Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria,  Bacteria release outer membrane vesicles (OMVs) that play important roles in pathogenesis and intercellular interactions. Here, Roier et al. provide evidence supporting that phospholipid accumulation in the outer leaflet of the outer membrane participates in OMV formation in Gram-negative bacteria. Haemophilus influenzae, Pasteurella multocida, Vibrio cholerae, enterotoxigenic Escherichia coli, Neisseria meningitidis, Bordetella pertussis and Salmonella typhimurium89101112Outer membrane vesicles (OMVs) are spherical, bilayered, membranous structures that are released naturally from the outer membrane (OM) of Gram-negative bacteria231251235Pseudomonas aeruginosa. It is hypothesized that PQS enhances anionic repulsions between lipopolysaccharide molecules resulting in membrane blebbing by sequestering divalent cations, which are important in forming stabilizing salt bridges between the negatively charged B-band lipopolysaccharide molecules. Recently, it was proposed that PQS induces OMV formation through a mechanism of asymmetric expansion of the outer leaflet of the OM237192021P. aeruginosa and therefore species specific. In summary, all these proposed models of OMV formation require either genetic manipulations, the presence of stress, or are thus far only applicable to a single bacterial species. It is currently unknown whether Gram-negative bacteria share a conserved general mechanism of OMV biogenesis that is amenable to regulation.Despite these important biological roles and applications of OMVs, research has only just begun to understand the mechanism of OMV biogenesis. Currently, three major models are discussed within the scientific community. The first model is based on either loss or relocation of covalent linkages  between the OM and the underlying peptidoglycan layer. These missing cross-links in combination with an OM that grows faster than the underlying cell wall allow the OM to protrude and thus initiate vesiculation237141523717H. influenzae and V. cholerae without compromising OM integrity. Concordantly, mutations in homologues of E. coli also exhibit increased vesiculation, as independently demonstrated by other groups -dependent downregulation of the VacJ/Yrb ABC transport system correlating with an increased OMV production in H. influenzae, V. cholerae and E. coli. Our results indicate that the disadvantage of an increased serum sensitivity caused by this downregulation can be overcome in vivo by an increased OMV production suggesting an important pathophysiological role of this system. We propose a novel and potentially highly conserved bacterial OMV biogenesis mechanism, which provides the opportunity of regulation and may represent a first general mechanism applicable to all Gram-negative bacteria.Here we show that disruptions within the VacJ/Yrb ABC (ATP-binding cassette) transport system increase OMV production in the human pathogens H. influenzae, an opportunistic pathogen of the human respiratory tractH. influenzae and have recently shown to be present in OMVs in detectable levelsE. coli in a previous screen for alterations in OMV productionE. coli, Salmonella, and V. cholerae1527mreB, dsbC and gmhA can be linked to OM stability or stress response, which have been previously implicated in OMV secretion2715yrbB) and HI_1086 (yrbE), which showed strong OMV and OMP dot blot signals suggesting an overall increase in vesiculation. These were the most promising candidates for further evaluation, as they have not been previously implicated in OMV formation and both genes are located within a gene cluster comprising three additional genes . In E. coli, homologues of these five gene products, together with the VacJ protein, have been implicated in maintaining the lipid asymmetry in the Gram-negative OMH. influenzae (NTHi)Shigella flexneriH. influenzae based on the E. coli Mla pathway modelH. influenzae as well as the overall genomic organization of their corresponding genes are conserved among Gram-negative bacteria  and 4.3-fold (ΔyrbE) higher vesiculation levels but similar OMV sizes compared with the wild-type. Similar to the complementation analysis in H. influenzae, the hypervesiculation phenotypes of the ΔvacJ and ΔyrbE mutants in V. cholerae were restored to wild-type levels by expression of the respective gene in trans . This observation is reinforced by a recent genome wide assessment of OMV production in E. coli indicating an increased vesiculation of mlaA (vacJ) as well as mlaE (yrbE) mutants, which was not further investigatedmla gene cluster (comprising mlaF to B) in the E. coli wild-type. Consistent, with the increase in vesiculation upon deletion of the Mla pathway, the overexpression resulted in decreased vesiculation compared with the empty vector control  in Rd KW20 grown under iron-restricted conditions mediated by addition of 2,2′-dipyridyl  compared with Rd KW20 grown under the same conditions , this model of OMV formation could be a general mechanism applicable to a variety of Gram-negative bacteria.Taken together, our findings allow us to propose a novel and potentially highly conserved OMV biogenesis mechanism in Gram-negative bacteria . Accordiin vivo, which might impact the bacterial pathophysiology. One example could be the transmission of H. influenzae into a new host. Upon initial colonization of the nasopharynx, H. influenzae must overcome mucosal immune defence mechanisms, including the protective effects of complement factors and secretory IgA antibodies39in vivo cause a downregulation of the VacJ/Yrb ABC transport system resulting in increased OMV production, which counteracts antibody and complement attacks. The importance of OMVs in bacterial serum resistance is also highlighted by a recent study by Tan et al.Moraxella catarrhalis OMVs are involved in the complement resistance of nasopharyngeal bacteria by binding and depleting complement factors. One could speculate that this might also be a protective feature of H. influenzae OMVs within the human nasopharynx. The hypervesiculation during initial colonization of a new host could provide an advantage for H. influenzae by lowering the selective pressure of local immune defence mechanisms and thereby facilitating proliferation in the nasopharynx. In this sense, our proposed OMV biogenesis mechanism could be very important for bacterial adaptation to a new host.Moreover, we show that this mechanism can be regulated by iron availability in a Fur-dependent manner. Although additional regulatory pathways may act on the PL transporter genes, it is likely that iron limitation causes the observed downregulation of the respective genes It should be emphasized that this OMV biogenesis mechanism based on PL accumulation can act in concert with all other OMV formation models proposed so far. As already briefly discussed in the introduction, previously reported OMV formation models are species-specific, require the presence of stress or depend on mutations affecting OM integrity. In contrast, the model proposed herein overcomes these limitations as it represents a first general mechanism that can account for OMV formation under all growth conditions, is applicable for a broad range of Gram-negative bacteria, and can be regulated by the microorganisms.ad libitum and monitored under the care of full-time staff and in accordance with the rules of the Institute of Molecular Biosciences at the University of Graz. All animals were acclimated for 1 week before any procedures were carried out and were 9–11 weeks old at the start of the experiment. Normal human serum was obtained and pooled from five healthy adult volunteers according to an approval of the University of Graz Ethics Commission (GZ. 39/31/63 ex 2012/13). All volunteers provided written consent.Female BALB/c (Charles River Laboratories) were used for all immunization experiments and female CD-1 IGS mice (Charles River Laboratories) were used for colonization studies in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the national ‘Bundesgesetzblatt für die Republik Österreich'. The corresponding animal protocol (39/53/00 ex 2012/13) has been approved by the Austrian Federal Ministry of Science and Research Ref. II/10b and the local Committee on the Ethics of Animal Experiments of the University of Graz. Mice were housed with food and water E. coli and V. cholerae, or in brain heart infusion (BHI) broth or on BHI agar supplemented with NAD and either protoporphyrin IX (PPIX) or hemin solution  in the case of H. influenzae. BHI–NAD–PPIX served as standard growth medium for H. influenzae. BHI–NAD–hemin was used only for the following experiments: construction of transposon insertion mutants, deletion mutants and complementation strains; quantification of OMVs under conditions of iron excess or depletion; and quantitative real-time RT-PCR experiments. Supplements were used in the following final concentrations: NAD, 10 μg ml−1; hemin, 20 μg ml−1; L-histidine, 20 μg ml−1; triethanolamine, 0.08%; and PPIX, 20 μg ml−1. When appropriate, streptomycin , chloramphenicol , kanamycin , or 2,2′-dipyridyl (100 μM) were added to H. influenzae growth media. E. coli and V. cholerae growth media were supplemented with streptomycin , kanamycin , ampicillin , sucrose (10%), arabinose (0.0002%), or isopropyl-β-D-thiogalactopyranoside , if appropriate.All bacterial strains and plasmids used in this study are listed in H. influenzae, OMVs as well as density gradient purified OMVs and OM were prepared as described previouslyV. cholerae and E. coli OMVs were isolated according to Schild et al.et al.H. influenzae) or 8 h (V. cholerae and E. coli). Bacterial cells were removed from the supernatants containing the OMVs by centrifugation and subsequent filtration through 0.45 and 0.2 μm pore size filters. OMVs were pelleted from the filtrate by ultracentrifugation  using a Beckman Coulter Optima L-100 XP ultracentrifuge. In case of lipidome and proteome analyses as well as transmission electron microscopy, H. influenzae OMVs were further purified by density gradient ultracentrifugation using an isopycnic OptiPrep-iodixanol (Sigma-Aldrich) density gradientH. influenzae cultures grown to late exponential phase for 13 h followed by purification steps via ultracentrifugation using a sucrose cushion and an isopycnic sucrose density gradientH. influenzae, V. cholerae or E. coli Bradford assays, an OMV preparation of Rd KW20, Vc AC53, or Ec BW whose protein concentration had been determined by a TrayCell measurement was used as a protein standard, respectively.For et al.E. coli RNA polymerase (NeoClone Biotechnology) was diluted 1:2,000. The horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Dianova), diluted 1:7,500, served as secondary antibody. Chemiluminescence detection was performed by incubating each membrane in an ECL solutionThe protein content of OMV and OM preparations was analysed by SDS–PAGEH. influenzae OMVs, five female BALB/c mice were intraperitoneally immunized at days 0, 14 and 28 with approximately 2 μg of an OMV preparation derived from Rd KW20 dissolved in 0.1 ml PBS (pH 7.4). Sera were isolated from blood samples collected between day 38 and 46 as described previouslyTo generate a specific polyclonal antiserum against H. influenzae was accomplished according to a method described by Schlör et al.10d-cat were performed in Rd AK01. Cm-resistant colonies were pooled and extracted chromosomal DNA was cut with XmaI. The digested DNA was retransformed into Rd KW20 resulting in 2,820 chloramphenicol-resistant transposon insertion mutants, which were subsequently screened by dot blot for altered OMV production and amounts of the OMPs P1, P2, P4, P5 or P6 in OMVs. According to Akerley et al.490) were measured and cells were subsequently pelleted . About 1 ml of pooled supernatant per transposon insertion mutant culture was filtered through a 0.2 μm pore size Supor (polyethersulfone) filter  by centrifugation  and each filtrate was stored at –20 °C until dot blots were performed. For dot blot analysis, filtrates were thawed and appropriate volumes were blotted onto six Amersham Hybond ECL nitrocellulose membranes  using a 96-well Whatman Minifold I Dot-Blot System . In addition, each membrane was spotted with the respective volume of a prior made and aliquoted wild-type (Rd KW20) filtrate (prepared in the same manner as the transposon insertion mutant filtrates) as a positive control and a sterile control filtrate as a negative control. Then, each membrane was dried, incubated in Tris-buffered saline (TBS)  for 2 min, and blocked in 10% skim milk in TBS for 2 h. Afterwards, the six membranes were incubated with either anti-OMV, -P1, -P2, -P4, -P5, or -P6 mouse antiserum  diluted 1:500 in 10% skim milk in TBS overnight. The next day, membranes were washed three times in TBS for 10 min, incubated with the secondary antibody  for 2 h, washed once in TBS-T , and twice in TBS for 10 min each. Finally, chemiluminescence detection was performed as described above. Relative intensities of dot blot signals derived from transposon insertion mutants were evaluated by eye through comparison with the respective wild-type signals in combination with the respective OD490 values. Representative examples for dot blots as well as the evaluation of relative signal intensities are provided in 10d-cat insertions were located by DNA sequencing of junction fragments generated by PCR using the transposon mutagenesis oligonucleotide primers, which are listed in 44Transposon mutagenesis in H. influenzae, overlap extension PCRs in combination with a Rd KW20 transformation protocol were performed as described previouslycat gene of pAKcat, and the npt gene of pACYC177, as well as primers for validation of correct construction are listed in In cis complementations were achieved by replacing the cat gene of a deletion mutant with the respective gene (upstream fragment containing upstream sequence and gene of interest) and the npt gene due to homologous recombination. For construction of deletion mutants in V. cholerae, isolation of chromosomal DNA, PCRs, purification of plasmids or PCR products, construction of suicide plasmids, as well as subsequent generation of deletion mutants were carried out as described previouslyvacJ, pCVDΔyrbE, pCVDΔfur, as well as the respective deletion mutants. The expression plasmids pvacJ and pyrbE were constructed in a similar manner. PCR fragments of the respective genes containing their own ribosomal binding sites were generated using oligonucleotide primer pairs digested with the respective restriction enzymes indicated by the name of the oligonucleotide primer, and ligated into the similarly digested IPTG-inducible expression vector pMMB67EH. To construct expression plasmids for the E. coli mlaF-B genes, a PCR fragment containing the respective genes generated using oligonucleotide primer pairs digested with the respective restriction enzymes indicated by the name of the oligonucleotide primer, was ligated into the similarly digested IPTG-inducible expression vector pTRC99A. From there, a fragment containing the mla gene cluster was cut by NcoI and XbaI and ligated into a similarly digested pBAD24 to generate the arabinose-inducible expression plasmid pBADmlaF-B. Expression constructs were transformed into DH5αλpir, and Apr colonies were characterized by PCR. All mutants and plasmids described herein were confirmed by PCR (data not shown). Used oligonucleotide primers are listed in For construction of deletion mutants and complementation strains in H. influenzae, V. cholerae and E. coli strains, OMVs were purified essentially as described above with slight modifications. Cultures were grown to late exponential phase, OD were measured, and 70 ml (H. influenzae and V. cholerae) or 140 ml (E. coli) of the sterile-filtered supernatant was ultracentrifuged  using a Type 45 Ti rotor (Beckman Coulter). The pellet comprising the OMVs was immediately resuspended in 100–110 μl PBS (pH 7.4) and stored at –20 °C. To quantify the protein content of OMVs, Bradford assays as described above were used. To quantify the lipooligosaccharide or lipopolysaccharide content of OMVs, purpald assays were performed as described previouslyD-manno-octulosonic acid (Kdo) (Sigma-Aldrich) as a standard. OMVs were quantified by back-calculating the respective protein and lipooligosaccharide/lipopolysaccharide content to 1 l original culture volume per OD unit (mg l−1 OD unit−1). In case of H. influenzae cultivated with BHI–NAD–PPIX an additional method for OMV quantification based on dot blot analysis as described above was used. To this end, the same volume of sterile-filtered supernatants derived from all strains to be tested were dot blotted on the same membrane and anti-OMV mouse antiserum was used as primary antibody. Dot blots were developed as described above and OMV signal intensities of dots were determined using the Quantity One software (Bio-Rad Laboratories) to calculate the respective artificial units (a.u.) per OD490 unit (a.u. OD490 unit-1).To compare the amount of OMVs produced by different H. influenzae, V. cholerae or E. coli OMV preparations and transmission electron microscopy was applied for OMV visualization using H. influenzae density gradient purified OMV preparations as described previouslyH. influenzae OMVs by transmission electron microscopy, preparations were diluted to 0.1 mg ml−1  in PBS (pH 7.4) and 3 μl thereof was allowed to adsorb onto a carbon-coated copper grid for 15 s. After removal of excess liquid, samples were negatively stained with 2% uranyl acetate for 15 s followed by a further removal of excess liquid. Micrographs were recorded using a Morgagni 268 (FEI) transmission electron microscope.Nanoparticle tracking analysis was used to measure the respective OMV size distribution of H. influenzae strains, the degree of cell lysis was assessed by dot blot analysis and minimal inhibitory concentrations of SDS and polymyxin B were determined. Dot blot analysis was performed as described above for the quantification of OMVs, except that a monoclonal antibody to the α-subunit of the E. coli RNA polymerase  was used as primary antibody to detect RpoA in the sterile-filtered supernatants. RpoA detection has previously been used as a cytoplasmic contamination and lysis control in H. influenzae490 of 0.1. Appropriate serial dilutions of SDS and polymyxin B were added and the plate was incubated at 37 °C for 16 h. Afterwards, the OD490 of each well was measured and compared with wells not containing bacteria (negative control). The minimal inhibitory concentration was defined as the lowest concentration of SDS or polymyxin B that inhibited bacterial growth (OD490<0.3).To compare the OM stability between different 18 microcolumns (Millipore) according to the manufacturer's manual, and resuspended in 8 μl 0.1% formic acid. Finally, samples were analysed using a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer (Waters) equipped with a nanoACQUITY ultra performance liquid chromatography system (Waters). The resulting MS/MS spectra were used for the identification of proteins by the Mascot search engine using the NCBInr database with the bacteria (eubacteria) taxonomy selected. Searches were carried out with the fixed modification carbamidomethyl (C), the enzyme selected as trypsin allowing up to 1 missed cleavages, a peptide tolerance of 1.2 Da, a MS/MS tolerance of 0.6 Da, a peptide charge of 1+, 2+ and 3+, monoisotopic mass values, and the instrument setting selected as ESI-QUAD-TOF. All data were searched with the significance threshold set to a P value of <0.05 resulting in an identity ion score threshold between 58 and 61. Only results with ion scores greater than or equal to the identity threshold were considered significant.Mass spectrometry was used to identify proteins from density gradient purified OMV and OM preparations. Sample preparation, mass spectrometry, and protein identification by the Mascot search engine  were performed as described previouslyet al.2SO4 (72%)/HClO4 , which were subsequently quantified colourimetrically as phosphomolybdate measured at 820 nm according to the standard procedure described by BroekhuyseLipids from density gradient purified OMV and OM preparations were extracted according to Folch vacJ, yrbE, fabB, fabD or fabH was determined by quantitative real-time RT-PCR (qRT-PCR). For this purpose, respective strains were grown to an OD490 of 0.8-1 in BHI–NAD–hemin medium supplemented with or without 100 μM 2,2′-dipyridyl in case of H. influenzae or to an OD600 of 0.7–0.9 in LB medium in case of V. cholerae and E. coli. Bacterial RNA extraction, DNase digestion, cDNA synthesis and qRT-PCR were performed as described previouslyvacJ and yrbE in H. influenzae under in vivo conditions, CD-1 mice were intranasally inoculated with Rd KW20 essentially as described previously8 c.f.u. per mouse. After 4 h, the mice were sacrificed, the nasopharynx of each mouse was removed by dissection and mechanically homogenized in 2 ml Trizol. RNA was subsequently extracted using chloroform extraction and precipitated with isopropanol. DNase digestion, cDNA synthesis and qRT-PCR were performed as described previouslyT method by normalizing the mean cycle threshold of each investigated transcript to the housekeeping gene rpoB and to one randomly selected reference sample.Expression of H. influenzae strains in serum bactericidal assays, cells were grown to an OD490 of 0.8–1 and diluted in Hank's buffer  to an OD490 of 0.1. About 100 μl of this suspension were mixed with 5 μl normal human serum  and optionally with 5 μl of appropriate dilutions of respective OMV preparations. This resulted in the following physiological final in vitro concentrations of OMVs based on the OMV quantification via purpald assays  of ∼0.6 mg l−1 OD490 unit−1 for Rd KW20-derived OMVs and 1.2 mg l−1 OD490 unit-1 for Rd ΔvacJ derived OMVs. In case of two-fold amounts of Rd KW20-derived OMVs, 10 μl were added, which result in a final concentration of approximately 1.2 mg l−1 OD490 unit−1. Hank's buffer was added beforehand to obtain a final reaction volume of 250 μl. The mixture was incubated at 37 °C for 45 min with rotation, the assay was stopped by cooling to 4 °C, and appropriate dilutions were plated on agar plates to determine viable counts. To calculate the percentage of survival, viable counts were compared with control samples in absence of normal human serum.To test P values of <0.05.Data were analysed using GraphPad Prism version 6.0f for Mac OS X (GraphPad Software). The statistical significance of differences between groups was examined using the unpaired t test or an ordinary one-way ANOVA followed by Sidak's multiple comparison post test. Differences were considered significant at Accession codes: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD003249.How to cite this article: Roier, S. et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat. Commun. 7:10515 doi: 10.1038/ncomms10515 (2016).Supplementary Figures 1-6, Supplementary Tables 1-4 and Supplementary References


Purpose. The aim of this study was to compare diffusion-weighted magnetic resonance imaging (DWI) with computed tomography perfusion (CTP) for preoperative detection of metastases to lymph nodes (LNs) in head and neck squamous cell carcinoma (SCC). Methods. Between May 2010 and April 2012, 30 patients with head and neck SCC underwent preoperative DWI and CTP. Two radiologists measured apparent diffusion coefficient (ADC) values and CTP parameters independently. Surgery and histopathologic examinations were performed on all patients. Results. On DWI, 65 LNs were detected in 30 patients. The mean ADC value of metastatic nodes was lower than benign nodes and the difference was statistically significant (P < 0.05). On CTP images, the mean value in metastatic nodes of blood flow (BF) and blood volume (BV) was higher than that in benign nodes, and mean transit time (MTT) in metastatic nodes was lower than that in benign nodes. There were significant differences in BF and MTT values between metastatic and benign LNs (P < 0.05). There were significant differences between the AUCs of DWI and CTP . Conclusion. DWI with ADC value measurements may be more accurate than CTP for the preoperative diagnosis of cervical LN metastases. Squamous cell carcinoma (SCC) is the most common malignancy of the head and neck region. It accounts for 5% of all malignant tumours worldwide . A meta-Although ultrasound, routine contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) allow the detection of enlarged cervical LNs, none of these methods can distinguish between benign and malignant causes of enlargement with any accuracy , as theySingle photon emission computed tomography (SPECT) and positron-emission tomography (PET) are imaging techniques that supply functional information, but they involve radiation exposure and are expensive with low availability and are hampered by relatively low spatial resolution –13.Recently, other functional imaging techniques such as MRI diffusion-weighted imaging (DWI) and CT perfusion imaging (CTP) have shown promise in detecting metastatic cervical LNs, and there is increasing experience in head and neck SCC. DWI is an MR technique that depicts molecular diffusion, which is the Brownian motion of water protons in biologic tissues. Examination of molecular diffusion using DWI performs with an EPI (echo planar imaging) sequence and a linear regression after a logarithmic transformation of the signal intensity was used to calculate the ADC values . To dateUnlike conventional contrast-enhanced CT, which is normally assessed visually, perfusion imaging requires quantification of the enhancement in tissue and blood at certain time points following intravenous injection. These enhancement data are used to calculate blood flow (BF), blood volume (BV), and mean transit time (MTT) for each voxel, depicted in a color-coded display. Data processing methods are based on the robust physiological principles of compartmental analysis or linear systems theory. In the compartmental modeling technique, analysis can be done by one compartment method which assumes the intravascular and extravascular compartment as a single compartment and allows measurement of tissue perfusion during the first pass of contrast , 16.Although DWI and CTP are increasingly used to detect LN metastases in head and neck SCC, comparison of the diagnostic value of the two imaging modalities has been rarely performed. The aim of this study was to compare the value of DWI and CTP for detecting metastatic LNs which were confirmed by pathologic diagnosis in patients with head and neck SCC.The study was approved by the local ethics committee and all patients signed review board-approved consent before participation.n = 9), tongue (n = 3), nasopharynx (n = 6), floor of mouth (n = 3), nasal cavity (n = 4), oropharynx (n = 4), and gingiva (n = 1). All patients underwent DWI and CTP before surgery.Sixty-five cervical LNs from 30 treatment-naïve patients with head and neck SCC were excised during surgery from May 01, 2010, to April 30, 2012. All LNs underwent pathological analysis. Among the 30 patients, 21 were men and 9 were women; their age ranged from 38 to 70 years, with a mean age of 53.6 years. The primary cancers were of the larynx  with a head and neck coil. Thirty patients underwent conventional MRI and DWI to include nodes from the base of the skull to the suprasternal notch. Before scanning, all patients were trained to avoid swallowing during the MRI examination.fast spin-echo (FSE) T2-weighted images  in the axial plane;fast spin-echo (FSE) T2-weighted images , in the coronal plane;fast spin-echo (FSE) T1-weighted images, with fat suppression  in the axial plane; b, 600 s/mm2) in the axial and coronal planes. Image of black and white reverse image was constructed.diffusion-weighted imaging with background body signal suppression (DWIBS) images . The ADC values were obtained by drawing regions of interest (ROIs) around the solid portions of nodes, avoiding necrotic-appearing areas. Two experienced radiologists analysed the results independently. Disagreements (controversy about positive nodes) regarding image findings were resolved with mutual accord.The thirty patients underwent preoperative routine CT and perfusion CT scans using a multidetector 16-slice CT scanner . Selection of the nodal targets was based on a plain CT scan; nonionic iodinated contrast agent   was injected at a flow rate of 5 mL/s via the antecubital vein with an injector  for dynamic perfusion CT scanning. The perfusion CT parameters were as follows: 120 kVp, 150 mAs, 16 × 1.5 detector collimation, 3 mm slice thickness, and a scanning speed of 1 s/rotation. Thus, we could evaluate flow perfusion in eight slices, including 24 mm from top to bottom.Choosing the common carotid or internal carotid artery as the input artery and internal jugular vein as the output vein, we obtained time density curves and calculated BF, BV, and MTT of the ROIs with perfusion software (deconvolution arithmetic) from the workstation . ROIs again were placed in solid areas of the LNs, avoiding calcified or necrotic-appearing areas. Two experienced radiologists carried out this procedure and the mean values were calculated.t-test. The two imaging techniques were compared using receiver operating characteristic curves (ROC curves). P < 0.05 was considered statistically significant. All statistical analyses were performed with the SPSS 17.0 software package.ADC values and BF, BV, and MTT of the LNs were compared using Student's b = 600 s/mm2), whereas on the black and white flip images they presented low signal (b = 600 s/mm2) on DWI images (−3 mm2/s (range: 0.738 × 10−3–0.960 × 10−3 mm2/s), lower than the mean value of the benign nodes ; this difference was statistically significant (P < 0.05) with t = 2.629   . The optZ = 4.612, P < 0.001) (< 0.001)  for diagDWI is an MR imaging-based technique whereby diffusion properties of water can be quantified using the ADC. Hypercellular tissue, such as solid tumour, is characterized by a low ADC, while more hypocellular tissue, such as normal tissue, is typically characterized by a higher ADC. As several studies reported, metastatic nodes showed a reduction of diffusivity, which can be attributed to hypercellularity, to an increased nuclear-to-cytoplasmic ratio, and to perfusion , 18. KinPathological states, such as malignant tumor cell, strongly affect water diffusion more than benign tumor cell and so influence DWI, and hence the technique has attracted considerable research attention for both benign and malignant disease processes.b values (0 and 800 s/mm2) and a 3 T MR unit, they established an optimal ADC threshold of 1.3 × 10−3 mm2/s for diagnosis. The authors of the other DWI investigation [b values of 0, 500, and 1000 s/mm2 at 1.5 T yielded a significant difference between benign and malignant lesions (P < 0.001). Also, an optimal ADC threshold of 1.25 × 10−3 mm2/s to help differentiate benign from malignant lesions was established, consistent with that of the previous study [One of the DWI investigations performed in the head and neck  showed ttigation  confirmeus study , and yieb = 600 mm2/s); conversely, benign nodes were hypointense (b = 600 mm2/s). We chose 0.960 × 10−3 mm2/s as the optimal ADC threshold value for distinguishing benign from metastatic nodes, with a sensitivity of 89.58%, a specificity of 76.47%, an accuracy of 86.15%, a PPV of 91.48%, and an NPV of 72.22%. Our data are not in agreement with those of Kato et al. [−3 mm2/s) to be higher than that in benign lymphadenopathies (0.89 ± 0.21 × 10−3 mm2/s).In our series, the evaluation with DWI showed that metastatic nodes appeared hyperintense  and the use of sequences that reduce the artefacts in order to make the measurement of the ROI more precise [Differences among these studies can be attributed to several causes. One is the choice of the  precise .CTP is a technique that allows quick qualitative and quantitative evaluation of tissue blood perfusion by generating maps of BF, BV, and MTT. Gandhi et al.  reportedIn our study, 13/17 metastatic LNs showed high BF and BV and short MTT. There were significant differences between metastatic LNs and benign LNs in BF and MTT. In metastatic nodes, BF is increased by newly developed vessels, while MTT is usually decreased by the presence of pathological arteriovenous shunts. Changes in vascular endothelium and in the function of vessels are induced by neoangiogenesis , 28.In our study, 15/48 metastatic LNs also had lower BF, reflecting the necrotic tumour elements , a quite common phenomenon in malignant tumours , 30. Thi−3 mm2/s. Our data was in agreement with the results mentioned above and obtained a sensitivity of 89.58%, a specificity of 76.47%, and an accuracy of 86.15%.DWI has been reported to be able to distinguish between malignant and benign LNs with sensitivities ranging from 52% to 98% and specificities ranging from 88% to 97% , 32. ADCP < 0.001). In addition, the patients undergoing CTP were injected with an iodinated contrast agent that has been confirmed to have some nephrotoxicity; meanwhile, patients inevitably suffer from radiation injury.In CTP, there were general differences between benign and metastatic LNs, but with some overlap . We obtaIn conclusion, DWI exhibited better sensitivity and specificity than CTP. DWI may be the preferred technique for the preoperative assessment of LNs in head and neck SCC.


Garcinia kola, and L-ascorbic acid (AA) are known to protect against oxidative damage in vivo. This study evaluates the protective capacity of KV and AA on chlorambucil-induced oxidative stress in the testes of rat. Twenty male Wistar rats (180–200 g) were randomized into four groups: I: control, II: chlorambucil (0.2 mg/kg b.w.), III: 0.2 mg/kg chlorambucil and 100 mg/kg KV, and IV: 0.2 mg/kg chlorambucil and 100 mg/kg AA. After 14 days of treatments, results indicated that chlorambucil caused significant reduction (P < 0.05) in testicular vitamin C and glutathione by 32% and 39%, respectively, relative to control. Similarly, activities of testicular GST, SOD, and CAT reduced significantly by 48%, 47%, and 49%, respectively, in chlorambucil-treated rats relative to control. Testicular MDA and activities of ALP, LDH, and ACP were increased significantly by 53%, 51%, 64%, and 70%, respectively, in the chlorambucil-treated rat. However, cotreatment with KV and AA offered protection and restored the levels of vitamin C, GSH, and MDA as well as SOD, CAT, GST, ACP, ALP, and LDH activities. Overall, kolaviron and L-ascorbic acid protected against chlorambucil-induced damage in the testes of the rat.Chlorambucil (4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid) is an alkylating agent, indicated in chronic lymphocytic leukaemia. Kolaviron (KV), a biflavonoid complex from The upsurge in the synthesis and use of anticancer agent has generated questions about the safety of these substances on normal tissues. Most drugs administered in cancer chemotherapy are well known to be associated with toxic side effects . Among tChlorambucil (4-[4-[bis (2-chloroethyl) amino] phenyl] butanoic acid), Chlorambucil and its derivatives form covalent bonds with proteins and DNA of neoplastic cells resulting in structural and functional damage to DNA . While cThe testes have evolved a formidable antioxidant defence to protect against the damaging effects of free radicals and oxidants which may have negative implication on spermatogenesis . These c Garcinia kola, containing Garcinia biflavonoids GB1 and GB2 and kolaflavanone , and thiobarbituric acid (TBA) were all purchased from Sigma Chemical Company . Assay kits for alkaline phosphatase (ALP) and acid phosphatase (ACP) were obtained from Randox Laboratories Ltd. . Assay kit for lactate dehydrogenase (LDH) was a product of Cypress Diagnostics, Belgium. All other reagents used were of analytical grade and were obtained from British Drug House (BDH), Poole, England.Chlorambucil (Leukeran) was a product of Aspen Pharm Trading Limited, Dublin 1, Ireland. 1-Chloro-2,4-dinitrobenzene (CDNB), 5′,5′-dithiobis-2-nitrobenzoic acid (DTNB), glutathione (GSH), epinephrine, hydrogen peroxide (H Garcinia kola (3.5 kg) and characterized according to the method of Iwu et al. [Kolaviron was extracted from the fresh seeds ofu et al. . Brieflyu et al. . ad libitum. The experimental protocol relating to animal handling conformed to the Guidelines of the National Institute of Health—Guide for the Care and Use of Laboratory Animals .Twenty inbred male rats (Wistar strain) weighing between 180 and 200 g were obtained from the animal holding unit of the Department of Chemical Sciences, Ajayi Crowther University, Oyo, Nigeria. The rats were acclimatised under laboratory conditions prior to the commencement of the study. The animals were housed in wire meshed cages maintained at standard conditions of temperature and humidity with an alternating light cycle (12 hr light/dark). They were fed with standard rat pellet  and were supplied waterThe rats were randomised into four experimental groups (I–IV) of 5 animals each. Group I (control) animals were administered distilled water. Animals in group II received chlorambucil at a dose of 0.2 mg/kg body weight (b.w.). Group III animals were coadministered chlorambucil (0.2 mg/kg b.w.) and kolaviron (100 mg/kg b.w.). Animals in group IV were coadministered chlorambucil (0.2 mg/kg b.w.) and L-ascorbic acid (100 mg/kg b.w.). The dose of chlorambucil administered to the animals was based on the recommended dose in the treatment of Hodgkin's lymphoma (0.1-0.2 mg/kg/day) , while dAnimals were sacrificed 24 hours after the last treatments by cervical dislocation and the testes were carefully excised from each animal for preparation of cytosolic fraction and histopathological analysis. Testicular samples for histopathological analysis were immediately fixed in Bouin's solution for 24 hours.The testes excised from rats were blotted of blood stains, rinsed in ice-cold 1.15% KCl, and homogenized in 4 volumes of ice-cold 0.01 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 12,500 g for 15 min at −4°C  and the supernatants, termed the postmitochondrial fractions (PMF), were aliquoted and used for biochemical assays.The protein concentration in the testicular homogenate was determined by the Biuret method of Gornall et al.  using boThe method of Misra and Fridovich  as descrTesticular reduced glutathione (GSH) level was determined according to the method of Jollow et al. . The chrTesticular acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) activities were determined using Randox diagnostic kits. ACP and ALP activities were determined by the method of Tietz . The p-nThe method of Baker and Silverton  was emplP < 0.05 was accepted as statistically significant.The results were expressed as mean of 5 replicates ± SD. Data obtained were subjected to one-way analysis of variance (ANOVA) and complemented with Duncan's multiple range test using SigmaPlot Statistical Software. A value of Following two-week exposure to chlorambucil, the testicular activities of SOD, CAT, and GST were significantly decreased by 47%, 49%, and 48%, respectively, in chlorambucil-treated animals in comparison with the controls . KolavirAs presented in The testicular acid phosphatase (ACP) activity  was signRepresentative photomicrographs of the testicular sections from rats are shown in This study investigated the protective role of orally administered kolaviron and ascorbic acid in chlorambucil-induced testicular toxicity in rat. Chlorambucil is an anticancer agent indicated for chronic leukaemia and may have the capacity to induce oxidative stress or deplete tissue antioxidant reserve during chemotherapy . The tesIn this study, we observed a significant reduction in testicular activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase as a result of CLB administration. SOD catalyzes the reaction involving a rapid dismutation of superoxide radical to hydrogen peroxide and dioxygen while CAT converts the hydrogen peroxide formed in this process and other cellular processes into water and molecular oxygen. Reduction in the activities of SOD and CAT by chlorambucil may predispose the testes to oxidative damage . GlutathAA is a vital antioxidant of the aqueous phase of the cell and rapidly scavenges free radicals. It also plays an important role in the regeneration of vitamin E—the membrane bound antioxidant . The AA Activities of testicular marker enzymes such as acid phosphate (ACP), alkaline phosphate (ALP), and lactate dehydrogenase (LDH) are considered functional indicators of spermatogenesis. The reduction in the activity of ACP by the administration of CLB was supported by Ananthan and Kumaran  where adThe degeneration of testicular histological structure by CLB may be a result of oxidative damage. This can also result in the degeneration of germ cells and poor sperm quality . KV and From our present study, it may be suggested that chlorambucil impairs testicular antioxidant system and causes degenerative changes in the germ cells. However, kolaviron and ascorbic acid positively modulate the effect of the drug on the antioxidant status and effectively prevent chlorambucil-induced testicular toxicity.


Despite the increasingly well-documented evidence of high genetic, ethnic, and linguistic diversity amongst African populations, there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system. With the high disease burden in southern Africa, HLA diversity data is increasingly important in the design of population-specific vaccines and the improvement of transplantation therapeutic interventions. This review highlights the paucity of HLA diversity data amongst southern African populations and defines a need for information of this kind. This information will support disease association studies, provide guidance in vaccine design, and improve transplantation outcomes. The human leukocyte antigen (HLA) complex on chromosome 6, also known as the major histocompatibility complex (MHC) in all mammals, consists of highly polymorphic genes whose protein products present antigens to T cells as part of an immune response to infections , 2. HLA The World Health Organization (WHO) indicates that there is a high burden of disease in southern Africa, especially communicable diseases such as HIV/AIDS, TB, and malaria . Despite cis/trans assignment of DNA bases in a heterogeneous sample [HLA typing methods have evolved from low resolution serology typing to high resolution DNA sequencing based technologies (SBT). Despite high resolution, SBT has limitations of mostly typing certain exons within the HLA loci . The ants sample , yieldins sample , 21, 22.s sample .African populations have been shown to be genetically diverse  and are There are an ever increasing number of HLA alleles, reflecting the rate of discovery of the diversity of the gene loci , 2. TherPrugnolle et al. suggested that up to 39% of observed HLA class I diversity was due to geographical distance (and consequently human migration history) from the source of modern humans (assumed to be Ethiopia in this study), with the unaccounted source of diversity most likely being from pathogen driven selection . GeneralThere is growing evidence for positive selection being responsible for maintaining HLA polymorphisms, most likely due to overdominant selection (heterozygote advantage) which maintains allelic lineages for much longer periods of time than neutral polymorphisms , 45–47. ∗41:21, HLA DQB1∗02:10, HLA QA1∗01:12); (b) deletion leading to frame shift (HLA A∗01:123N); (c) intralocus gene conversion ; and (d) interlocus gene conversion (HLA C∗07:294) [Other mechanisms of HLA diversity generation include point mutations : gene conversion  and gene cross over . Gene cross over, which is a form of recombination that can be intra/inter-HLA loci during meiosis, enables exchange of genetic material linked to the generation of novel alleles in offspring as described by Carrington . Other r∗07:294) . It is i∗07:294) .The human immune system uses HLA's uniqueness in every individual to recognize self from nonself; hence the body only mounts an immune response against foreign cells/molecules under normal conditions. Transplantation as a therapeutic intervention matches donor and recipient HLA molecules to decrease the likelihood of rejection . The likGVHD is an immunocompetent donor T cell mediated response against the recipient's immune system which is common in unmatched donor-recipient pairs. Acute GVHD can be reduced by donor T cell depletion, but this increases the risk of rejection, malignant disease relapse, and impaired immune recovery , 64. In ∗67 and -B∗15 have been associated with fatal outcomes and B∗07 and B∗14 have been associated with nonfatal Ebola infections [The high disease burden in southern Africa  offers afections .Haplotype analysis gives information on disease/condition associated alleles, which are assumed to be inherited as blocks due to strong linkage disequilibrium . HLA allThere is documented evidence of geographical distribution of human genetic variation, which helps to understand human evolution, migration and adaptation to different environments and pathogens . SeveralThere are marked differences in HLA diversity distribution globally, with geographically separated regions showing varying degrees of diversity , 42, 48.http://dx.doi.org/10.1155/2015/746151). http://www.allelefrequencies.net/) [http://www.allelefrequencies.net/) [To highlight the paucity of HLA diversity data in southern Africa, this review used a comprehensive literature search for previously published work on HLA diversity together with the Allele Frequency Net Database (AFND) to determine the information in the public domain. The key search terms for articles were “HLA AND genetic diversity AND southern Africa”. Allele frequency data from AFND was extracted for sub-Saharan African countries, from which southern African data was compiled   used in es.net/) , 48. Fores.net/) , 48. Thees.net/) , 48, res∗30 and its derivatives  are common in black populations . Caucasians and Tamils had a completely different HLA-A allele frequency distribution compared to the other populations. HLA-A∗02:01:01 was most frequent (0.26) in South African Caucasians, as has been reported by Solberg et al. (HLA-A∗02:01) in European (27%) and white American (20%) populations [∗02:01 allele and its derivatives being restricted mostly to white populations. For the HLA-B locus, B∗58  was most common in Mozambicans, Black South Africans (including Zulus and Tswanas) as highlighted in Tables ∗06:02 being commonly high in Black South Africans and Tamils. Although more than ten years old (2004), the study by Cao et al. identified A∗02:02, A∗34:02, A∗36:01, A∗74:01, B∗15:03, B∗42:01, B∗53:01, B∗57:03, and B∗58:02 as unique African alleles. Recently, diverse and novel HLA alleles have been reported in sub-Saharan populations, for example, HLA class II as reviewed in Ayele et al. [∗30:01:02, A∗30:02:02, A∗68:27, B∗42:06, and B∗45:07) were reported in a recent South African study [∗02:01, -A∗34:02, and -B∗58:02 in HIV negative controls in Zimbabwe [South Africa had the highest number of HLA data sets from the AFND compared to other southern African countries . Some soulations . This sues A∗30:0, A∗30:02es A∗30:0, A∗30:02e et al.  and HLA e et al. , to furtan study . AdditioZimbabwe  compared∗01:01:01 (0.355) in Shona Zimbabweans and DPB1∗13:01 (0.148) in Black South Africans; and least frequent DPB1∗01:01:02, DPB1∗02:02, DPB1∗62:01, DPB1∗65:01, and DPB1∗80:01 (0.002) in Shona Zimbabweans. No alleles were reported for the DPA1 and DQA1 loci. The DQB1 locus was reported only in Botswana, Black South Africans, Shona Zimbabweans, and Venda South Africans. DQB1∗06 in Black South Africans was the most frequent (0.555) with DQB1∗06:15 in Shona Zimbabweans being least frequent (0.002). DRB1 alleles were reported in all the studied populations except in some South Africans . The most frequent allele was DRB1∗11 (0.366) in Black South Africans, while the least frequent ones were DRB1∗16 (0.002) in Mozambicans and DRB1∗03, DRB1∗04:04, DRB1∗12:04, DRB1∗13, and DRB1∗15:01  in Shona Zimbabweans.The AFND reports very few HLA class II alleles amongst southern African populations; only Zimbabwean Shonas and Black South Africans  had HLA-http://www.allelefrequencies.net/) [The number of classical HLA alleles  varies ges.net/) , 48. IntThe number of southern African HLA studies in the AFND is relatively low, reflecting the underrepresentation of this region. The data currently available is mostly low resolution with low sample numbers and is not a true reflection of HLA diversity in the southern African context. This highlights the need for continual submission of southern African HLA diversity data to centralized databases like the AFND. The few studies from southern Africa also highlight the knowledge gap on HLA diversity in this region in this era of high resolution typing. Several HLA disease association studies with allele frequency data have been reported in the region , 93–95; ∗27 restricted CD8 T cells had variable viral replication inhibition capabilities in HIV controllers versus progressors due to a modulation by specific T cell receptor clonotypes [There is limited data on HLA diversity in southern Africa, with most having been generated from disease association studies and which is therefore not a true reflection of the general population. It is often difficult to assign causality of a specific HLA allele to an infection/condition, because of linkage disequilibrium and other factors such as selection pressure, which are dependent on the condition/infection and the other arms of the immune system which are HLA independent . As evidonotypes . There aonotypes , 48 despHLA diversity data forms the cornerstone of population-specific vaccine development, and taking into consideration the high disease burden in southern Africa, information of this nature is particularly important in this region . This reFurthermore, advancement in HLA typing methods such as NGS will help to finely investigate HLA diversity, as previous strategies have targeted a few exons per locus thereby missing some of medically important variants outside the typed regions.+ T cells induce B cells leading to an antigen specific humoral immune response [An understanding of HLA diversity will provide insight into allele frequency dependent selection fitness which varies between populations. This might help understand the high disease burden  and form the basis of vaccine development for the many infectious diseases as well as in the planning of vaccine clinical trials in the region. The paucity of HLA data from this region is a major hurdle in vaccine design . Brumme response . HLA claresponse , active response , and HIVresponse . The comresponse . HLA divresponse , mechaniresponse , 109, anresponse . Posteraresponse .It is generally easier to match donor-recipient pairs from populations with known HLA genotypes than in areas with information gaps , highligIt is thus important to fully understand HLA diversity in the southern African context, to establish HLA-disease associations, to use this data for the informed design of population-specific vaccines against the many diseases, and to improve donor-recipient matching.A Microsoft Excel spreadsheet listing all classical HLA alleles, their frequencies as reported by the AFND and a limited number of disease association studies in southern African populations has been made available online as supplementary Material (S1)


Purpose. To identify retinal pigment epithelium (RPE)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care. Methods. Differential gene expression of over 34,000 well-characterized mouse genes in the RPE/choroid of 6-week-old C57BL/6J mice was analyzed after intravitreal steroid injections at 1 week and 1 month postinjection, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpring GX 12.5 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. Results. Both triamcinolone and dexamethasone caused differential activation of genes involved in “Circadian Rhythm Signaling” pathway at both time points tested. Triamcinolone (TAA) uniquely induced significant changes in gene expression in “Calcium Signaling” (1 week) and “Glutamate Receptor Signaling” pathways (1 month). In contrast, dexamethasone (Dex) affected the “GABA Receptor Signaling” (1 week) and “Serotonin Receptor Signaling” (1 month) pathways. Understanding how intraocular steroids affect the gene expression of RPE/choroid is clinically relevant. Conclusions. This in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in RPE/choroid during intravitreal steroid injections, which likely has consequences in the dysregulation of RPE function and neurodegeneration of the retina. Intravitreal injections of dexamethasone (Dex) and triamcinolone (TAA) are a mainstay in a clinical retinal practice to treat a wide range of pathologies, such as cystoid macular edema secondary to retinal vein occlusion, diabetic macular edema, choroidal neovascularization in macular degeneration, and noninfectious uveitis –6. TheseGlucocorticoids have powerful anti-inflammatory action, but the exact underlying mechanism of this effect remains poorly defined. Additionally, intravitreal administration of glucocorticoids may lead to side effects such as cataract progression, development of steroid-induced glaucoma, and central serous chorioretinopathy , 8. in vivo, since they too are affected by both disease pathogenesis and intravitreal steroidal treatment at clinically relevant time points of 1 week and 1 month postinjection. The purpose of this study was to evaluate the gene expression effects that intravitreal Dex and TAA have on the RPE/choroid and the differences between them at clinically relevant time points.Clinical decision making may be greatly enhanced with a better appreciation of the differential gene expression induced by intravitreal steroids. In a previous study using a mouse model, we demonstrated that intravitreal steroids affect the expression of several genetic pathways and alter the balance between neuroprotective and neurodegenerative processes in retina . In this ad libitum. Three groups of animals with 4 animals per group were used for each experiment.This study protocol was approved by the Institutional Animal Care and Use Committee at the University of California Davis before initiation. The study was conducted according to the approved protocol and in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Six- to eight-week-old male C57BL/6J mice  were used for all experiments. Mice were housed at 21°C, under a 12-hour light/12-hour dark cycle, with food and water suppliedOne mL of ketamine (100 mg/mL), 0.1 mL of xylazine (100 mg/mL), and 8.9 mL of sterile water were mixed to prepare a final solution for injection. Animals were anesthetized by injecting a 0.1 mL/10 g of body weight intraperitoneally.μL of solution. In group 1 (n = 4) animals received balanced salt solution (BSS), group 2 (n = 4) dexamethasone (10 μg), and group 3 (n = 4) TAA (25 μg). The solvent for Dex and TAA was BSS. All mice included in this study were injected on the same day at the same time. At weeks 1 and 4 after the injections, the eyes were harvested for ocular dissection and total RNA isolation.Intravitreal injections were performed transconjunctivally in anesthetized C57BL/6J mice with Hamilton 33 g needles delivering 1 2 chamber and the RPE/choroid tissue was harvested using published procedures [μL of RNALater, and stored at −20°C.At days 7 and 30 postinjection mice from each group were sacrificed using a COocedures . Sample QIAGEN's RNeasy isolation kit was used to isolate total RNA. As a first step tissue was placed in Qiazol and pushed through syringe with 17 g needle several times so the pigmented cells of RPE would be released, while sclera stayed intact as a clear sheet and was removed from the tube with needle. Since the choroid tends to be firmly attached to the inner sclera, it is likely that most of it stayed attached. RNA samples were run on an Agilent BioAnalyzer microfluidics chip RNA Nano 6000 to assess quality and quantity. Out of four samples, three samples having the highest quality (RNA Integrity Number (RIN) ≥ 7 value) were labeled as probes for the Affymetrix GeneChip microarrays .RNA samples were labeled using 0.100 ug of total RNA, following the manufacturer's protocol for GeneChip Target Labeling 3′ IVT Express Kit . After labeling, probes were hybridized to the Affymetrix Mouse Genome 430 2.0 GeneChip microarrays. This array contains 45,101 probe sets, analyzing the expression of 39,000 transcripts and variants from over 34,000 well-characterized mouse genes. Hybridization was performed in the UC Davis Genome Center Microarray Core Facility using a standard procedure . The total data set included 18 GeneChip microarrays.Microarray data were analyzed using GeneSpring GX 12.5 microarray analysis software and Ingenuity Pathway Analysis (IPA) software for biologically relevant changes of expression in genes and related pathways of the RPE/choroid.p ≤ 0.01. The Benjamini-Hochberg post hoc correction method was too conservative for microarray results with limited number of biological replicas such as this. No probe sets met this significance level of testing. Therefore, a different set of stringency requirements was applied to reduce the risk of false positives. To identify biologically relevant gene expression changes for each of the time point/treatment conditions, Volcano plot analysis [t-test and asymptotic p value computation. The Volcano plot is a scatter plot of the fold change versus the p value . It is commonly used to simultaneously depict the p value and the fold change for gene selection cutoff schemes in microarray data. It may be viewed as a summary of “statistical” significance and “biological” significance over a large number of genes. We employed the standard approach of using a p value (p ≤ 0.05) as the primary criterion followed by fold change (−1.5 ≥ FC ≥ 1.5) as the secondary criterion to select differentially expressed genes. This approach ensures control of false-positive error and preserves the desired biological significance [One-way ANOVA was used in GeneSpring to identify statistically significant genes at a significance level of analysis  was perfificance .http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=%20GSE49872). Supplementary Material (available online at http://dx.doi.org/10.1155/2016/9856736) attached to this paper contains the complete list of the differentially expressed genes identified by ANOVA at p ≤ 0.01 and Volcano plot at (−1.5 ≥ FC ≥ 1.5) p ≤ 0.05.The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus , 14 and Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software . The lists of candidate genes identified by GeneSpring analysis were uploaded to the IPA site and IPA core analysis was applied to give us the list of activated pathways and gene networks with the highest degree of significance.Quantitative PCR (qPCR) was used as a second method for validation of the results of microarray data analysis for several candidate genes . All gene expression assays  were conducted in the real-time PCR Research and Diagnostic Core Facility at University of California Davis. Control genes  were also included in order to validate the results of the qPCR.For the genes used for qPCR validation of the microarray data, threshold cycle (Ct) values were used to determine the quantity of gene copies. For each gene, the mean of three samples was calculated and used for analysis. The fold change for each gene was determined compared to the mean of all 3 control genes used.p value of p ≤ 0.05 and 430/36,136 at p ≤ 0.01 when the entire 18-chip sample set was considered.Data was analyzed using GeneSpring 12.5. Raw data was imported as  .cel files and underwent RMA normalization with using baseline values of median of all samples. Next step was filtering by expression on the raw data for the signals between 20% and 100%, to eliminate too weak signals which were on the borderline of background noise. The signals for the probes were retained only if they were present in at least 1 out of 6 conditions within the range. The number of 36,136 probe sets passed out of total of 45,101 probe sets on the GeneChip. Statistical analysis by one-way ANOVA identified 1,868 out of 36,136 probe sets differentially expressed at a t-test identified a set of common genes similarly regulated by both steroids, as well as, a set of unique genes differentially regulated by each steroid. Figures p ≤ 0.05. A list of common genes and unique genes for each steroid/time point/regulation group is presented in Supplementary Tables S1–S6. The full lists of differentially expressed genes by ANOVA at p ≤ 0.01 and p ≤ 0.05, as well as t-tests −1.5 ≥ FC ≥ 1.5, p ≤ 0.05, can be found in the Supplementary Tables S7 and S8.Comparison of treatment versus control for each time point using an unpaired p ≤ 0.01. From this data set the top candidate pathway affected was “Circadian Rhythm Signaling” . Biological networks and pathway analysis was performed utilizing Ingenuity Pathway Analysis (IPA) software to identify integral biological pathways that are commonly or uniquely affected by the steroids. Statistical analysis (ANOVA) of the entire 18-chip sample set identified 430 probe sets differentially expressed at gnaling” . An ANOVp values. Mammalian circadian clock generates molecular circadian rhythm through coupled transcription/translation feedback loops which involves core clock genes: Period (Per) 1 and Per 2, cryptochrome (Cry) 1 and Cry 2, Clock, and Aryl hydrocarbon receptor nuclear translocator-like  genes. Recently, it has been shown that the transcription factor Npas2 (Mop4) has been able to functionally substitute for Clock in the mouse forebrain, thus adding the total number to 7 genes [ 7 genes . The cir 7 genes . The glu 7 genes , 18. At Molecular timekeeping in cells is synchronized and sustained by interneuronal neuropeptidergic signals. Genes do not work alone, but in an intricate network of interactions. Ingenuity Pathway Analysis is a hypothesis generating tool that helps interpret the data in the context of biological processes, pathways, and networks. While each gene can be interpreted as a one-dimensional representation of data, we can think of a pathway (linked list of interconnected genes) as a two-dimensional representation of data, while gene networks represent a multidimensional representation of the data. p ≤ 0.05) taking into account only genes that have the highest fold change was done for each steroid/time point to identify additional signaling pathways unique for each steroid/time point. In this section are highlighted changes in gene expression of genes of interest for ophthalmic diseases, neurodegeneration and neuroprotection, and related signal transduction pathways. The top 6 relevant canonical pathways for each time point with the significance of representation identified by IPA are presented in Figures An alternative analysis by Volcano plot in GeneSpring  .++ signaling pathway , Trdn, triadin (FC = −2.09), Asph, aspartate-beta-hydroxylase (FC = −1.87), Ptger3, prostaglandin E receptor 3 (FC = −2.25), SLN, and sarcolipin (FC = −2.56).The most affected pathway for TAA in week 1 postinjection was Ca pathway . Ca++ siinopathy  and in aammation . The genInterestingly, the most upregulated gene was Lcn2, lipocalin 2, an innate immunity protein, with a role in cellular response to oxidative stress, drug insult, IL-1 response, and TNF-alpha response (FC = 19.3). The most downregulated ones were crystallins , genes whose protein products play a role in development of the eye, in visual perception, and have a role as heat-shock proteins (Table S2). It shows that expression of signal transduction genes was influenced by TAA. The genes Arntl, Per1, Gnb3, and Arrdc2 were upregulated (FC = 1.55–2.33), while Abra, Ptger3, Dpp4, Chrna1, Ifi204, Murc, Itsn1, Prokr1, and Ddit4l were downregulated (FC = −2.42 to −1.56).Only three genes were found in common for both Dex and TAA: Myh8, Tpm3, identified as self-antigen in patients with Behcet's disease with posterior uveitis , and EloTen genes had significantly altered expression following Dex at 1 month postinjection (Table S4). One of the upregulated genes was Slc18a2, a solute carrier with a role in neurotransmitter transport, as well as Serotonin and Dopamine Receptor Signaling , 24. The++ ion transport via voltage gated Ca++ channel activity.There were eight affected genes in this group (Table S5), the most interesting being the downregulation of glutamate receptor Grm7. This receptor has a role in neuroprotection, synaptic transmission, and regulation of CaAs a general observation and contrary to the data of steroid effects in the retina , there wt-test with FC = 1.5, p < 0.05 (Table S6). That gene was Fibrillin2 (Fbn2), an extracellular matrix constituent, with Ca++ ion binding capabilities, mildly upregulated for both steroids (FC = 1.5–1.6).Only one gene was common for both steroids after a Independent qPCR validation of representative differentially expressed genes was performed using commercial gene expression assays . The fold changes and their direction were confirmed for the chosen genes, thus corroborating the microarray data analysis results. Data are presented in Retinal pigment epithelial cells are responsible for the health and maintenance of retinal cells. RPE dysfunction plays a role in many ocular disorders including age-related macular degeneration, central serous chorioretinopathy, white-dot syndromes, and retinitis pigmentosa, which can lead to permanent visual distortion and blindness. One of the main functions of the RPE is regulation of disc shedding by photoreceptors, which is a rhythmic process essential for photoreceptor health and proper function. This process has been shown to be under circadian clock control –28. The The cyclic pattern of expression of circadian proteins is dependent on a number of external cues or “zeitgebers” including light exposure, feeding pattern, and exercise. The primary and the strongest zeitgeber is light, which starts the process, and then there are many secondary ones, conveying the information to peripheral tissue. Secondary ones start with serotonin and melatonin and then there are more downstream ones, such as insulin and glucocorticoids , 39. GluIn our study “Circadian Rhythm Signaling” was the primary identified pathway by IPA. This finding suggests that intravitreal injection of steroids has a significant effect on the RPE/choroid, causing dysregulation of the retinal circadian clock, which is observable at 1 week postinjection and continues through 1 month postinjection. Disruption of circadian rhythm has been demonstrated to promote inflammation –47 and hOn the other hand, it has been reported that the release of glucocorticoid hormones happens in an hourly or ultradian rhythm in a pulsating manner and that levels of free glucocorticoid hormones are highly synchronized between blood, subcutaneous tissue, and the brain. By administering a bolus of steroids, this fine regulation is disrupted, and the loss of the ultradian rhythm may subsequently result in gene expression changes in downstream target tissues . Our resThe most affected pathway for Dex week 1 postinjection is Phototransduction Pathway . Why genTo verify whether some of the rod and cone genes are actually expressed in RPE, we checked for the presence of several phototransduction genes listed in the tables from Newman et al.  , leading to glutamate receptor activation [++ concentration in neurons and astrocytes and protect neurons against glutamate toxicity [The main signal transduction pathways that appear to be activated by steroids in the RPE are glutamate and Cativation , 58. A rtoxicity .L-glutamate is the major excitatory neurotransmitter in the central nervous system that activates both ionotropic (NMDAR) and metabotropic (GRM) glutamate receptors. Glutamanergic neurotransmission is involved in most aspects of normal neural function, such as intracellular signal transduction, axonal targeting, and synaptic clustering. 7, with a role in neuroprotection, synaptic transmission, and regulation of Ca++ ion transport via voltage gated Ca++ channel activity, has been shown to confer susceptibility to many neuropathologic conditions such as age-related hearing impairment [Our results show upregulation of Grm7 with Dex and downregulation with TAA at 1 week. At 1 month postinjection we see downregulation of Grm7 for both steroids. Grm7, glutamate receptorpairment , 60, 61,pairment , 62, andpairment –65. MostTwo genes that were consistently downregulated at both time points for TAA were Rbfox1 and neuronal mRNA splicing factor Fox-1, which splices mRNAs encoding proteins important in synaptic transmission and membrane excitation . The secOur results from differential gene expression of over 34,000 well-characterized mouse genes of the RPE/choroid of C57BL/6J mice after intravitreal steroid injections at 1 week and 1 month postinjection suggest that dysregulation of glutamatergic and dopaminergic neurotransmission signaling may represent a novel target for therapeutic approaches in the treatment of steroid-induced glaucoma.In conclusion, in our previous study , we haveSupplemental material contains the complete list of the differentially expressed genes identified by Volcano plot at -1.5≥ FC ≥1.5; p≤0.05 (Tables S1-S6), and ANOVA at p≤0.01 and p≤0.05 (Tables S7 and S8).


Similar differences were observed for erythrocyte DHA. We conclude that DHA concentrations of Chinese pregnant and lactating women are higher in coastland and lakeland regions than in inland areas. DHA status in the study population appears to be stronger than populations from other countries studied to date.Few studies have examined docosahexaenoic acid (DHA) in pregnant and lactating women in developing countries like China, where DHA-enriched supplements are increasingly popular. We aimed to assess the DHA status among Chinese pregnant and lactating women residing areas differing in the availability of aquatic products. In total, 1211 women in mid-pregnancy (17 ± 2 weeks), late pregnancy (39 ± 2 weeks), or lactation (42 ± 7 days) were enrolled from Weihai (coastland), Yueyang (lakeland), and Baotou (inland) city, with approximately 135 women in each participant group by region. DHA concentrations were measured using capillary gas chromatography, and are reported as weight percent of total fatty acids. Mean plasma DHA concentrations were higher in coastland  and lakeland women  than inland women  ( Docosahexaenoic acid DHA, 22:6n-3), as a fundamental constituent in cell membranes, is indispensable to the structure and function of the retina and central nervous system ,2. DHA i2:6n-3, aet al. observed an obvious positive correlation between fish consumption and DHA levels. DHA status may also vary across ethnicities. In a study [et al. reported significant ethnic differences in maternal DHA status despite controlling for fish intake. Besides, in an earlier study [et al. observed consistent decreases in the DHA weight percentage of total fatty acids as women progress from early pregnancy to delivery.The availability and consumption of aquatic products plays an important role in DHA status. In a study  conducte a study  compariner study  involvinIn China, fish availability varies considerably in populations, and is greatest in coastland and lakeland regions in contrast to inland areas. In this study, we aimed to examine DHA status in a diverse population of Chinese pregnant and lactating women from coastland, lakeland, and inland areas.The DHA Evaluation in Women (DEW) study was a cross-sectional survey conducted from May to July 2014 in three cities of China: Weihai (selected to represent the coastland population), Yueyang (selected to represent the lakeland population) and Baotou (selected to represent the inland population). Weihai is surrounded on three sides by the Huang Sea. Yueyang is near Dongting Lake, the second largest freshwater lake in China. Baotou is a typical inland city in the Mongolian Plateau. A total of 1211 apparently healthy women who were at mid-pregnancy , late pregnancy , or lactation (42 ± 7 days postpartum) were recruited approximately equally from the three regions, with on average 135 (127–138) women in each group per region. Eligible women were 18–35 years old, were local permanent residents, and had singleton pregnancies. An additional inclusion criterion for the lactating group was current breastfeeding. Women were excluded if they had been diagnosed with any cardiovascular, metabolic, and renal diseases, mental disorder, or aquatic food allergy; or had participated in other research projects in the past 30 days. Women with severe vomiting after 16 weeks of gestation were also excluded for the mid-pregnancy group. The research protocol was approved by the Institutional Review Boards/Human Subjects Committees at Peking University Health Science Center , and all participating women signed informed consents.Participants were enrolled from four local hospitals: one located in Weihai, one in Yueyang, and two in Baotou. Trained obstetricians or nurses from the hospitals completed enrolment and data collection. A structured questionnaire was used to collect maternal characteristics, including birthdate, ethnicity, height, pre-pregnancy weight, and educational attainment. For pregnant women, gestational age at enrolment was calculated according to the date of the last menstrual period. For lactating women, self-reported gestational age at delivery and parity were also collected.Fasting venous blood (~5 mL) was collected from the antecubital vein into ethylenediaminetetraacetic acid (EDTA)-containing tubes. Samples were kept in the refrigerator at 5 °C for at least 30 min and then centrifuged at 3000× g for 10 min to separate plasma and erythrocytes. The erythrocytes were washed out with normal saline. Both plasma and erythrocyte samples were stored at −20 °C in the hospital for approximately 10 days, and then were transported on dry ice frozen at −80 °C to the central laboratory where samples were stored at a −80 °C freezer. Notably, the temporal storage of blood samples at −20 °C might have somewhat compromised DHA in erythrocyte ,12.To ensure data quality and to standardize data collection methodologies across sites, study staff attended training workshops and each site had a designated investigator who oversaw the standardized data collection procedures. In addition, senior investigators met weekly and provided additional oversight.et al. [The extraction and derivatization of total lipids in plasma and erythrocyte samples were carried out using a modified method of Folch et al. . The intT-tests were used to examine the differences between women in inland and lakeland/coastland as well as between women in mid-pregnancy and late-pregnancy/lactation. Additionally, we explored whether DHA concentrations varied across subgroups based on maternal age , pre-pregnancy BMI , and education attainment  by using multiple linear regression with adjustments for covariates including region and participant group.DHA concentrations are presented as means ± SDs. One-way analyses of variance were performed to compare overall differences in DHA concentrations among participant groups and regions. To illustrate the relationship between plasma and erythrocyte DHA, we performed several sets of Pearson correlation analyses. We first estimated the overall correlation coefficient between plasma and erythrocyte relative DHA concentrations. Because the scatterplot indicated an obviously different correlation pattern between individuals with erythrocyte DHA concentrations ≥3% and those <3%, separate correlation analyses for the two subgroups were then performed. We also repeated the above-mentioned correlation analyses within the 9 subgroups defined by region and participant group.p < 0.05. All statistical analyses were performed by using SPSS version 20.0 .Significance was set at p values < 0.001). In the lactating group, primiparous women accounted for 87.5%, 81.5%, and 86.0% in coastland, lakeland, and inland, respectively, and corresponding mean gestational age at delivery in the three regions was 39.3, 39.3, and 39.1 weeks, respectively.Mean plasma and erythrocyte relative DHA concentrations of 9 region- and group-specific subgroups ranged 1.67%–3.19% and 5.06%–7.59%, respectively; corresponding absolute values ranged between 60.5 and 146.2 μg/mL and 89.3–127.7 μg/mL, respectively .p values < 0.001). The concentrations were higher in coastland and lakeland women than in inland women. Similar regional differences in DHA absolute concentrations were observed ; the concentrations were higher in mid-pregnancy than in late pregnancy and lactating women across regions . Similar patterns were also observed for erythrocyte DHA relative concentrations, although the difference was not significant among the three groups of lakeland women. In contrast, the plasma DHA absolute concentrations were highest in late-pregnancy women, followed by in mid-pregnancy, and lowest in lactating women. The erythrocyte DHA absolute concentrations were relatively higher in mid-pregnancy in inland, in mid- and late-pregnancy in lakeland and coastland women . The correlation profile was visually examined via a scatterplot and an obviously different correlation pattern was detected between women with erythrocyte DHA concentration ≥3% and those <3% , but not in those <3% . After excluding women with erythrocyte DHA concentrations <3%, most of region- and group-specific correlation coefficients were substantially augmented by 18%–64%  [versus 2.56% in plasma and 6.07% versus 6.80% in erythrocyte) [FADS genotypes influence maternal DHA concentrations [Consistent with a previous study , we founet al. , DHA conet al.  or Germaet al. , and cloet al. ,23,24,25 plasma)  and Cubahrocyte) . Besidestrations ,29.r = 0.63). The correlation was even stronger (r = 0.73) in women with erythrocyte DHA ≥3%, but not at all in those with erythrocyte DHA <3% ; interestingly, the plasma concentrations in the two subgroups of women (2.25% versus 2.04%) did not differ materially. One explanation regarding the inflection point in the correlation between plasma and erythrocyte DHA was that erythrocyte could serve as a reservoir and its DHA could be transported into plasma for body need in case of a lower DHA status [As expected, we observed a moderate to high level of positive correlation between plasma and erythrocyte DHA  was the largest compared to previous similar studies (~50). However, our study also has several limitations. Firstly, the study was not longitudinal, and was conducted only in urban areas of China, possibly confining the generalization of findings. Secondly, the mean erythrocyte DHA concentrations might be slightly lower than the true values due to the temporary storage of blood samples under −20 °C ,12. ThirIn summary, DHA concentrations of Chinese pregnant and lactating women are higher in coastland and lakeland regions than in inland areas. DHA status in our population appears to be stronger than populations from other countries as reported in the literature. DHA concentrations varied by region and participant groups, which is likely due to differences in consumption of aquatic products or changes in physiological needs for DHA.


Allergic rhinitis is a morbid condition that is frequently overlooked by patients and physicians. This type of atopy has not been adequately investigated in the United Arab Emirates.This cross-sectional, population-based observational study was conducted in the seven Emirates . It used the European Community Respiratory Health Survey (ECRHS II) to screen for allergic rhinitis in people living in this region.p ≤ 0.001). Dust was non-seasonal (p ≥ 0.121) and animal allergy was worse in the winter (p = 0.024) and spring (p = 0.044). Spring symptoms were less common in people living in the inner city (p = 0.003).Symptoms of allergic rhinitis were present in 85 (7%) of the 1,229 study population. Only 33 (39%) patients received treatment. Seventy-six (89%) patients had asthma. Thirty-seven (44%) patients were poly-sensitized. Symptoms were aggravated by dust (59%), grass/pollens (44%) and proximity to animals (21%). Winter was the peak season (37%), followed by spring (30%), autumn (18%) and summer (15%). Grass/pollen allergies were clustered in the winter, spring and summer  of these patients had environmental triggers and remained untreated. Allergic rhinitis awareness and measures to control allergens and dust are needed. The impact of preventing allergic rhinitis on other common atopies in the region deserves future studies. Allergic rhinitis is an IgE-mediated inflammation of the nasal mucosa that is frequently triggered by inhaled allergens . The symhttp://www.who.int/gard/publications/GARDGMreport2013.pdf). A reported prevalence of self-reported seasonal and perennial nasal symptoms in the United States is 30% [Prevalence of allergic rhinitis has been recently estimated by the World health organization  were random representatives of the inner cities, suburbs, small towns, villages, and farms. The study sample size was calculated based on the “UAE Census Estimation - 2009”, as previously described . There wECRHS II was used to identify individuals with nasal or eye symptoms that were consistent with allergic rhinitis. A face-to-face community survey was conducted across the seven Emirates  between January 2010 and March 2010. All participants were informed about the voluntary nature of the study and a signed written informed consent was obtained from each participant. There were no exclusion criteria. The research protocol was approved by the Clinical Research Ethics Authority of Dubai.The ECRHS II screener questionnaire was translated to Arabic and the interviews were conducted using a side by side bilingual (English/Arabic) version  in the screener questionnaire were invited to answer the modified, bilingual version (English and Arabic) of ECRHS II main questionnaire  software version 19.0 for Windows was used. Logistic regression analysis was used to identify independent predictors of triggers  and seasons of symptoms. One hundred eighty-eight (15%) of the 1,229 participants answered “yes” to Question 1, 4, 5, or 7, in the screener questionnaire (see above). Of those, 85 (7%) patients answered “yes” to Question 7, and were diagnosed as having allergic rhinitis. Twenty-two (27%) patients were ≤19 years of age, 46 (55%) were 20–44 years of age, and 15 (18%) were ≥45 years of age; two patients did not report their ages. Fifty-eight percent of the patients lived in inner cities, 25% in suburbs, 8% in small towns, 7% in rural areas and 2% in farms.Seventy-six (89%) of the 85 patients had asthma. Sixty-one percent of the patients did not receive any treatment, 13% received steroid nasal spray, 5% received oral antihistamines and 14% received steroid nasal spray plus antihistamines.Thirty-seven (44%) patients were poly-sensitized. House-hold dust was a common trigger; 50 (59%) patients reported worsening of symptoms in dusty areas of their homes or near dusty pillows, rugs or duvets. Grass/pollens were triggers in 37 (44%) patients and proximity to animals in 18 (21%) patients  and 30% reported worsening of symptoms in the spring (March-May). Summer and autumn were less frequent seasons, since many people traveled outside the country in the summer and returned in early autumn Table . Three (p ≤0.001), Table p ≥0.121) and animal allergy was in the winter (p = 0.024) and spring (p = 0.044), Table p ≥0.374), and spring symptoms were less common in people living in the inner city (p = 0.003), Table Grass/pollen allergies were clustered in the winter, spring and summer  is the most common allergen in Turkey [The prevalence of physician-diagnosed allergic rhinitis in five Middle East countries  is 9%, with dust being the main trigger . These rn Turkey .The prevalence of allergic rhinitis in school children from Saudi Arabia is 24%, Turkey 16%, Kuwait 41%, Lebanon 45%, and Al-Ain city (Abu Dhabi) 36% ,15-18. BConstruction produces numerous amounts of fine dust in the air, which is a major source of airway irritation. Consistently, dust is shown here to be a main airway irritant Tables  and 2. BMany (44%) patients were poly-sensitized. Thirty-seven (58%) of the patients with a specific trigger had grass/pollens allergy. Dust alone was a trigger in only 22 (26%) patients Table . Seasonsp = 0.003).A high prevalence of allergies in urban  areas was reported in Germany, Poland, and Turkey -26. Our Only 46% of the patients made efforts to minimize exposure to dust and dust-mites; only 39% received treatment. Other studies have shown low prevalences of allergic rhinitis treatment  [This study employed the standard ECRHS II for identifying patients with allergic rhinitis. The questionnaire did not address severity of symptoms and did not include a physician diagnosis. Furthermore, this survey was performed in a population with little awareness about allergic rhinitis. Thus, the symptoms might have been underreported. The number of patients was small for sub-analysis by individual Emirates. This important limitation should be addressed in a future study. Follow-up studies should include physician documentations of the allergic symptoms and patients’ response to interventions.Prevalence of allergic rhinitis in the UAE is at least 7%. Dust and grass/pollens are common triggers. Improving public awareness and implementing preventive measures are necessary for controlling allergic rhinitis in the region.The authors declare that they have no competing interests.BM have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and drafting the manuscript and revising it critically for important intellectual content. SAH have made substantial contributions to analysis and interpretation of data, drafting the manuscript, critical revision for important intellectual content, statistical analysis and rewriting the manuscript according to the reviewers' opinions. VPP, AAR and DMV have made substantial contributions to statistical analysis, acquisition of data, and have given their final approval of the version to be published. NS have made substantial contributions to analysis and interpretation of data, and have given final approval of the version to be published. MSB have made critical revision for important intellectual content, drafting the manuscript, and have given final approval of the version to be published. All authors read and approved the final manuscript.Screener Questionnaire.Click here for fileMain Questionnaire.Click here for file