Gingival overgrowth (GO) is a common side effect of the chronic use of cyclosporine (CsA), an immunosuppressant widely used to prevent rejection in transplant patients. Recent studies have reported elevated levels of specific cytokines in gingival overgrowth tissue, particularly TGF-beta, suggesting that this growth factor plays a role in the accumulation of extracellular matrix materials. The effectiveness of azithromycin, a macrolide antibiotic, in the regression of this undesirable side effect has also been demonstrated.In this study, we created an experimental model for assessing the therapeutic effect of roxithromycin in GO and the expression of transforming growth factor beta (TGF-beta2) through immunohistochemistry. We used four groups of rats totaling 32 individuals. GO was induced during five weeks and drug treatment was given on the 6th week as follows: group 1 received saline; group 2 received CsA and was treated with saline on the 6th week; group 3 received CsA and, on the 6th week, ampicilin; and group 4 received CsA during 5 weeks and, on the 6th week, was treated with roxithromycin.The results demonstrated that roxithromycin treatment was effective in reducing cyclosporine-induced GO in rats. Both epithelial and connective tissue showed a decrease in thickness and a significant reduction in TGF-beta2 expression, with a lower number of fibroblasts, reduction in fibrotic areas and decrease in inflammatory infiltrate.The present data suggest that the down-regulation of TGF-beta2 expression may be an important mechanism of action by which roxithromycin inhibits GO. Gingival overgrowth (GO) is a common side effect of certain drugs, such as phenytoin, calcium channel blockers and cyclosporin ,2.Cyclosporine (CsA) has been widely used to prevent organ transplant rejection and to treat various immunodiseases . It seleFibrosis, one of the most important finding in GO, is the result of a variety of biochemical signals from many cell types including inflammatory cells and fibroblasts, which stimulate fibroblast proliferation and extracellular matrix production . The traRattus novergicus albinun), weighing 100 to 150 g. were randomly selected and divided equally into four groups of eight animals each. The control rats (group 1) were daily injected subcutaneously with saline. The experimental rats (group 2) were treated with CsA injected subcutaneously in a daily dose of 10 mg/kg for six weeks and, at the beginning of the 6th week, they were injected subcutaneously with saline. Group 3 was similar to group 2, but in the last week it was treated with 50 mg/kg ampicillin  via gastric feeding. In group 4, the animals received CsA daily for 6 weeks and in the last week they were treated with 40 mg/kg roxithromycin by gastric feeding. The rats were weighed weekly and the dosage was adjusted accordingly. Diet and drinking water were given ad libitum during the experiment.Thirty-two 6-week-old male Wistar rats (® - 10 mg/kg and Vetanarcol® - 90 mg/kg). To investigate gingival alterations over time, stone models were made from silicone impressions of the maxillary anterior region. The higher buccal-lingual width and mesio-distal width of the anterior segment of the maxilla were measured using a digital caliper.At the end of the experimental period, all the animals were sacrificed by means of an overdose of anesthesia  for at least 24 hours. After fixation, the samples were gradually dehydrated in increasing concentrations of ethanol (70% to 100%), cleared in xylene, soaked and embedded in paraffin, according to routine histological methods.The paraffin-embedded fragments were cut with an "820" Spence microtome and 4 μm thick sections were obtained. All gingival samples were included in paraffin with the same orientation. The sections analysed always showed the epithelium, the connective and muscular tissue. The histometry measurements were performed since the ephitelium until the muscular tissue. The histological slides were kept in an incubator to dry, and then the sections were stained with hematoxylin and eosin for histological analysis. Histomorphometry was performed using images captured and evaluated by a computerized Axion Vision  image capture system. Images were captured from four randomly chosen microscopic fields for each histological slide, using the digital camera (400× and 100× magnification) of an Axiostar Plus microscope  and the number of TGF-betapositive cells were counted.The images were stored and submitted to a count of inflammatory cells and of all fibroblast-like cells. Linear measurements were made to obtain the higher diameter of epithelial and gingival connective tissue, using digital marking.Paraffin sections (4 μm of thick) of formalin-fixed tissue were obtained at varying depths from each tissue block. The presence of TGF-beta2 protein in the gingival samples was investigated in paraffin sections by using the avidin-biotin-peroxidase complex procedure (ABC) with an additional step for antigen retrieval with a retrieval solution . In brief, the sections were incubated with polyclonal rabbit anti-TGF-beta2 antibody diluted 1/20 and incubated for one hour at room temperature  followed by a goat anti-rabbit IgG biotinylated antibody . Controls for the ABC procedure were performed by replacing anti-TGF-beta2 antibody with normal rabbit serum, or by omitting the anti-TGF-beta2 antibody. The sections were analyzed via light microscopy, and photomicrographs were taken with an Axiostar Zeiss microscope . The number of the TGF-beta2 immunoreactive cells by microscopic field (400 × magnification) was counted in connective tissue.t-test. The significance level adopted was p < 0.05.Data were expressed as means and standard deviation. ANOVA was used for statistical evaluation and the Bonferroni post-hoc test was used after ANOVA. The gingival mucosa width, the number of inflammatory cells, all fibroblast-like cells and TGF-beta2 immunoreactive cells were compared between groups by using the Student's The manuscript has been performed with the approval of ethics committee in experimental research on animals by Federal University of Juiz de Fora/Brazil (protocol n° 039/2005).All cyclosporine treated rats presented GO that was evident in all gingival localizations. Graphic 1 shows macroscopic measurements of the buccal-lingual width and mesio-distal width p < 0.05). The macroscopic analysis revealed a significant reduction of the buccal-lingual width and mesio-distal width in the roxithromycin treated rats group 4) when compared with group 2 and group 3 (graphic 1) (p < 0.05). The histopathological analysis revealed that GO was a result of the enlargement of epithelial and connective tissues figures . A moder when com. The macTolypocladium inflatuns gams. It specifically acts in suppressing the immune response mediated by T cells. It goes through biotransformation in the liver, resulting in 14 metabolic products, of which 90% is excreted in feces and 10% eliminated by the kidneys [Cyclosporine (CsA) is a hydrophobic neutral cyclic polypeptide composed of eleven amino acids and made from the fungus  kidneys .Gingival overgrowth is one of the side effects caused by the chronic use of cyclosporine (CsA), which is employed to prevent rejection of transplanted organs and to treat several autoimmune diseases, such as diabetes mellitus, Behcet's disease, psoriasis, multiple sclerosis, erosive lichen planus, systemic lupus erythematosus, bullous pemphigoid, rheumatoid arthritis, myasthenia gravis, uveitis, and various glomerulopathies ,24,25.Several studies have demonstrated elevated levels of specific cytokines in overgrowth gingival tissue, especially TGF-beta, a multifunctional inflammatory mediator, which suggests that this growth factor plays a role in the accumulation of the extracellular matrix ,25,26, iGO treatment includes removing bacterial plaque, maintaining adequate oral hygiene, and also includes invasive procedures, such as gingivectomy. Treatment for this complication, until recently, was only surgical. Nowadays, several studies have been conducted to determine the effect of antibiotic treatment on the regression of gingival overgrowth. In 1995, Wahlstrom, Zamora and Teichmann coincidentally used azithromycin - a semi synthetic antibiotic, derived from the macrolide erythromycin - to treat respiratory infections in two renal transplant patients who had cyclosporine induced GO. They observed gingival reduction after using it. The daily treatment with 500 mg for five days is simple, cheap, conservative, quickly effective, and avoids gingival surgery. It acts mostly against gram-positive and negative bacteria, has quick oral absorption, does not alter serum levels of cyclosporine or levels of creatinine. Azithromycin is well tolerated and associated an efficient oral hygiene program induce a gingival overgrowth reduction , but canRecently, we used roxithromycin in four renal transplanted patients and the results suggest that roxithromycin may be an important therapeutic tool used to reduce cyclosporine-induced GO . Yamabe In the present study, the results demonstrated that roxithromycin treatment was effective to reduce cyclosporine-induced GO in rats. Both epithelial and connective tissues showed a decrease in thickness in roxithromycin treated rats in comparison with animals from the control groups. In addition, the significant reduction in TGF- beta2 expression in roxithromycin treated rats was associated with a lower number of fibroblasts, with reduction of fibrotic areas and decrease in inflammatory infiltrate. Taken together, our data suggest that the down-regulation of TGF-beta2 expression may be an important mechanism of action by which roxithromycin inhibits GO.AMP: ampicillin; CsA: ciclosporine; GO: gingival overgrowth; MAP quinase: quinase protein actived by mithogen; NF-kB: nuclear factor kappa B; ROX: roxithromycin; TGF-beta1: transforming growth factor beta1; TGF-beta2: transforming growth factor beta2; UFJF: Federal University of Juiz de ForaThe authors declare that they have no competing interests.SC have been involved in drafting the manuscript, acquisition and analysis of data and carried out the immunohistochemistry. MB participated in the design of the study and coordination such as performed the statistical analysis. BV analysis and interpretation of data. IM helped in the experimental phase, inducing gingival overgrowth. LV carried out the immunohistochemistry. FA participated in the design of the study and coordination and have given final approval of the version to be published. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/9/33/prepub


Mesenchymal Progenitor/Stem Cells (MSC) respond to homing cues providing an important mechanism to deliver therapeutics to sites of injury and tumors. This property has been confirmed by many investigators, however, the efficiency of tumor homing needs to be improved for effective therapeutic delivery. We investigated the feasibility of enhancing MSC tumor targeting by expressing an artificial tumor-binding receptor on the MSC surface.in vitro in cell binding assays and in vivo using two model systems: transient transgenic mice that express human erbB2 in the lungs and ovarian xenograft tumor model. The levels of luciferase-labeled MSCs in erbB2-expressing targeted sites were evaluated by measuring luciferase activity using luciferase assay and imaging.Human MSC expressing an artificial receptor that binds to erbB2, a tumor cell marker, were obtained by transduction with genetically modified adenoviral vectors encoding an artificial receptor (MSC-AR). MSC-AR properties were tested in vitro, compared to unmodified MSCs. Furthermore, we have tested the properties of erbB2-targeted MSCs in vivo and demonstrated an increased retention of MSC-AR in lungs expressing erbB2. We have also confirmed increased numbers of erbB2-targeted MSCs in ovarian tumors, compared to unmodified MSC. The kinetic of tumor targeting by ip injected MSC was also investigated.The expression of AR enhanced binding of MSC-AR to erbB2-expressing cells These data demonstrate that targeting abilities of MSCs can be enhanced via introduction of artificial receptors. The application of this strategy for tumor cell-based delivery could increase a number of cell carriers in tumors and enhance efficacy of cell-based therapy. Some of the key factors for the success of this type of therapeutic delivery have been established, such as the means and efficiency of cell loading with a therapeutic payload, and the nature of therapeutics that the cells can carry. However, the issue of biodistribution of injected cell carriers in vivo still remains an important aspect of cell-based delivery that has yet to be fully investigated. Importantly, different types of cell vehicles may have specific biodistribution or cell homing patterns and, therefore, may provide a special advantage to achieve site-specific or targeted delivery of therapeutics.In the last few years, cells have been increasingly used as vehicles for the delivery of therapeutics. The cell-based approach is particularly attractive for the delivery of biotherapeutic agents that are difficult to synthesize, have limited tissue penetrance, or are rapidly inactivated upon direct The ability of injected cells to either passively concentrate in specific organs or actively home to disease sites supports the rationale for targeted delivery of therapeutics by cell vehicles. There is growing evidence that sites of injury or growing tumors favor active homing of endogenous and exogenous stem or progenitor cells ,2. The fin vitro [in vivo [Along with using native cell homing properties, modification of the cell membrane by expressing appropriate receptors was also proposed as a means to obtain targeted cell vehicles. Much of the groundwork for such targeting approaches has previously been established for immune cells , where lymphocyte populations were modified to express artificial receptors (T-bodies) with distinct binding specificities to target cells. Artificial or chimeric receptors (AR) have been derived from the binding domains of antibodies  or T-cell receptors. An array of chimeric receptors, mostly with specificity for different tumor markers, has been tested for biological function in vitro ,20 and i[in vivo ,22. This[in vivo ,24.Despite showing its potential, the AR-based strategy has not been translated to other cell types that may serve as promising cell vehicles. Only a few applications have demonstrated the feasibility of using AR as a binding moiety in non-immune cell contexts ,26. In oin vitro. Furthermore, we tested erbB-2 targeting of MSC-AR in model systems in vivo and demonstrated that addition of the AR increased retention of circulating MSC-AR in erbB2-expressing sites. We also confirmed an increased concentration of MSC-AR, compared to MSC, in erbB2 positive ovarian tumors.The present study tested the hypothesis that artificial receptors with affinities to target sites can be added to cell vehicles and the new cell binding properties can be utilized to increase cell vehicle levels in the target sites. Specifically, we investigated the possibility of increasing the number of MSCs in ovarian tumors by expressing a tumor antigen-binding receptor on the MSC surface. This would provide an additional means to increase the number of tumor-associated MSCs beyond their native tumor homing potential. To this end, we have created MSCs that express an artificial receptor (AR) that binds to erbB2, a frequent marker of tumor cells (MSC-AR). We have shown that these AR-expressing MSCs (MSC-AR) have enhanced binding to erbB2-expressing cells These data show that the number of tumor-associated MSCs can be increased via affinity-based targeting, which can potentially serve to improve therapeutic delivery. Broadly, we demonstrated that an artificial cell targeting strategy can be beneficial to MSC-based cell vehicles and suggests that this strategy could also have potential for other cell types that lack native homing abilities.Anti-HA antibody conjugated to horse radish peroxidase (HRP) clone HA-7  was used for detection of artificial receptor expressed on MSCs membrane. Anti-erbB2 (HER-2/neu) antibody, clone AM-2000-01  was used to test expression of the erbB2 protein. Goat anti-mouse IgG1 (HRP conjugated) was used as a secondary antibody . CFDA-CE and SP-DiI fluorescent dyes (green and red fluorescence correspondingly) for cell labeling were from Molecular Probe .2.The human ovarian carcinoma cell line SKOV3ip1 was obtained from Dr. Janet Price . K562 cells - were obtained from ATCC the American Type Culture Collection  and cultured as recommended. Cells were maintained in DMEM/F-12, containing 10% fetal bovine serum (FBS)  and 2 mM glutamine at 37°C in a humidified atmosphere of 5% CO2 and used for experiments at passages 2-8.Primary human MSCs were obtained from bone marrow draw leftovers (screen filters with bone marrow cells remaining) from several individuals undergoing bone marrow harvest for allogeneic transplantation at the UAB Stem Cell Facility under an approved IRB protocol. MSCs were isolated and cultured as previously described . Cells wAdenoviral vectors having either wild type or genetically modified Ad5 fibers were used for experiments to load MSC with the targeting moiety and reporter genes. The following viruses were used in the study: AdCMV.AR, Ad.RGD.AR, Ad.RGDpK7.ARluc, Ad.RGDpK7.GFPluc. All viruses were replication-incompetent recombinant adenoviral (Ad) vectors having either single transgene or double cassette of transgenes in the E1 region under control of two CMV promoters. Coding sequences of AR, firefly luciferase and GFP were amplified by PCR from the plasmids pDisplayAR, pGL3  and pTrack  correspondingly and cloned into pShuttle plasmid.AdCMV.AR has a wild type Ad5 fiber; AdRGD.AR has a fiber protein with an integrin binding motif (CDCRGDCFC) inserted in the HI loop ; both AdThe lentiviral vector used in the study to obtain K562-erbB2 was constructed as described previously . The plain vitro and in vivo experiments MSCs were transduced with adenoviral vectors as described previously [MSC-AR were obtained by transduction of MSCs with adenoviral vectors encoding the artificial receptor (AR). The artificial receptor to target MSC to ovarian carcinoma was first constructed using the pDisplay mammalian expression vector  that allows display of proteins on the cell surface. An anti-erbB2 scFv C6.5  as bindieviously  at MOI 5ex vivo MSC transduction have luciferase gene in the context of double expression cassettes: GFPluc and C6.5luc. This simultaneous loading of the transgene with a luciferase reporter allows the use of luciferase expression for quantitative comparison of MSC-AR and MSC targeting.MSCs expressing scFv C6.5 with anti-erbB2 specificity are labeled throughout the text as MSC-AR. MSC transduced with isogenic viral vector AdRGDpK7.GFPluc were used as an appropriate counterpart for AR-transduced cells in all experiments and labeled as MSC-GFP. Both recombinant Ad vectors (AdRGDpK7.C6.5luc and AdRGDpK7.GFPluc) for MSC-SKOV mixed assay. Binding properties of MSC-AR were tested using SKOV3ip1 cells that abundantly express erbB2. MSCs and MSC-AR were labeled with the green fluorescent dye CFDA, whereas the SKOV3ip1 cells were labeled with the red dye SP-DiI according to the reagents' manuals. Labeled cells were lifted with Versene, washed and counted. MSCs and SKOV3ip1 were mixed in different ratios in 300 μl of PBS at 500,000 total cells/sample and incubated in solution under agitation. After washing, cell populations were separated by flow cytometry and the percentage of double-labeled cell population that corresponded to MSC-SKOV conglomerates was determined by gating on the GFP-PE population.in vitro binding was tested using K562 cells that artificially express erbB2. K562 expressing erbB2 were obtained via lentiviral transduction. To test cell-cell interaction, the suspensions of K562 or K562-erbB2 labeled with a green fluorescent dye (CFDA) were added to MSCs or MSC-AR cultured on a plastic. After 1 hr incubation, K562 cells were washed out and all cells in the wells were trypsinized. The cell mixture was subjected to flow cytometry and the percentage of bound fluorescent cells was determined in each well. Each experimental group was assayed in triplicates.MSC-K562 ELISA-based assay. K562 are non-adherent cells and allow the possibility to perform ELISA-like analysis of cell-cell interaction. MSC-AR 6 cell/mouse. Mice were followed after MSC injection by live non-invasive imaging at several time points. Upon sacrifice, luciferase expression was measured by imaging of the whole animal, imaging of the excised lungs and by luciferase expression analysis of lung lysates. To compare MSC numbers in the lungs and compensate for potential differences in luc expression in injected samples of targeted and untargeted MSC, the amount of RLU/cell was calculated for MSC-GFP and MSC-AR.Transient transgenic model. To create a state of transient expression of erbB2 in mouse lungs, we injected h-CAR transgenic mice  with AdC6 SKOV3ip1 cells. After 14 days of tumor development, mice received intraperitoneally MSC-GFP or MSC-AR at 2x106 cells/injection. At designated time points after MSC injection, mice were subjected to non-invasive imaging for luciferase expression. The animals were subsequently sacrificed; tumor nodules, liver, spleen, kidney and part of the intestine were collected, imaged in a Petri dish and proceeded to prepare tissue lysates for conventional luciferase analysis. All visible tumor nodules in the peritoneum were collected for imaging and combined as one tumor sample for luciferase analysis.Ovarian xenograft model. Female CB17 SCID mice  6-8 weeks of age were used to establish human ovarian xenografts. Intraperitoneal tumors were established in mice by i.p. injection of 5x10Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of UAB.in vivo optical imaging was performed with a custom-built optical imaging system with a liquid-nitrogen cooled lKB digital CCD camera . Mice were anesthetized with 2% isoflurane before intraperitoneal injection of d-luciferin. D-luciferin potassium salt, the substrate for firefly luciferase, was purchased from Molecular Imaging Products . Each mouse received an injection of 2.5 mg of d-luciferin diluted in 100 μl of PBS. Mice  were placed in the supine position within the imaging chamber with continuous isoflurane sedation. Whole body luminiscent images were obtained during the 5-10 min interval after injection of the substrate. Luminescence images and brightfield images were acquired with an exposure time of 60 and 0.02 sec respectively using WmView/32 software (Roper Scientific) without a filter at f/16. Index color image overlays were performed in Photoshop 7. 0 . The range of acquisition signal was kept constant at all imaging time points. The gray scale photographic images and bioluminescence color images were superimposed using the Adobe Photoshop 7.0 software. Statistics on bioluminescent signal intensity was obtained using WinView software according to the software instruction. For comparison of tumor targeting of two cell populations, total intensity of bioluminescence signal acquired from collected tumors were normalized per tumor area. Obtained value of relative light units per area (RLU/cm2/min) is proportional to the number of cells present on tumor surfaces.An Tumors and selected organs  after imaging were used to prepare tissue lysates. Organs collected after sacrifice were homogenized using Mini Beadbeater (BioSpec Product Inc) in 500 ul of 1x tissue/cell lysis buffer (Promega). Luciferase expression in tissue lysates was determined using luciferase assay system  according to the manufacturer protocol. The luciferase activities were measured in a Lumat LB 9507 luminometer  in relative light units (RLU) and normalized by the protein concentration in cell or tissue lysates (Bio-Rad DC Protein Assay kit). To account for the potential differences in luciferase expression of the injected MSC populations (targeted and untargeted), we normalized tumor luciferase activity (RLU) by luciferase activity of the MSCs (RLU/cell) and presented data as MSC numbers per mg protein.in vivo data are presented as mean values ± standard deviation. Statistical differences among groups were analysed in a two-tailed Student's t-test using GraphPad Prizm Software .All Although MSCs have the native ability to home to tumors, we attempted to enhance their tumor-targeting abilities by adding an additional tumor-targeting element: an artificial receptor (AR) with specificity to erbB2 Fig. . Expressin vivo. Two Ad vectors were created for our studies: Ad.RGDpK7.AR.Luc and Ad.RGDpK7.GFP.Luc. MSC transduction with Ad.RGDpK7.AR.Luc enabled us to obtain erbB2-targeted luc-labeled MSCs (MSC-AR) to further test our strategy in vitro and in vivo. As an appropriate counterpart to MSC-AR, we used cells transduced with an isogenic Ad vector, in which AR gene in double cassette was substituted by GFP. These cells are labeled MSC-GFP throughout the text.In addition, a double expression cassette incorporated into Ad vectors allowed simultaneous loading of MSCs with a targeting moiety and a reporter gene for MSC detection in vitro in cell-cell binding assays. The SKOV3ip1 ovarian tumor cell line expresses high levels of erbB2, and was, therefore, used to test MSC-AR binding. MSC, MSC-AR, and SKOV3ip1 were labeled with different fluorescent dyes CFDA-CE (green) and SP-DiI (red) respectively. Both MSCs and SKOV3ip1 cells are highly adherent to plastic; therefore, binding interactions were performed in solution after cell dissociation with Versene. Cell interaction resulted in the formation of small aggregates, which were detected as a double-positive (CFDA-SP) population by flow cytometric analysis. Increasing ratios of MSC-AR: SKOV3ip1 cells consistently resulted in increased percentages of the double-positive population ; this was in contrast to a control mixture (MSC and SKOV3ip1), where the double-positive population never exceeded 8% . In this experiment an increased concentration of MSC-AR in the lungs was detected starting at 8 hrs and persisted until 32 hrs after cell injection. This trend was visualized using total body images Fig.  as well We have previously tested MSC homing to ovarian tumors using the SKOV3ip1 ovarian tumor xenograft model, where preferential homing of MSC to ip tumors was demonstrated, compared to other organs in the peritoneal cavity . In thisIn a pilot experiment, tumors were collected 24 hrs after ip injection of MSC-GFP and MSC-AR and luciferase expression was measured in tumor lysates Fig. . The estTo confirm this initial observation and to investigate the kinetics of cell accumulation in tumors, we conducted another experiment with a broader time line, which included evaluation of cell numbers in tumors at 2, 6, 24, and 48 hrs after MSC injection Fig. . MSC-GFPWhole body imaging typically revealed discrete zones of luciferase activity in the peritoneum, starting from the earliest time point (2 hrs) tested. The signal has a more diffuse pattern at 2 hours after cell injection, compared to the more localized pattern observed at 24 hrs in both groups (data not shown). The whole body imaging signal approximated tumor localization, however, quantitation of the signal in whole body images  was not performed, as initially planned. We noted that the signal intensities in the whole body images were greatly influenced by body positioning, tumor localization in the cavity, and the extent of tumor masking by other organs.To attribute the obtained signals to particular organs, excised tumors and organs of peritoneal cavity were imaged separately in a Petri dish. MSC biodistribution to these organs was quantitatively assessed by measuring the bioluminescent signal intensities of individual organs and luciferase activity in corresponding tissue lysates. In both groups tested, this analysis demonstrated a clear tumor preference of injected MSC Fig. . As earlThe intensity of the signal in tumors grew over time, reached its maximum at 24 hrs, and declined at 48 hrs Fig. . We did A growing number of studies utilize engineered MSCs as a tool to track malignant tissues and deliver anticancer agents within the tumor microenvironment. MSC homing to tumors has been confirmed in a variety of experimental models, however the homing efficiency is clearly model-dependent and generally modest ,10. Addiin vitro experiments, MSCs grafted with anti-erbB2 artificial receptors demonstrated increased binding to cells overexpressing erbB2 . The only available study that investigated similar erbB2-based cell binding interactions [Our study applied affinity-based targeting to cell vehicles that lack immune recognition. To date, only a few applications have demonstrated the feasibility of using scFvs as binding moieties in non-immune cell contexts. One example is where an artificial chimeric receptor was applied to primary human monocytes to target monocytes to CEA-expressing tumor cells . Anotherractions  reportedin vivo tumor localization advantage, compared to unmodified cells. Given complexity of the processes of biodistribution and homing of injected cells, we reasoned that an effect of engineered cell targeting would be more pronounced and better detectable in model systems. For instance, an isolated heart model was used to detect the difference of MSC homing to normal versus infarcted myocardium [The next important question was whether the enhanced MSC-AR binding ability would translate to an ocardium . Thus, focardium . Expressin vivo cell-cell interactions result in a detectable cell retention effect.It is not accidental that most studies detecting tumor homing of intravenously introduced MSC were performed on lung tumor models ,13,45,46A more relevant and stringent model to test potential benefits of additional MSC targeting is the ovarian tumor model. We have previously demonstrated the native ability of MSC to home to SKOV3ip1 xenografts . The higWe detected preferential tumor localization of MSCs in both MSC and MSC-AR groups, which confirmed the native tumor homing abilities of these cells and assured that these functions are not perturbed by AR expression. In two separate experiments we detected an increase in the number of tumor-associated MSC-AR versus MSC-GFP starting from 24 hrs using two methods of luciferase quantitation.The analysis of MSC distribution in the peritoneum demonstrated that intraperitoneally developed tumors are the major cell homing sites in both groups and across all time points tested. The ability to localize to even minor tumor metastasis at the surface of organs is a remarkable property of MSCs that can be exploited for diagnostic or treatment endpoints. Although addition of the AR might not influence the actual process of MSC moving towards a chemotactic homing gradient, it was able to "strengthen" binding to tumors and resulted in increased tumor-associated cell numbers and the tumor/liver ratio. The mechanism of these effects is potentially mediated by increased adherence to tumor cells, which affected both the efficacy (number of cells attached to tumors) as well as the specificity (ratio of tumor-bound versus other organ bound cells).In both models we capitalized on the accessibility of targeting tumor markers to cells expressing AR. The necessity of the accessibility may dictate a careful selection of the marker and corresponding targeting moiety. For instance, under systemic injection, circulated cells are most likely to have physical contact with endothelial cells, supporting the targeting of specific endothelium markers. Such selective binding of circulating cells to key neovasculature markers has been described ,52. An aAnother important issue concerning the therapeutic use of cells as vehicles is the quantity of cells reaching the desired destination, as well as an understanding of how these numbers translate to therapeutic benefits. Some applications might need a maximum possible cell number to achieve a therapeutic benefit ,7,53, whThe majority of studies exploiting MSC tumor homing have only demonstrated the presence of labeled MSCs in the tumor parenchyma ,11,13,29Among the studies that attempted quantitation of MSC numbers in particular sites, only moderate cell numbers in the targeted tissues were reported ,10. MSC The major purpose of our study was to investigate the differences that AR-modified cells achieve in tumor targeting. Therefore, thorough quantitative evaluation of absolute cell numbers per tumor or other organs was not performed. Of note, some features of the ovarian tumor model influence accurate quantitative estimation of tumor cell homing and have to be accounted for. Multiple tumor nodules and metastasis hamper accurate collection of the entire tumor sample, which results in underestimation of total MSC tumor homing levels. On the contrary, metastases to organs , if not identified and dissected out, may incorrectly attribute MSC homing to these organs, especially using bulk assays such as luciferase activity of organ lysate. This leads to an overestimation of MSC distribution to off-tumor sites, while, in fact, this is also tumor-related homing. Therefore, accurate quantitative analysis would require more attention to both, the procedure of organ collection for analysis, and the ability to better visualize tumor nodules.Nevertheless, the importance of quantitative assessment, as well as developing an accurate methodology for determination of absolute cells numbers in organs has been recognized. Thus, tumor homing data were presented as cells per mg of protein, which gives a ballpark estimation of cell numbers present in tumors at these conditions. The level of native MSC tumor targeting was roughly estimated as 10% of the injected cell dose, while addition of the artificial receptor increased this efficiency 1.5-2 times. Based on this estimation, we believe that MSC-based therapeutic delivery has more practical utility after tumor debulking. In the residual disease it may provide much higher vehicle/tumor cell ratio than cases in which large primary tumors exist. Also, a marker targeted by MSC-AR will be better exposed on small tumor nodules without prominent stromal component or on patches of disseminated tumor cells.Our study confirmed that modification of cell carriers via expressing artificial receptor mediates the numbers of injected cell carriers in the organ or site of interest. We have demonstrated the practical relevance of our strategy in an ovarian tumor model, and showed that the number of modified MSC carriers increased in intraperitoneal tumors. Artificial receptor strategy can be applicable to other cell types, especially to circulating cells lacking native homing abilities.The authors declare that they have no competing interests.SK, LP carried out all study including in vitro and in vivo experiments needed to test porposed strategy. JR helped with in vitro studies, drafting and editing the manuscript. RA carried out general supervision regarding ovarian model used, involved in drafting the manuscript. DTC oversaw the project, have made contribution to study design and discussion of ideas and results. LP have made the major contribution to developing the concept of cell targeting, carried out all study design, acquisition and interpretation of data, wrote and edited the manuscript. All authors read and approved the final version of the manuscript.


Coexisting conditions and previous antimicrobial drug exposure predict colonization. Escherichia coli and Klebsiella spp., and 29 (25%) had a subsequent ESBL-positive clinical culture. Multivariable analysis showed the following to be statistically associated with ESBL colonization at admission: piperacillin-tazobactam , vancomycin , age >60 years , and chronic disease score . Coexisting conditions and previous antimicrobial drug exposure are thus predictive of colonization, and a large percentage of these patients have subsequent positive clinical cultures for ESBL-producing bacteria.Extended-spectrum β-lactamase (ESBL)–producing bacteria are emerging pathogens. To analyze risk factors for colonization with ESBL-producing bacteria at intensive care unit (ICU) admission, we conducted a prospective study of a 3.5-year cohort of patients admitted to medical and surgical ICUs at the University of Maryland Medical Center. Over the study period, admission cultures were obtained from 5,209 patients. Of these, 117 were colonized with ESBL-producing  Clinicians, microbiologists, infection control practitioners, and hospital epidemiologists are concerned about ESBL-producing bacteria because of the increasing incidence of such infections, the limitations of effective antimicrobial drug therapy, and adverse patient outcomes .The primary objective of our study was to identify factors predictive of colonization with ESBL-producing bacteria at admission to an intensive care unit (ICU). In addition, we identified the percentage of patients colonized with ESBL-producing bacteria who had a subsequent positive clinical culture for the same species of ESBL-producing bacteria. Understanding risk factors for colonization is important for several reasons. First, understanding the potential causal mechanisms of colonization can lead to successful infection control, involving antimicrobial stewardship and public health interventions aimed at controlling the emergence of ESBL-producing bacteria. Second, such knowledge can help identify which patients should be receiving empiric ESBL-targeted antimicrobial therapy. Some hospitals have used active surveillance culturing for antimicrobial drug–resistant, gram-negative bacteria to help guide empiric therapy . This study was approved by the Institutional Review Board of the University of Maryland, Baltimore. Informed consent was not required by the Institutional Review Board because perianal specimens were cultured as part of infection control quality improvement involving active surveillance culturing for vancomycin-resistant enterococci.Escherichia coli or Klebsiella species by using API 20E identification strips . All E. coli and Klebsiella isolates underwent ESBL confirmatory testing by disk diffusion for ceftazidime and cefotaxime with and without clavulanic acid as recommended by the Clinical Laboratory Standards Institute’s guidelines  with 2 μg/mL of ceftazidime added to the cooled agar before the plates were poured , and the infectious disease–specific CDS (CDS-ID)  to 1.0 (indicating perfect prediction). In addition, we calculated the sensitivity, specificity, positive predictive value, and negative predictive value for patients with or without all dichotomous variables in the final model. Statistical analysis was performed with SAS Version 9.1 .Initial bivariable statistical comparisons were conducted by using the χDuring the study period, 5,209 (84%) admitted patients had results of admission perianal cultures and were included in this study, 4,398 patients had 1 ICU admission, and 618 patients had repeat admissions. Ninety-one percent of the surveillance cultures were obtained within the first 12 hours of ICU admission. The cross-sectional patient cohort consisted of 2,096 (40%) admissions to the medical ICU and 3,113 (60%) admissions to the surgical ICU. The mean age of the patients was 55 years. The mean comorbidity score as measured by the CDS–ID was 2.73 and 2.42 as measured by the Charlson Comorbidity Index. Based upon International Classification of Diseases, 9th revision (ICD-9) codes, 1,285 (25%) had diabetes, 1,344 (26%) had cancer, and 193 (4%) were HIV positive; 1,594 (31%) of patients had been transferred from another healthcare facility, and 1,693 (33%) had been previously admitted to the same hospital within the past year.E. coli or Klebsiella species bacterium on ICU admission. Specifically, 76 (65%) patients were colonized by an ESBL-producing E. coli, 55 (47%) were colonized by an ESBL-producing Klebsiella species, and 14 (12%) patients were colonized by both. Stratified bivariable analyses by organism are as follows: for E. coli, zosyn , vancomycin , age , and coexisting conditions ; for Klebsiella spp., zosyn , vancomycin , age , and coexisting conditions, as measured by CDS-ID . Stratified analysis results for those patients who were in the hospital at least 24 hours before ICU admission are as follows: zosyn , vancomycin , age , and coexisting conditions as measured by CDS-ID (OR 95% CI 1.01–1.44).We examined patient characteristics, coexisting conditions, and previous antimicrobial drug exposures to identify factors potentially associated with colonization by an ESBL-producing bacterium on ICU admission  or vancomycin-resistant enterococci (VRE). Among the 5,092 patients not colonized with ESBL-producing bacteria, 33 (0.6%) had a subsequent positive ESBL clinical culture with the same bacterial species between the time of ICU admission surveillance culture to the date of hospital discharge.For the 117 patients identified as colonized with ESBL-producing bacteria, we assessed their history of culture positivity with ESBL-producing bacteria as well as other antimicrobial drug–resistant bacteria . Of the E. coli and Klebsiella spp. at ICU admission. We identified age >60 years, comorbidity as measured by the CDS-ID, previous in-hospital piperacillin-tazobactam use (current admission), and previous present admission in-hospital vancomycin use (current admission) as independent risk factors. We also quantified the ESBL colonization/clinical culture positivity rate among these patients and addressed the question of whether patients colonized with ESBL had a history of colonization with MRSA and VRE.In this study, we identified risk factors for colonization with ESBL-producing The risk factors identified are potentially important because they can help determine which patients may need empiric antimicrobial drug therapy targeted to the ESBL-producing bacteria. Carbapenem antimicrobial agents may be preferred as empiric choice for patients at risk for ESBL-producing bacteria (,–,We hope that our risk factor study and other risk factor studies in the area of antimicrobial drug resistance will be used in future antimicrobial agent stewardship intervention studies and future infection control intervention studies. Previous risk factor studies have led to antimicrobial agent stewardship intervention studies aimed at controlling ESBL-producing bacteria (,,,The risk factors identified may be causally related to the outcome of ESBL-colonization or may only be statistically associated. Age >60 years and the presence of coexisting conditions have validity and biologic plausibility for a causal association with colonization status (We found a ratio of colonization to clinical culture positivity that was the same order of magnitude as for VRE and MRSA (Enterobacteriaceae. Many studies have not analyzed the specific antimicrobial drug resistance mechanism and thus are not directly comparable to our study. A study from Canada determined that several antimicrobial drugs were risk factors for multidrug resistant Enterobacteriaceae (2 receptor antagonists (Several studies. performed worldwide, have analyzed risk factors for colonization with multidrug resistant A limitation of our study is that we did not have access to records of the antimicrobial drugs that patients may have received as outpatients before their hospital admission. However, relevant to the question of empiric therapy, most intensive care clinicians do not have access to records of outpatient antimicrobial drug use when they are empirically choosing antimicrobial agents. Another limitation of the study is that we did not have access to the subsequent ESBL-positive clinical isolates and thus were unable to compare them by molecular epidemiologic methods, such as pulsed-field gel electrophoresis, to see whether they were identical to the ESBL-colonizing isolates identified previously. We did not perform chart review; thus, the subsequent clinical cultures with ESBL-producing bacteria could have represented either clinical infection or colonization, based on definitions from the Centers for Disease Control and Prevention (In this study, we identified risk factors for ESBL-producing bacterial colonization among ICU patients. These data may be useful for identifying which patients may warrant empiric ESBL-targeted antimicrobial drug therapy. We also demonstrate that subsequent infections with ESBL-producing bacteria develop in a large percentage of ICU patients colonized with ESBL-producing bacteria.


Patients with tuberculosis (TB) frequently produce anti-citrullinated protein antibodies (ACPA). The objective of this study is to characterize the citrulline-dependence of the ACPA reactivity in sera of patients with mycobacterium infections.Serum samples of 134 patients with untreated mycobacterium infections  were tested for antibodies against both the citrullinated (Cit) and the non-citrullinated (Arg) form of 2 cyclic synthetic peptides. In 33 patients, a follow-up sample was tested six months after starting anti-mycobacterial drugs.A substantial proportion of patients with mycobacterial infections demonstrated antibodies against 0401Cit, 0401Arg, 0722Cit and 0722Arg. Fourteen patients demonstrated anti-0401Cit, 83 anti-0401Arg, 22 anti-0722Cit and 61 anti-0722Arg, while none of these antibodies were detected in the 20 healthy controls. All the patients but one, who were anti-0401Cit and anti-0722Cit positive, demonstrated reactivity against the respective Arg peptide. In the subset of 33 patients with a follow-up test six months after starting treatment, the mean levels of antibodies to 0401Cit, 0401Arg, 0722Cit and 0722Arg significantly decreased after treatment. All the patients who were anti-0401Cit and anti-0722Cit positive turned negative after treatment. The presence of anti-0401Cit/Arg and anti-0722Cit/Arg was found to be significantly correlated with the presence of HIV.ACPA may be found in patients with TB. In most of the cases, the reactivity is citrulline independent. A positive cyclic citrullinated peptide (CCP) test in these patients should therefore be interpreted with care, and preferably followed by a control ELISA with a non-citrullinated antigen. A group of autoantibodies, anti-citrullinated protein antibodies (ACPA), has been described in patients with rheumatoid arthritis (RA) . The speIt has recently been reported that anti-CCP2 antibodies can be detected in 9% of patients with type 1 autoimmune hepatitis (AIH-1) in the absence of recognizable rheumatoid arthritis overlap, and in some cases with high titres, comparable to those observed in RA . HoweverWe have reported increased levels of anti-CCP2 in up to 32% of patients with tuberculosis (TB) . These pThe objective of our study was to characterize the observed ACPA reactivities in TB, especially regarding their dependence on the citrulline moiety, as is the case in RA, as well as their presence after treatment. For this purpose, we tested mycobacterial sera for reactivities with citrullinated peptides as well as the corresponding arginine-containing controls.Mycobacterium. A questionnaire was used to determine data on the clinical features of the disease, such as duration of symptoms, the presence of fever, cough, as well as rheumatological manifestations such as arthralgia/arthritis, myalgia, rash, mucocutaneous symptoms, sicca symptoms, spontaneous abortion, history of thrombosis, and familial history of autoimmune diseases. All the patients were tested for HIV, hepatitis B and C. Data on the resistance of the mycobacterium was collected. Serum samples were collected before starting treatment for TB or NTM infection in all patients and six months after starting treatment with anti-tuberculous drugs in 33 subjects. The samples were frozen at -20°C and subsequently tested.One hundred and thirty-four consecutive patients with recently diagnosed active mycobacterial infections (122 with mycobacterium tuberculosis and 12 with nontuberculous mycobacterium (NTM)) participated in the study. All were admitted to the hospital department of tuberculosis, with clinical symptoms and radiological signs as well as positive cultures for 2SO4 10 minutes later to stop the staining reaction. Optical density (OD) values were measured on an ELISA-reader at 450 nm.Antibodies against both the citrullinated (Cit) and the non-citrullinated (Arg) form of 2 cyclic synthetic peptides  were determined by ELISA. These peptides were synthesized with a C-terminal spacer and biotin tag. The 0401 peptides consist of 18 amino acids with the Cit/Arg at the sixth position, whereas the 0722 peptides consist of 13 amino acids with the Cit/Arg also at the sixth position. Cyclization was achieved by coupling the side chain of a cysteine residue at position 13 (of both peptide sets) to the amine at the N-terminus. Streptavidin-coated pre-blocked microtiter plates  were coated with 10 μg/ml peptide diluted in PBS/0.1% BSA at room temperature for one hour. The coated plates were incubated with 100 μl/well serum for one hour (200 times diluted in PBS/1% BSA/0.05% Tween-20). This and the subsequent incubation step were performed at 37°C in a humidified atmosphere and were followed by washing steps with PBS/0.05% Tween-20. Antibodies were detected after incubation for one hour with 100 μl/well rabbit-anti-human IgG HRP-conjugated antibody  (1:10000 in PBS/1% BSA/0.05% Tween-20). Bound antibodies were visualized using either 100 μl/well of 3,3',5,5'-tetramethylbenzidine (TMB) solution mixed 1:1 with urea peroxide as a substrate, followed by 100 μl/well 2 M HA positive control consisting of an RA serum known to be positive for both citrullinated peptides, was included on each plate to determine similarity between plates coated with these peptides. Cut-off values to determine whether a peptide was recognized or not, were defined on the basis of the reactivity of sera from healthy control subjects. Cut-off values for the citrullinated and arginine-containing peptides were defined as the mean plus two times the standard deviation of the values of 10 control subjects, which were included on each plate.The presence of anti-citrullinated protein antibodies in the TB sera was also monitored using the Immunoscan CCPlus kit  and the citrulline-dependency of these reactivities was determined in parallel by ELISA using plates coated with the corresponding arginine-containing peptides.The study was approved by the ethics committee of the medical center. Appropriate informed consent was obtained from all patients, and the clinical research was conducted in accordance with guidelines for human experimentation specified by the Tel Aviv Sourasky Medical Center.P < 0.05 was considered significant.Statistical analysis was performed by the SPSS software, using Student's t test and the chi square test to compare antibody titers or positivity rates, respectively, between TB patients and controls. The Pearson correlation coefficient was used for correlations between clinical and laboratory data. Table To investigate the ACPA reactivity in the sera of these TB and NTM patients, first two pairs of synthetic peptides were used. One of these, 0401Cit and 0401Arg, corresponds to the cfc1-cyc peptide used in the first generation CCP test (CCP1) and its arginine-containing equivalent cf0-cyc ; the seqThe mean levels of Anti 0401 Cit/Arg and Anti 0722 Cit/Arg were significantly increased in patients with mycobacterial infections in comparison with healthy controls.Next, ACPA reactivities in TB patient sera were determined by a commercially available CCP2 test (CCPlus). To investigate the citrulline-dependence of the antibodies detected by this test, the sera were analyzed in parallel with plates coated with the same peptides in which the citrulline residues were replaced by arginine residues (ArgPlus). The results of these analyses confirmed that ACPA are relatively frequently present in TB sera and that these reactivities are not citrulline-dependent Figure .To demonstrate that individual patient sera are reactive with both the citrullinated and non-citrullinated peptides, the results for a selection of sera (the most highly reactive sera) are depicted in correlation diagrams in Figure Patients were treated with anti-tuberculous drugs to eradicate the mycobacteria and the reactivity with the (citrullinated) peptides was assessed six months after starting treatment. Serum samples from 33 patients were available for these analyses. The mean levels of antibodies to 0401Cit, 0401Arg, 0722Cit and 0722Arg significantly decreased after treatment (Table P < 0.001). Extra-pulmonary involvement as well as African origin correlated with the presence of anti-0722Cit (P = 0.024) (Table The results in Table 4) Table .Factors such as age, gender, length of symptoms, persistence of positive cultures, NTM, hepatitis C, hepatitis B, multi-drug resistance TB and pulmonary cavitations did not correlate with anti anti-0401Cit/Arg and anti-0722Cit/Arg antibodies.The present study confirms our previous observation that ACPA are relatively frequently found in the serum of patients with mycobacterial infections. A total of 10 to 16% of patients with mycobacterial infections demonstrated antibodies against citrullinated peptides. However, almost all of these patients also developed reactivity against the non-citrullinated peptides. Interestingly, the frequency by which antibodies to the non-citrullininated peptides were detected was much higher than that of the antibodies reactive with the citrullinated peptides. These results strongly suggest that the antibodies produced by TB and NTM patients are reactive with at least one epitope in the citrullinated peptides, for which the citrulline residue is not critically important. Instead, the presence of an arginine at the position of the citrulline in these peptides appeared to enhance the recognition of this epitope by the patient antibodies.Our results are consistent with those of Kakumanu et al. who reported that up to 37% of TB patients developed anti-CCP1 antibodies, while many of them reacted to the unmodified arginine-containing peptide as well . The proInterestingly, we could show that the levels of anti-citrullinated and anti-arginine-containing peptide antibodies significantly decreased after anti-TB treatment. This further supports the hypothesis that the presence of ACPA, like other autoantibodies that have been detected in TB patients, probably reflects a polyclonal non-specific activation of the prolonged inflammatory process .A significant correlation was found between the anti-0401Cit/Arg and anti-0722Cit/Arg antibodies and the presence of HIV. Humoral immunological abnormalities are frequent in HIV and include cryoglobulins, Rheumatoid Factor, antinuclear antibodies, antiphospholipid antibodies and ANCA in 17 to 51% of patients ,14. Becaafter treatment sample was available, was strongly reactive with the 0401Cit and 0722Cit, as well as the CCPlus peptides, but was not or was only very weakly reactive with the 0401Arg, 0722Arg and ArgPlus peptides, respectively. For the other patient, only the after treatment sample displayed strong reactivity with 0722Cit and CCPlus, whereas none of the other peptides was recognized. Both of these patients did not show signs of arthritis, but it might be worthwhile to follow these patients to monitor whether they will develop arthritis and whether they remain ACPA positive.For two of the patients citrulline-specific reactivities were observed. One of these patients, for which no Previously, a similar citrulline-independent ACPA response has been reported in type 1 autoimmune hepatitis, AIH-1 . HoweverIn conclusion, ACPA are significantly increased in patients with untreated TB, but in contrast to RA this response is not citrulline-dependent. The results of these studies indicate that ACPA detection in at least a subset of infectious and other inflammatory diseases should be performed by using a combination of citrullinated and the corresponding non-citrullinated antigens.ACPA: anti-citrullinated protein antibodies; AIH-1: type 1 autoimmune hepatitis; Arg: arginine; CCP: cyclic citrullinated peptide; Cit: citrulline; NTM: nontuberculous mycobacterium; PAD: peptidylarginine deiminase; RA: rheumatoid arthritis; RF: rheumatoid factor; TB: tuberculosis.The authors declare that they have no competing interests.OE was responsible for initiative, writing of the protocol and Helsinki approval, statistical analysis and writing of the manuscript. RS and DB recruited patients and sera. RvU was responsible for anti-CCP2 and corresponding arginine control peptide analyses. CO gave technical assistance for ELISA analyses. GP supervised the study, interpreted data, and wrote the manuscript.


Observational outcome studies of patients with obstructive sleep apnea (OSA) require adjustment for co-morbidity to produce valid results. The aim of this study was to evaluate whether the combination of administrative data and self-reported data provided a more complete estimate of co-morbidity among patients referred for sleep diagnostic testing.A retrospective observational study of 2149 patients referred for sleep diagnostic testing in Calgary, Canada. Self-reported co-morbidity was obtained with a questionnaire; administrative data and validated algorithms (when available) were also used to define the presence of these co-morbid conditions within a two-year period prior to sleep testing.Patient self-report of co-morbid conditions had varying levels of agreement with those derived from administrative data, ranging from substantial agreement for diabetes (κ = 0.79) to poor agreement for cardiac arrhythmia (κ = 0.14). The enhanced measure of co-morbidity using either self-report or administrative data had face validity, and provided clinically meaningful trends in the prevalence of co-morbidity among this population.An enhanced measure of co-morbidity using self-report and administrative data can provide a more complete measure of the co-morbidity among patients with OSA when agreement between the two sources is poor. This methodology will aid in the adjustment of these coexisting conditions in observational studies in this area. Obstructive Sleep Apnea (OSA) is a disorder characterized by periods of cessation of breathing during sleep with intermittent hypoxemia and sleep fragmentation. Population-based studies estimate the prevalence of OSA to be approximately 3 to 7% for adult males and 2 to 5% for adult females in the general population -4. FurthGiven the increased morbidity associated with OSA, observational studies of patients with OSA must also adjust for these co-morbid conditions to determine the independent effect of OSA on the outcomes of interest. The use of self-reported data through questionnaires or interviews is a common method of determining the presence of co-morbid conditions due to its efficiency and relative low cost. However, the reliability and accuracy of this data is questionable -21. In aAdministrative data is another source from which to determine the presence of co-morbid conditions. While agreement between self-reported medical conditions and that obtained from administrative databases also varies -27, combThis project is part of a larger retrospective study investigating health care utilization among patients with OSA. We included all adult patients (> 18 years old) referred for sleep diagnostic testing at either a hospital location in Calgary, Alberta, or private home care facilities within the Calgary Health Region between July 2005 to August 2007. Virtually all sleep diagnostic testing for the city of Calgary and surrounding areas (population of approximately 1.3 million) is conducted in these facilities. All patients who underwent polysomnography (PSG) or ambulatory monitoring for the presence of OSA were invited to participate in the study. We excluded non-Alberta residents, patients previously diagnosed with OSA, and those referred but did not undergo diagnostic testing.We used polysomnography (PSG) and ambulatory monitoring to identify OSA within participants. Although PSG is considered the 'gold standard' diagnostic test for OSA, an ambulatory monitoring device has proven to have excellent agreement, sensitivity and specificity with PSG . In addiWe stratified patients by OSA severity, based on their sleep test results, using the respiratory disturbance index (RDI). The RDI was defined as the number of apneas and hypopneas per hour of sleep. Apnea was defined as a cessation of airflow for at least 10 seconds. Hypopnea was defined as an abnormal respiratory event lasting 10 seconds or more, with at least a 30% reduction in thorocoabdominal movement or airflow compared to baseline, and associated with at least a 4% oxygen desaturation. OSA severity categories included: no OSA (RDI <5 event/hr), mild OSA (RDI 5–14.9 events/hr), moderate OSA (RDI 15–29.9 events/hr) and severe OSA (RDI ≥ 30 events/hr). This classification system is well accepted in both clinical practice and within the medical literature ,40. The Baseline clinical and demographic information was collected for all participants prior to sleep diagnostic testing. This included: age, sex, height, weight, body mass index (BMI), neck circumference, and postal code. Each participant also completed the Epworth Sleepiness Scale (ESS) , a self-Using the patient's unique Provincial Health Number (PHN), the cohort was linked to two Alberta Health and Wellness administrative databases, the hospitalization discharge database, and the physician claims database. For each patient, all hospitalization and physician claims information was obtained for a two-year period prior to sleep diagnostic testing.The hospital inpatient data source contains details regarding hospitalizations including admission date, discharge date, length of stay, 25 diagnostic codes (ICD-10), and 10 procedure codes for each admission. The physician claims registry contains information on physician services including dates and location of the visits, diagnostic codes (ICD-9-CM), provider specialty, and include the majority of residents in the province except a small proportion of special population groups , and federal inmates – accounting for approximately 1% of the total population) .Co-morbid conditions were identified within the Alberta Health and Wellness administrative databases using the International Classification of Diseases (ICD-9-CM and ICD-10) definitions for the nine specific co-morbidities. When available, validated algorithms were used to define each co-morbid condition  were reported. Means and proportions were compared using analysis of variance and chi-square tests respectively. In addition, proportions of patients presenting with specific co-morbidities, identified in the questionnaire, were calculated.To assess the agreement between self-reported co-morbidity and administrative databases, we calculated the proportion of subjects with each co-morbid condition based on: self-report only, administrative data sources only, both self-report and administrative data, and either self-report or administrative data. To evaluate consistency between self-report and administrative data the Kappa (κ) statistic and 95% confidence intervals were calculated. The Kappa statistic is an index of the degree of agreement between two raters, and can be thought of as the chance-corrected proportional agreement; possible values range from +1 (perfect agreement) to 0 (no agreement above that expected by chance). Kappa values were defined as: < 0.40 as poor or fair agreement, 0.40–0.60 as moderate agreement, 0.61–0.80 as substantial agreement, and 0.81–1.00 as almost perfect agreement .In addition, the McNemar's test of paired proportions was determined. This is a statistical procedure to compare two dependent or correlated proportions, and is a test of marginal homogeneity that compares agreement between discordant pairs. A statistically significant McNemar's test would indicate a difference between the proportions compared. Finally to assess the validity of the enhanced measures of co-morbidity an analysis was also performed in which patients were stratified by severity of OSA to determine trends in the prevalence of the co-morbid conditions. All statistical analysis was conducted using STATA 10.0 software .From July 2005 to August 2007, 2295 patients were referred for sleep diagnostic testing, of whom 78 (3.4%) patients refused to participate and 42 1.8%) patients were from out of province and were therefore excluded. Of the remaining 2175 patients, 26 (1.2%) were excluded because they were not present in the Alberta Health and Wellness registry file, for a final study population size of 2149  in Manitoba, Canada. Agreement between the two sources was highest for diabetes (κ > 0.70) and hypertension (κ > 0.50), and lowest for non-specific heart disease (κ = 0.38) . CricellWe found very poor agreement between self-report and administrative data for the presence of cardiac arrhythmia and stroke. Underreporting of cardiac arrhythmia likely occurred because respondents are not aware of the diagnoses, or lack of familiarity with this medical term found on the self-report questionnaire . Though The measure of co-morbidity using the enhanced combination of data sources found that as OSA severity increased, the prevalence of hypertension, diabetes, and myocardial infarction also increased. This dose-response relationship for these specific conditions by OSA severity has been documented in previous studies ,10,53-55The results of our study should be interpreted in context of the study limitations. First, for three of the conditions of interest , validated administrative algorithms were unavailable. Using an algorithm of at least one physician claim or hospitalization in a two-year period may have resulted in some misclassification and an over-reporting of these conditions. Secondly, we did not have a gold standard to determine whether the enhanced measures are more valid than a single data source alone. However, the increasing prevalence of conditions by OSA severity, consistent with that in the literature, does provide evidence of face validity. Finally, our study was limited to a single geographic region  and only included patients referred for sleep diagnostic testing. These patients likely represent those with more severe morbidity and will limit the generalizability of these results to other clinic-based sleep cohorts in North America.We found that administrative data in combination with survey data has the potential to create a more complete measure of the co-morbidity among patients referred for sleep diagnostic testing, particularly when agreement between survey and administrative data is poor. Given the resources required to obtain clinical data, use of data enhancement with administrative data may be valuable to other researchers. Although, future studies are required to validate co-morbidities based on data enhancement, these results suggest that this methodology can aid in the adjustment of these coexisting conditions in observational studies in this area.The authors declare that they have no competing interests.PER Study design, data analysis, and manuscript preparation. WHT Study design, interpretation of results, manuscript preparation. HQ: Interpretation of results, manuscript preparation and critical review. PF: Data analysis, interpretation of results, and critical review. BRH: Study design, data analysis, and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/9/50/prepub


MicroRNA (miRNA) signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool.Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set.A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81). Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR.Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma. For many human cancer entities, there is still a lack of high-performing biomarkers. In the past years, different tumor markers have been identified not only in tissue but also in blood, urine, or saliva of cancer patients. Several types of biomarkers can be distinguished. Prognostic biomarkers differentiate between "good outcome" and "bad outcome" tumors. Predictive biomarkers assess the probability for a treatment response, and pharmacodynamic biomarkers can be used to guide dose selection for certain drugs . FurtherRecent proof-of-principle studies indicate that analysis of miRNA expression in sera and peripheral blood cells is a promising approach for a blood-based diagnosis of cancer and other diseases -9. We reIn this study, we describe a highly specific miRNA expression profile for melanoma patients. Malignant melanomas represent the most aggressive form of skin cancer. According to the World Health Organization (WHO) the number of melanoma cases continues to increase in incidence, faster than any other type of cancer. Melanoma accounts for about 4% of skin cancer cases but for as many as 74% of all deaths of skin cancer. The 5-year survival rate is as low as 5% for patients with advanced melanoma .Currently, there is no promising standard therapy available for the treatment of patients with melanoma in an advanced stage. In order to improve prognosis it is crucial to detect melanoma in a very early stage, especially with metastasis occuring very early in the progression of the disease.Several studies described altered miRNA expression fingerprints in melanoma with the majority of these studies analyzing miRNA expression in formalin fixed paraffin embedded cancer tissue -15 and fAs of now there is, however, no evidence for altered miRNA expression in peripheral blood samples of melanoma patients. Here we used the Geniom Real Time Analyzer (GRTA) microarray platform  to analyze all human miRNAs as annotated in the Sanger miRBase version 12.0 -21.In total, we analyzed 35 blood samples of melanoma patients and 20 blood samples of healthy individuals. The 35 melanoma samples include a test set of 24 samples and an independent validation set of 11 melanoma samples.As analysis tools we employed different well known statistical measures, including t-test, Wilcoxon Mann-Whitney test (WMW), a linear model with p-values computed by an empirical bayes approach (limma) ,23, AreaOur study provides evidence for a novel and complex miRNA expression profile in blood cells of melanoma patients.The study was conducted in compliance with the Helsinki Declaration. The local ethics committee ("Ärztekammer des Saarlandes") approved the study. All participants of this study have given written informed consent. The 35 blood samples of melanoma patients were collected in two independent institutions. We used the 24 blood samples from one institution as test set and the 11 blood samples from the second institution as validation set. The control samples were obtained from 20 healthy volunteers. Information on age and sex of all blood donors and detailed clinical informations for all melanoma patients is given in the Additional Files .The microarray data were deposited in the publicly available database Gene Expression Omnibus , RNA from blood of melanoma patients that generate miRNA intensity values below t as false negatives (FN), RNA from blood of healthy subjects that generate miRNA intensity values below t as true negatives (TN), and RNA from blood of healthy subjects that generate miRNA intensity values above t as false positives (FP). Likewise for all thresholds, specificity (TN/(TN+FP)) and sensitivity (TP/(TP+FN)) were computed. The Receiver Operator Characteristics (ROC) curve shows the sensitivity as function of one minus the specificity. AUC values can range from 0.5 to 1. An AUC of 0.5 for a miRNA means that the distribution of intensity values generated by RNA from blood of melanoma patients and healthy subjects cannot be distinguished. The more the AUC value of a miRNA differs from 0.5, the better this miRNA is suited to separate between the two groups of blood donors . An AUC of 1 corresponds to a perfect separation.First, we analyzed the miRNA expression to detect miRNAs that show a different expression in different groups of blood donors. To this end, we applied different statistical measures to monitor differences between these measures. The set of approaches contains parametric t-test , Wilcoxon Mann-Whitney test , a linear model with p-values computed by an empirical Bayes approach (limma) ,23, the For all hypothesis tests, the resulting p-values were adjusted for multiple testing by Benjamini-Hochberg ,28 adjusWe carried out a hierarchical clustering approach to detect clusters of miRNAs and blood samples. In detail, we applied bottom up complete linkage clustering and used the Euclidian distance measure.In addition, we carried out a standard principal component analysis (PCA) and provide scatter plots of the first versus second principal component ,30.s miRNAs with lowest p-values were computed on the training set in each fold of the CV, where s was sampled from 1 to 866 . The respective subset was used to train the SVM and to carry out the prediction of the test samples. As result, the mean accuracy, specificity, and sensitivity were calculated together with the 95% Confidence Intervals (95% CI) for each subset size. To check for overtraining we applied permutation tests. Here we sampled the class labels  randomly and carried out classifications using the permuted class labels. All statistical analysis was performed using R [In addition to the single biomarker analysis and unsupervised clustering we also carried out classification of samples using miRNA expression patterns by applying Support Vector Machines  as impl using R .® MicroRNA Assays (Applied Biosystems). The qRT-PCR was performed on ten melanoma samples and ten samples of healthy individuals. We used RNU48 as endogenous control.To validate the microarray results we performed quantitative Real Time-PCR (qRT-PCR). We analyzed 13 miRNAs that showed significant deregulation in the microarray experiments, including hsa-miR-106b, hsa-miR-107, hsa-miR-1280, hsa-miR-151-3p, hsa-miR-17*, hsa-miR-18a, hsa-miR-199a-5p, hsa-miR-20a, hsa-miR-20b, hsa-miR-30a, hsa-miR-362-3p, hsa-miR-550*, and hsa-miR-664, using TaqManUsing the Geniom Realtime Analyzer microarray platform, we analyzed the expression of 866 miRNAs in blood cells of 20 healthy volunteers and 35 patients with melanoma. Out of these 35 patients, 31 (88.57%) had melanoma of clinical stages 0, IA, IB, IIA, or IIB. One patient (2.86%) had a stage IIIB melanoma, and three patients (8.57%) had stage IV melanoma , and a linear model with p-values computed by an empirical bayes approach (limma). We considered all miRNAs with adjusted p-value below 0.001 to be significant. All tests combined identified 213 miRNAs that showed a different expression in blood of melanoma patients as compared to the blood of healthy individuals. A more detailed view on the results of the three hypothesis tests is presented as three-way venn-diagram in Figure To further specify our search for miRNAs differentially expressed between blood of melanoma patients and of healthy controls, we added two additional filters. First, we only considered miRNAs that were at least 2-fold up- or down-regulated in their expression level in blood cells of melanoma patients compared to blood cells of healthy controls. Second, we calculated the median intensity values combined for all melanoma and combined for all normal blood samples and excluded miRNAs, with a combined median intensity value below 100 in either melanoma or in normal blood samples. Median intensity values lower than 100 are likely to be due to background noise. By using this high stringent threshold we tried to avoid false positives. Using these two empirically determined thresholds we identified 51 differentially expressed miRNAs, including 21 miRNAs downregulated and 30 miRNAs upregulated in blood cells of melanoma patients compared to blood cells of healthy individuals. Table We further compared the fold quotients in both the 24 melanoma blood samples that were used as test set and the 11 melanoma blood samples that were used as validation set. To reduce the noise, we only considered miRNAs with a median intensity level of at least 50 in any of the two sets. In this step we used a less stringent threshold of 50 to obtain more data points and little background noise has a less stringent influence on the correlation calculations. We computed for each melanoma set  the fold quotient versus the controls and determined the correlation. The scatter-plot in Figure -16 for this clustering using two-tailed Fisher's Exact test.We also analyzed the miRNA expression profiles of the melanoma test set, the melanoma validation set and the set of healthy controls by hierarchical clustering. Since many miRNAs contributed mostly noise to the clustering, we used only the 50 miRNAs with the highest data variance for clustering. As shown in the dendrogram in Figure To provide a low-dimensional visualization of the high-dimensional data we carried out a principal component analysis. Investigating the eigenvalues of the first principal components, we found that the first component contained the highest overall data variance while the first and second principal component combined contributed to approximately half of the overall variance. A plot of the first versus the second principal component is shown in Figure To confirm the results of the unsupervised cluster analysis, we employed a supervised statistical learning approach. We carried out SVM classification together with a feature subset selection relying on t-test p-values. In detail, we applied radial basis function SVMs that have been evaluated using 10-fold CV. The CV runs have been repeated 100 times to estimate the classification variance. To test for data overfitting, we carried out 100 permutation tests, i.e., we applied the same statistical approach to a data set with randomly assigned class labels for melanoma and control samples. The best classification accuracy has been obtained by using a subset that consists of 16 miRNAs including hsa-miR-186, hsa-let-7d*, hsa-miR-18a*, hsa-miR-145, hsa-miR-99a, hsa-miR-664, hsa-miR-501-5p, hsa-miR-378*, hsa-miR-29c*, hsa-miR-1280, hsa-miR-365, hsa-miR-1249, hsa-miR-328, hsa-miR-422a, hsa-miR-30 d, and hsa-miR-17*. By using these 16 miRNAs, we separated melanoma from healthy controls with a high accuracy, specificity and sensitivity of 97.4%, 95.0% and 98.9%, respectively. The results of all 100 CV runs and 100 permutation tests are provided as box-plots in Figure An example of a classification result is shown in Figure One of the major challenges towards an improved melanoma treatment is the identification of appropriate markers for a most early detection of the primary lesion. For patients with stage I melanoma the overall 5-year survival rate exceeds 90% but can fall below 10% for stage III or IV melanoma. It is especially important to detect melanoma before metastasis that occurs early during melanoma progression. Equally important are the prognosis of the patients' outcome and the prediction of the response to treatment. Since melanoma is a very heterogeneous disease, complex biomarker profiles appear to be best suited for the task of early tumor detection and the monitoring of high-risk patients.In our study we investigated the miRNA expression of almost all currently known human miRNAs and miRNA star sequences in peripheral blood cells of melanoma patients. The majority (88.57%) of the melanoma patients had a melanoma in clinical stage 0, IA, IB, IIA, or IIB. Comparing miRNA expression profiles in blood cells of melanoma patients and in blood cells of healthy donors, we detected 51 differentially expressed miRNAs. A total of 30 miRNAs was upregulated in blood cells of melanoma patients, whereas 21 miRNAs were downregulated. We used highly stringent selection criteria for the identification of deregulated miRNAs to reduce the false discovery rates, a problem that has recently been addressed by McCarthy et al. .http://202.38.126.151/hmdd/mirna/md/[We compared our results with the data deposited in the Human miRNA and Disease Database . Most nmirna/md/. The miRmirna/md/. Howevermirna/md/. They alOut of the 51 miRNAs that were deregulated in blood of melanoma patients four miRNAs, namely hsa-miR-99a, hsa-miR-365, hsa-miR-30a, and hsa-miR-146a, were deregulated in non-cancer skin diseases ,38. The While these first results do not allow any conclusion on the disease specificity of any of the addressed miRNAs, future miRNA profiling of a larger number of different human diseases will contribute to the identification of miRNAs that play a crucial role in specific human diseases. Furthermore, the identification of miRNAs will contribute to the overall understanding of the molecular alterations underlying disease development including melanoma progression.In addition, and in keeping with the focus of this study, miRNA expression profiling especially of human blood has the potential to serve as future tumor biomarker. Applying SVM with a feature subset selection method we used a set of 16 miRNAs to differentiate melanoma patients and healthy blood donors with high accuracy (97.4%). Recently, we used a subset of 24 miRNAs to discriminate between blood cells of patients with lung cancer and healthy controls with an accuracy of 95.4% . Except In this study we provide first evidence for the potential of miRNA expression profiles to distinguish patients with melanoma from healthy control subjects based on the analysis of peripheral blood cells. Until now it is not known which type of blood cells is responsible for the differences in the miRNA expression pattern. It is conceivable that the cancer miRNA signatures arise as part of a cancer-associated immune response. However, any hypothesis about the origin of the miRNA signatures generated from blood awaits experimental confirmation. Identifying the blood component responsible for specific miRNA signatures will likely contribute not only to our understanding of the mechanism underlying the pattern, but also to an improved prediction and prognosis of a disease.Finally, it remains to be proven which cancers or non-cancer diseases also show a specific miRNA expression pattern that might be used to tell these diseases apart form controls and possibly apart from each other.Using a subset of 16 significant deregulated miRNAs, we distinguished melanoma patients from healthy individuals with an accuracy of 97.4%. The high specificity and sensitivity of the miRNA signatures generated for blood cells of melanoma patients underlines the potential of this approach for future diagnostic applications. Blood based miRNA signatures may be ideally suited as prognostic, predictive, or pharmacodynamic biomarkers not only for melanoma but also for other tumors.AUC: area under the receiver operator characteristics curve; CV: cross validation; FP: false positive; FN: false negative; GRTA: Geniom Realtime Analyzer; HMDD: Human miRNA and Disease Database; limma: linear model with p-values computed by an empirical bayes approach; miRNA: microRNA; MPEA: microfluidic-based enzymatic on-chip labeling of miRNAs; PCA: principal componenet analysis; ROC: receiver operator characteristics curve; SVM: Support Vector Machines; TP: true positive; TN: true negative; WMW: Wilcoxon Mann-Whitney testAK: salary (febit), patent (febit); AB salary (febit), patent (febit); MS salary (febit); FW salary (febit); AW salary (febit);PL contributed to study design and developed the protocol for miRNA extraction, extracted the miRNAs from blood cells, and contributed in paper writing. AK contributed to study design, carried out the statistical analyses, initiated the study and contributed in paper writing. AB performed the miRNA screening. JR, KR, and SUJ collected the blood samples and contributed in paper writing. HPL contributed in the bio-statistical analysis. EM contributed in study design and development of miRNA extraction, supervised the study and contributed in paper writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/262/prepubAdditional Table S1: Detailed information on all blood donors, i.e. melanoma patients and healthy individuals.Click here for file