that the DNA pol α-primase, RPA and topo I were readily inhibited by low concentration of psammaplin A whereas the SV40 DNA replication assay still showed about more than 80% DNA replication in the presence of 125 μM psammaplin A. In our opinion, at least three points could account for this discrepancy. First, the cell extract we have used to support in vitro replication, the significant inhibition of psammaplin A on the viability of macrophage RAW264.7 cells occurred at a relatively low concentration. There is the possibility that DNA replication might not be the single or primary event that affected by psammaplin A. It should be evident that the event of DNA replication in a living cell is more complicated than that in the crude extract system because of many existing cell cycle signals. So, it is still vague whether the ability of psammaplin A to inhibit cellular viability is correlated with its ability to inhibit DNA replication. In order to make it clear, it is necessary to check the effects of psammaplin A on other macromolecular synthesis (RNA synthesis and protein synthesis). Although the results in this paper do not clearly define the mechanism of cytotoxicity of psammaplin A, they have convincingly shown that psammaplin A possesses the abilities to inhibit DNA replication and some important replication proteins.Different from the effect of psammaplin A on SV40 DNA in vitro assay conditions in this study, we can find that all these assays were performed in similar conditions . Given the mildness and tolerance of these reaction mixtures, we guess that psammaplin A is stable in the assays. Of course there is the second possibility that psammaplin A was reduced in the reaction, but the reduction product had nearly the same inhibition effect on topo I as the original compound. Further investigation aimed at this question need to be carried out.Because of the disulfide bridge linking two identical subunits in the structure of psammaplin A, in this study, we also paid attention to the potential reduction effect of DTT present in the topo I assay. Different from the report of Nicolaou , we didnBased on our results, we suggest that the cytotoxicity of psammaplin A might be related to the inhibitory effect it has on the fundamental cellular process-DNA replication, and one of the main target molecules of psammaplin A could be pol α-primase.The authors declare that they have no competing interests.YHJ and DKK conceived and designed the experiments and wrote the manuscript. YHJ performed all of the experiments. JHJ provided psammaplin A sample. EYA, SHR, JSP, HJY, SY, BJL and DSL participated in the conception, supervision, coordination and guidance of the study and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The use of herbal supplements in the United States has become increasingly popular. The prevalence of herbal use among primary care patients varies in previous studies; the pattern of herbal use among urban racially/ethnically diverse primary care patients has not been widely studied. The primary objectives of this study were to describe the use of herbs by ethnically diverse primary care patients in a large metropolitan area and to examine factors associated with such use. The secondary objective was to investigate perceptions about and patterns of herbal use.Data for a cross-sectional survey were collected at primary care practices affiliated with the Southern Primary-care Urban Research Network (SPUR-Net) in Houston, Texas, from September 2002 to March 2003. To participate in the study, patients had to be at least 18 years of age and visiting one of the SPUR-Net clinics for routine, nonacute care. Survey questions were available in both English and Spanish.A total of 322 patients who had complete information on race/ethnicity were included in the analysis. Overall, 36% of the surveyed patients (n = 322) indicated use of herbs, with wide variability among ethnic groups: 50% of Hispanics, 50% of Asians, 41% of Whites, and 22% of African-Americans. Significant factors associated with an individual's herbal use were ethnicity other than African-American, having an immigrant family history, and reporting herbal use by other family members. About 40% of survey respondents believed that taking prescription medications and herbal medicines together was more effective than taking either alone. One-third of herbal users reported using herbs on a daily basis. More Whites (67%) disclosed their herbal use to their health-care providers than did African-Americans (45%), Hispanics (31%), or Asians (31%).Racial/ethnic differences in herbal use were apparent among this sample of urban multiethnic adult primary care patients. Associated factors of herbal use were non-African-American ethnicity, immigrant family history, and herbal use among family members. Whereas Hispanics and Asians reported the highest rates of herbal use, they were the least likely to disclose their use to health-care professionals. These findings are important for ensuring medication safety in primary care practices. The use of complementary and alternative medicine (CAM) in the United States gained greater popularity in the 1990s. Two national telephone surveys of 1,539 and 2,005 adults, respectively, demonstrated an increasing trend in the use of CAM, including relaxation techniques, herbal medicine, massage, chiropractic, and acupuncture[Efficacy studies of herbal supplements are on the rise, but most data published to date are preliminary and do not provide strong evidence for the clinical effectiveness of herbs. Nevertheless, about 15 million American adults 18%) are thought to use prescription medications concurrently with herbal or vitamin products, and as % are thoThe prevalence of herbal use among racially/ethnically diverse primary care patients varies from study to study-7,10-12,We implemented this cross-sectional study within the Southern Primary-care Urban Research Network (SPUR-Net) from September 2002 to March 2003. SPUR-Net is a practice-based research network in Houston, Texas, that consists of five constituent member organizations affiliated with a county health system, a managed care organization, or a private practice clinic. SPUR-Net clinicians provide care to patients from diverse ethnic and socioeconomic backgrounds, with approximately one million patient visits per year. A total of six primary care clinics were included in this study that varied according to socioeconomic status (SES) of their patients as measured by income level and insurance type. For the purposes of this study, we defined "clinic SES" according to the insurance status of the majority of patients; "high SES" means that most patients have insurance , and "low SES" means most patients are indigent . Human subject approvals were obtained from the Institutional Review Boards at all of the SPUR-Net constituent organizations. Permission to conduct the study was also obtained from the medical directors and applicable patient advisory groups at each of the six participating clinics.To be eligible for participation in the study, patients had to be at least 18 years of age and to be visiting one of the participating clinics for routine, nonacute care. A target of 50 surveys in each of the six clinics was collected from a convenience sample of patients. The decision regarding the number of patients to be surveyed was limited by our resources, including availability in funding and personnel. A research assistant approached potential subjects in the clinic setting to determine their willingness to complete a 23-item questionnaire about herbal use in either English or Spanish. Those patients who consented to participate were either given the survey to complete on their own or had the survey administered to them by the research assistant. Research assistants were available on-site to answer any questions the patients had, helping to improve patients' understanding of the terms used in the survey. Recruitment methods were the same in all of the participating clinics. The research assistants stopped recruiting patients when a minimum of 50 surveys was collected in each clinic.Survey questions were adopted and modified from previously developed and validated surveys on CAM use, including national telephone surveys conducted by Eisenberg et al.,2,13, a The final survey instrument had three components. First, all participating patients answered questions regarding sociodemographic characteristics . Immigrant family history and spoken language were elicited with the following questions: "Are your family members immigrants to the United States (Y/N)?," and "Do you speak another language other than English?" After completing the demographic questions, respondents answered a series of questions regarding their belief in herbal use and their herbal information sources. The questions pertained to their personal use of herbs (Y/N); their belief in the benefit of herbal remedies (Y/N), the source of their herbal information ; their preferred content of herbal information , and their preferred methods for obtaining herbal information from physicians or pharmacists . Patients who reported using herbal supplements answered additional questions related to their patterns of and reasons for herbal use. In open-ended questions, the participating patients were asked about the herbs they specifically used and the health conditions for which they took the herbal products. Related questions included frequency of herbal use ; duration of use ; expenditure on herbal products; reported concomitant use of prescription medications; disclosure of herbal use to physicians or pharmacists; and any experiences of adverse reactions from using herbs.For the purposes of this study, we used the definition of dietary supplements stipulated in the 1994 Dietary Supplement and Health Education Act (DSHEA) to differentiate herbs from vitamins and minerals. Herbal use was defined as having ever used herbal products or natural medicines for health maintenance or treatment of health conditions. To measure herbal use, we asked the following question: "Do you use any of the following?" Response options included: herbs/herbal products or natural medicine , folk medicine or home remedy, vitamins, minerals, or none. Herbal use did not include the use of folk medicine, home remedies (such as honey), vitamins, or minerals.Data from the paper-based survey were entered into an ACCESS database and were imported into SAS 9.1 for Windows. The study variables were summarized by using one-way frequencies to examine the sociodemographic characteristics of the study sample, the belief in and information source for herbal use, and the patterns of and reasons for herbal use among urban multiethnic primary care patients. The frequencies of use of specific herbs were counted, and the health conditions for which herbs were used were further coded into three types—acute, chronic, and health maintenance.p value ≤ 0.05 was considered to be statistically significant. In order to assess factors associated with herbal use, all hypothesized variables  were included in both the univariate and the multivariate logistic regression analyses. These independent variables were entered as dichotomous variables in the model: gender , age , ethnicity , education (less than college vs. college and greater), immigrant family history (yes vs. no), herbal use by family members (yes vs. no), and clinic clientele (high SES vs. low SES). Significant variables identified by backward elimination of the main effects from the multivariate analysis were further evaluated in two-way interactions. Thus, the final model contained all of the significant main effects and the two-way interaction terms. Odds ratios and 95% confidence intervals were calculated to determine the effects of the significant variables on herbal use. Since the sample size for Asians was small, Asians were not included in the logistic regression analyses. Furthermore, the language variable was excluded from the regression analyses because the survey question was not clearly answered by many patients; for example, 10 Spanish-language forms had "no language other than English" indicated. In addition, some answers were possibly indicative of an exclusive language other than English instead of the bilingual capability of the respondent.Based on findings from previous studies, we used the following independent variables as reference variables for both the univariate and multivariate logistic regression analyses: male gender, age less than 30 years, African-American ethnicity, less than a college education, no immigrant family history, no herbal use by other family members, and visiting a high SES clinic. A Chi-square test of proportions was used to determine the association between herbal use and each of the independent variables related to demographic characteristics; a Of the 327 patients who agreed to participate in the survey, only 322 completed the race/ethnicity information and were included in the analysis. The characteristics of the study sample are summarized in Table p < 0.05). Nearly half of the patients who reported using herbs (46%), particularly Hispanics (63%) and Asians (57%), also reported taking prescription medications concomitantly with the herbs . Patients who reported using herbs indicated that they received information about those herbs mainly from family members and relatives. Nevertheless, most patients reported that they preferred receiving herbal information  through handouts or brochures from their physicians or pharmacists, followed by having access to a consultation service or a Web site. About 40% of all of the survey respondents, but especially Asians (55%) and Whites (47%), believed that taking prescription medications and herbal medicines together was more effective than taking either alone. About 41% of Hispanic respondents believed that herbal medicines were superior to prescription medications, as compared to 12% of Whites. These differences in beliefs about herbal use among the ethnic groups were found to be statistically significant (p < 0.05) with herbal use were ethnicities other than African-American, immigrant family history, and herbal use by other family members (Table p < 0.05). In addition, interactions between immigrant family history and herbal use by other family members were found to be significant terms in the model , the desire to try alternative therapies (33%), and preference for having their own methods to care for their health (20%). Among the herbal users, 32% reported taking herbs on a daily basis, and 60% reported using herbs for longer than three years. Usage varied by race/ethnicity; for example, 48% of Whites reported taking herbs on a daily basis, and 79% of Hispanics reported using herbs for longer than three years.Even though Hispanics and Asians used herbs more frequently, they were the least likely to disclose their herbal use to their physicians or pharmacists. More Whites (67%) told their health-care professionals about their herbal use than did the African-Americans (45%), Hispanics (31%), or Asians (31%). The reasons given for nondisclosure generally fell into two main categories: 1) "They (the provider) never asked," and 2) "It wasn't important for them to know." While few respondents (5.3%) reported having experienced an adverse reaction to herbs, many of them (43%) did not inform their physicians of it.The specific herbs used by the patients covered a wide spectrum and varied by ethnicity. The herbs used most commonly by White patients were echinacea (32.1%), St. John's wort (21.4%), ginkgo biloba (14.3%), and chamomile (14.3%). Hispanic patients most often reported using chamomile (61.2%), aloe vera (44.9%), and garlic (20.4%). African-American patients reported primarily using garlic (40%), ginseng (30%), and ginkgo biloba (10%). The herbs used by Asian patients were garlic (50%), ginkgo biloba (30%), and ginger (30%). Other herbs that were reported by patients—albeit infrequently—included Yun Zhi, black cohosh, dong quai, guggle phosphate, bee pollen, cat claws, and "a shot of whiskey." The patients who reported using herbs used them for a wide range of health problems, such as boosting the immune system, improving memory, and treating insomnia, depression, or diabetes. For conditions considered to be chronic, 44% of the White patients reported herbal use versus 32% of African-American patients. For conditions considered to be acute, 71% of Hispanic patients used herbs versus 10% of Asians. For health maintenance, 50% of Asian patients used herbs versus 16% of Hispanic patients.Our data show that herbal use is common 36%) among urban multiethnic primary care patients, but has a wide variability among racial/ethnic groups. Hispanics and Asians reported the highest rates of herbal use 50%), and African Americans reported the lowest 22%). Previous research conducted in the western United States found that the prevalence of herbal use among racially/ethnically diverse primary care patients varies[2%. Previ6% among 0%, and AAs expected, factors associated with herbal use included race/ethnicity, having an immigrant family history, and herbal use by other family members. In addition, we found interactions between having an immigrant family history and herbal use by other family members. Previous studies did not examine such interactions and found age to be predictive of herbal use,6,7. Unl® (warfarin sodium) and cause an increase in bleeding time[®, Parnate®) and potentiate the effects of selective serotonin reuptake inhibitors [® (digoxin)[® [We found that nearly half of herbal users (46%) reported taking herbal medicines and prescription medications concomitantly. More importantly, 43% of herbal users reported not disclosing their herbal use to their physicians or pharmacists. Interestingly, Hispanics and Asians used herbs the most frequently but disclosed their herbal use to their physicians or pharmacists less often than did Whites and African Americans. This lack of communication about herbal use is an area of concern because of the potential for medication errors and untoward reactions to herb-drug interactions. Adverse drug-herb interactions pose a great danger for patients. For example, ginkgo biloba, garlic, and ginseng all may interact with Coumadinding time,16. Echiding time,18. The ding time-23. St.  Zoloft®). Moreove(digoxin). In addi(digoxin) and has prazolam). As thesprazolam)-33.Two-thirds of the patients we surveyed reported wanting to receive information on herbal medicines from their physicians or pharmacists, preferably in the form of a handout or a brochure. These findings suggest that future studies are warranted to develop and test educational materials to 1) deepen our understanding of racial/ethnic variation in herbal use among primary care patients; 2) educate health-care professionals about the variations in the use patterns and the rationales for use that may help to reduce medication errors and increase the quality and safety of medical care; and 3) educate patients regarding evidence-based herbal information and encourage patients to communicate their herbal use to their physicians/pharmacists.Our study results should be interpreted in the context of several limitations. First, our estimates of herbal use frequency are imprecise because we used a convenience sample instead of identifying patients by randomized sampling. Secondly, even though we adopted the DSHEA definition of herbs, some patients had difficulty understanding this definition. Specifically, a small group of patients thought that herbs were equivalent to prescription medications such as digoxin and aspirin; the patients' level of understanding of herbs was improved after the research assistants provided further explanation and clarification. Third, we discovered that asking questions, such as "What do you take when you run out of your medications?," was more effective in eliciting answers from the study subjects than when asking them, "Do you use herbs, herbal products or natural medicine?." For these reasons, we had research assistants on-site to help facilitate the survey process. Fourth, the patients surveyed reported their concomitant use of herbs with prescribed medications based on their perceptions and memories. Last, we did not include measures of quality of life or questions about patient satisfaction with herbal use, which would be helpful in future studies, especially when comparing multiethnic and socioeconomically diverse patient groups.Despite these limitations, our findings confirm the increasing frequency of herbal use as reported in previous studies. Our study also gives a unique perspective by focusing on factors associated with reported herbal use among an urban multiethnic primary care patient population. In particular, we found that patients with immigrant family history—especially those with family members who use herbs—are most likely to report herbal use. Perhaps most disconcerting was our finding that while an increasing number of primary care patients report taking herbal medicines concomitantly with prescription medications, many of them do not disclose their herbal use to their physicians or pharmacists. These findings suggest that primary care clinicians need to understand the extent and patterns of herbal use by their multiethnic patients and efforts to elicit information from patients about herbal use may be warranted. Future studies are needed to develop effective interventions for primary care health-care professionals and patients to improve medication safety by eliminating potential adverse herb-drug interactions and medication errors.The author(s) declare that they have no competing interests.GMK conceived of the study, designed the survey questionnaires, coordinated and managed the data-collection process, directed data analysis, and drafted the manuscript. STH participated in drafting the manuscript and helped with data analysis. LTW performed the statistical analysis and participated in drafting the manuscript. RB helped with data analysis. RJV reviewed the questionnaires and data analysis, and participated in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In response to the Kosovo crisis, Canada received 5,500 Albanian Kosovar refugees in 1999 as part of the emergency humanitarian evacuation and settlement effort. This study attempts to describe the experiences of service providers at the international, national, and local levels, involved in the organization and delivery of health and settlement services in Canada for the Kosovar refugees.A qualitative case study design using key informant interviews was used. Nominated sampling was used to identify 17 individuals involved in the organization and delivery of health and settlement. Key themes were identified and recommendations made to provide a framework for the development of policy to guide response to future humanitarian emergencies.Six themes emerged: (1) A sense of being overwhelmed, (2) A multitude of health issues, (3) critical challenges in providing health care, (4) access to health and settlement services, (5) overall successes and (6) need for a coordinated approach to migration health.For those involved, the experience was overwhelming but rewarding. Interviewees' major concerns were the need for a more comprehensive and coordinated approach to the flow of medical information and handling of specific health problems. The Kosovo crisis resulted in the largest population displacement in Europe since World War II. During this time, over 300,000 Albanian Kosovars were expelled from Kosovo,  resultinCanada received approximately 5,500 refugees in 1999, with approximately 500 arriving to the city of Hamilton, in southern Ontario. Some Kosovars stayed temporarily in military bases and arrived after sponsorship housing had been arranged, while others arrived directly to temporary reception "houses" established at two Hamilton hotels. The reception in Hamilton included the organized provision of health services by physicians, nurses, dentists and optometrists, and settlement services from the Settlement and Integration Services Organization (SISO).This HEP was unique for many countries, including Canada, and involved a complex coordinated effort on the part of international, national, and local organizations traditionally involved with immigrants and refugees, settlement, and health. This event was an important opportunity for health and settlement organizations operating at international, national, and local levels to critically examine and reflect on their efforts to organize and deliver health and settlement services to new immigrants and refugees.The goal of this study was to explore the main challenges and successes of the Kosovar arrival, from international, national, and local perspectives and to develop recommendations to advise and guide planning of the future complex humanitarian emergencies, using interviews with key informants.A qualitative case study methodology was used with semi-structured interviews of key informants who were involved in the humanitarian evacuation at the international, national and local levels in Hamilton, Canada. This method was deemed most appropriate to provide a framework for understanding the challenges and successes associated with Complex Humanitarian Emergencies and generate recommendations for future efforts. The background of research team members was diverse. Medicine, nursing, anthropology, sociology and settlement were represented. Some members had been directly involved with the Kosovar settlement process at the local level .A purposive sampling strategy was used, in which appropriate key informants known to the researchers were approached for participation first. Snowball sampling, whereby additional participants were identified by the initial respondents, was used to increase the diversity of respondents. These sampling approaches are used when certain known individuals are likely to have in-depth information on a topic . IndividInterviews were conducted in English between April and July 2001. The interviewer (EM) took extensive field notes to supplement the taped and transcribed interviews. Of the 17 key informant interviews, the three international and national ones were conducted by telephone, and 14 were in person at the workplace of the interviewee. Six participants were male and 11 were female. Interviews were approximately 30 minutes in length. The interviews were based on an interview guide and the main part of the interview focused on the organization and delivery of health services to the Kosovars. Questions were further refined during the study. Specific probes were used to follow up on open-ended questions, where appropriate.Consultation with the local settlement agency (SISO) took place throughout the research process. Key informants were told that the purpose of the study was to gather information that would help in planning for similar events in the future. All participants provided verbal consent and were assured of confidentiality of responses. All interviews were tape recorded with the participants' permission and all but one  was transcribed. Ethics approval for the study was received from McMaster University Research Ethics Board.Questions pertained to the participants' involvement in the process, challenges, flow of information and communication, and involvement in health care services and settlement.Two research team members (EM and NF) initially independently reviewed the transcripts, coded categories and themes, and then compared results. A third researcher with expertise in qualitative research, who was not involved in the project, also reviewed the transcripts using NVivo software version 2.0  and compared results. The interviewer's field notes were also used for comparison with the data. Ambiguities were resolved and themes were developed from categories through discussion among the research group members and re-reading of transcripts. This process continued until no new themes emerged from the interviews. It was felt that saturation was achieved after 17 interviews, and data collection was ended. The process was iterative, thus the later interviews probed new emerging themes identified in previous interviews.Six core themes emerged from the data analysis.There was agreement among the participants that the Kosovo crisis in 1999 was very unusual in nature. Participants stressed that there was a lot of "scrambling" to prepare the infrastructure to receive a large number of people in a very short period of time. Respondents/informants were surprised by the magnitude, immediacy, scope and scale of the response required. Most described feeling overwhelmed.Although the local settlement agency (SISO) had begun preparations by meeting with various local agencies and organizations weeks in advance, they were given only three days notice by national immigration authorities that the Kosovars were arriving locally. One Hamilton Social and Public Health Services representative noted "We had many community people come together to talk about how we could plan for this big influx of refugees... we knew were in dire straits". One settlement worker called it "organized chaos". One local health care professional explained the triage process and it was evident that a major organizational effort was required to process the arrivals."We had to do our own triage at the hotel to find out whether there was anybody needing medications, anyone with heart conditions, anyone with diabetes ... we had to scan the place to find out, and even then we didn't have medical records that came with them... We just went around asking: Do you know about anybody who is pregnant? Do you know about anybody who has heart conditions? Do you know about anybody who is diabetic? ... And we tried to identify those people or they presented in the little clinic".Participants described facing a multitude of challenges in responding to the health needs of Kosovar refugees. Women's health services, including pregnancy care and abortion services were required. Dental care, mental health services and general curative services were also required. Participants were struck by the poor oral health of the Kosovars. Many Kosovars had been deprived of any curative or preventative care in Kosovo for a number of years prior to the 1999 crisis. One local health care professional observed,"I noticed with the Kosovars...that a lot of them had not had any health care for a long time so they had many of the things that we take for granted. Immunizations being up to date and those sorts of things had not been done.... They only saw doctors if they were absolutely on their deathbed. So sometimes, you sort of had a lot of catching up to do in terms of getting their health up to date"Chronic and poorly managed conditions such as diabetes, hypertension and renal failure were common. An interviewee with an international perspective noted,"I think the world, from a health point of view, went into Kosovo thinking that all refugees were like the Great Lakes and Rwanda...The challenges [in Kosovo] were more of chronic diseases, diseases of socialization, hypertension, diabetes, renal failure...I think that people have learned to be a bit more comprehensive in their approach to conflicts and emergencies."The participants expressed three main challenges which made their work more difficult: (i) tuberculosis screening, (ii) the lack of medical records and tuberculosis test results and (iii) mental health issues.Several participants noted that the tuberculosis screening process was not optimally organized. One local healthcare professional lamented,"I was reassured, but without any documentation to back it, that everyone had been screened for TB...When I contacted Public Health, they had not received any notification ...Certainly I didn't know where or how to get that information, and it seemed to me that Public Health didn't either".Communication to local health care providers about test results (tuberculosis and radiographs) was often described as sub-optimal. Immigration officials in Europe attempted to keep medical information flowing. One interviewee from an international perspective explained that,"We provided by fax and e-mail a summary of the medical conditions on the aircraft, so that they can be dealt with appropriately on arrival in Canada...We also used colour-coded cards and things so that (when) people who got off the plane (who needed care) they did receive expedient care".However, many local respondents expressed concern over the location of medical records and the inability to access this information in a timely manner. A local healthcare professional expressed,"At no point did I receive any medical documents about people with serious or chronic medical conditions that required care...if that information was available, I never got it".It was clear among the participants that mental health concerns were prevalent and service providers struggled with the delivery of appropriate mental health services. One local healthcare professional explained,"...things like headaches presenting when really the underlying condition was one of stress and anxiety, distress. Most of them came under the umbrella of what we might call, presenting with trivial complaints, but really what was beneath it all was stress and anxiety..".The rapid settlement of the Kosovars resulted in the local collaboration and coordination of health and settlement services at a single geographic site. This multi-agency collaboration was thought to enhance access and provision of services. One local healthcare professional described,"[The Kosovars] had, I thought, very well organized access...The SISO organization provided chauffeurs and translators, and administrators to pull all those areas together. So...if a refugee needed to go to a lab, they were driven, they were translated for, and they were brought back...".Since translators organized through SISO were available at the local health care sites, language was not perceived as a significant challenge by local health care providers.There was an awareness that the care provided was transitional in nature. This created some hesitancy in initiating treatment, as frequently there was uncertainty about the medical follow-up arrangements. Mental health issues and the lack of opportunity for identification and communication with future care providers were identified as concerns.While the Kosovars received medical coverage through the Interim Federal Health (IFH) program, local health care providers felt that the amount and nature of coverage was inadequate for services such as home care, dental care and optometry. One local healthcare professional described the issues surrounding home care,"One access issue that came up was the fact that under their IFH coverage, IFH does not cover home care, and without special arrangements to be in place...they don't qualify for it...There were a lot of people...who had walked miles and miles...and I saw [some] who had really bad foot conditions, infections and ulcers requiring daily treatment, soakings, dressings, bandage changes... Normally, those are things in the community that we would involve home care in...".In several instances, both the optometrist and dentist interviewed provided services that were not covered, free of charge, but in general, there was much confusion and uncertainty regarding the payment scheme for professionals. A dentist explained,"..we were not informed as a health professional that if we were approached by people from Kosovo that plans were available for treatment...we knew absolutely nothing until we started asking the questions..".Specific examples of successes included provision of comprehensive onsite health care integrating both health and settlement services, the use of nurse practitioners to allow physicians to focus on more complex cases, the policy to keep families intact, and the positive media coverage that contributed to an atmosphere of acceptance. A local settlement provider observed,"The Kosovars was something that the whole nation took on...We saw what kind of role the media can play in making the host community aware what other people are going through, explaining that refugees are not to be seen as invaders but as people who are in need of welcoming, and the Kosovars received one of the best, I think at least in my experience, one of the warmest welcomes...".Services available to the Kosovar refugees were deemed better compared to that provided to other refugee groups. This was a concern to many participants. Some stated that all refugees should be offered the same standard of high quality settlement services as those made available to the Kosovars. Another local settlement provider noted,"Refugees, regardless from where, should be treated the same way because it creates resentment not just in other refugee communities that have come to Canada, but [it] creates resentment among the workers that are providing services and sometimes struggling to get resources for a group of people...The Kosovars should be an example of how refugees should be treated in general".A number of participants suggested that we should learn from this experience with the Kosovars and prepare to put a contingency plan in place for similar events in the future.Better communication and organization were repeatedly stressed. One participant used the term 'Migration Health' to describe such an overall coordinated approach. Several participants commented on the importance of the coordinated approach as a necessary societal investment. One interviewee from an international perspective noted,...a lot of people who are working on the receiving end are simply following the process for receiving refugees.... there may not be the resources to look at the longer term issues: primary health care, health education, explaining how the health systems work, looking at some of the parameters that may influence longer term mortality and morbidity: dietary counselling, smoking cessation counselling, primary preventive health care procedures that we do in Canada that may not take place in the developing world."Most participants suggested that health care and settlement providers need to enhance their cultural sensitivity and cultural competence and better understand the health conditions of the displaced individuals in their country of origin.Despite organized attempts to coordinate efforts at different stages of the migration process, communication gaps and the sheer size of the influx resulted in challenges at the local level. Officials and service organizers at the international/national levels were unaware of these local gaps at the time of the evacuation. The flow of medical information and health records is an example. Primary care health workers needed to have easy access to targeted health information about the Kosovars, however this information was not available. If this information had been more easily available, health care services may have been more streamlined, and unnecessary duplication of lab tests and radiographs could have been avoided.Health care providers did not know what to 'expect'. Mental health, dental care and communicable diseases  were identified as requiring further specialized planning. This finding is similar to other refugee experiences in Australia  and CanaThere was a consensus among participants that this international evacuation represented an improved approach and a good foundation on which to organize refugee health and settlement in the future. Informants' concerns about local preparedness and the need for future advanced planning was consistent with recent United Nations High Commission for Refugees (UNHCR) findings . ParticiA potential limitation of this study is the time elapsed between the refugees' arrival and the interviews. Asking people to recall events that took place approximately 18 months previously may have influenced the nature and detail of the information obtained. However, it may also have enabled informants to recover from the initial emotional reaction and to see these events from a different perspective.It must be stated that the 'success' as defined by key informants may be very different than 'success' from the refugees' perspective. This study examined the perceptions of those involved with the Kosovars. The majority  of the local health professionals and settlement workers interviewed had worked in the local 'settlement houses' site where there was a shorter notice of arrival of the refugees. This may have contributed to the sense of 'chaos' echoed in many of the comments.'Operation Safe Haven'). Triage questionnaires for primary health care based on International guidelines were developed [Operation Safe Haven produced a template for refugee "acute health response" system. Using the Australian template combined with findings from this study, we propose the following attributes for an International Rapid Response System (IRRS):Many of the participants in this study made observations about the diverse nature of complex emergencies. The importance of logistics and planning has also been described in other studies -10. Someeveloped . Operati1. Establish lead organizations at the different levels 2. Clearly define roles for different organizations involved3. Establish communication linkages between lead organizations at different levels4. Identify strategies for flow of information ("situation reports") from authorities to local organizations5. Establish a protocol for triage/rapid assessment of health, settlement needs and cultural preferences6. Establish a system of medical charts that follow individual refugees though the process7. Establish links to primary and specialized health care especially for urgent communicable disease and mental health issues8. Identify a plan for the provision of urgent dental and eye care9. Establish a surveillance/data-tracking system to collect essential health information, tack service use, and provide the ability to conduct quality assurance assessments10. Use information technology – key internet links; background data; briefing; high quality background and cultural information about refugee groups11. Introduce better training of professionals who will be dealing with refugees. Includes increasing cultural competence of health care and settlement providers, women's health, mental health, chronic illness, dental care and current health coverage for refugees. Global health issues should be introduced into health school curricula.12. Local health care professionals need access to better information regarding the background, circumstances and organizational arrangements relating to refugees. Regular, ongoing information sharing sessions with health professionals involved with refugees, public health and government would facilitate communication when the next refugee crisis occurs. There must be political commitment at all levels.For those involved in the Kosovar Humanitarian Evacuation Program, the experience was both overwhelming and rewarding. Many perceived that a superior effort was made for the Kosovars compared to other groups of refugees and that positive media coverage contributed to a warm and effectively organized reception. Interviewees' major concerns were the need for a more comprehensive and coordinated approach to the flow of information and handling of specific health problems.Complex Humanitarian Emergency: CHEUnited Nations High Commission for Refugees: UNHCRHumanitarian Evacuation Program: HEPSettlement and Integration Services Organization: SISOTuberculosis: TBInterim Federal Health: IFHInternational Rapid Response System: IRRSThe author(s) declare that they have no competing interests.NF and LRC designed and implemented the study, analysed the data and critically revised the manuscript. SR, MJ, MH, JK, SR critically revised the manuscript, EM conducted the interviews, assisted with study design and implementation, data analysis, and drafted the manuscript.


There is analytical potential for multiple cause of death data collected from death certificates. This study examines relationships of multiple causes of death as a function of factors available on the death certificate .Data from 326,332 Minnesota death certificates from 1990–1998 are examined. Underlying and non-underlying causes of death are examined (based on record axis codes) as well as demographic and death-related covariates. Associations between covariates and prevalence of multiple causes of death and conditional probability of underlying compared to non-underlying causes of death are examined. The occurrence of ischemic heart disease or diabetes as underlying causes are specifically examined.Both the probability of multiple causes of death and the proportion of underlying cause compared to non-underlying cause of death are associated with demographic characteristics of the deceased and other non-medical conditions related to filing death certificate such as place of death.Multiple cause of death data provide a potentially useful way of looking for inaccuracies in reporting of causes of death. Differences across demographics in the proportion of time a cause is selected as underlying compared to non-underlying exist and can potentially provide useful information about the overall impact of causes of death in different populations. In their 1986 paper Israel, Rosenberg, and Curtin . The resAcknowledgment of the potential for multiple cause of death data analysis is increasing in other countries as well ,4. For eMultiple cause of death data has been used to look at trends in certain diseases, e.g. HIV ,6 and luThe purpose of this article is to examine some straightforward relationships of multiple causes of death as a function of factors available on the death certificate . Using all death certificates issued by the state of Minnesota between 1990 and 1998 , the current study documents the relationship between these factors and the associated frequency of reporting of multiple causes of death as well as the associated frequency that a cause is considered underlying (after data processing) given that it is mentioned on the death certificate. The implication being that differences found are either due to actual differences in causes of death in these groups or due to systematic biases in the reporting of causes of death, or a combination of both. The study will not be able to discern which is the cause but hopes to contribute at the very least by providing an example of the potential relationships which can be examined with the rich multiple cause of death data.The data used are from the Minnesota Department of Health Mortality Database and include entries from 326,332 individual death certificates, which represent all deaths occurring in Minnesota during the period of 1990–1998. Record axis codes (those codes which have been completely data processed) are used for all analyses in this paper rather than entity axis codes.A brief description of the entity and record axis coding is given here. The translation of causes of death listed on the death certificate . When we refer to a cause of death and do not want to distinguish if it is underlying or non-underlying we will refer to it as a "mentioned" cause of death.In addition to listing one underlying and up to 14 non-underlying causes of death, each death certificate also contains information about the demographics of the deceased, including age, gender, race, marital status, and educational attainment. Also, other conditions related to the death are recorded – place and time of death, who completed the death certificate, if autopsy has been performed and type of body disposal. Minnesota Laws and guidelines govern the process for who and how a death is certified under different circumstances in Minnesota. For example when an unattended death occurs (e.g. at a persons residence) a medical examiner's investigator must arrive at the scene. The medical examiner will contact the last attending physician asking about past medical history of the decedent and most likely cause of death. When an attending physician has seen the decedent within 90 days and the death is natural, jurisdiction is usually given to the physician to certify the death. Sudden or unexpected deaths due in part to any factor other than natural disease must be referred to the medical examiner's office. Autopsies are performed at the discretion of the medical examiner but can also be performed for any death at the request of the immediate family.th revision of International Classification of Disease (ICD). The specific ICD9 codes are grouped into standard reporting of cause of death categories resulting in a total of 107 different causes of death. In this study, individuals are dichotomized as having multiple causes of death (i.e. at least one non-underlying cause) or not having multiple causes of death (i.e. only having an underlying). In addition, because heart disease is the leading cause of death and diabetes is a good example of a disease which often shows up as a non-underlying cause of death, this research investigates two sub -populations: 1) Individuals that have ischemic heart disease (ICD-9: 410 – 414) mentioned as a cause of death , and 2) Individuals that have diabetes mellitus (ICD-9: 250) mentioned as a cause of death . For both sub-populations, a dichotomous variable is created to indicate whether the mentioned disease is coded as the underlying cause of death or not.The underlying and non-underlying causes of death derived from the death certificate, in this study, are coded according to the 9Descriptive statistics including total numbers and proportion of all deaths  in each of the covariate categories are reported as well as proportions of people within each covariate category who have multiple causes of death. In order to examine the association between each covariate and the dichotomous outcome of multiple causes of death, logistic regression is used to mutually adjust each factor for the others. 95% confidence intervals of odds ratios are reported. Trends in multiple cause of death reporting across time are investigated graphically.Similarly, descriptive statistics including total numbers and proportions will be presented for the two sub-populations with ischemic heart disease  or diabetes mellitus  mentioned either as underlying or non-underlying cause of death. Logistic regression is used and 95% confidence intervals are reported to examine factors that are associated with each of these diseases being reported as underlying cause of death rather than non-underlying.Overall, 68.9% of the 326,332 deaths from 1990–1998 had at least one non-underlying cause of death in addition to the underlying cause (i.e. have multiple causes). There was a noticeable decreasing trend of reporting multiple causes of death over the 9 year period from 1990 to 1998 with 74.0% in 1990 consistently dropping down to 64.8% in 1996 and remaining around 66% until 1998. Table For places of death, hospital in-patient 73.4%) and nursing home (70.6%) had the highest probability of reporting multiple causes of death, and residence had the lowest percentage (58.7%) Table . Between.4% and nThe results presented so far explored how covariates may be correlated with multiple causes of death being reported. The following results pertain to the conditional probability that a particular cause of death (ischemic hear disease or diabetes) is selected as underlying given that it is mentioned. Results for the subpopulations with ischemic heart disease or diabetes mentioned are shown in Table Table Unlike Ischemic heart disease, only 29.3% of deaths with diabetes mentioned on the certificate had it selected as the underlying cause of death. While only 2.3% of deaths with diabetes mentioned occurred in the youngest age group 0–44 years), , followed by cerebrovascular disease (2.7% of the time underlying), then pneumonia (1.5% of the time underlying). When we focus on the population that has diabetes mentioned on the death certificate, as mentioned above 29.3% of the time diabetes is selected as the underlying, and the second most common underlying cause selected is ischemic hear disease at 25.4%, followed by cerebrovascular disease at 7.75% then followed by Other diseases of the heart 2.9%.Distinct differences in the frequency of multiple causes of death were found across time, age, race, disposal method and place of death. Definitive explanations for the differences cannot be given based on this study, but it is of interest to consider plausible explanations which may motivate further investigation. The increased reporting of non-underlying causes of death as the age of the decedent increases is likely due to actual increases in co-morbidity with age, hence would be explained by actual differences in the causes of death.th most commonly reported cause of death to be the only cause of death listed on the certificate. This reporting of it as the only cause of death pushes it up to be the overall 12th leading cause of death. If almost any other cause would be listed simultaneously on the death certificate, this code would not end up as underlying.The differences found in reporting of multiple causes of death for the other factors may be partly due to systematic reporting biases. According to the NCHS All Mortality Altas , The decreasing trend in reporting multiple causes over the decade may be reflective of a gradual change in the procedures of death certification. It would be of interest to consider this trend across different states and longer periods of time including shifts from one ICD coding system to the next.Previous literature offers various plausible explanations to what contributes to the inaccuracy of reporting causes of death. The cause of death reported on the death certificates depends on a person's disease history that leads to death . If a peGender and race can also play a role in the accuracy of reporting. Lloyd  showed tSimilar to the case of reporting multiple cause of death, the selection of ischemic heart disease and diabetes as underlying compared to non-underlying differs across the several factors considered. The implications of these differences across demographics are that mortality rates would be differentially affected when underlying cause of death is used compared to any mention cause of death. For example, for diabetes we might conclude that diabetes is being under-reported in Whites compared to Blacks, Native Americans and Hispanics if only underlying cause of death were considered since the proportion of diabetes as underlying to mentioned is substantially lower in Whites. This is not to say there is any inaccuracy in the way it is being coded but it points out where multiple cause of death reporting will provide a different perspective than underlying.Nevertheless, studies have shown the sensitivity and positive predictive value of the death certificate are particularly poor with regard to stroke and diabetes . FurtherAs mentioned in the introduction, one limitation of this research is the fact that there is no outside panel of experts who decide independently what the true causes of death are for each decedent, thus whether the associations we found are due to actual differences or reporting bias cannot be discerned. Therefore, this study cannot provide sensitivity or specificity per se, but it aims to identify factors that are associated with variability in reporting multiple cause of death and that perhaps contribute to inaccuracy in reporting underlying cause.There is much to be learned from multiple cause of death data. It provides ways of looking at mortality data that go well beyond the typical examination of underlying cause of death. Future research is needed to understand further what the greatest concerns are about the accuracy of reporting causes of death. Multiple cause of death data have the potential to help point out potential concerns in the accuracy as well as provide a more complete picture of mortality for causes which are frequently not recorded as the underlying cause of death.The author(s) declare that they have no competing interests.MW conceived of the study and wrote most of the manuscript. JH performed the statistical analysis and wrote part of the manuscript. JO provided access to the data and collaboration regarding processes underlying data collection. DD provided details about cause of death reporting and collaboration regarding processes underlying data collection. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: