Accurate assessment of suicidality is of major importance in both clinical and research settings. The Scale for Suicidal Ideation (SSI) is a well-established clinician-rating scale but its suitability to adolescents has not been studied. The aim of this study was to evaluate the reliability and validity, and to test an appropriate cutoff threshold for the SSI in a depressed adolescent outpatient population and controls.218 adolescent psychiatric outpatient clinic patients suffering from depressive disorders and 200 age- and sex-matched school-attending controls were evaluated by the SSI for presence and severity of suicidal ideation. Internal consistency, discriminative-, concurrent-, and construct validity as well as the screening properties of the SSI were evaluated.Cronbach's α for the whole SSI was 0.95. The SSI total score differentiated patients and controls, and increased statistically significantly in classes with increasing severity of suicidality derived from the suicidality items of the K-SADS-PL diagnostic interview. Varimax-rotated principal component analysis of the SSI items yielded three theoretically coherent factors suggesting construct validity. Area under the receiver operating characteristic (ROC) curve was 0.84 for the whole sample and 0.80 for the patient sample. The optimal cutoff threshold for the SSI total score was 3/4 yielding sensitivity of 75% and specificity of 88.9% in this population.SSI appears to be a reliable and a valid measure of suicidal ideation for depressed adolescents. Accurate assessment of suicidality is of major importance in both clinical and research settings. Adolescent suicide occurs usually in the context of an active, often treatable, but unrecognized or untreated mental illness -3. The iSuicide attempts are complex acts for which no single set of clinical features can be expected to be a good predictor . SuicidaThe Scale for Suicidal Ideation (SSI)  was desiThe psychometric properties of the SSI have been evaluated in adult population and in inpatient children. Both in a sample of adult psychiatric inpatients and in a sample of inpatient children the internal consistency of the scale was good ,13. SSI The predictive validity of the SSI has been studied in a sample of hospitalised patients, where the SSI scores of those who committed suicide were not significantly higher than the scores of inpatients that did not . In a saSome instruments have evolved from the SSI, for example the Modified scale for suicidal ideation (MSSI)  that wasThe SSI has been used widely in adult psychiatric populations e.g. ,21], but, but21]]The study population consisted of two samples; a psychiatric outpatient sample of 218, and an age- and sex-matched control sample of 200 school-attending adolescents. The outpatients suffered from depressive mood disorder, were of ages 13 through 19, and took part in the Adolescent Depression Study (ADS). They were recruited between 1.2.1998 and 31.12.2001 from a consecutive sample of patients attending the outpatient clinics of the Department of Adolescent Psychiatry of Peijas Medical Health Care District covering approximately 210,000 inhabitants and comprising the cities of Vantaa and Kerava in the Helsinki metropolitan area, southern Finland.Of the eligible  660 outpatients, 624 (94.5%) were screened during their first consultation visit by the Beck Depression Inventory (BDI)  and the The control sample was drawn from the enrollment lists in four schools in the corresponding geographical area. It was a random sample of age- and sex-matched students equating the distribution of the educational level of the outpatients.1) The Scale for Suicide Ideation (SSI) is a clinician-rating scale and is presented in a semi-structured interview format . It cons2) The Schedule for Affective Disorders and Schizophrenia for School-Aged Children-Present and Lifetime (K-SADS-PL)  is a widThe K-SADS-PL is considered internationally reliable and valid diagnostic instrument for adolescent population . It has 3) Clinical suicidality assessment (CSA): A three-point mutually exclusive grouping of suicidality  is a simplified version of the 5-item "Spectrum of Suicidal Behavior Scale" . It has After a description of the study, a written informed consent was obtained from the subjects. For subjects less than 18 years consent was also asked from the parents or other legal guardians. For the community sample the K-SADS-PL and the SSI were performed at the same day by an expert clinician. For the outpatient sample the K-SADS-PL was performed within variable time from the SSI rating. The CSA was performed for the patient sample by clinicians during the beginning of the treatment.Central tendencies of some data were reported using medians and quartiles because of non-normal distribution. Mann-Whitney U test was used to assess the significance of differences between the two samples.Internal consistency of the SSI was evaluated by calculation of Cronbach's α for the whole scale.Concurrent validity of the instrument was examined by comparing it with the K-SADS-PL with the CSA classifications. SSI total scores were first assessed in 5 classes of increasing suicidality derived from the K-SADS-PL responses in the following way: 1-no suicidal ideation or acts, 2-mild suicidal ideation (score 2 on item-1), 3-severe suicidal ideation (score 3 on item-1), 4-mild suicidal acts (score 2 on any of items 2–4 regardless of ideation), 5-severe suicidal acts (score of 3 on any of items 2–4 regardless of ideation).Then the SSI total scores were measured in 3 classes of increasing suicidal ideation severity, regardless of possible suicidal acts, derived from the K-SADS-PL responses on item 1: 1-no ideation, 2-mild ideation, and 3-severe ideation. Severe ideation (score 3) in this item was considered as "clinically significant suicidal ideation".Finally the SSI total score was assessed in the three classes of the CSA: 1-no suicidality, 2-suicidal ideation, 3-suicidal or self-harming acts.The statistical significance of the between-class differences was evaluated by Kruskal-Wallis test. For the analyses of concurrent validity only SSI-measurements in a range of 30 days from the K-SADS-PL and the CSA were included.Construct validity was measured by performing a principal component analysis (PCA) with varimax rotation in the outpatient sample. The internal consistencies (Cronbach's α) of the extracted components as well as the originally reported factors  were calROC-analysis was performed to evaluate the screening properties of the SSI, and the cutoff threshold for the instrument was defined by optimal trade-off between sensitivity and specificity (Youden's index ). The K-SPSS 11.0  was used for the statistical analysis.Eighteen percent (n = 40) of the outpatient sample were boys and 82% (n = 178) girls, in the community sample the percentages were 18.6% (n = 37) and 81.4% (n = 162), respectively. The subjects' mean age was 16.4 (SD 1.6) in the outpatient sample and 16.5 (SD 1.6) in the community sample. The median SSI total score for the patient sample was 0 (Q1–3 = 0–6) and for the community sample 0 (Q1–3 = 0-0) . The median SSI total score for subjects aged 13–15 was 0 (Q1–3 = 0–1) and those aged 16–19 0 (Q1–3 = 0–1) . The median time distance between SSI and K-SADS-PL was 21.5 days (Q1–3 = 9–36) for the patient sample and 0 days (Q1–3 = 0-0) for the control sample . The median time distance between SSI and the CSA was 6 days (range 0–35).Forty-seven (21.6%) outpatients and one  control subject had current clinically significant suicidal ideation (p = 0.000) according to the K-SADS-PL.Cronbach's α was 0.95 for the whole sample, 0.81 for the community sample and 0.95 for the outpatient sample.146 (67%) of the outpatients and 199 (99.5%) of the controls were included in the analyses for concurrent validity, as their measurements were within the required range of 30 days.2 = 111.6, df 4, p = 0.000).The median SSI sum scores in the five suicidality classes derived from the K-SADS-PL were class-1 = 0 (Q1–3 = 0-0); class-2 = 5.5 (Q1–3 = 0–8); class-3 = 13 (Q1–3 = 0–18.5); class-4 = 4 (Q1–3 = 0–17.3); class-5 = 8 (Q1–3 = 0–13). The differences were significant .The median SSI sum scores in the three classes of suicidal ideation derived from the K-SADS-PL were class-1 = 0 (Q1–3 = 0-0); class-2 = 4 (Q1–3 = 0–8); class-3 = 13 (Q1–3 = 4–18). The differences were significant .The median SSI sum scores in the three clinical suicidality evaluation classes (only the outpatient sample) were class-1 = 0 (Q1–3 = 0–1); class-2 = 10 (Q1–3 = 5–18); and class.3 = 15 (Q1–3 = 13.3–16.6). The differences were significant  and the sample of patients was large compared to earlier similar studies in adult populations, and probably representative of adolescent psychiatric outpatients with depressive disorders. The main finding was that the SSI appeared to be a reliable and valid instrument for evaluation of suicidal ideation in a depressed adolescent population. Its internal consistency and different aspects of validity were good and similar to what has been reported among adults.The construct validity of the SSI was checked by Principal Component Analysis, which yielded 3 theoretically meaningful and coherent factors, only slightly different from the original ones, with good internal consistencies. This suggests good construct validity. The first factor  was nearly identical to Beck's original one . The secThe SSI converged theoretically meaningfully with both the three-class K-SADS-PL suicidal ideation-item and the clinical suicidality assessment (CSA); growing SSI scores were found within categories with increasing severity of suicidality. As to the convergence with the 5-class K-SADS-PL suicidality instrument, the results were more complex. The Kruskal-Wallis test yielded significant differences between the SSI scores in the different categories as expected, but the SSI-scores in the K-SADS-PL classes 4 and 5, with the supposedly most severe suicidality were not higher than in class 3. Classes 4 and 5 inquire about suicidal acts, and may represent a partly separate domain from suicidal ideation, which may be related to the presence of comorbid personality traits or conduct disorders. The SSI was designed to tap suicidal ideation and it may not satisfactorily tap features related with suicidal acts. In accordance with the theory of multidimensional nature of suicidality , severe The authors are not aware of previous empirical estimations of clinically relevant cutoff for the SSI in adolescents. In this study ROC analysis of the whole sample yielded a reasonable result, but the validity coefficients for the different cutoffs of the SSI were somewhat difficult to interpret. In the community sample, there was only one subject with K-SADS-PL-diagnosed clinically significant suicidal ideation, which may have biased the analyses made with the whole sample. The results for both the whole sample and the patient sample suggest that a cutoff threshold score of four or more might be optimal for adolescents. Depending on the purpose the SSI is used, however, the emphasis between sensitivity and specificity may change, and a different threshold may be useful. For example, if the purpose is to detect the maximum number of potential suicides the cutoff threshold should be lowered to minimize the number of false negatives.Several methodological limitations should be noted, some suggesting caution in interpreting the findings. Inter-rater and test-retest reliabilities, which would have given a complete picture of the reliability of the SSI, could not be evaluated in our setting; they would have required repeated SSI measurements for each subject. However, the alpha-coefficients are a marker of internal consistency, which is one indicator of reliability.Although large and representative, the sample was a pure outpatient sample with age- and sex-matched controls, and females were over-represented. The absence of inpatients may have caused us to see the spectrum of suicidal ideation narrower than in real clinical situations. The sample was limited to an urban area in southern Finland, the generalizability of our findings to rural areas, or to other countries, is not known.The use of K-SADS-PL as a standard for clinically significant suicidal ideation and behavior may be criticized, as the authors are not aware of a data on its validity. It is used, however, as one of the best available standards in adolescent mood disorder diagnostics, and taps suicidality with 4 relevant items.The same rater rated the K-SADS-PL and the SSI, which is a weaker test of concurrent validity than correlating measures rated by separate raters.The SSI can safely be used to evaluate suicidal ideation in adolescents where it seems to perform as well as in adults, where it is considered to be well established. When screening clinically significant suicidality in adolescents, a total score threshold of 3/4 may be useful.Suicidal acts may occur among adolescents with only "mild" suicidal ideation. Thus, prevention of suicidal acts cannot rely solely on the SSI, which does not seem to tap them accurately. Furthermore, questionnaires should be only an adjunct to the clinical evaluation of suicidality.SSI appears to be a reliable and a valid measure of suicidal ideation at depressed adolescents, with a cutoff threshold value of four or more of total SSI score being an appropriate for detecting significant suicidal ideation.The author(s) declare that they have no competing interests.MMH analyzed the data and wrote the paper. MP interviewed patients, participated in planning the study and analyses, and writing the paper. LK, TR, HH and VT participated in planning the study, interviewed patients, and commented on the manuscript. OK participated in planning the study and the analyses, and commented on the manuscript. MM supervised the study, interviewed patients and participated in planning of the study and analyses, and writing the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 × 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is “focused” across the center of the bilayer. Voltage-gated potassium (Kv) channels play a key role in electrically excitable cells . Recent In support of the VS domain as an independent unit, homologous domains have been found in two non-Kv proteins: a voltage-sensitive phosphatase (Ci-VSP)  and a voThe VS is composed of four transmembrane (TM) helices, S1–S4. The isolated VS domain of KvAP revealed that the S3 helix was distorted into two distinct helical segments, namely S3a and S3b, where the C-terminal S3b segment and the N-terminal portion of the S4 helix  simulations provide a computational approach to exploring structure/function relationships of ion channels and related membrane proteins  and the 1ORS). This was evaluated by measuring the root-mean-square deviation (rmsd) of the Cα atoms from their starting coordinates. Each simulation shows an initial jump followed by a slower rise to a plateau of ∼1.8 Å for all Cα atoms, and of ∼1.5 Å for core fold Cα atoms  show that the S3b-S4a region of the paddle exists in different orientations with respect to other components of the VS. It is also interesting to note that the motions of the two helical segments (S3a and S3b) which comprise the S3 helix do not appear to be significantly correlated in either the VS/PC or VS/PCPG simulations  of each Cα atom from its time-averaged coordinate. The overall profiles were similar, with the rmsf values being significantly higher for the interhelical regions relative to the α-helical core regions see . There iions see .Overall, one may conclude that the VS domain protein is conformationally stable in a bilayer environment simulation on a 50 ns timescale. Thus, it behaves like a stable canonical membrane protein, even though it is an isolated domain containing a positively charged S4 helix. Perhaps the stability of the isolated domain is not surprising, given that the two-state model of membrane protein folding  implies The nature of the interactions between the VS  and the lipid head groups is of particular interest. Simulation studies of an isolated S4 helix  and of tAs a first step, we have combined visualization of simulations with analysis of the location  of the P atoms of the phospholipid head group A. Examin+ channel KirBac3.1, which when simulated for 20 ns in a PC bilayer does not lead to localized lipid bilayer compression  distance out from the center of the protein see . It is eAlthough the charged S4 helix is significantly (∼50%) exposed to the lipid bilayer environment during the course of each simulation, the VS domains remain conformationally stable. It is of some interest, therefore, how these gating charges are accommodated over the course of each simulation B. It wasFurther analysis also reveals that the lipid head groups in the mixed PC/PG have the propensity to form a greater number of H bonds with the positively charged residues located along S4 than do the lipid head groups of the PC simulation . This fiEach of the charged residues located along the S4 helix is involved in forming H bonds with water molecules over the course of the simulations, indicating that individual water molecules extensively penetrate the VS. However, in contrast to the gating charge arginines, the centrally located Arg133 forms a limited number of H bonds with waters. It can also be observed that the lipid head groups can form H bonds with all of the gating charge residues located toward the N terminus of S4  and the two charged residues located toward the C terminus of S4 (Asp146 and Lys147). Such H-bonding interactions may help stabilize the VS and the highly charged S4 helix within a bilayer environment.Inspection of the average partial density plots for each simulation reveals a reduction in water density toward the center of the S4 helix and the marked absence of water density at a central constriction point . InteresThe partial density plots also reveal an asymmetric distribution of the lipid head groups, with the phosphorus atoms sitting lower in the lipid leaflet accommodating the gating charges. This location bias suggests that the gating charges form more favorable interactions with lipid head groups than do the charged residues located toward the C terminus of S4.Analysis of the radius profile along the VS crevice reveals that there is a central constriction which (when averaged over the course of each simulation) is significantly narrower than the radius of a water molecule see B. The coElectrostatic potential calculations  was assessed by calculating the mean-square fluctuation (msf) of the lipid phosphorus atoms over the last 10 ns of each simulation . In geneVisual inspection of the equilibrated portion of each trajectory revealed that some of the lipid head group H-bonding interactions to the VS were facilitated by interchanging water molecules. It was also clear that some of the more mobile lipids could maintain continual H-bonding interactions with the VS throughout the simulation period. This is best understood by considering the lipid head group that binds to R123 and over time migrates down to R126 B.Our analysis has examined the interactions of the isolated VS domain of KvAP in lipid bilayer environments. These simulations, conducted over a timescale that allows reasonable sampling of lipid/protein interactions, reveal how it is that the isolated VS domain of KvAP can form a stably folded domain in either a PC or a mixed PC/PG lipid bilayer environment. This provides further support for the contention that the VS domain can subsist independently of the pore-forming domain of Kv channels.The VS domain and, moreover, the charged S4 helix, appear to be accommodated in both a PC and a PC/PG bilayer through substantial H-bonding interactions of charged residues with water molecules and lipid head groups. These favorable H-bonding interactions lead to two phenomena occurring, namely the extensive penetration of water molecules into the VS and the significant local perturbation of the lipid species within close proximity of the VS. This latter process is the consequence of favorable H bonding between lipid head groups and charged residues situated in the crevices of the VS. Such interactions effectively draw the coordinated lipid species into the VS and effectively reduce the bilayer width (∼7 Å over 50 ns) within the immediate vicinity of the VS domain.Lateral lipid mobility analysis revealed another interesting phenomena occurring in the lipid bilayer: discrete clusters of slow lipids within the immediate vicinity of the VS and in the bulk lipid bilayer environment. Favorable nonbonding interactions between the VS and the lipids (head groups/tails) may account for the decreased lateral mobility of the lipid species that comprise these slow islands.Thus, these simulations reveal that the lipid bilayer does not simply function as an unstructured fluid into which the protein is embedded; indeed, they reveal a picture of a structured yet dynamical lipid bilayer that can reorganize to accommodate the (charged) protein. A similar situation was seen in simulations of the isolated S4 helix in a bilayer . FurtherThese simulations also reveal that the S3b-S4a region is significantly more mobile in PC than in PC/PG. This suggests that the S3b-S4a region exhibits a degree of flexibility that can be attenuated by its environment. The sensitivity of the S3b-S4a region to its environment may account for the different orientations of the S4 helix observed in the Kv channel structures elucidated thus far .Another important finding from these simulations is that there is a central constriction in the apparent VS cavity which is too narrow to facilitate the passage of water molecules through the VS. The constriction is centered on a salt bridge formed between Asp62 and Arg133 and the hydrophobic band of residues that sit beneath this bond . Electrostatic potential calculations reveal that the lipid bilayer potential is focused across this highly circumscribed hydrophobic region. Given that the isolated VS of KvAP is broadly consistent with a range of biophysical and physiological data for an open state of the channel, it is possible that this salt bridge and the hydrophobic occlusion it maintains may be where the voltage is focused in the open conformation under physiological conditions.1ORS). However, it should also be noted that the conformation of this domain is different in the two structures of the intact KvAP channel (PDB ID codes 1ORQ and 2A0L). This may reflect a degree of distortion (or adoption of a “minor” conformation) of the KvAP channel conformation in the absence of a lipid bilayer environment. The isolated VS domain structure is rather closer in conformation to that of the VS domain in the Kv1.2 structure  structure of the isolated VS domain from KvAP   was taken from the 1.9 Å isolated voltage-sensor crystal structure (PDB ID code 1ORS) . Side-ch WHAT IF  combined WHAT IF . Thus, thttp://moose.bio.ucalgary.ca) for the VS/PC simulations. To set up the 3:1 PC/PG bilayer, lipids of the PC bilayer were “mutated” into PG species. To mutate a PC into PG, the choline group needs to be replaced with a glycerol group. A glycerol group already exists in PC which connects the phosphate group to the fatty chains. For each choline group, the rmsd of all possible choline/glycerol pairs in the bilayer was calculated after least-square-fitted superimposition. The choline group of each lipid was replaced by the glycerol group of the pair with the lowest rmsd. This ensured that the final bilayer was as close to an equilibrated state as possible.An equilibrated PC bilayer comprised of 128 lipid molecules (64 lipid molecules per leaflet) was obtained, courtesy of D.P. Tieleman  protein atoms were harmonically restrained with an isotropic force constant of 1000 kJ molhttp://www.gromacs.org). The GROMOS96 force field was used for both simulations  method, with a 10 Å cutoff for the real space calculation . A cutofbarostat  at 1 barlgorithm  was usedlgorithm .


The first target of antimicrobial peptides (AMPs) is the bacterial membrane. In the case of Gram-negative bacteria this is the outer membrane (OM), the lipid composition of which is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is made up solely from lipopolysaccharides (LPSs). LPS, therefore, represents the first target of AMPs. The binding and intercalation of polycationic AMPs is driven by the number and position of negatively charged groups of the LPS. Also, proteins other than cationic AMPs can interact with LPS, e.g. leading eventually to a neutralization of the endotoxic effects of LPS. We compared different biophysical techniques to gain insight into the properties of the electrical surface potentials of lipid monolayers and aggregates composed of LPSs and various phospholipids and their interaction with peptides and proteins.The net negative charge calculated from the chemical structure of the phospholipid and LPS molecules is linearly correlated with the adsorption of calcium to two-dimensional lipid monolayers composed of the respective lipids. However, the ζ-potentials determined by the electrophoretic mobility of LPS aggregates can only be interpreted by assuming a dependence of the plane of shear on the number of saccharides and charged groups. Various peptides and proteins were able to displace calcium adsorbed to monolayers.To characterize the electrical properties of negatively charged phospholipids and LPSs and their electrostatic interaction with various polycationic peptides/proteins, the adsorption of calcium to and displacement from lipid monolayers is a suitable parameter. Using the calcium displacement method, the binding of peptides to monolayers can be determined even if they do not intercalate. The interpretation of ζ-potential data is difficulty for LPS aggregates, because of the complex three-dimensional structure of the LPS molecules. However, the influence of peptides/proteins on the ζ-potential can be used to characterize the underlying interaction mechanisms. Cell membranes composed of lipids and proteins are an essential part of all living organisms. The interaction between peptides or proteins and lipid membranes is driven by entropic effects and in many cases also by electrostatic forces. The architecture of membranes differs for various cell types and with that also the specificity of the interaction between lipids and peptides/proteins.The cytoplasmic membrane of mammalian cells has an asymmetric lipid distribution , in partThe important influence of electrostatic interactions is even more pronounced for the interaction between bacteria and antimicrobial peptides (AMPs) of the innate host defense system. Membranes of Gram-positive as well as of Gram-negative bacteria express a high amount of negatively charged lipids on the outer leaflet of the membrane which is in direct contact with the extracellular environment.Gram-positive bacteria have a cytoplasmic membrane containing negatively charged diphosphatidylglycerol   and lipoTo investigate the interactions between negatively charged lipids and polycationic peptides/proteins on a molecular level, the knowledge of the number of charges per lipid molecule or the surface charge density of the lipid aggregates is of importance. A number of techniques to determine these parameters is available. Some of the methods can be applied for whole cells or bacteria (for review see ), for liFour different methods to determine values which depend on the number of charges per molecule and the size of the molecules are compared in this paper for phospholipids and, in particular, for LPS:(i) Calculation of the net charge per molecule which is based on the chemical structure as determined in mass spectrometric experiments.(ii) Determination of innermembrane potentials of monolayers ,19 or pl(iii) Determination of the electrophoretic mobility of cells and lipid aggregates. From the electrophoretic mobility, the ζ-potential can be calculated which is correlated with the surface charge density .45Ca2+ as a marker for negatively charged lipids.(iv) Utilization of marker molecules, which bind to specific lipids, e.g. annexin V as a marker for PS in apoptotic cells, or to charged residues, e.g. calcium doped with small amounts of the β-active isotope 45Ca2+. The measurement of the adsorption of calcium to monolayers and also its displacement . Due toTo investigate the influence of lipid structures, in particular the presence of negatively charged groups, on the surface potential, we determined the electrophoretic mobility and calculated the respective ζ-potentials of various aggregates made from LPSs or phospholipids. The respective values are given in Tab. In the case of phospholipids, the absolute values of the ζ-potential increase linearly with increasing negative net surface charge as it could be proposed form the ζ-potential theory. The ζ-potential and the surface charge density of PG and DPG is almost the same, because the net charge as well as the surface area occupied by a DPG molecule is twice of that of a PG molecule. For the various LPSs used, we did not find any correlation between the calculated net charge and the ζ-potential. This might be explained by incorrect assumptions for the calculation of the ζ-potentials from the electrophoretic mobility. The determination of the ζ-potential is based on the Smoluchowski approximation  assumingPhospholipids are described to form multilamellar vesicles. Aggregates formed by LPS are also described to be multilamellar in the presence of di- or polyvalent ions at a temperature of 25°C ,30. It cd1 between the aggregate surfaces and the planes of shear are in the range between 0.20 nm (LPS R7) and 0.42 nm (LPS R60)  = ΨI exp (- κ x) + ΨO exp (- κ (x - 1.1 nm)) for x ≥ 1.1 nm(Eq.1)Ψx is the distance from the inner charged plane and κ is the reciprocal Debye- length )/κ.(Eq. 2) S. minnesota LPS used in this study are given in Tab. M1 [The respective values for the  Tab. M1 ,32 and P Tab. M1  showed tIt should, however, be pointed out that our approach to describe the surface potential by the superposition of two distinct planes is only a simplified model. For example, the charges of the two phosphate groups of the lipid A and the two Kdo were assumed to be in one plane, however, the diglucosamine backbone of the lipid A moiety of the LPS is tilted with respect to the aggregate surface . Thus, tSeveral advanced theories, taking a rough surface of the aggregate into account, have been published (for review see ). Howeve45Ca2+) was titrated in amounts of up to 25 μM into the subphase underneath the monolayers composed of the identical number of the respective lipid molecules. The β-intensity was measured and plotted against the calcium concentration. To further analyze the data, the plots were fitted by the equation:To investigate the adsorption of calcium to lipid monolayers made of various phospholipids and LPSs, calcium (doped with radioactive (Eq. 3) I(c) is the β-intensity I as a function of the calcium concentration c, Imax the maximum β-intensity at complete saturation of the lipid monolayer with calcium and c0 is the calcium concentration at which 1-1/e (~63%) of the monolayer is saturated with calcium. In Fig. where Imax is plotted versus the net charge q of all lipids used for monolayer preparation. The respective values are given in Tab. Imax increases with increasing negative values of q and that Imax and q are linear correlated with R = -0.98. The dependence of Imax on q is given by Imax = (-3.1 ± 21.0) cps + (-89.3 ± 6.5) cps/e0 q.In Fig. q and Imax that calcium ions intercalate into the oligosaccharide portion of the LPS, also binding directly to the charged groups of the lipid A part of the molecule. This is supported by calcium displacement experiments using fragments of rabbit and human cathelicidin peptide [These results indicate that the amount of calcium adsorbed to a lipid monolayer depends solely on the number of negative charges and is not influenced by the 3D structure of the monolayer-forming lipid. Furthermore, it can be assumed from the linear correlation between  peptide , in whic-1, and the β-intensity was monitored.To check the influence of the lateral pressure on calcium adsorption, we used a Langmuir-Pockels film balance equipped with a movable barrier. LPS F515 monolayers were prepared and compressed to a final lateral pressure of 40 mN m-1, the lateral pressure increases almost linearly with decreasing area, above 20 mN m-1 the isotherm shows a typical non-linear dependence between area and lateral pressure. In contrast to the latter, the β-intensity increases linearly with decreasing film area  were calculated. The most potent substances were the proteins lactoferrin and the bactericidal/permeability-increasing protein (rBPI21) and a fragment of the 18 kDa rabbit cationic antimicrobial protein (rCAP18106–137) which were active already at submicromolar concentrations. The IC50 values of the recombinant human serum albumin (rHSA) and hemoglobin were by two orders of magnitude higher. Interestingly, rCAP18, PMB, BPI, and lactoferrin are well-known LPS-neutralizing peptides [To compare the various peptides/proteins in their ability to displace calcium from an LPS monolayer, the ICpeptides , in casepeptides . Thus, t2+ or Ca2+. Thus, the binding of LPS to proteins being involved in the LPS-induced activation of mononuclear cells is inhibited.Divalent cations lead to a cross linking between the LPS individual molecules  and lead-1 [In many cases, the intercalation of peptides/proteins decreases with increasing lateral pressure. Therefore, to guarantee a maximal interaction between the monolayers and the peptides/proteins, experiments were done at a relatively low lateral pressure as compared to that in a natural cell membrane which is discussed to be in the range from 20 to 30 mN m-1 ,42.-1, no significant change of the film area was observed  underneath an LPSF515 monolayer at a constant lateral pressure of 27 mN mved Fig. , indicatved Fig. , demonst45Ca2+ which is linearly proportional to the calculated net negative charge of the respective phospholipids or LPSs. Thus, calcium ions seem to bind to all negatively charged groups of LPS, even to those which are located close to the LPS backbone. Larger probe molecules might not be able to reach these groups. The peptide/protein-induced displacement of calcium ions is a usable parameter for the determination of the adsorption of molecules to lipid monolayers. The film balance experiments provide information on i) the adsorption of peptides at the monolayer surface caused by electrostatic interactions determined by calcium displacement and ii) the intercalation into the monolayer caused by electrostatic and hydrophobic interactions. In contrast to this, in calorimetric and surface plasmon resonance experiments it is difficult or in many cases not possible to distinguish between binding and intercalation.The characterization of the electrical potential of lipid membranes and of electrostatically driven interactions between lipids and peptides/proteins are important aims for an understanding of the molecular interaction mechanisms. The adsorption of calcium ions to lipid monolayers can be determined by measuring the β-intensity of The determination of the ζ-potential provides valuable information for the characterization of the electrostatic interaction between peptides/proteins and phospholipid or LPS aggregates. However, due to the complexity of the LPS molecules, the theories, which are based on the assumption of flat surfaces, fail to describe the potential of LPS aggregates. The calculation of the respective planes of shear demonstrates that the ζ-potentials depend on the number of saccharides and charged groups. As we showed earlier , the innIt has been shown in many publications that the intercalation of peptides/proteins into lipid monolayers and bilayers can be different. However, according to Schoch et al. ,44 the eIn summary, measurements of calcium adsorption and displacement provide additional useful information for the characterization of the electrostatic interaction between negatively charged monolayers and peptides/proteins.Salmonella enterica serovar Minnesota (S. minnesota) strains R595 (LPS R595), R4 (LPS R4), R7 (LPS R7), Rz (LPS Rz), R5 (LPS R5), R345 (LPS R345), and R60 (LPS R60) as well as deep rough mutant LPS from Escherichia coli strain F515 (LPS F515) and Proteus mirabilis strain R45 (LPS R45) were used [Rough mutant LPS from ere used . LPS wasPhosphatidylcholine (PC) from egg yolk lecithin, phosphatidylglycerol (PG) from egg yolk lecithin , and synthetic diphosphatidylglycerol (DPG) were purchased from Avanti Polar Lipids  and used without further purification.1–193 (rBPI21) was a kind gift of XOMA . The rabbit CAP18106–137 was a kind gift provided by J.W. Larrick .Polymyxin B (PMB) and its nonapeptide (PMBN), lactoferrin from human milk, lactalbumin from bovine milk, lysozyme from chicken egg white, and human hemoglobin were purchased from Sigma Aldrich , and recombinant human serum albumine (rHSA) from Welfide Corporation . Recombinant BPIFixed charges within the headgroups of lipid molecules cause an electric potential at the lipid bilayer surface with respect to the surrounding bathing solution, the surface potential ,52. The r ε0)(Eq. 4) ζ = μ η/(ε-3 kg m-1s-1), ε0 the dielectric constant of vacuum (8.854·10-12 A2s4kg-1m-3) and εr the dielectric permittivity of water (78.54).where ζ is the ζ-Potential, μ the electrophoretic mobility, η the viscosity  at 25°C and with a driving electric field of 19.2 V cm2, pH7). Briefly, the lipid dispersions were sonicated for 20 min at 60°C, cooled down to 4°C for 30 min and temperature-cycled twice between 60°C and 4°C (30 min each). The dispersions were equilibrated overnight at 4°C. Prior to ζ-potential measurements, lipid dispersions were diluted to a final concentration of 0.01 mM. Presented values  at a constant and (ii) at a variable film area. In both types of experiments the phospholipids were dissolved in chloroform and the LPSs in a 10:1 (v:v) mixture of chloroform and methanol at a concentration of 1 mM. All lipids were deposited in the given amounts on the subphase and the solvents were allowed to evaporate for 5 min.45Ca2+  were added to the subphase, resulting in a final calcium concentration of 25 μM. Calcium ions bind to the negatively charged headgroups of the lipids, and the low-energy β-radiation originating from the bound 45Ca2+ is not absorbed by the hydration shell anymore and, therefore, an increase in β-intensity was observed using a β-counter   Itot is the β-intensity originating from the monolayer and the subphase and Isub is the β-intensity of the pure subphase. These and the following experiments were performed at a subphase temperature of 20°C instead of 37°C to avoid condensation at the β-counter.where 2+ ions from LPS F515 monolayers, a subphase containing 12.5 μM Ca2+ doped with radioactive 45Ca2+ (resulting in a relative β-activity of 250 Bq/ml), buffered with 5 mM HEPES at pH7 was used. Also in the displacement experiments, the number of LPS molecules was kept constant. The agents were added to the subphase at a constant total film area and relatively low lateral pressure of the monolayer (~10 mN m-1). This way, the final pressure after saturation of the intercalation of agents was still in the range of lateral pressures in biological membranes [Irel(c) in dependence on the peptide/protein concentration c was calculated fromTo investigate the capacity of various peptides/proteins to displace divalent Caembranes ,42. The (Eq. 6) Itot(c) is the absolute β-intensity, Isub the β-intensity originating from the pure subphase, and Imono the β-intensity originating from the monolayer. From the displacement curves the concentrations at which 50% of the calcium were displaced  by the peptides/proteins were determined and summarized in Tab. where All presented values are mean values with standard derivations resulting from 3 to 4 independent experiments.2. The solvent was allowed to evaporate at zero pressure for 5 min. Then the monolayers were isothermally compressed at a speed of 1.5 mm2 s-1 to a lateral pressure of 40 mN m-1.A Langmuir-Pockels film balance equipped with a Wilhelmy system  was used to determine the lipid pressure/area isotherms of LPS F515 monolayers. The lateral pressure, area, and the β-intensity were determined. For the experiments, 36 μl of the LPS F515 were spread on the buffer surfaces of 290 cmAMP – antimicrobial peptidesAra4N – 4-amino-4-deoxyarabinoseBPI – bactericidal/permeability increasing proteinDPG – diphosphatidylglycerolENP – endotoxin-neutralizing proteinKdo – 3-deoxy-alpha-D-manno-oct-2-ulosonic acidLPS – lipopolysaccharideLTA – lipoteichoic acidOM – outer membranePC – phosphatidylcholinePE – phosphatidylethanolaminePEG – poly(ethylene glycol)PG – phosphatidylglycerolPMB – polymyxin BPMBN – nonapeptide of PMBPS – phosphatidylserinerBPI21 – recombinant 21 kDa fragment of BPIrCAP18 – 18 kDa rabbit cationic antimicrobial proteinrHSA – recombinant human serum albuminSOH designed and carried out the adsorption binding assay and drafted the manuscript. MUH carried out the ζ-potential experiments and helped to draft the manuscript. AW participated in the film balance experiments. US participated in the design of the study and helped to draft the manuscript. TG participated in the design and coordination of the study, performed some of the monolayer experiments and helped to draft the manuscript. All authors read and approved the final manuscript.


Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 μg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 μg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 μg/ml or inhibition at 10 μg/ml. In the absence of vitamin D3, type I collagen alpha 1 chain expression was inhibited by 10 μg/ml of gal-3. The vitamin D3induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.In this study we examine the extracellular role of galectin-3  in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 μg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D Osteoarthritis (OA) accounts for 40% to 60% of degenerative illnesses of the musculoskeletal system. On the whole, approximately 15% of the population suffers from OA. Of these, approximately 65% are 60 years of age and over. The high incidence of this illness is rather disturbing since its frequency increases gradually with the aging of the population.It is well known that age is a primary risk factor for the development of OA, but the mechanisms by which aging contributes to an increased susceptibility to OA are poorly understood . The endMechanisms by which synovitis exacerbates structural damage in OA are complex. Synovitis induces alterations in chondrocyte function and in subchondral bone turnover and enhances angiogenesis ,14. CytoThe exact role of gal-3 in articular tissues is not yet known. It is a soluble animal lectin of 30 kDa that preferentially recognizes lactosamine and N-acetyllactosamine structures ,19. Intrin vivo effect in mice having received an intra-articular injection of gal-3, and further investigated its effect on cells from two OA articular tissues: cartilage and subchondral bone.In the present study, we further investigate the role of extracellular gal-3 in joint tissues. To this end, we first examined its ad libitum. Recombinant human gal-3  was prepared in our laboratory and sterilized on a 0.2 μm filter. As the amino acid sequence of rh-gal-3 shows 85% identical homology and 91% positive homology with murine gal-3, we injected rh-gal-3 into the knees of wild-type mice. Mice were distributed into 4 groups receiving 100 ng, 1 μg or 10 μg of gal-3 or vehicle (PBS) alone according to previous established protocols 2 D3  in combination or not with gal-3. To evaluate signalling pathways involved in vitamin D3-stimulated osteocalcin production that are inhibited by gal-3, cells were pre-incubated for 2 h with specific inhibitors and then incubated for 22 h in the presence of the inhibitors and vitamin D3 in combination or not with gal-3. The inhibitors used were KT5720 , KT5823 , Genistein , Taxifolin , wortmannin (inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase); final concentration 250 nM), PD98059 (inhibitor of mitogen-activated protein kinase kinase-1 (MEK-1) activation; final concentration 10 μM), and SB202190 . All inhibitors were purchased from Calbiochem .The overlying cartilage was removed from the tibial plateaus, and the trabecular bone tissue was dissected from the subchondral bone plate. Primary subchondral osteoblasts were released as previously described . BrieflyRNA extraction and real time RT-PCR were performed as previously described . PrimersThe assay measured only intact human osteocalcin and was performed on human osteoblast-conditioned media using a specific enzyme immunoassay (EIA) kit with a sensitivity of 0.5 ng/ml .Cells were lysed in 0.5% sodium dodecylsulfate and proteins quantified with the bicinchoninic acid assay .t-tests for animal experiments and cell culture, respectively. Results of p < 0.05 were considered significant.Data are expressed as mean ± SEM or median (range). Statistical analyses were the Mann-Whitney U and the two-tailed Student's p ≤ 0.0002 versus D0) , a significant difference was still seen when D2 was compared to D0 (p < 0.004). Values gradually returned to the basal conditions. Gal-3 exacerbated and extended the swelling; thus, at D2, gal-3 injections of 0.1, 1, and 10 μg significantly induced higher swelling than the vehicle alone . This effect was sustained the third day post-injection . Finally, at D4, values tended to return to those of the control group, although gal-3-induced joint swelling was still statistically significant (p < 0.006) with the highest gal-3 concentration used.As Ohshima and colleagues  showed t) Figure . Althougp < 0.04 versus control), 10.5 (8.5 to 12.5) (p < 0.02 versus control) and 13 (p < 0.04 versus control) in the gal-3-injected group with 0.1, 1, and 10 μg gal-3, respectively  and 8 (p < 0.04 versus control) in the gal-3-injected group with 0.1, 1, and 10 μg gal-3, respectively , 4.0 (3.0 to 5.0) (p < 0.04 versus control) and 5 (p < 0.04 versus control) in the gal-3-injected group with 0.1, 1, and 10 μg gal-3, respectively ) in the control group was 5.0 (3.5 to 6.0) whereas it reached 9.5 (7.0 to 12.5) , but not at 10 μg/ml . Alkaline phosphatase, which is upregulated by vitamin D3, tended to be increased with gal-3 at 1 μg/ml (p < 0.07). A significant difference in alkaline phosphatase expression was found between osteoblasts treated with vitamin D3 in the presence of 1 μg/ml gal-3 and vitamin D3 in the presence of 10 μg/ml gal-3 (p < 0.03).The effects of gal-3 on human osteoblasts were evaluated in the presence or absence of vitamin Dl Figure . In cont3, osteocalcin expression was maintained at a minimal level, and gal-3 had no effect on osteocalcin expression  and 6.5 (p < 0.0001) times the basal level, respectively. These results were confirmed at the protein level by analyzing osteocalcin concentration in conditioned media using an EIA. Osteocalcin production was inhibited by around 40% and 85% at gal-3 concentrations of 1 and 10 μg/ml, respectively  for osteoblasts treated with vitamin D3 alone, 67.6 ± 7.9 for those treated with 1 μg/ml rh-gal-3 in the presence of vitamin D3 and 2.4 ± 0.9 for cells treated with 10 μg/ml rh-gal-3 in the presence of vitamin D3. In addition, we made a truncated isoform of gal-3 (Gly108 to Ile249) corresponding to the carbohydrate recognition domain (CRD). This truncated isoform is known to be incapable of multimerizing and it is unable to reproduce the effects of whole gal-3. Results obtained with an EIA were 130.2 ± 16.5  for osteoblasts treated with vitamin D3 alone, 158.5 ± 22.6 for those treated with 1 μg/ml CRD in the presence of vitamin D3 and 163.4 ± 26.1 for those treated with 5 μg/ml CRD in the presence of vitamin D3. As expected, CRD was not able to downregulate the osteocalcin production.As previously described, in the absence of vitamin Dn Figure . In conty Figure , insert.3-stimulated osteocalcin production with 5 μg/ml gal-3, which resulted in an inhibitory effect closer to 50%  and SB202190 (40%), indicating that tyrosine kinases and p38 mitogen-activated protein kinase may be slightly involved , indicating that gal-3 did not induce these pathways. The combination of gal-3 with either KT5720 or KT5823 also significantly inhibited osteocalcin production compared to their respective controls , indicating that neither protein kinase A nor protein kinase G are involved in gal-3-inhibited osteocalcin production. This result was confirmed by the fact that gal-3 alone and gal-3 in the presence of KT5823 did not produce results with a significant difference. In contrast, PD98059 prevented further inhibition of osteocalcin production by gal-3. This result indicates that Erk1/Erk2 kinases are also involved to some extent in gal-3 signalling transduction. Taxifollin, an antioxidant flavonoid, also seemed to prevent gal-3 inhibition of osteocalcin production, but this inhibitor had the weakest effect. The most spectacular result was obtained with an inhibitor of PI 3-kinase, wortmannin, which totally prevented the inhibition of osteocalcin by gal-3.As 10 μg/ml gal-3 almost entirely inhibited osteocalcin production, we further examined the signalling cascades of gal-3 inhibition of vitamin D% Figure . Vitamind Figure . However3, 10 μg/ml of gal-3 inhibited 50% of type I collagen α1 chain expression (p < 0.02) but this inhibitory effect was partly reversed by vitamin D3  was unaffected (data not shown). In addition, the level of tissue inhibitor of MMP-1 (TIMP-1), a natural protein inhibitor produced by chondrocytes, also remained stable (data not shown). We show that ADAMTS-5 was more sensitive than MMP-3 to gal-3, since its expression was stimulated with very low concentrations of gal-3, unlike MMP-3, which required higher concentrations for stimulation. The regulation of ADAMTS-5 is crucial since it was recently demonstrated by two independent groups (using knock-out mouse models) that ADAMTS-5 is the major aggrecanase responsible for proteoglycan degradation in cartilage destruction [It is interesting to note that two major enzymes responsible for proteoglycan degradation were stimulated by gal-3. This finding corroborates the truction ,42. On t3-stimulated osteocalcin level [3-stimulated osteocalcin levels, the PI 3-kinase appears to be a key enzyme. This could be related to the implication of integrins, since it has recently been shown that several biological functions of osteoblasts are regulated via the integrin/PI 3-kinase pathway [Gal-3 not only modulated chondrocyte-expressed genes but also those of osteoblasts. More particularly, production of osteocalcin, which is an osteoblastic marker , was strin level . One hyp pathway ,46.3 prevented the inhibition of type I collagen expression. This latter finding raised the potential role of gal-3 in preventing osteoid matrix formation during the inflammatory process, particularly in individuals with low or depleted levels of vitamin D3 since it has been shown that vitamin D3 analogues have immunomodulatory effects [Unlike osteocalcin, type I collagen α1 chain expression was downregulated only with a high gal-3 concentration. However, vitamin D effects .The presence of extracellular gal-3 in the vicinity of chondrocytes and osteoblasts causes deleterious effects by both downregulating the anabolic processes and upregulating the catabolic processes. In fact, this factor may participate in cartilage destruction and subchondral bone erosion, particularly during the highly inflammatory phases of OA.ADAMTS-5 = a disintegrin and metalloproteinase with thrombospondin type 1 motif; CRD = carbohydrate recognition domain; D = day; DMEM = Dulbecco's modified Eagle's medium; EIA = enzyme immunoassay; FBS = fetal bovine serum; Gal-3 = galectin-3; MMP = matrix metalloproteinase; OA = osteoarthritis; PBS = phosphate buffered saline; PI 3-kinase = phosphatidylinositol 3-kinase; rh-gal-3 = recombinant human gal-3.The authors declare that they have no competing interests.in vitro study, analyzed the data and drafted the manuscript. CB participated to the animal study design, analyzed the data and drafted the manuscript. MG participated in the in vitro study. FP, JPP, ND, JMP, PR contributed to the study design. FP, JPP, JMP, PR contributed to the revision of the final manuscript.AJM contributed to the


According to the innate immunity concept , animalsWhat might bacteria derive from producing this type of lipid A, and what do animals gain from recognizing it? A survey of diverse lipid A structures found that the best-recognized configuration is produced by most of the aerobic or facultatively anaerobic Gram-negative bacteria that can live in the gastrointestinal and upper respiratory tracts. We hypothesize that the CD14–MD-2–TLR4 mechanism evolved to recognize not just pathogens, but also many of the commensals  and colonizers that can inhabit the body's most vulnerable surfaces. Producing this lipid A structure seems to favor bacterial persistence on host mucosae, whereas recognizing it allows the host to kill invading bacteria within subepithelial tissues and prevent dissemination. A conserved host lipase can then limit the inflammatory response by removing a key feature of the lipid A signal, the secondary acyl chains.Gram-negative bacteria that inhabit water, soil, plants, or insects display impressive diversity in their lipid A structures see . AlthougBordetella and Haemophilus species to persist in the respiratory tract [Neisseria gonorrhoeae to survive within epithelial cells [Pseudomonas aeruginosa lives in water, produces a predominantly pentaacylated LPS, and does not colonize the mucosae of normal humans. When P. aeruginosa colonize the airways of children with cystic fibrosis, however, the bacteria often adapt by producing hexaacylated (and even heptaacylated) lipid A [Shigella and Salmonella, pathogenic Escherichia coli [Aeromonas species, Plesiomonas shigelloides, and Vibrio cholerae O1. In Salmonella and some others, a PhoP/PhoQ-regulated transcriptional program promotes lipid A palmitoylation (heptaacylation) along with other changes that increase resistance to CAMPs [Campylobacter jejuni) or more of them: heptaacyl (Moraxella) or octaacyl (V. cholerae O139). Those that produce less hydrophobic lipid A seem to be special cases: the pentaacyl LPS of Chlamydia species is found in spore-like elementary bodies, and Helicobacter pylori, with tetraacyl LPS, is adapted to live in the stomach.Mucosal secretions contain numerous cationic antimicrobial peptides (CAMPs) . As notery tract –7 and Neal cells . Pseudom lipid A . Mucosalhia coli , Aeromonto CAMPs ,11–13. OLegionella , Burkholderia pseudomallei (soil and water), Yersinia pestis , Coxiella burnetti , Leptospira , and Francisella tularensis . These pathogens usually enter vertebrate tissues via insect bites or cuts, within inhaled droplets, or across the conjunctivae. Brucellae (livestock), which inhabit macrophages yet are typically acquired via ingestion, also produce a nonmucosal LPS [Many other disease-associated Gram-negative bacteria have nonmucosal habitats. Their lipid A moieties differ from the typical mucosal structure by having shorter or longer acyl chains, unsaturated acyl chains, only four or five chains, or only one phosphate see : Legioneosal LPS .P. aeruginosa LPS. Further discrimination is performed by MD-2 [Whereas bacterial peptide resistance and outer membrane impermeability seem to vary directly with the number of acyl chains, the inflammation-inducing CD14–MD-2–TLR4 sensory mechanism best recognizes lipid A that has the hexaacyl mucosal lipid A structure –20 Figu. In supp by MD-2 . The sam by MD-2 .E. coli mutant that was unable to attach the secondary myristate at position 3′ and could not stimulate human endothelial cells. Having a hexaacyl LPS also enhances other responses to intact bacteria, including the induction of tumor necrosis factor by Salmonella in vivo [S. flexneri [E. coli [Evidence that lipid A structure influences the recognition of intact bacteria by host cells came from mutating enzymes that attach secondary acyl chains to the backbone. Somerville et al.  found an in vivo , the iniflexneri , and the[E. coli . These a[E. coli .Shigella invasion through the colonic epithelium prompts intense local inflammation, for example; the fact that Shigella possess hexaacyl LPS may help the host confine infection to the intestine (bacteremia rarely occurs) [Haemophilus and Bordetella species to the respiratory tract, most strains of N. gonorrhoeae to the urethral mucosa, and E. coli to the bladder [Salmonellae [Although the TLR4-based mechanism for sensing LPS has been highly conserved , how it  occurs) ,34. TLR4 occurs) . Similar bladder . In contmonellae  avoid reY. pestis [B. pseudomallei [F. tularensis [L. pneumophila [C. burnetti [H. pylori [Brucellae [Leptospira [Most mucosal Gram-negative bacteria that enter the bloodstream are rapidly killed. In many instances, LPS sensing is required for effective elimination ,36. Few . pestis  and B. pdomallei  produce domallei –42. As sdomallei ,40,42, tlarensis ,44, L. pumophila , C. burnburnetti , H. pylo. pylori , Brucellrucellae ,49, and ptospira  species.Neisseriae. Both N. meningitidis and N. gonorrhoeae produce a mucosal lipid A and colonize mucosal surfaces. How they invade the bloodstream is not known [The most obvious exceptions are the pathogenic ot known , but theDictyostelium discoideum produces several lipid A–deacylating enzymes, only one has been found in mammals. Acyloxyacyl hydrolase (AOAH) removes only the secondary chains from lipid A; it cleaves saturated, short secondary chains, as are found in the mucosal lipid A structure, more rapidly than it removes long unsaturated ones [Whereas ted ones ,53. A phted ones , and theted ones  and the ted ones In vertebrates, AOAH is produced by neutrophils, dendritic cells, renal cortical epithelial cells, and monocyte-macrophages. AOAH treatment greatly reduces LPS sensing via TLR4 , and LPSThe host response to LPS also has noninflammatory, immunostimulatory elements. Lipid A analogs that lack the optimal configuration for inducing inflammation may be excellent adjuvants, enhancing acquired immune responses in ways that mimic those induced by LPS itself ,58. The Porphyromonas gingivalis, the pentaacylated monophosphoryl LPS of Bacteroides fragilis seems to be sensed principally by TLR2 [LPS recognition by CD14–MD-2–TLR4 has received intensive study because it initiates the inflammatory response to so many disease-associated Gram-negative bacteria. Less is known about how animals sense their far more abundant flora of strictly anaerobic Gram-negative bacteria, although doing so may be important for establishing beneficial mutualism between bacteria and host ,60. Like by TLR2 –63 and c by TLR2 ,65.An immune system that only recognizes pathogens would leave animals vulnerable to the commensal and colonizing microbes that enter subepithelial tissues at sites of microtrauma throughout life . An innaIf the synthesis proposed here is correct, it would not be surprising to learn that other elements of innate immunity also sense commensal microbes. Animals may also have conserved enzymatic mechanisms for extinguishing microbial signals that are sensed via other receptors ,68.Table S1(208 KB DOC)Click here for additional data file.Table S2(25 KB DOC)Click here for additional data file.http://www.ebi.ac.uk/swissprot) primary accession numbers discussed in this paper are AOAH (P28039), CD14 (P08571), MD-2 (Q9Y6Y9), and TLR4 (000206).The SwissProt (


Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae.We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans.There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation. Cyanobacterial LPS is attributed with a range of pathological effects in humans, from gastro-intestinal illness, cutaneous signs and symptoms, allergy, respiratory disease, headache and fever. This review will present the studies of cyanobacterial LPS, and will attempt to place the knowledge of these products within the broader understanding of LPS from Gram-negative heterotrophic bacteria. The paper will present an overview of the mechanisms of toxicity of Gram-negative bacterial LPS, discussing the history of its discovery and the present perception of its pathogenicity.Table Several authors note that the health implications of cyanobacterial LPS are poorly understood and the topic requires more research -15. OnlyThe relationship between cyanobacterial LPS and illness is discussed in language ranging from cautious: "...may be responsible..." , "possibThe references in the literature to the association between cyanobacterial LPS and this rather diverse range of symptoms are not based on any research evidence specific to cyanobacterial LPS. As will be discussed later in this review, the few toxicological investigations that have been carried out to date are mostly limited to the end-points of lethality and the local Shwartzman reaction, in which sequential subcutaneous and intravenous injections of LPS produce a dermonecrotic lesion in rabbit skin. Rather, symptomatology attributed to contact with cyanobacterial LPS appears to be something of a default diagnosis for illnesses that are not otherwise explained by the current knowledge of cyanobacterial exotoxins, most of which are somewhat specific to their target organ system.The most likely explanation for the ready attribution of these illnesses to cyanobacterial LPS lies in the realisation that LPS from Gram-negative heterotrophic bacteria are implicated in significant morbidity – and mortality – so cyanobacteria, which are widely but somewhat inaccurately accepted as Gram-negative bacteria may equally be responsible for illness because they contain LPS. Codd makes such a proposition, suggesting that: "The LPS of other bacteria are associated with gastroenteritis and inflammation problems and it is thought that cyanobacterial LPS may contribute to waterborne health incidents although this possibility has not been adequately investigated" .There is a risk that speculative attribution of symptoms in humans to environmental exposure to cyanobacterial LPS will continue without an appropriate and specific research foundation. This review will attempt to examine more closely the mechanisms by which LPS from Gram-negative heterotrophic bacteria is associated with morbidity, and compare and contrast cyanobacterial LPS and heterotrophic bacterial LPS.Anabaena variabilis they investigated was Gram-positive, "like other blue-green algae...". Drews [Most references to cyanobacteria describe these organisms as Gram-negative prokaryotes ,22-26. W". Drews  also cla". Drews  reviewed". Drews ,32, and ". Drews  and Gole". Drews  suggest ". Drews , electroAphanizomenon flos-aquae as an "alkaloid endotoxin". Gentile & Maloney [While most references to endotoxin in the cyanobacteria literature , a core oligosaccharide featuring an outer and inner region, and an acylated glycolipid (termed lipid A) – is seen in such ecologically diverse bacteria as hizobium . The O-shizobium , while lhizobium   in two ester-linked fatty acids [R. sphaeroides and R. capsulatus is of pharmacological interest because of their LPS-antagonist properties; these lipid A structures and some synthetic derivatives have been shown to be potent LPS antagonists in vitro and to protect against LPS-induced morbidity and mortality in animal models [Rhodopseudomonas viridis and Rhodopseudomonas palustris are also reported to lack endotoxic activity [Rubrivivax gelatinosus, is reportedly associated with high lethality in mice – at similar doses that cause lethality in Salmonella – and high pyrogenicity in rabbits [R. gelatinosus is apparently due to the presence of six fatty acid groups in the lipid A structure [Chlamydia trachomatis and Legionella pneumophila and the non-pathogenic hyperthermophile Aquifex pyrophilus reportedly possess little or no LPS agonist or antagonist properties [Although the basic structure of lipid A is seen in phylogenetically diverse Gram-negative bacteria, variations in the nature and location of acyl groups or alterations in the hydrophilic backbone can result in partial or total loss of biological activity ,63,82,83tivation ,52,63,84l models ,63,78,85activity ,84, but  rabbits ,84. The tructure . Lipid ABacteroides spp., which are common gut and periodontal commensals, have low endotoxic activity compared to that of Salmonella, with a greater than 500-fold higher mouse LD50 and 100 to 1,000-fold reduced ability to stimulate production of IL-1 in monocyte cultures [Bacteroides fragilis LPS inhibits E. coli LPS-induced endothelial adhesiveness for polymorphonuclear leucocytes, although B. fragilis LPS is reported to be directly toxic to endothelial cell cultures at high concentrations [B. fragilis stimulated IL-1β, IL-6 and IL-1ra at levels comparable to Gram-positive bacteria, where 100 to 1,000-fold more organisms are required to produce similar concentrations of cytokines than with common pathogenic Gram-negative bacteria. Wilson et al [B. fragilis may be such a weak cytokine inducer because, being a dominant organism in normal gut flora, it has evolved mechanisms to downregulate the synthesis of inflammatory cytokines in order to optimise their niche in the host gut. B. fragilis has five fatty acyl groups in the lipid A moiety, and other structural differences to Salmonella and E. coli LPS, such as a monophosphorylated disaccharide backbone and longer fatty acyl chains [cultures ,88. Bacttrations . In a sttrations  showed ton et al  suggest l chains .Neisseria, Haemophilus and Bordetella have developed unique surface glycolipids lacking O-antigens, which some workers call lipooligosaccharides [Some pathogenic bacteria have LPS which reflects the highly specific niches they inhabit: enteric Gram-negative bacteria have long, hydrophilic and neutral O-specific polysaccharide chains which protects the organism from solubilisation by bile acids and intestinal enzymes, whereas organisms that colonise the mucous membranes of the respiratory and genital tracts have outer membrane surfaces that are hydrophobic and can be solubilised by bile ,93. Gramcharides ,92,93.E. coli, Salmonella typhimurium, Haemophilus influenzae and Neisseria meningitidis were shown to have reduced activities in a series of tests relating to endotoxic potential, in some cases by greater than two orders of magnitude, and deacylated Neisseria LPS demonstrated some antagonistic activity towards Neisseria and Salmonella LPS [The presence of one or more secondary acyl chains appears to be essential for lipid A to stimulate some endotoxic reactions ,88. One ella LPS ,95,96.E. coli lipid A, is unable to induce cytokines in human cells [R. capsulatus) conforms to lamellar structures, whereas endotoxic lipid A adopts exclusively cubic or hexagonal structures.The lipid A analog, compound 406, which lacks the two secondary fatty acids of an cells ,51,75. RNetea et al  suggest R. gelatinosus, which has high endotoxic activity [Chromobacterium violaceum forms a cylindrical geometry and is reportedly three orders of magnitude less active than E. coli lipid A [C. violaceum lipid A had markedly lower cytokine-inducing capacity than an E. coli-based synthetic lipid A. These findings are in contrast to some other reports, which describe C. violaceum lipid A as having a high agonist activity [C. violaceum and R. gelatinosus are very similar  [C. violaceum lipid A is an LPS antagonist, whereas R. gelatinosus lipid A expresses high agonist activity. Seydel et al[Campylobacter jejuni, which has low endotoxic potential, shows a very slight tendency to adopt a conical/concave shape, whereas the lipid A of E. coli clearly adopts a conical/concave form [More recent work by Seydel and collaborators has led to the realisation that the biological activity of specific lipid A structures can be determined by an understanding of their supramolecular structure, which is a function of the monomeric conformation, which in turn is largely determined by the primary molecular structure ,99,100. activity ,84, was activity . Lipid A lipid A . Ulmer e lipid A  also fouactivity ,75. Of idel et al suggest ave form .As a result of these studies, the ability of a lipid A monomer to adopt a conical shape  has been described as a prerequisite for endotoxicity . Seydel The discussion above has contrasted the endotoxic potential of lipid A structures from the most widely studied forms, those of the Enterobacteriaceae, with some unusual lipid A complexes from photosynthetic bacteria and synthetic lipid A analogs. The reason that cyanobacterial LPS has not been discussed here is simply that the required research has not been done as yet. No cyanobacterial lipid A structures have been published, therefore no inferences can be deduced as to their likely endotoxic potential, or lack of it. But with the knowledge that endotoxic potential can vary in the most fundamental way across Gram-negative bacteria, from agonistic to weakly active to inactive to antagonistic, it should be incumbent on the cyanobacteria research community to cease attributing biological activity and clinical symptoms to cyanobacterial LPS without specific research evidence. Cyanobacteria may not be typical Gram-negative organisms because of their unusual cell wall architecture, and cyanobacteria will have experienced very different selection pressures to gut-dwelling Gram-negative bacteria, which may be reflected in different lipid A structures.in vitro studies of isolated cell lines or animal and human studies where LPS is exposed to the circulation by parenteral injection.Much research into LPS and lipid A has understandably concentrated on the severe, life-threatening host responses to circulating LPS that constitute Gram-negative septicaemia and septic shock. Many experimental models have utilised either The matter of the degree to which cyanobacterial LPS/lipid A can stimulate mammalian cytokine networks under experimental conditions, which remains largely unanswered, is only one aspect in the understanding of cyanobacterial LPS and the gastro-intestinal, respiratory and cutaneous illnesses which have been attributed to contact with it. The other side of the story that needs to be considered is the mechanism by which cyanobacterial LPS might (and might not) stimulate endotoxic responses by the various natural exposure routes: ingestion, inhalation and contact. The following discussion will briefly review the association between acute gastrointestinal illness and Gram-negative bacterial LPS. Cutaneous responses to LPS will be briefly discussed.Infectious diseases caused by bacteria are characterised by several discrete steps: bacterial adhesion to the host, colonisation within or on the host, and evasion of host defences ,49. The • Rank -109 put Microcystis aeruginosa is the causative organism of the mostly tropical, water-exposure-related invasive disease rhinosporidiosis. However, this theory has been disputed; convincing and probably conclusive evidence appears to place the eukaryotic protist Rhinospiridium seeberi as the causative organism .Most public health workers would presumably place cyanobacteria-related illnesses in the context of environmental exposures, as distinct from familiar diseases due to communicable infectious bacteria, which in many cases feature transmissible illnesses, secondary to the original reservoir of infection. Cyanobacteria-related illness is viewed as intoxication, rather than infection, usually on the basis of a sudden onset of symptoms occurring soon after exposure (i.e. without an incubation period), and lack of secondary cases . GieseckNausea and vomiting are normal physiological responses to the ingestion of toxic substances; they are essential defences because they are the end result of the actions of sensorimotor systems that operate to identify and rapidly expel hazardous substances from the upper G-I tract -117. TheE. coli LPS is a potent emetic stimulant. In a series of experiments using piglets, Girod et al [Circulating od et al  showed tod et al .The area postrema is reported to be the primary sensory area involved in nausea as well as vomiting, although nausea is accompanied by autonomic excitation, whereas vomiting is a somatic process independent of the autonomic nervous system ,121. PreTo investigate vomiting associated with exposure to cyanobacteria, the appropriate research efforts should determine whether cyanobacterial LPS is capable of stimulating gut chemoreceptors, or if cyanobacterial LPS can gain access to the circulation and stimulate nausea and vomiting centrally. Another point of interest should be directed towards the cyanobacterial exotoxins, as to their capacity to stimulate either gut mucosal chemoreceptors, vascular emetic chemoreceptors, or whether they induce vomiting through the activity of endogenous mediators. Bacterial exotoxins such as staphylococcal enterotoxins (SEs) are known to stimulate emesis via gut chemoreceptors ,117,124.One inference that can be drawn from the references that posit cyanobacterial LPS as the cause of G-I illnesses  to LD80 concentrations of 10–23 mg/kg [Literature searches using PubMed and Web of Science with the search terms  AND (LPS OR endotoxin) revealed 17 publications that describe the extraction ± purification of cyanobacterial LPS. The Westphal hot phenol/water method was used in 15 of these studies (described in ). Jürgen23 mg/kg .Agmenellum quadruplicatum LPS, Schizothrix calcicola LPS and Anabaena flos-aquae LPS, but not by Oscillatoria tenuis LPS [A. quadruplicatum LPS led the study authors to describe Agmenellum LPS as "a less effective endotoxin than Salmonella LPS" [A. flos-aquae LPS was reportedly "weakly positive" [A. nidulans LPS for pyrogenicity in rabbits, reporting a tenfold lower response than that seen from E. coli LPS. Schmidt et al [Synechococcus strains for pyrogenicity, with maximum increases in rabbit body temperature of 1.5°C after injection of up to 1 mg/kg. The authors reportthat these doses are some three orders of magnitude higher than those required of Salmonella LPS to achieve the same effect. LPS extracted and purified by author IS [E. coli LPS. Cyanobacterial LPS doses of 7 mg/kg also initiated transient hypothermia and sickness behaviour, although at markedly lower intensity and duration than was seen with E. coli LPS at 4 mg/kg [In addition to lethality, various other toxicity endpoints were examined in some of the studies listed in Table nuis LPS -169. Thenuis LPS ,171. Howlla LPS" . The Shwositive"  tested Adt et al  also tesuthor IS  and alsouthor IS  were inv 4 mg/kg .S-transferases (GSTs) in Zebra fish embryos. They reported that cyanobacterial LPS reduced microsomal and soluble GSTs in vivo to a greater extent than LPS from E. coli or Salmonella typhimurium. The authors suggested that such reduction in GST availability may deleteriously affect the ability of organisms to detoxify microcystins, presumably through decreased utilisation of glutathione (GSH) for conjugation reactions. This would seem to be a reasonable assumption, although other interpretations of this finding are possible. While the GSH system is an essential intracellular redox buffer, preventing oxidative injury, it is also an important participant in many cellular functions, including DNA and protein synthesis, metabolism, cell growth and amino acid transport [Most workers concluded that the cyanobacterial LPS they examined were weakly toxic when compared to the activity of positive control heterotrophic bacterial LPS. The exception was the work of Best et al , who invransport ,176. Manransport . GSH is ransport -184. It ransport ,185-187.ransport ,189. Gluransport ,190. Drö"It is clear that GSH is one of the limiting factors that determine the magnitude of immunological functions in vitro and in vivo...we must be prepared to reevaluate one of the central dogmas concerning the role of GSH. GSH was viewed mostly as an important anti-oxidant that protects cells against oxidative stress" .in vitro and in vivo. Animals treated for three days showed two-fold-plus increases in the same enzymes [So the findings of Best et al , where c enzymes ,193. We  enzymes , perhapsIn addition to the studies cited above, nine reports describe the isolation and purification of cyanobacterial LPS for various biomedical, biochemical or structural studies, but no toxicological endpoints were investigated ,194-200.Limulus amoebocyte (LAL) assay. This is a highly sensitive test, though there are trade-offs in terms of specificity, with other bacterial products such as peptidoglycan and glucan capable of registering positive responses [in vivo responses [E. coli LPS, and several were below the assay's detection limits. This suggests that the lipid A structures of these LPS have some significant and fundamental structural differences to endotoxic lipid A, as the LAL assay does not react to some unusual or modified lipid A structures [In addition to work done on isolated and purified LPS, some reports discuss the activity of cyanobacterial LPS by use of the esponses ,202,203.esponses . Seydel esponses  suggest esponses  had someructures .S. platensis has a long history of use as a foodstuff, dietary supplement and livestock feed additive, with its probable use dating back to the ninth century in Africa, and the 14th century in Mexico [Spirulina is classified taxonomically under the genus Arthrospira, order Oscillatoriales; A. maxima and A. fusiformis are grown commercially in mass culture, but usually designated as "S. platensis" [Spirulina was briefly reviewed along with that of other edible microalgae by Kay [Spirulina feeding were seen in multigenerational studies and mutagenicity tests. However, this appears to be a misinterpretation on the part of Kay [Spirulina is not harmful, and enhances various immune functions [n Mexico . Spiruliatensis" . The useatensis" , who cone by Kay , who cite by Kay  in statit of Kay , as Cifet of Kay  describet of Kay  were unounctions -214.Spirulina, presumably on a regular basis. Liver function tests showed a significant deterioration over the following three weeks, after which he was hospitalised. Although a physical examination was unremarkable, a liver biopsy revealed some degenerative changes. Serological studies for a range of viruses were negative. His medications and Spirulina were withdrawn, after which his hepatic function rapidly returned to normal. Hepatotoxicity in this case was attributed to consumption of Spirulina, on the basis of temporal relationships between liver function abnormality and recovery with consumption and withdrawal of Spirulina, although possible interaction effects with the medications would have been worth considering. This may have been the case with simvastatin, the cholesterol-lowering agent this individual was taking. Simvastatin causes increased proinflammatory cytokine production, and it can potentiate inflammatory responses induced by bacterial products [Spirulina pills in Canada in the early 1980s, though there was an indication that one individual's tablets had some bacterial contamination [A single case report is the exception to the rest of the literature. Iwasa et al  describeproducts . A briefmination .A. fusiformis in Kenyan soda lakes is capable of producing the cyanobacterial exotoxins microcystin-YR and anatoxin-a [A. fusiformis is the principal food source for these animals [Anabaena and Microcystis are also found in these lakes, and periodically dominate the phytoplankton profile [Recent work has demonstrated that atoxin-a ,219. The animals ,221. Com profile , so presArthrospira spp. illuminates the importance of considering the route of exposure in toxicology studies, and the dangers in this case of presumptive inference of disease from the findings of parenterally administered LPS. Tornabene et al [Spirulina platensis LPS in the range of 400 mg/kg (i.p. mouse), although those findings are not supported by the work of Stewart, where Spirulina LPS i.p. injections of 350 mg/kg failed to induce either thermoregulatory change or sickness behaviour signs [Spirulina were highly toxic to mice when administered by intraperitoneal injection.The long-standing and widespread consumption of ne et al  reportedur signs . Howeverur signs  reportedAphanizomenon flos-aquae was over a decade ago reportedly "consumed by thousands of people without incident" [Microcystis spp., and some A. flos-aquae end-product batches contaminated with microcystins have since been found [Aphanizomenon, Spirulina and unidentified cyanobacteria) but did not present the results according to the component genera, so it is not clear whether Spirulina products  contained microcystins [Nostoc commune, a terrestrial cyanobacterium, has a long history of use in China and Scandinavia as food and medicine [Other cyanobacteria are consumed as foods, medicines and dietary supplements. Wild-harvested ncident" , althougen found -227. A Cocystins . Nostoc medicine . The widPseudomonas spp., colonised water spray systems in the facility, and increased endotoxin in bio-aerosols was linked to the illnesses.In our opinion, the sole natural exposure route that might explain aquatic cyanobacterial LPS-related illness is via inhalation of aerosolised cells or fragments. Extrapolating from the understanding of Gram-negative bacterial LPS on the respiratory system  in the bronchial tree. Microcystin-LR appears to be able to efficiently gain access to the circulation by both intranasal and intratracheal routes -248, butCyanobacterial exotoxins may have the capacity to generate respiratory illness in non-atopic individuals, with endotoxins from cyanobacteria or commensal bacteria possibly augmenting the symptoms. The potential for cyanobacterial and/or contaminant endotoxin alone to produce symptoms by inhalation exposure remains open, given the observation that LPS can produce measurable airway function changes in animal models and in some healthy individuals ,252-254.Rubrivivax gelatinosus, as discussed above. A similar example is given by another group of non-pathogenic bacteria, Rhizobium spp., the LPS from some of which are comparable to that of enterobacterial LPS in lethal toxicity and cytokine-inducing activity [Lipid A, the endotoxic moiety of LPS, was in previous decades thought to remain constant across different Gram-negative bacteria . This isactivity ,256. DetIn vivo studies of oral exposure to cyanotoxins would be well served by use of a vomiting-capable model, i.e. non-rodent experiments.From the discussion in this review, we will put the hypothesis that oral consumption of non-toxic cyanobacteria, i.e. absolutely or essentially free of any of the known cyanobacterial exotoxins, will not result in either vomiting or diarrhoea. This hypothesis would be falsified by experiments that show isolated cyanobacterial LPS or non-toxic crude extracts can cause gastrointestinal signs and/or pathology in a suitable model. Our impression is that reports of G-I symptoms in humans exposed to cyanobacterial products are indications of innate defences being signalled by exotoxins that have breached the intestinal barrier. Once this occurs, and gut permeability is sufficiently disrupted, LPS may well synergise the pathology of cyanotoxins, especially the hepatotoxins. From what little is known to date about the toxic potential of cyanobacterial LPS, i.e. that they are weakly toxic compared to those of the Enterobacteriaceae, gut-derived LPS would seem to be the more likely candidate for augmenting the pathology of cyanotoxins. There does not appear to be good evidence that cyanobacterial LPS are likely to initiate cutaneous reactions in healthy people exposed in recreational or occupational settings. Cutaneous reactions to cyanobacteria are discussed in detail elsewhere -259.Exposure to bio-aerosols containing cyanobacterial endotoxins may be worthy of investigation, but we are not convinced that cyanobacteria-related acute respiratory illness in non-atopic, non-allergic individuals is not equally or more likely to be explained by inhalation of cyanobacterial exotoxins. If some of the exotoxins turn out to possess ligands that stimulate innate immune responses (discussed further in ), then tIn conclusion, LPS of the Enterobacteriaceae are potent immunomodulatory and immunotoxic bacterial products that stimulate a wide variety of responses in mammals, not least of these being a desire to wax lyrical on the topic. Thus:"Endotoxins possess an intrinsic fascination that is nothing less than fabulous. They seem to have been endowed by Nature with virtues and vices in the exact and glamorous proportions needed to render them irresistible to any investigator who comes to know them" .And:"The dual role of LPS as effector and target makes it a fascinating molecule which...still hides many miracles. It intrigues at the same time clinical, biological, chemical, and biophysical researchers...".Facetiousness aside, these workers are pointing out that there is much to learn about the LPS of the most widely studied Gram-negative bacteria, these being the Enterobacteriaceae. The understanding of cyanobacterial LPS is utterly miniscule by comparison, and we urge caution before continuing to attribute such a disparate range of symptoms in humans to contact with these materials without the required research evidence. Weckesser, Drews and Mayer wrote in 1979 that:"...the picture obtained with the Enterobacteriaceae cannot be assigned to other Gram-negative bacteria without detailed investigations. Considering the broad spectrum in morphological and physiological diversity of the many taxonomic groups of both photosynthetic bacteria and cyanobacteria, there is a wide open field for studies on the composition of their cell wall." .Ressom et al  stated t"Given the enormous heterogeneity in LPS from Gram-negative bacteria there is every reason to suspect that the same will apply to cyanobacterial LPS and, due to their taxonomic distance apart, cyanobacterial LPS are likely to be different from those found in Gram-negative bacteria."We agree with these statements.CYP cytochrome P450DNA deoxyribonucleic acidG-I gastrointestinalGSH glutathioneS-transferaseGST glutathione IgE immunoglobulin EIL interleukini.p. intra-peritonealLimulus amoebocyte lysate assayLAL assay LBP lipopolysaccharide-binding proteinLD lethal doseLPS lipopolysaccharide/sSEs staphylococcal enterotoxinsTh cell T-helper cellTLR Toll-like receptorTNF-α tumour necrosis factor-alphaThe author(s) declare that they have no competing interests.IS conducted the review; PJS and GRS supervised the work and contributed to redrafting the paper. All authors read and endorsed the final manuscript.