Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.The microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.Strong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides . Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.The multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment. With the discovery of anti-citrullinated protein/peptide antibodies (ACPAs), the interest in autoantibodies has increased during the last decade, from both a diagnostic and a prognostic RA-perspective. In the former American College of Rheumatology (ACR) 1987 classification criteria for rheumatoid arthritis (RA) , the impCommercial ACPA tests generally aim to identify collectively as many antibodies against citrullinated epitopes as possible. However, on the peptide level, the ACPA response in RA patients has been shown to be heterogeneous, as different RA patients show reactivity against different citrullinated peptides -8. AlthoMost studies on ACPA fine specificity have so far focused on individual antibody responses to epitopes on three citrullinated autoantigens identified in rheumatoid joints: fibrin/fibrinogen ,13, vimeMost commercial ACPA tests investigate only the reactivity against citrullinated antigens, and only very few tests take into account the possibility of reactivity against the protein/peptide backbone by simultaneous investigation of reactivity against the arginine-containing noncitrullinated counterpart. Although the ACPA response in RA has been shown to be citrulline specific, a number of studies have shown false ACPA reactivity with concomitant reactivity to arginine-containing control antigens in inflammatory diseases like pulmonary tuberculosis ,23, hepaIn this article, we describe the first version of a new microarrayed platform allowing the simultaneous investigation of more than 100 specific antibodies. In the present form, the platform is used for investigation of the ACPA response against 11 previously described citrullinated peptides and their arginine-containing equivalents, as well as one novel peptide, found to be citrullinated in RA synovial membrane and not described before. The peptides represent epitopes from five human autoantigens , including multiple epitopes from the four antigens  that have been found to be citrullinated in RA joints. We show that the heterogeneous ACPA response can be investigated with this technique in a serum-saving way. We foresee that this assay can be used in studies on RA patients in both prognostic and diagnostic contexts.For this study, 984 RA patients and 472 healthy controls from the Epidemiological Investigations in Rheumatoid Arthritis (EIRA) case-control study were investigated. EIRA cases have newly diagnosed (within 12 months of first symptoms) RA according to the 1987 ACR classification criteria .Serum samples were drawn at the time of inclusion in the EIRA study and thereafter stored frozen at -70°C. All patients and controls had given written informed consent before participating in the study, which had been ethically approved by the regional ethics committee at Karolinska Institutet.Serum samples were analyzed for the presence of ACPA-specific IgG by using a custom-made microarray, based on the ImmunoCAP ISAC system , containing 12 different citrullinated peptides and their native arginine-containing counterparts. All peptides except Fibβ60-74, Fibα621-635, CCP-1/Fil307-324, and citC1 were obtained at ≥ 95% purity from Innovagen AB . The CCP-1/Fil307-324 peptide was obtained from the same source at 80% purity. The triple helical citC1 peptide was synthesized as described in , analyzeThe performance of the microarray was investigated by comparison with individual ELISAs for the 12 included citrullinated peptides. For each peptide, suitable serum samples (between 40 and 100 samples per peptide) encompassing a broad range of specific reactivities against the individual citrullinated peptide were defined by specific ELISAs, whereupon the same samples were investigated with the microarray. ELISAs for 10 of the peptides were performed at the Department of Rheumatology, Karolinska Institutet, as described in . Eight EAnalyses were performed in two ways for each separate citrullinated peptide. In a first investigation, gross data for the results for the 12 citrullinated peptides in the 927 RA patients and 461 healthy controls were compared. In the second evaluation, net ACPA results were calculated by subtracting the fluorescence intensity for the arginine-containing control peptide from each citrullinated peptide for all RA patients and controls, whereupon these net values were used to construct ROC curves and to compare performance. This calculation of net ACPA reactivity was performed for all peptides, except for citCI, for which only uncorrected values were used, as the argC1 peptide is an autoantigen in its own right, and with conformational epitopes differing from the citC1 peptide ,21.To allow stringent comparison between performances for the different citrullinated peptides, the cut-off point for each investigation was defined as the fluorescence intensity corresponding to a diagnostic specificity of 98.0%, calculated from the fluorescence intensities of the investigations for the 461 healthy controls.ACPA responses are known to show a bimodal distribution clearly separating ACPA-positive and ACPA-negative subjects, and studies on the correlation between microarray and ELISA results were therefore performed with the nonparametric Spearman Rank Test.Receiver operator characteristic (ROC) curves were constructed by using the Analyze-it software , and correlation between ELISA and microarray results was calculated by using the JMP software .When the laser-scanned slides were processed with the Microarray Image Analysis software, the sites where the citrullinated peptides had been spotted in triplicate were clearly visualized after treatment with serum positive for the corresponding autoantibodies. Examples of a positive RA serum and a negative control serum are shown in Figure Comparison of the fluorescence intensities in response units (RUs) with corresponding ELISA units yielded satisfactory correlations between the two techniques, with Spearman ρ values typically between 0.75 and 0.90. Three representative examples are shown in Figure The inter- and intraassay coefficient of variation (CV%) was investigated by using serum samples with high , intermediate , and low  levels of antigen-specific antibodies targeting representative citrullinated peptides and their arginine-containing counterparts. The inter- and intraassay CV% for samples with high levels of specific antibodies ranged from 8.3 to 16.8 and 5.3 to 17.0, respectively. For the intermediate samples, the CV% was 9.5 to 22.8 and 10.3 to 19.3, respectively. The corresponding figures for low-level samples were 5.0 to 22 and 13.3 to 31.4.Besides the difference in reactivity between RA patients  and the healthy controls, a difference also was found in the size of signals against the individual peptide backbones that was common to both citrullinated peptides and to arginine-containing control peptides, as well as to both RA patients and controls. This fact manifested itself in different cut-off values for the individual peptides: with the highest values for Fibβ580-600  and Vim60-75  and the lowest for Vim2-17 (256 RU) and Fibβ36-52 (222 RU). Peptides with generally high fluorescence intensities in the microarray also showed generally high OD levels in the parallel ELISA investigations (data not shown).Staining of the slides with some serum samples yielded a fluorescence signal also from the glass surface surrounding the triplicate areas with spotted antigen. This happened in 57 (5.1%) of 984 of the RA sera and in 11 (2.3%) 472 of the control sera. As this phenomenon of nonspecific binding might conceal weakly reactive spots, we decided to exclude samples with this reactivity pattern from our further analyses. Exclusion of samples with unspecific binding had, however, very limited effect on the performance at group level and on the appearance of ROC curves for the individual citrullinated peptides (data not shown).When ROC curves were constructed for the individual citrullinated peptides, they generally showed a high specificity pattern, with alignment with the Y axis, as is also seen for anti-CCP2 . Three eIn parallel to evaluating the diagnostic properties of the crude fluorescence intensities against the citrullinated peptides, we also performed the same evaluation after the fluorescence intensities for the control peptides containing arginine had been subtracted for all samples from RA patients and controls. In some cases, this correction increased the number of RA cases that were positive in the assay : for reactivity against Vim60-75, the number of positive patients increased from 31.9% to 40.0%, and for Fibα621-635, a corresponding increase was found from 26.9% to 29.0%. For some peptides, subtraction of the arginine control values instead decreased the number of positive sera: for Vim2-17, from 10.8% to 8.0%, and for Fibβ36-52, from 45.2% to 41.9%. For the other investigated peptides, the corresponding differences were smaller, and of the 11 peptides investigated, five showed increased, and six showed decreased frequencies of positive reactions among the RA patients after arginine correction. The performance data for all investigated citrullinated peptides are shown in Table Among the 927 investigated RA patients, the median number of citrullinated peptide reactivities against the 12 investigated peptides was 2, and the mean, 3.33.When the patients were split according to anti-CCP2 status, a marked difference between the groups was evident, as the corresponding figures were 7.0 and 6.48 for the anti-CCP2-positive patients and 0.0 and 0.93 for the anti-CCP2-negative patients. The median number of positive reactions among the healthy controls was 0.0 . The distributions of the number of positive ACPA reactions in the different groups are graphically shown in Figure In this report, we describe a microarray in which specific reactivities against multiple citrullinated peptides can be investigated in parallel. Notwithstanding the microarray approach, each one of the individual autoantibody reactivities showed good correlation to results obtained with parallel ELISAs for the individual peptides. With a large validation cohort of RA patients and controls, followed by ROC curve analyses, we could show that the individual analyses showed high diagnostic specificity. At a defined specificity level (98%), the individual peptides showed marked differences in diagnostic performance, with some individual peptides performing similarly to the anti-CCP2 assay.When the RA patients were split into those positive and negative for anti-CCP2, a large difference was shown in the number of peptide reactivities detected in the two groups. This difference was particularly pronounced concerning individuals with multiple reactivities. However, a few patients also in the anti-CCP2-negative group showed multiple reactivities against the citrullinated peptides in the multiplex assay. This indicates that the microarray might be useful for analyses of residual ACPA reactivities that are not covered by the conventional ACPA tests like anti-CCP2. The value of this assay to generate hypotheses in a diagnostic setting remains to be evaluated by using larger sets of sera from different cohorts of RA patients and disease controls (patients with infections and other noninfectious inflammatory diseases). Of special interest will be the use of the assay to estimate the risk of RA development in patients with early undifferentiated arthritis  and in oIn the current design, the microarray uses very small quantities of serum (< 10 μl). This is an advantage when available serum volumes are small and when replacement samples are not available . We are currently performing such studies on the development of individual ACPA responses in sera from RA patients retrieved from historic biobanks from the years preceding the development of RA. In such studies, an imperative demand exists to use small serum quantities very efficiently.Certain sera yielded high background reactivities that might obscure the readout for the individual antigens. However, this is not unique to this specific microarray. It is well known, among developers of addressable laser bead immunoassays (ALBIA) and ELISA assays, that certain sera show unspecific binding. Given the setup of most commercial ELISAs, in which each serum is investigated only in wells coated with the antigen in question but without serum-specific control wells, this unspecific binding will often pass unperceived. Because we can readily assess the overall nonspecific binding in the used assay, we have excluded sera with apparent unspecific binding, but have also noticed that inclusion of such sera had very little effect on the performance at group level of the citrullinated peptides used in this investigation.th percentile for the healthy controls, implying that 2% of the healthy controls showed a positive response to each individual peptide. The discriminatory property of each individual citrullinated peptide in this multiplex assay is thus consistent with that of the anti-CCP2 assay when comparing RA patients and healthy controls. For stochastic reasons, this results in a higher number of healthy controls having a positive signal for any of the 12 analyzed peptides. These positive signals were, however, mostly only slightly above the threshold, and in only a very few of the healthy controls was multiple reactivity seen, as was the case in most anti-CCP2-positive RA patients.We defined the cutoff levels as the 98Our initial pilot studies indicated a gain in diagnostic sensitivity when subtracting the fluorescence intensities from the arginine control peptides. However, in this validation, by using a large set of RA patients and controls, this was not a universal finding, and similar numbers of peptides showed decrease or increase in diagnostic performance after correction for reactivity to control peptides. We conclude that the issue of arginine subtraction still is an open question, and that the most-suitable approach has to be decided for each antigen/peptide. This is also reflected by the different approaches used in currently available commercial ACPA tests, in which some assays, like the anti-Sa assay from Euroimmun, include sample-specific control wells with noncitrullinated antigen, whereas most assays, like the anti-CCP2 test, do not.et al. [et al., and a different setup of peptides was investigated. Thus, several features differ between the two multiplex approaches, in which our method may have certain advantages in allowing more antigens to be investigated in parallel (up to 170 per chip). However, a straight head-to-head comparison of the two approaches to multiplexing will be needed to harmonize the interpretation of ACPA multiplex assaying.In a recent study by Chandra et al. , anotherAs in our present investigation, the authors of the previous report  used no et al. [A particular issue in any effort to measure antibodies against several different epitopes on citrullinated autoantigens is the degree of cross-reactivity between these different epitopes. Previous ELISA-based investigations have addressed this issue by peptide-absorption experiments and concluded that a large fraction of the antibodies against epitopes included in our assay are not cross-reactive, whereas a smaller fraction of antibodies cross-react between different epitopes ,8,33. A et al. , have muet al. , reactivet al. . FurtherRA subgroups with different clinical prognosis are characterized by different autoantibody profiles. Besides the ACPA (and RF)-associated RA phenotype associated with poor long-term prognosis, we have, for example, defined an acute-onset RA phenotype associated with high levels of antibodies against native human CII ,35, wherOur long-term aim for the currently described multiplex assay platform is further to expand the number of peptides and protein fragments included, and further to validate the current and future antibody targets in additional clinical cohorts. Such extensions of numbers of targets will be feasible, as abundant positions are left for antigen spotting.We have developed a microarray for the simultaneous measurement of multiple specific ACPA responses. The individual analytes have been quantitatively validated against specific ELISAs and show a high specificity comparable to that of the anti-CCP2 assay. We foresee that this assay will have a broad range of applications in studies of prediction, diagnosis, and prognosis of arthritis, as well as in studies of specific ACPA responses before and during conventional and experimental therapies in RA.ACPA: anti-citrullinated proteins/peptide antibody; ACR: American College of Rheumatology; ALBIA: addressable laser bead immunoAssay; AU: arbitrary unit; CCP: cyclic citrullinated peptide; CCP-1: cyclic citrullinated filaggrin peptide encompassing amino acids 307 through 324 (Fil307-324); CEP-1: cyclic citrullinated α-enolase peptide encompassing amino acids 5 through 21 (Eno5-21); citC1: collagen type II peptide encompassing amino acids 359 through 369; CII: type II collagen; CII359-369: collagen type II peptide encompassing amino acids 359-369; CV: coefficient of variation; DMARD: disease-modifying anti-rheumatic drug; EIRA: Epidemiological Investigations in Rheumatoid Arthritis; ELISA: enzyme-linked immunosorbent assay; Eno5-21: cyclic citrullinated α-enolase peptide encompassing amino acids 5-21 (CEP-1); ERC: European Research Council; Fibα621-635: fibrinogen α-chain peptide encompassing amino acids 621-635; Fibβ36-52: fibrinogen β-chain peptide encompassing amino acids 36-52; Fibβ563-583: fibrinogen β-chain peptide encompassing amino acids 563-583; Fibβ580-600: fibrinogen β-chain peptide encompassing amino acids 580-600; Fibβ60-74: fibrinogen β-chain peptide encompassing amino acids 60-74; Fibβ62-81a: fibrinogen β-chain peptide encompassing amino acids 62-81, citrullinated at position 72; Fibβ62-81b: fibrinogen β-chain peptide encompassing amino acids 62-81 citrullinated at position 74; Fil307-324: cyclic citrullinated filaggrin peptide encompassing amino acids 307-324 (CCP1); IMI: Innovative Medicines Initiative; ISAC: immuno solid phase antigen chip; NSAID: nonsteroidal antiinflammatory drug; OD: optical density; RA: rheumatoid arthritis; RF: rheumatoid factor; ROC: receiver operating characteristic; RU: response unit; Vim60-75: vimentin peptide encompassing amino acids 60-75; Vim2-17: vimentin peptide encompassing amino acids 2-17.The project is part of the Innovative Medicines Initiative (IMI) project BeTheCure, in which Karolinska Institutet is a scientific partner and Phadia AB/ThermoFischer is a commercial partner. The project follows the rules for IMI projects.Thomas Schlederer, Linda Mathsson, Per Matsson, and Mats Nystrand are employees of Phadia AB/ThermoFischer.Lars Klareskog, Vivianne Malmström, and Rikard Holmdahl are co-founders of a company, Curara AB, which collaborates with Phadia/ThermoFischer concerning certain technical aspects of the multiplex assay. This development is done with partial support from an ERC Proof of Concept Grant (pRActice) to LK.Rikard Holmdahl is the inventor of patent US7/148,020B2, describing the diagnostic use of the C1 epitope.Karin Lundberg is co-inventor of patent US12/524,465, describing the diagnostic use of the CEP-1 epitope.As co-inventors of international patents about ACPA held by BioMérieux Cy and licensed to Eurodiagnostica Cy and Axis-Shield Cy, Leonor Nogueira and Guy Serre receive parts of the royalties paid to the Toulouse III University.Johan Rönnelid has received remuneration for lectures and for the salary for Linda Mathsson from Phadia AB during the time she was employed in his laboratory. He has also obtained reagents from Phadia AB for the investigation on other rheumatoid arthris cohorts, as well as partial allowance for costs for attending congresses, in accordance with the rules established between Läkemedelsindustriföreningen  and the public Swedish health care system.The other authors declare that they have no competing interests.MH planned the study and performed laboratory work with the ISAC chip and validation ELISAs. LM planned the study and performed laboratory work with the ISAC chip. TS performed the spotting of ISAC chips. LI established and performed validation ELISAs. PM planned the study. LN provided fibrinogen peptides and performed validation ELISAs. PJJ provided fibrinogen peptides. KL provided α-enolase peptide and participated in ELISA validation. VM participated in ELISA validation. GS provided fibrinogen peptides. RH provided collagen peptides. MN planned the study and participated in the laboratory work. LK planned the study. JR planned the study, performed statistical analyses, and drafted the manuscript. All authors read, commented on, and approved the final manuscript.


In humans, different B-cell subpopulations can be distinguished in peripheral bloodand other tissues on the basis of differential expression of various surface markers.These different subsets correspond to different stages of maturation, activation anddifferentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, iswidely used in the treatment of various malignant and autoimmune diseases. Rituximabinduces a very significant depletion of B-cell subpopulations in the peripheral bloodusually for a period of 6 to 9 months after one cycle of therapy. Cells detectedcirculating during depletion are mainly CD20 negative plasmablasts. Data on depletionof CD20-expressing B cells in solid tissues are limited but show that depletion issignificant but not complete, with bone marrow and spleen being more easily depletedthan lymph nodes. Factors influencing depletion are thought to include not only thetotal drug dose administered and distribution into various tissues, but also B-cellintrinsic and microenvironment factors influencing recruitment of effector mechanismsand antigen and effector modulation. Available studies show that the degree ofdepletion varies between individuals, even if treated with the same dose, but that ittends to be consistent in the same individual. This suggests that individual factorsare important in determining the final extent of depletion. In humans from birth all new B cells originate from common precursors in the bonemarrow. In the bone marrow, peripheral blood and secondary lymphoid tissues, differentB-cell subpopulations can be distinguished corresponding to different stages ofmaturation, activation and differentiation. B-cell subpopulations are characterisedmainly by the differential expression of different cell surface markers that includevarious cluster of differentiation (CD) molecules and different surface immunoglobulinisotypes (B-cell antigen receptor). B-cell development can be separated into an earlierantigen-independent phase, which takes place in the bone marrow, and a laterantigen-dependent phase that takes place mainly in secondary lymphoid tissues. In asimplified way, the different B-cell lineage subsets include pro-B cells, pre-B cells,immature and transitional B cells, mature naïve B cells, memory B cells,plasmablasts and plasma cells Figure . Plasmab+ memory B-cell subsets [B-cell precursor subpopulations are found in the bone marrow. In the peripheral blood,transitional, naïve mature and memory B cells and plasmablasts, and more rarelyplasma cells, can be identified. Plasma cells are more frequently seen in the bonemarrow and peripheral lymphoid tissues. Centrocytes and centroblasts are found insecondary lymphoid tissues where germinal centre reactions take place, and are not foundcirculating in peripheral blood. Marginal zone B cells can be found in the marginal zoneof the spleen and similar populations are described in particular locations in othersecondary lymphoid tissues . Margina subsets ,2.+IgM+CD27+ memory B cells,IgD-IgM+CD27+ memory B cells (IgMonly memory Bcells), post-switch IgA+CD27+ and IgG+CD27+memory B cells and also IgA+CD27- andIgG+CD27- memory B cells [Immunophenotyping of B cells with multiparameter flow cytometry has allowedidentification of an increasing number of different subpopulations, increasing ourknowledge of normal B-cell biology and, in particular, changes associated with differentdisease states. For example, different memory B-cell subsets have now been described inperipheral blood including subsets that do not express CD27, a marker previously thoughtto be present on all memory B cells ,4. Memor B cells . These m B cells . A poten B cells . A propo B cells ,8-14.®,Rituxan®; Roche, Basel, Switzerland) was licensed for the treatmentof follicular lymphoma in 1997/98 and later for diffuse large non-Hodgkin's lymphoma andchronic lymphocytic leukaemia. In 2006 rituximab was licensed for the treatment ofrheumatoid arthritis (RA). Rituximab is also used off-license for the treatment of otherB-cell malignant diseases, in transplantation and for the treatment of a variety ofother autoimmune diseases, predominantly diseases associated with the presence ofautoantibodies. Various other therapeutic anti-CD20 mAbs are either available on themarket , undergoing clinical trials or underdevelopment [Anti-CD20 mAbs were developed in the late 1980s and in the 1990s for the treatment ofnon-Hodgkin's lymphoma of B-cell origin. Rituximab  orterminally differentiated plasmablasts and plasma cells Figure . The CD2Because haematopoietic stem cells are not directly depleted by anti-CD20 antibodies, onecourse of treatment with rituximab is followed by B-cell repopulation of the peripheralblood starting usually within 6 to 9 months-but it can take several months or even yearsfor total B-cell numbers in the peripheral blood to recover to pretreatment levels.Repopulation occurs mainly with naïve B cells, with increased frequency and numbersof transitional B cells similar to that seen after bone marrow transplantation ,17. The The fact that plasma cells are also not directly depleted by anti-CD20 antibodiesexplains why, in the majority of patients, serum total immunoglobulin levels remainwithin the normal range after treatment with one course of rituximab. Several studieshave shown that serum levels of several autoantibodies decrease after treatment withrituximab  and do so proportionallymore than total immunoglobulin levels or anti-microbial antibodies -23. Thisinvivo. Depletion in the peripheral blood is frequently higher than 99% butdepletion in other tissues has been less well studied, with several studies documentingthat depletion in solid tissues with rituximab is frequently not complete and can showconsiderable variation between individuals. In vitro, rituximab depletesmalignant B cells by antibody-dependent cellular cytotoxicity, complement-mediatedcytotoxicity and induction of apoptosis. In vivo, rituximab is thought to actmainly by inducing antibody-dependent cellular cytotoxicity with activation ofcomplement also contributing [Treatment with rituximab is associated with major depletion of normal B cells ributing . One of ributing ,25,26. IBecause depletion is achieved by binding of the mAbs to the cell surface CD20 molecules,the final extent of depletion will necessarily depend on the relationship between totalnumber of B cells and total dose of rituximab administered, on accessibility of the drugand effector immune cells to the tissues where B cells are located, on intrinsic orextrinsic factors that may influence B-cell survival and on the efficacy of recruitedhost immune mechanisms responsible for depletion.Former small dose-ranging studies in lymphoma and in animal models have shown that Bcells in the peripheral blood are readily killed by anti-CD20 antibodies but that higherdoses and higher serum levels are needed for depletion in extravascular sites ,24,25.Factors influencing antigen and effector modulation are thought to be important indetermining the final extent of depletion achieved Table  18,27,2,227,28. Intrinsic B-cell factors that may influence depletion include high expression ofcomplement regulatory proteins as seen in chronic lymphocytic leukaemia . In cynoIn animal models, certain subpopulations have been shown to be more resistant todepletion with anti-CD20 antibodies but this varies with the mice strain used andwhether they were studies using human CD20 transgenic mice treated with anti-human CD20mAbs or non-transgenic mice treated with anti-mouse CD20 mAbs ,33. Popuin vivo is not known.Different B-cell malignancies deriving from B cells at different stages ofdifferentiation and different tumour locations are also associated with differentialresponses to treatment with anti-CD20 mAbs but susceptibility of the correspondentnormal human B-cell sub-populations is expected to be substantially different. Whetherthere are any differences in susceptibility to depletion of autoreactive human B-cellclones when compared with nonautoreactive ones, as suggested by mouse models [To what extent the differential susceptibility of various B-cell subsets demonstrated insome of the animal models reflects what happens in humans e models , and wheIn addition, administration of chimaeric anti-CD20 mAbs such as rituximab can beassociated with formation of human anti-chimaeric antibodies that can influence drugaction and clearance. Although most large studies show no association between thepresence of human anti-chimaeric antibodies and clinical response or depletion, thisassociation has been described, for example, in small studies in systemic lupuserythematosus patients ,37.in vivo during theperiod of depletion. Are they eventually depleted later on, particularly if theyrecirculate in peripheral blood? Are they functionally impaired? Are they able to expandin an environment with less competition and raised BAFF levels? Kamburova and colleaguestried to address some of these issues by studying the in vitro effects ofincubation with rituximab on proliferation, activation and differentiation ofnondepleted human normal peripheral blood B cells [- naïve Bcells but not of CD27+ memory B cells and this was associated with a relativeincrease of B cells with an activated naïve phenotype. B cells stimulated in thepresence of rituximab induced stronger T-cell proliferation and the T-cell populationshowed a more Th2-like phenotype. These results suggest that B cells which are exposedto rituximab but are not depleted may have altered function and that naïve andmemory B cell populations may be differentially affected. Whether any of these phenomenaoccur in vivo and what their implications would be are unclear. Interestingly,and similar to what happens after bone marrow transplantation, the residual B cells arenot able to expand and repopulate the peripheral blood, even in the presence of abundantBAFF.With evidence showing that not all B cells that bind rituximab are depleted there is aninterest in knowing what exactly happens to these cells  B cells . They reAdministration of rituximab is usually associated with a rapid and profound depletionof circulating B cells in the peripheral blood . Major dThe very small numbers of circulating B cells that can be detected during periods ofdepletion usually show a phenotype of plasmablasts but cells with memory or evennaïve B cells have also been reported ,40,41,43+IgD+)was larger than thedecrease in the switched memory B-cell population (CD27+IgD-) [Repopulation of the peripheral blood after treatment with a standard dose ofrituximab usually starts 6 to 9 months after treatment with predominantlytransitional and naïve B cells as previously mentioned. Frequently, repopulationwith larger numbers of memory B cells and/or plasmablasts has been associated withearlier relapse ,40,45. A27+IgD-) . However+memory B cells before treatment [In RA, nonresponse has been associated with higher numbers of plasmablasts beforetreatment and early relapse has been associated with higher numbers of CD27reatment ,45. Agai- plasmablasts and plasma cells to depletion [Unfortunately, there are limited data on the degree of depletion of normal B cells insecondary lymphoid organs and other solid tissues in human individuals treated withrituximab, and hardly any data on differential susceptibility to depletion ofdifferent subpopulations in different tissues except for the expected resistance ofCD20epletion . Animal epletion ,48,49. Tepletion ,20,48,49- plasma cells has been observed asexpected [In bone marrow samples of RA patients treated with rituximab a relatively high numberof B-cell precursors subpopulations can be seen -52. Thisexpected ,51. In texpected ,52. Onceexpected ,52.The presence of cells of B-cell lineage that presumably should be expressing CD20 hastherefore been well documented and rituximab is probably still present and binds tothe CD20 molecule, preventing its detection in flow cytometry as discussed above ,51. AlteIn a study of autopsy samples of lymph node and spleen of patients with lymphomatreated with rituximab monotherapy or with rituximab and chemotherapy, a substantialreduction of B-cell populations was documented-with only three out of eight patientsshowing any reactivity for markers of cells of B-cell lineage in the lymph nodes andonly one out of eight in the spleen by immunohistochemistry . SimilarVaccination studies in patients treated with rituximab can provide indirect data onB-cell subpopulations that may be resistant to depletion with anti-CD20 mAbs.However, published data are difficult to interpret because of the small number ofpatients, effects of concomitant therapy and the background disease itself on thehumoral response to vaccines and, in particular, because studies included patients atvarious stages of B-cell depletion or repopulation at the time of vaccination. Moststudies have looked at responses to influenza vaccines and showed absent or decreasedhumoral responses to vaccination in patients previously treated with rituximab whencompared with normal controls or patients not treated with rituximab -64. Some- plasma cells [In patients with RA, several studies have documented significant but variabledepletion of B cells in samples of synovial tissue of involved joints and persistenceof CD20ma cells -68. Varima cells . In a stma cells . A previma cells . In a stma cells .In summary, although there are several studies looking at the degree and duration ofB-cell depletion induced by rituximab in the peripheral blood, there is very littleinformation on the exact degree of depletion in solid tissues - and, in particular, fewdefinite data on whether different subtypes of CD20-expressing B cells are more or lesssusceptible to depletion by anti-CD20 antibodies. The data available suggest that thereis variability between individuals on the extent and duration of depletion induced andthat this may have clinical correlations with response and duration of response inautoimmune diseases. Understanding what underlies this variability - and, in particular,whether drug clearance and antigen and effector modulation phenomena are involved - hasthe potential to lead to more effective B-cell depleting strategies and to increasingour understanding of the role that different B-cell subtypes play in the pathogenesis ofthe different autoimmune diseases.BAFF: B-cell activating factor; CD: cluster of differentiation; mAb: monoclonalantibody; RA: rheumatoid arthritis; Th: T-helper type.The author has received consultancy fees and funding to attend international medicalmeetings from Roche Pharmaceuticals and consultancy fees and research funding fromGlaxoSmithKlein.Arthritis Research & TherapyVolume 15 Supplement 1, 2013: B cells in autoimmune diseases: Part 2. Thesupplement was proposed by the journal and content was developed in consultation withthe Editors-in-Chief. Articles have been independently prepared by the authors and haveundergone the journal's standard peer review process. Publication of the supplement wassupported by Medimmune.This article has been published as part of


Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma.Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper.Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization.The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four  or three  reference miRNAs is recommended for normalization. MicroRNAs (miRNAs) belong to a class of small noncoding RNAs of 19 to 24 nucleotides that are known to regulate signaling pathways for various cell functions. Not surprisingly, changes in miRNA expression have been associated with several diseases, including cancer Several methods such as bead-based flow cytometry, microarray, deep sequencing, and real-time quantitative PCR (RT-qPCR) allow fast, high-throughput, and sensitive profiling of miRNAs. RT-qPCR produces specific, sensitive, and reproducible quantification of nucleic acids. To overcome experimental variations in RT-qPCR analyses , relative quantification of miRNAs of interest based on the normalization to reference genes is the approach of choice to prevent errors within a dataset Because of our general interest on miRNAomes in urological tumors and the increasing incidence of urothelial cancer Bladder cancer is the fourth most common cancer in Western industrialized countries In the present study, we aimed to systematically identify suitable reference genes for normalizing RT-qPCR assays of miRNA expression in urothelial carcinoma tissue. Using miRNA microarray analyses, we first identified invariant miRNAs that showed stable expression in both nonmalignant and malignant bladder tissue samples as candidate reference miRNAs. RT-qPCR analyses were subsequently performed for validating these miRNAs from the microarray experiments and the above mentioned small RNAs RNU6B, RNU48, and Z30 as putative reference genes. Appropriate reference miRNAs were identified by geNorm, NormFinder, and BestKeeper, and the results of unsuitable normalization are illustrated with invalid normalizers.All bladder cancer patients went through radical cystectomy or transurethral resection at the University Hospital Charité in Berlin between 2008 and 2009 and gave written informed consent for the use of representative tissue specimens for research purposes. The study was approved by the Ethic Committee of the University Hospital Charité (File: EA1/153/07). The samples were collected immediately after surgery in liquid nitrogen and stored at −80°C until further analysis. Tumor staging was performed in conformity with the International Union Against Cancer Frozen histologic sections were prepared, stained with hematoxylin/eosin, and examined by uropathologists . Only tissue specimens with more than 80% tumor cells were included in the study as tumor samples. Tissue cryotome sections  were treated with 350 µl of lysis buffer and total RNA including miRNAs was isolated using the miRNeasy Mini Kit  with 30 to 50 µl of elution buffer according to the manufacturer’s protocol. An additional DNase I digestion step on the RNA binding silica gel membrane of the spin column was performed. RNA concentration and the 260 nm to 280 nm absorbance ratios were measured on the NanoDrop 1000 spectrophotometer . The quality of isolated RNA was determined by the RNA integrity number (RIN) with a Bioanalyzer 2100 . Only samples with RIN values >5 were used. The RNA samples  isolated from nonmalignant as well as from low-grade and high-grade tumor tissue samples showed comparable median 260/280 absorbance ratios  and RIN values .Microarray analyses of eight samples each from nonmalignant tissue and low and high grade tumor specimens were performed. One-color hybridizations on human catalog 8-plex 15 K microRNA microarrays (AMADID 019118) from Agilent encoding probes for 723 human and 76 viral microRNAs from the Sanger database v10.1 were used. All reaction steps were carried out as previously described in detail PLUS  was used, employing a generalized and universally applicable quantification model based on efficiency correction, error propagation, and multiple reference gene normalization RT-qPCR analyses of miRNAs were carried out with TaqMan microRNA assays  according to the manufacturer’s protocol and the MIQE guidelines Statistical analyses were performed using GraphPad Prism Version 5.04 . Non-parametric tests  were used to analyze significant differences between independent groups. The Spearman correlation coefficients were applied to calculate the relationships between the miRNAs as well as between the clinical variables and the expression of candidate reference miRNAs. P values <0.05 (two-tailed) were considered statistically significant.Plus as an implementation of the software qBasePLUS  The assessment of the putative reference genes for normalization was evaluated by the computer programs geNorm To identify putative reference miRNAs in the miRNA microarray data obtained from the eight samples of each tissue group, the following criteria were used: (a) miRNAs had to be detected in Genespring GX11 software as “present” in all examined 24 samples to filter out signals that did not reach a minimum of intensity, (b) the absolute fold change between the nonmalignant and the two cancerous groups had to be lower than 1.2-times with (c) no significant differences (P>0.05) between the groups. Based on the total of 723 human miRNA species located on the Agilent microarray chip according to the miRBase version 10.1, we identified 101 miRNAs that were flagged as “present” in all of the examined groups . Eight oTo increase the statistical power to find suitable reference miRNAs, in addition to the 24 analyzed samples in the microarray experiments, we included nine nonmalignant and 25 malignant tissue samples as described in the section “Patients and tissue samples” to validate the aforementioned 16 candidate reference miRNAs in more detail by RT-qPCR. Furthermore, the set of candidate reference miRNAs was extended by the small RNAs RNU6B, RNU48, and Z30 that were commonly used for expression normalization in the literature as stated in the Introduction. First, to determine if reliable quantification of these putative normalizers is feasible by RT-qPCR, three RNA pools were prepared containing equal amounts of RNA from the samples used in the microarray analysis. miR-15a, miR-20b, miR-107, miR-513a-5p, and miR-939 showed Cq values >35 in the pools and were excluded from further analyses because accurate quantification would be questionable. By this preselection, 11 putative reference miRNAs , sex , or tumor stage . Differences in expression between low-grade and high-grade tumors were found for miR-29c , miR-874 , miR-181a , and miR-181b , while all other miRNAs were not differentially expressed .The expression of the 11 miRNAs and three small RNAs was not associated with age , which is a criterion for the optimum number of reference genes  were recommended as an optimum reference miRNA set for normalization; the best combination of two reference miRNAs was miR-125a-5p and miR-151-5p, and the best single reference miRNA was miR-151-5p. After excluding the potentially deregulated reference miRNAs mentioned above, geNorm analysis was repeated. However, under these conditions, geNorm calculated a VNF value >0.15 and therefore did not recommend a normalization set.To identify suitable reference genes for the normalization of miRNA expression, we applied the aforementioned three computer programs geNorm, NormFinder, and BestKeeper. GeNorm, an implementation of the new software qBasece genes . The proSimilar to geNorm, NormFinder identified genes with the lowest M values as the most stably expressed reference targets . NormFinBestKeeper considers all genes in all observed groups. First, BestKeeper determines the geometric mean and coefficient of variance. Genes with a standard deviation greater than 1 were assumed to be inconsistent. In the second step, the inter-gene relationships were examined by pairwise correlation analysis. This calculation is used to determine whether the gene expression exhibits a similar behavior. Candidate reference genes that highly correlate with each other are included in the BestKeeper-Index calculation. The program provides only an analysis of ten candidate reference genes simultaneously. Therefore, we excluded the reference targets with the lowest M values as determined by geNorm (miR-181a) and NormFinder (miR-424) and also excluded miR-29c . Under these conditions, BestKeeper ranked RNU48 as the best reference gene, followed by miR-874, miR-151-5p, and Z30.th by geNorm, 12th by NormFinder, and 9th by BestKeeper , 11 invariant miRNAs were identified to be putative reference miRNAs. Since miR-151-3p and miR-151-5p derive from the same pre-miRNA and miR-151-5p exhibited slightly higher expression in examined samples, we included only miR-151-5p in further analysis. A data search in the miRNA bladder cancer studies mentioned in the Introduction showed that miR-29c, one of these 11 invariant, stably expressed miRNAs, was found to be down-regulated in two microarray studies The putative reference miRNAs identified by the microarray analyses, except miR-151-3p as mentioned, were included with the additional RNAs RNU6B, RNU48, and Z30 in the geNorm, NormFinder, and BestKeeper analysis. Differences in expression observed in the subsequent RT-qPCR measurements between nonmalignant, low-grade, and high-grade tumor samples as well as co-expressions of genes did not exclude candidate reference genes. However, as comprehensively described in the Results section, geNorm, NormFinder, and BestKeeper did not always recommend the same reference miRNAs for normalization . The lacThe differences between the recommended reference miRNAs by the three programs prompted us to validate their suitability in clinical samples . The impIn summary, the present study was the first systematic investigation to identify suitable reference miRNAs in a transparent and comprehensive manner for the relative quantification of the microRNAome in urothelial carcinoma. It was based on a four-step approach with microarray analyses, RT-qPCR validation, reference miRNA selection through computer software, and proof of principle with different miRNA expression levels. Starting with 16 putative reference miRNAs from the microarray analysis and three additional small RNAs from the literature, we validated several combinations of reference miRNAs for miRNA expression studies in bladder cancer. We believe that these are robust methods that will allow future studies on the functional roles of miRNAs as regulators in signal transduction and metabolic pathways that are associated with small expression changes.Table S1Description of the experimental details of the RT-qPCR analyses according to the checklist of the MIQE guidelines.(PDF)Click here for additional data file.Table S2TaqMan assays for microRNAs and small nuclear and nucleolar RNAs.(PDF)Click here for additional data file.Table S3List of the 101 miRNAs from the microarray for the identification of candidate reference miRNAs.(PDF)Click here for additional data file.Table S4s) between the candidate reference genes.Spearman rank correlation coefficients (r(PDF)Click here for additional data file.Methods S1qPCR validation experiments according to the MIQE guidelines with respect to the calibration curves and the dynamic range of measurements.(PDF)Click here for additional data file.


Pediatric infectious disease clinicians in industrialized countries may encounter iatrogenically transmitted HIV, hepatitis B virus, and hepatitis C virus infections in refugee children from Central Asia, Southeast Asia, and sub-Saharan Africa. The consequences of political collapse and/or civil war—work migration, prostitution, intravenous drug use, defective public health resources, and poor access to good medical care—all contribute to the spread of blood-borne viruses. Inadequate infection control practices by medical establishments can lead to iatrogenic infection of children. Summaries of 4 cases in refugee children in Australia are a salient reminder of this problem. Blood-borne viruses (BBVs) have benefitted from internal political strife, migration, prostitution, intravenous/injection drug use, and defective public health resources in some Central Asian republics and Southeast Asian and sub-Saharan African countries. Iatrogenic transmission of HIV in children in Romania , where medical procedures have resulted in transmission of HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV). Among the case-patients are 2 children with BBV co-infection.<12 years of age) from high-prevalence countries were notified to the Australian National HIV Registry through the end of 2011. The mother of 1 child was reported as HIV negative. No information was available about the HIV status for the mothers of the 7 other children .Detecting possible iatrogenic BBV infections in children relies on a careful history. However, accurate histories are difficult to obtain because, in many cases, details are acquired through interpreters and facts are lost in translation. Nevertheless, several encounters with a family, during which family members are encouraged to tell their life stories  Infectious Diseases Clinic for assessment and management. Some children are not screened on arrival, and undiagnosed BBV infections may be discovered later.BBV infection acquired through injection of blood or blood products and for which vertical (mother-to-child) infection (including breast milk and surrogate breast-feeding) was ruled out by serologic testing of the mother and by history of sexual abuse and evidence of tattooing, piercing, and scarification. Iatrogenic infection, a form of horizontal transmission, usually involves mediation through injection of the BBV by a third party who might or might not be infected with the virus(es) in question. For this study, I defined an iatrogenic BBV infection according to all of the following criteria: BBV infection in a refugee child <18 years of age who was seen at the WCH Infectious Diseases Clinic during 2008–2010, who had been exposed to medical hollow-bore needles and blood products, who had acquired a BBV infection that was absent in his/her mother, and who had not been breast-fed by a surrogate or sexually abused but for whom other modes of horizontal infection could not be completely ruled out.Internet searches of databases, including PubMed and Google Scholar, were performed to obtain peer-reviewed and other journal articles by using the following search terms: iatrogenic blood-borne virus infection, refugee children, blood-borne viral infections in war and transition zones, HIV, HBV, HCV. In addition, case reports of 4 refugee children seen in the WCH pediatric infectious diseases clinic during January 2008–December 2010  are summAn 8-year-old girl was brought for treatment in early 2010. She was born vaginally in a hospital in Uzbekistan after a full-term normal pregnancy. There were no neonatal concerns. At 18 months of age, severe hematemesis developed. Blood loss required admission to a clinic in Andijan and transfusions of donated plasma and of blood donated by her father. At 7 years of age, she underwent a tonsillectomy in the same clinic. She had no illness typical of HIV seroconversion. Her mother was hepatitis B surface antigen (HBsAg) positive and HIV negative. The girl’s 2 younger siblings were BBV negative but anti-HBs positive. As part of migrant health screening on arrival in Australia in 2009, family members underwent serologic tests for HIV, HBV, and HCV. The girl was anti-HIV-1 and HBsAg positive. Her HIV load was 1,010 HIV RNA copies/mL, and HBV DNA was not detectable (<20 IU/mL) (HBeAg and anti-HBe negative) in 2009. Her father was negative for BBV markers. Her CD4 count was 260 cells/μL (19%), indicating severe immunosuppression. Examination was largely unremarkable except for severe dental caries and an ulcer on the lower gums (PCR positive for herpes simplex type 1). She began antiretroviral therapy soon after diagnosis and has responded well.On arrival to Australia in 2010, a 9-year-old boy was discovered to be anti-HIV-1 positive on arrival. Both parents tested negative for anti-HIV-1 and anti-HCV. Through an interpreter, it was established that he had exposure to medical needles in the hospital. In 2004, he had undergone an appendectomy at the same Andijan clinic mentioned above; in summer 2008, he was admitted with “hepatitis”  to an Andijan hospital, where he received several injections (possibly vitamin K) by medical and nursing staff, but as far as is known, received no blood products and had no seroconverting-like illness. He had been asymptomatic. His prenatal history was unremarkable; he was born at term by normal vaginal delivery. He was vaccinated in Uzbekistan but to an unknown extent. He looked reasonably well. There was no lymphadenopathy. He had severe dental caries. He began antiretroviral therapy soon after his HIV-1 infection was diagnosed and has responded well.This child, the younger sibling of case-patient 2, arrived in Australia with his family in 2010 when he was 5 years 9 months of age. At arrival screening, he was anti-HIV-1 negative and anti-HCV positive. He had attended the clinic in Andijan in November 2007 for “management” of jaundice, presumably associated with hepatitis A infection acquired from his sibling (case-patient 2), who had contracted hepatitis A and received the described treatment ≈40 days before case-patient 3 received his. Case-patient 3 was in the Andijan clinic for 10 days and received daily injections (the nature of which is unknown)—presumably the source of the HCV infection. When first seen at WCH on referral, he was asymptomatic. There were no abnormal physical findings. He was repeatedly anti-HCV positive and HCV RNA negative, indicating resolved infection.This boy was born in Andijan in 2002. Immigration serology screening was not performed. He was first seen at WCH as a refugee in 2008 at 6 years of age after his general practitioner found him to be anti-HCV positive (anti-HIV-1 and HBsAg testing was not performed at the time). His mother was anti-HCV negative. He was born at 38 weeks’ gestation and had some neonatal problems for which he received multiple intravenous injections and blood transfusion(s) in an Andijan hospital. His 2 younger siblings were anti-HCV negative. Examination findings were normal except for serious dental caries requiring extractions. His liver function showed slightly elevated alanine aminotransferase . Initially, nonquantitative HCV RNA was detectable in his peripheral blood, and in early 2011, his viral load was 623 IU/mL, at which time his liver function was entirely normal. In 2012, because of rising alanine aminotransferase (57 U/L) and increasing viral load , he was referred to a pediatric gastroenterologist, who noticed that anti-HIV and HBsAg/antibody tests had never been performed; results were positive for anti-HIV-1 (HBsAg and anti-HBs negative). The patient’s viral load was 18,300 RNA copies/mL. His CD4 count was 810 cells/μL (27%). Examination indicated no abnormality. His siblings and parents are anti-HIV negative.Vertical transmission of the viruses involved in these 4 cases was ruled out by the mothers’ negative serostatus, as well as by exclusion based on history of surrogate breast-feeding and sexual abuse. Notwithstanding a small risk for horizontal transmission, the most likely mode of transmission is iatrogenic. Incomplete documentation made other cases speculative. One case-patient had an additional risk factor of rape. Three others with HBV infection in whom the serostatus of the mothers was tested and had revealed anti-HBs positivity could imply vertical transmission with maternal infection resolution and natural immunity. The presumptive cases demonstrate intended or unintended effects of nonfunctional infection control in the chaos left by political collapse and/or civil war on health systems and the blood supply. Such recent events have affected Central Asian countries, including Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and to a lesser extent, Uzbekistan  showed an incidence of hepatitis B of 8.1% with no cases of HIV or HCV infection  to increase in prevalence (through prostitution and intravenous/injection drug use) and therefore form an environment for possible iatrogenic transmission. Hamers and Downs  emerged in the second half of the 20th century. Almost certainly it was triggered and fed iatrogenically by the increasing use of injections and blood transfusion. In industrialized countries, the introduction of anti-HCV screening for blood donors sharply decreased the incidence of iatrogenic hepatitis C, but HCV continues to spread in developing countries, where the virus is still transmitted through unscreened blood transfusions and nonsterile injections (In 2004, Shisana et al. (No data exist on sharing of contaminated equipment during self-injection in children and adolescents in countries of high BBV prevalence (Information about modes of transmission in HIV-infected African children who have HIV-uninfected mothers is generally lacking. Since 1984, horizontally acquired HIV infection in African children who have HIV-negative mothers has been reported from 13 sub-Saharan countries (In a 2005 serosurvey, Rehle et al. (Estimates of up to 160,000 HIV, 4.7 million HCV, and 16 million HBV infections each year are attributable to unsafe injections (


This study sought to determine whether ethnic differences in diabetes, dyslipidemia, and ectopic fat deposition account for ethnic differences in incident cardiovascular disease.Coronary heart disease risks are elevated in South Asians and are lower in African Caribbeans compared with Europeans. These ethnic differences map to lipid patterns and ectopic fat deposition.Cardiovascular risk factors were assessed in 2,049 Europeans, 1,517 South Asians, and 630 African Caribbeans from 1988 through 1991 (mean age: 52.4 ± 6.9 years). Fatal and nonfatal events were captured over a median 20.5-year follow-up. Subhazard ratios (SHR) were calculated using competing risks regression.Baseline diabetes prevalence was more than 3 times greater in South Asians and African Caribbeans than in Europeans. South Asians were more and African Caribbeans were less centrally obese and dyslipidemic than Europeans. Compared with Europeans, coronary heart disease incidence was greater in South Asians and less in African Caribbeans. The age- and sex-adjusted South Asian versus European SHR was 1.70  and remained significant  when adjusted for waist-to-hip ratio. The African Caribbean versus European age- and sex-adjusted SHR of 0.64  remained significant when adjusted for high-density lipoprotein and low-density lipoprotein cholesterol . Compared with Europeans, South Asians and African Caribbeans experienced more strokes , and this differential was more marked in those with diabetes .Ethnic differences in measured metabolic risk factors did not explain differences in coronary heart disease incidence. The apparently greater association between diabetes and stroke risk in South Asians and African Caribbeans compared with Europeans merits further study. Cardiovascular disease (CVD) is now the leading cause of death globally . Marked Both ethnic minority groups have more insulin resistance (IR) and diabetes than Europeans, but although South Asians display classical dyslipidemia and central obesity associated with IR, African Caribbeans have favorable lipoprotein profiles and less central obesity than Europeans. We hypothesized that diabetes and associated metabolic disturbances, measured in midlife, would account for ethnic differences in incident fatal and nonfatal CVD in a unique tri-ethnic community-based United Kingdom cohort followed for 20 years.The SABRE  study examined a tri-ethnic community-based cohort from North and West London. Details of the cohort have been published . BrieflyParticipants attended a baseline clinic after an overnight fast. They underwent blood pressure measurements, electrocardiography, and anthropometry and completed a health and lifestyle questionnaire . Height Fasting bloods were drawn, and those not known to have diabetes underwent an oral glucose tolerance test . Bloods Baseline IR was estimated using the homeostasis model assessment 2 calculator . BaselinSince baseline, participants have been flagged for death by the Office for National Statistics. From 2008 through 2011, survivors were invited to take part in a morbidity follow-up. This included a health and lifestyle questionnaire, primary care medical record review, attendance at our clinic at St. Mary's Hospital, London, or a combination thereof. Hospital Episode Statistics were obtained for traced participants since baseline.All participants gave written informed consent. Approval for the study at baseline was obtained from Ealing, Hounslow, and Spelthorne, and University College London research ethics committees, and at follow-up from St. Mary's Hospital Research Ethics Committee (ref. 07/H0712/109).1Cause of death included any of the following: angina, myocardial infarction or its sequelae, or atherosclerotic heart disease using International Classification of Disease-Ninth Edition (ICD-9) codes 410 through 415 or ICD-Tenth Edition (ICD-10) codes I200 through I259.2Primary care data were reviewed independently by 2 senior physicians blinded to participant ethnicity and identity. A CHD event was identified if both physicians agreed on definite or probable diagnosis of myocardial infarction or acute coronary syndrome, according to pre-determined criteria used in the ASCOT  , based o3Hospital Episode Statistics: for causes of death, diagnostic ICD-9 codes 410 through 415 or ICD-10 codes I200 through I259 were used, in addition to any of the following operation codes from the Office of Populations and Surveys classification of interventions and procedures: K401 through K469, K491 through K504, K751 through K759, or U541 (coronary revascularization interventions or rehabilitation for ischemic heart disease).For CHD, a composite endpoint comprised the first event after baseline identified from any of the following sources:1Cause of death included ICD-9 codes 430 through 439 or ICD-10 codes I600 through I698.2Primary care data were reviewed in a similar manner to CHD, with definite or probable diagnosis of stroke made according to pre-determined criteria based on symptoms, duration of symptoms, and magnetic resonance image or computed tomography imaging.3Hospital Episode Statistics: diagnostic ICD-9 codes 430 through 439 or ICD-10 codes I600 through I698.4Participant report of physician-diagnosed stroke and duration of symptoms in excess of 24 h.For stroke, the first event after baseline identified from any of the following sources:Coronary heart disease or stroke that occurred before baseline was identified from participant report (at baseline) of physician-diagnosed disease (or presence of major Q waves on baseline electrocardiography for CHD).t) or nonparametric (Wilcoxon rank-sum or chi-square) statistical tests as appropriate.Primary analyses related to participants with follow-up data from any source. We combined first post-baseline fatal and nonfatal CHD or stroke events as primary outcomes. Baseline characteristics were stratified by sex; ethnic group comparisons were made with Europeans as the reference group within each sex, using parametric (Student Competing risk regression (competing risk = death from other causes) based on the proportional subhazards methods of Fine and Gray  was usedWe assessed linearity of associations using tertiles of continuous covariates; no initial analyses suggested departure from linearity, and we included these as linear terms, with exceptions of blood pressure, where we used 4 categories (those receiving treatment in the top category and the remaining into thirds by ascending level of blood pressure). We grouped measures of glycemia and IR similarly, with diabetes in the top category. There were no significant sex and ethnicity interactions in association with stroke or CHD events, and we show results for men and women combined to maximize statistical power. We investigated potential mediators of observed ethnic differences in univariate analyses. Multivariable models included covariates that had the greatest positive or negative effects on ethnic differentials. We examined the composite Framingham  and INTETo assess the integrity of proportional hazards assumptions, we tested interactions between covariates and follow-up time. We plotted cumulative incidence curves for each ethnic-diabetes group and examined Schoenfeld-like residuals.We compared baseline characteristics of 661 people lost to follow-up with those of 4,196 people followed-up. We repeated the above analyses as follows: (1) excluding 47 people who migrated directly from West Africa; (2) using data derived from direct follow-up only ; (3) separately for fatal and nonfatal events; (4) excluding participants with baseline CHD or stroke; and (5) including diabetes identified during follow-up and before events in multivariable analyses and stratification. All analyses were conducted in Stata software version 12.0 . Statistical significance was accepted as p < 0.05.We traced 4,534 participants (93%) to a United Kingdom address. Follow-up data were obtained for 4,196 participants (>92%) . ParticiFollow-up data were available for 2,049 Europeans (24% women), 1,517 South Asians (17% women), and 630 African Caribbeans (45% women). At baseline, the mean age of participants was 52.4 years . Baseline stroke was infrequent in all ethnic groups, and African Caribbean men had less CHD than European men. As expected, South Asians and African Caribbeans had more diabetes and hypertension than Europeans. Although they had lower body mass indices than European men, South Asian men were more centrally obese and dyslipidemic. African Caribbeans were less dyslipidemic than Europeans and African Caribbean men were less centrally obese than European men. South Asians and African Caribbeans smoked less and consumed less alcohol than Europeans .There was more loss to follow-up in African Caribbeans (21%) than in Europeans (13%) or South Asians (11%). Baseline characteristics of participants lost to follow-up were similar within the ethnic group when compared with those who were followed up .Coronary heart disease events occurred in 1,256 (30%) participants. Fatal CHD was the first recorded follow-up event in 159 participants. South Asians were most and African Caribbeans least at risk for CHD . IncidenThe age- and sex-adjusted subhazard ratio (SHR) for CHD between South Asians and Europeans was 1.70  . Of all CHD risk was lower in African Caribbeans than Europeans . Our multivariate model, including favorable and unfavorable risk factors , did not account for the African Caribbean protection from CHD  . BaselinFindings from sensitivity analyses were similar to those presented here.Stroke events occurred in 401 participants during follow-up. African Caribbeans had the highest and Europeans the lowest rates of stroke .In South Asians and African Caribbeans, the age- and sex-adjusted SHR, 1.45  and 1.50 , respectively, were strongly but incompletely attenuated on adjustment for baseline diabetes .Diabetes was predictive of stroke in all ethnic groups , but mosIn people with diabetes, the age-adjusted SHR in South Asians versus Europeans, at 1.96 , was little affected on adjustment for any measured risk factors. Multivariable adjustment  had a small attenuating effect, whereas Framingham and INTERHEART adjustments did not explain the South Asian excess in those with diabetes. In African Caribbeans with diabetes, the age-adjusted SHR  was little changed on multivariable adjustment. The most marked attenuation was obtained by the INTERHEART adjustment . In those without diabetes, there was only a modest and nonsignificant ethnic excess in risk of stroke .The diabetes and ethnicity interactions also were strong on analyses of fatal stroke events only. Date of diagnosis of pre-baseline diabetes was available for 440 (76%) participants with baseline diabetes; however, adjustment for age at diagnosis in these participants did not alter the ethnic differentials in stroke events. Sensitivity analyses demonstrated similar magnitude and direction of ethnicity-associated excess stroke risk and similar diabetes and ethnicity interactions. Inclusion of new diabetes (identified during follow-up and before stroke) in addition to baseline diabetes in multivariable analyses confirmed the additional risk of stroke in South Asians and African Caribbeans with diabetes compared with Europeans with diabetes, with age- and sex-adjusted SHR as follows: South Asians: 2.10 , and African Caribbeans: 2.17 .People of European, South Asian, and African Caribbean origin vary markedly in CVD risk, in parallel with differences in metabolic factors such as IR, dyslipidemia, and central adiposity. However, the between-ethnic group differences in CVD remained even after adjustment for conventional cardiometabolic risk factors measured in midlife. It is well recognized that explanations for disease risk within a group may not explain differences between groups .In 20 years of follow-up, CHD rates were significantly elevated in South Asian migrants compared with British Europeans. Multivariable adjustment for baseline risk factors explained little of the excess risk. In African Caribbeans  rates of CHD continue to be 35% lower than in comparable Europeans, with adjustment for favorable midlife risk factors, such as HDL and LDL cholesterol, accounting for little of this protection. Rates of stroke remain elevated in both ethnic minority groups and more markedly so in people with diabetes in midlife, being 2.0 times greater in African Caribbeans and 1.7 times greater in South Asians compared with Europeans with diabetes.There are few population-based studies comparing cardiovascular risk in South Asians and Europeans . To our Our previous analyses hinted at a potential excess stroke mortality associated with diabetes in African Caribbeans . We haveTo our knowledge, this is the largest tri-ethnic cohort in the United Kingdom with a 20-year follow-up between middle and older age, thus providing valuable and unique prospective ethnicity-specific information on CHD and stroke incidence. Our combined data sources for the primary analyses provide follow-up data on more than 90% of the original study population, and although loss to follow-up was more frequent in African Caribbeans (21%), baseline characteristics of those lost to follow-up were similar to those participants who were followed up. Although this is a relatively large cohort, the numbers of stroke events were small, in particular and with the exception of incident diabetes, our baseline measurements were limited to those made on only 1 occasion 20 years ago, meaning that we cannot account for changes in other risk factors during the follow-up period or in earlier life. We were unable to differentiate reliably between stroke subtypes, which may vary by ethnicity, although it is reported that the risks of all stroke types are elevated in South Asian and African Caribbean populations and that ischemic strokes are most frequent in all 3 groups . ClassifMorbidity and mortality resulting from CHD are elevated in South Asians and are lower in African Caribbeans compared with European-origin populations. These differences were not explained by conventional risk factors measured in midlife. Factors across the life course, in particular the mismatch between early and later life environments in migrant cohorts, may be key . This is


Maxillomandibular advancement (MMA) is a surgical option for obstructive sleep apnea (OSA). MMA involves forward-fixing the maxilla and mandible approximately 10 mm via Le Fort I maxillary and sagittal split mandibular osteotomies. We retrospectively reviewed outcomes from 24 consecutive OSA patients who underwent MMA at our institution. MMA resulted in an 83% reduction in the group mean apnea-hypopnea index (AHI) per polysomnography an average of 6.7 months after surgery. Forty-two percent of patients achieved a post-MMA AHI of less than 5 events/hour sleep and 71% achieved an AHI less than or equal to 10 events/hour sleep. The Epworth Sleepiness Scale score decreased by an average of 5 post-surgery. No parameters predictive of cure for OSA by MMA were identified.  Obstructive sleep apnea (OSA) is a common disorder  associatThe role of upper airway surgery for OSA has been contentious , 12. MosA surgical procedure with an apparently greater efficacy for lowering the AHI is maxillomandibular advancement (MMA) which enlarges the upper airway and decreases upper airway collapsibility  by forwaIn an effort to further define the role for MMA in OSA, we sought to review our experience with MMA. In doing so, we also wanted to address some of the limitations in the previously performed studies, such as controlling for body mass index (BMI) pre- and post-MMA and the percent of time spent supine in the pre- and post-MMA polysomnograms, examining changes in the pre- and post-MMA subjective sleepiness, and presenting tiered AHI outcomes. In addition, we sought to identify pre-surgical parameters predictive of surgical success.After obtaining approval from the Mayo Clinic Institutional Review Board, we retrospectively reviewed records of consecutive patients, 18 years or older, who underwent MMA for the indication of OSA and had pre- and post-MMA polysomnograms (PSGs) between November 1, 2004 and June 30, 2008. Patients with persistent OSA despite previous surgical interventions as well as surgery naïve patients were included. Pre- and post-MMA clinical, demographic, and PSG data were collected by review of each patient's comprehensive electronic medical record.Patients reported herein were accrued from routine clinical practice and typically had moderate to severe OSA for which they could not or would not use CPAP. All underwent a multidisciplinary preoperative evaluation, including cephalometric radiographs and examinations by an oral and maxillofacial surgeon (CFV) and an ear, nose, and throat (ENT) specialist , and a sleep specialist. The ENT exam included flexible fiberoptic nasal endoscopy and laryngoscopy. On the basis of clinicoradiologic data, patients who eventually underwent MMA were not felt to have significant nasal or palatal obstruction and were free of significant co-morbidities that might negatively impact surgical risk, such as unstable cardiopulmonary disease.z-Cz, Cz-Oz, C4-M1, or C3-M2); electrooculography ; submental and anterior tibialis electromyography; snoring by laryngeal microphone; electrocardiography; pulse oximetry; respiratory effort . Until August 2006, airflow was analyzed by nasal pressure transducer. From September 2006 onward, airflow was analyzed by both oronasal thermal sensor and nasal pressure transducer. Obstructive apnea was defined as cessation of airflow for at least 10 seconds despite respiratory effort. Hypopnea was defined by a ≥30% reduction in airflow for at least 10 seconds accompanied by at least a 4% drop in SpO2. Until July 2007, arousals were scored according to American Sleep Disorders Association criteria ) treated with MMA and with pre- and postoperative PSG data were identified and analyzed. The mean age at the time of surgery was 48.3 ± 10.8 years. All patients were Caucasians.P < 0.001) for an average AHI reduction of 83%. Ten (42%) patients achieved an AHI < 5 events/hour sleep; 17 (71%) patients achieved an AHI ≤ 10 events/hour sleep; 21 (87.5%) patients achieved a 50% AHI reduction and/or an AHI ≤ 20 events/hour sleep. The mean ESS score decreased by 35% after MMA. The percentages of stages N3 and REM sleep, the LSAT, and the respiratory-related arousal index improved during post-MMA PSG. Neither post-MMA BMI nor the percentage of supine position sleep during the post-MMA PSG was significantly different from pre-MMA values.Group pre- and post-MMA PSG data are provided in 2), and his oropharynx configuration was Grade II per modified Mallampati technique [All but one patient enjoyed an AHI reduction. This patient was a 61-year-old man with OSA and multifactorial insomnia. His BMI was normal (24 kg/mechnique . The preComparing various parameters between those cured of OSA by MMA (defined by postoperative AHI < 5 events/hour sleep) versus those with residual sleep disordered breathing, no significant differences were found with respect to age, gender, pre- or postoperative BMI, prior upper airway surgery, amount of maxillomandibular advancement, or cephalometric measurements .This study adds to the literature that MMA results in a substantial reduction of the AHI in the majority of nonrandomized patients described in case series. In our study population of patients with generally severe OSA, there was an 83% drop in the mean AHI, a value congruent with the 87% reduction by MMA per a recent review of upper airway surgeries for OSA sponsored by the AASM  and the There has been a call  for use The MMA meta-analysis  found thDetermining the role for any upper airway surgery in the management of OSA has been complicated by methodologic limitations in the surgical literature, including irregularities in the performance and interpretation of PSG (such as controlling for the percent sleep time in the more vulnerable supine position), lack of secondary outcome data beyond AHI (such as sleep architecture and ESS), and failure to control for the potential confounding of peri-procedural changes in BMI , 13. ThiThis study shares a number of limitations common in the OSA surgical literature. Our patients were not randomized to MMA, although there are significant practical hurdles to randomization in surgical trials for OSA, especially with an invasive surgery with cosmetic ramifications such as MMA. Not all patients who underwent MMA at our institution during the data collection period underwent postprocedural PSG. We did not have pre- and post-MMA matched data for every parameter for every patient either because the pre-MMA PSG was performed elsewhere or the documentation was incomplete, a problem inherent in retrospective analyses. In the case of patients who had their pre-MMA PSG at another institution, a repeat pre-operative study was not performed because it was either deemed clinically unnecessary or logistically impractical. Systematic documentation of post-MMA complications was not performed so adverse outcomes are not reported herein.Our study population included 8 patients who had previously undergone surgeries for OSA, mostly UPPP, which limits our ability to discern the specific impact of MMA. The patient numbers were too small to determine the impact of the specific previous upper airway surgeries on response to MMA. However, the frequency of previous surgery likely reflects, at least in part, the enduring paradigm of a step-wise surgical approach to OSA in which site-specific phase I surgery addresses areas deemed susceptible to obstruction in the naso-, oro-, or hypopharyngeal regions, while phase II surgery consists of facial skeletal advancement  for residual disease . Debate CPAP remains the standard treatment for most patients with OSA . However


In primary care patients with suspected coeliac disease, what is the accuracy and utility of point-of-care (POC) testing for coeliac disease compared to standard practice?Coeliac disease is a malabsorption syndrome precipitated by gluten ingestion, and characterised by inflammation of the small intestine.Serological tests for IgA anti-tissue transglutaminase antibody (tTGA) and anti-endomysial antibody (EMA) have high sensitivity and specificity for coeliac disease.Two POC devices available on the market were identified.There are two versions of the test, a home test and a professional test; a ‘total IgA measuring system’ is included in the professional kit. The Biocard requires a drop of whole blood (finger-prick) and provides results within 10 minutes.Anti-tTG IgA antibodies bind to antigen in the test strip to form a visible line. A positive test result shows two lines, while only one line appears if the test is negative. If there is no line, IgA deficiency should be suspected.Both are one-step tests detecting IgA, IgG, and IgM antibodies against human tTG; and the CD2 test also detects anti-gliadin antibodies. The tests use serum instead of whole blood, which limits their applicability to ambulatory settings. Results are available within 10 minutes.Patients in whom coeliac disease is suspected because of signs or symptoms  symptoms such as nausea and vomiting, prolonged fatigue, recurrent abdominal pain, cramping or distension, sudden/unexpected weight loss, unexplained iron-deficiency anaemia).2Patients with coexisting conditions associated with coeliac disease, such as autoimmune thyroid disease, dermatitis herpetiformis, irritable bowel syndrome, type 1 diabetes, or who have first-degree relatives with coeliac disease.2The Biocard test is not suitable for children <5 years of age or for patients with IgA deficiency.The prevalence of coeliac disease in the UK is estimated to be 0.8%–1.9% in the general population, and 4.5%–12% among first-degree relatives.Four case-control studies used the Biocard test in a population of biopsy-confirmed patients with coeliac disease and laboratory controls.7The earliest study was based on 121 consecutive samples from patients and 107 controls who had normal villous morphology on pathology.Subsequently, the Biocard test was evaluated in 24 untreated patients with coeliac disease and 19 controls compared to duodenal biopsy, with a sensitivity of 92% and specificity of 79%.4The diagnostic accuracy of the Biocard test was also assessed in a cross-sectional study of 150 patients from a tertiary clinic with suspected but unconfirmed coeliac disease.n = 2676) and offered biopsy if any result was positive. Coeliac disease was confirmed in 32 children (1.2%). When compared to biopsy plus follow-up, Biocard test sensitivity was 78% and specificity was 99.8%.One study assessed the Biocard test for screening.One studyOne prospective multicentre study evaluated the accuracy of Stick CD1 and CD2 test in 113 children with confirmed coeliac disease.The diagnostic accuracy of the Biocard test is high (sensitivity between 90–97% and specificity between 79–100%), but this may be an overestimate due to the study designs and the selective populations. Sensitivity in asymptomatic children was much lower (65–79%), which means that the ability to rule out coeliac disease in these children is reduced. There is limited evidence available for the Stick coeliac disease tests.Patient self-testing raises other concerns: those who self-diagnose may begin gluten-free diets without confirmatory testing, appropriate nutritional advice, or medical investigation for complications or comorbid conditions associated with coeliac disease.Given that 8% of patients with coeliac disease are IgA deficient, the Biocard test may give false negative results in this group, leading to delays in seeking medical attention.Use of the Biocard test by professionals may be of value in situations where venepuncture is difficult, for example, in children.Early-case identification through POC testing has the potential to prevent gluten-related morbidity and reduce costs. However, one UK studyThe 2009 NICE guideline CG 86 recommends that self-tests and/or POC tests for coeliac disease should not be used as a substitute for laboratory-based tests, and that patients with positive self- or POC-tests are sent for further serological testing.2Although the evidence is limited, point-of-care testing may be helpful in the diagnostic work-up of coeliac disease in primary care settings by increasing speed of results or access to testing in some settings, as sensitivity and specificity are comparable with laboratory-based serology. However, a negative result does not safely rule out coeliac disease.www.madox.org.Standardised methodology was applied in writing this report, using prioritisation criteria and a comprehensive, standardised search strategy, and critical appraisal. Full details of these are available from


Currently, there are over 1,800 annotated human miRNAs, many of which have tissue-specific expression. Numerous studies have highlighted their role in haematopoietic differentiation and proliferation, acting as master regulators of haematopoietic stem cell function. Aberrant expression of miRNAs has been observed in haematological cancers, exhibiting unique expression signatures in comparison to normal counterparts. Functional and target analyses as well as animal models have attempted to annotate how different miRNA may contribute to the pathophysiology of these malignancies from modulating cancer associated genes, functioning directly as oncogenes or tumour suppressor genes or acting as bystanders or regulators of the epigenetic mechanisms in cancer. miRNAs have also been shown to play a role in modulating drug resistance and determining prognosis between the various subtypes of blood cancers. This review discusses the important role that miRNAs play in haematological malignancies by exploring associations that exist between the two and trying to examine evidence of causality to support the tantalising possibility that miRNAs might serve as therapeutic targets in blood cancers. The haematological malignancies are a diverse group of neoplastic diseases. They are representing the fifth most common cancer in the UK and are among the twenty most common causes of cancer deaths in the UK . A combiIn vivo mice studies have illustrated that disruption of miRNA biogenesis through depletion of Dicer leads to embryological and developmental arrest and subsequent incompatibility with life ; no; no75]; d cancer , but alsd cancer , 84, abs 13q14.3  and/or m 13q14.3 , 21, evi 13q14.3 , 21, and 13q14.3 , 77, hasmiR-17-92 cluster and the tumour suppressors miR-15a/16-1 have been shown to be proangiogenic and antiangiogenic, respectively [Understanding how miRNAs contribute to the six hallmarks of cancer may also provide solid evidence for their role in tumourigenesis. This review makes it clear that miRNAs greatly contribute to enhanced proliferation, differentiation, and reduced apoptosis in haematological malignancies. Angiogenesis has been highlighted as an important feature of haematological malignancies, providing a specialised environment for HSC proliferation and differentiation through provision of GM-CSF, G-CSF, and IL-6 shown to be important in the proliferation and survival of malignant cells . The oncectively , 87. If ectively ; these dmiR-34 due to promoter hypermethylation has been identified in primary samples of CLL, myeloma, and NHL [MIR-34a gene is located at chromosome 1p35, which is subject to loss of heterozygosity in various cancers [Additional support for the role of miRNAs in cancer comes from their association with epigenetic changes. DNA CpG island hypermethylation of tumour suppressor genes is a well-characterised aberration in malignant cells, along with global genomic hypomethylation and histone modification disturbances . Over ha and NHL . The MIR cancers . Loss of cancers  for bonamiR-29b may target the DNA methylation agents DNMT1 and DNMT3 [miR-29b in AML cells resulted in global DNA hypomethylation and subsequent upregulation of tumour suppressor genes [Paradoxically, miRNA may also directly target epigenetic machinery . Evidencnd DNMT3 . Reexpreor genes . Furthermir-155 upregulated in patients with CN-AML was associated with lower remission rates and higher overall survival rates [miR-155 was interestingly associated with genes widely known to be deregulated in AML supporting the role of this miRNA in AML pathogenesis [let-7a showed enhanced chemosensitivity of AML cell lines overexpressed with let-7a [miR-363, was further characterised to target the genes RGS17 and HIPK3, which have previously been associated with therapeutic response in AML patients [Importantly, miRNAs are increasingly recognised as prognostic biomarkers and therapeutic targets in various types of haematological malignancies. Briefly focusing on AML, a few recent studies have attempted to support this notion. It was found that al rates . The higogenesis . Recent h let-7a . A miRNApatients . These sin vitro. Additionally, in some studies cell lines were used that were not indicative of the primary disease. The colonic cancer RKO cell line was used to investigate miR-10a in CML by Aggire et al. [miR-15a/16-1 and BCL2 used nonlymphoid cell lines [miR-155 and miR-106-25 have shown effect early in disease progression of lymphomas and myeloma, respectively [There are caveats and difficulties faced within the field of miRNA research. The benefits of investigating primary cells and patient cohorts are often reduced due to small sample size, reducing external validity. As mentioned in AML and ALL, differences in cytogenetics and therefore miRNA expression may be age dependent within the same malignancy. Often studies broadly look at disease without separating findings to a particular cytogenetic subtype or age group. A study looking at differences of miRNA expression between adult and childhood acute leukaemias  shows the et al. , and manectively , 76. ThemiR-181 and miR-17-92 illustrates how miRNA can function both as tumour suppressors and promoters dependent on biological and cellular context. The well-characterised functions of miR-15a/16-1 demonstrate that a single miRNA can have far reaching and multiple effects in cells and in cancer progression, targeting a variety of cancer associated genes. Furthermore, expression analysis has revealed that certain miRNAs, such as miR-155, are deregulated in all haematological malignancies, suggesting that certain miRNAs may be fundamental to tumourigenesis.It is apparent that there is accumulating evidence for the role of miRNA in haematological malignancies, with different patterns of expression correlating with distinct types and subtypes. In this review, evidence from miR-328 in CML-BC [miR-15a/16-1 showing that in addition to posttranscriptional silencing, miR-16 may function to target AU-rich elements (AREs) in target mRNA, mediating their decay [Our understanding of these small noncoding RNA is continually evolving and more recent research highlights areas of miRNA regulation and function not previously considered or identified. miRNAs were initially discovered as posttranscriptional silencers of protein-coding genes, and this still remains true. However, their function and mechanisms by which they mediate their effects extend further than this. As well as acting through the RISC complex, they have been shown to mediate protein inhibition through epigenetic modifications of proteins involved in tumourigenesis or tumour inhibition . Evidencn CML-BC . Addition CML-BC . Finallyir decay . Of hugeir decay , which hir decay  and haveir decay . This prir decay , which m