Differential microRNA expression between decidual and peripheral blood natural killer cells in early pregnancy.

STUDY QUESTION: Have decidual natural killer (dNK) cells a different microRNA (miRNA or miR) expression pattern compared to NK cells circulating in the peripheral blood (pb) of healthy pregnant women in the first trimester of gestation? SUMMARY ANSWER: dNK cells have a unique miRNA profile, showing exclusive expression of a set of miRNAs and significant up- or down-regulation of most of the miRNAs shared with pbNK cells. WHAT IS KNOWN ALREADY: dNK cells differ from pbNK cells both phenotypically and functionally, and their origin is still debated. Many studies have indicated that miRNAs regulate several important aspects of NK cell biology, such as development, activation and effector functions. STUDY DESIGN, SIZE, DURATION: Decidua basalis and peripheral blood specimens were collected from women (n = 7) undergoing voluntary termination of gestation in the first trimester of pregnancy. dNK and pbNK cells were then highly purified by cell sorting. PARTICIPANTS/MATERIALS, SETTING, METHODS: miRNAs expression was analysed by quantitative RT-PCR (qRT-PCR)-based arrays using RNA purified from freshly isolated and highly purified pbNK and dNK cells. Results from arrays were validated by qRT-PCR assays. The bioinformatics tool ingenuity pathway analysis (IPA) was applied to determine the cellular network targeted by validated miRNAs and the correlated biological functions. MAIN RESULTS AND THE ROLE OF CHANCE: Herein, we identified the most differentially expressed miRNAs in NK cells isolated from peripheral blood and uterine decidua of pregnant women. We found that 36 miRNAs were expressed only in dNK cells and two miRNAs only in pbNK cells. Moreover, 48 miRNAs were commonly expressed by both NK cell preparations although at different levels: 28 were upregulated in dNK cells, while 15 were downregulated compared to pbNK cells. Validation of a selected set (n = 11) of these miRNAs confirmed the differential expression of nine miRNAs: miR-10b and miR-214 expressed only in dNK cells and miR-200a-3p expressed only in pbNK cells; miR-130b-3p, miR-125a-5p, miR-212-3p and miR-454 were upregulated while miR-210-3p and miR-132 were downregulated in dNK cells compared to pbNK cells. IPA network analysis identified a single network connecting all the miRNAs as well as their significant involvement in several classes of functions: 'Organismal injury, Reproductive system disease, Inflammatory disease' and 'Cellular development'. These miRNAs target molecules such as argonaute 2, tumour protein p53, insulin and other genes that belong to the same network and significantly influence cell differentiation and pregnancy. LIMITATIONS, REASONS FOR CAUTION: In the present study, the cellular network and biological functions modulated by miRNAs differentially expressed in dNK and pbNK cells were identified by IPA considering only molecules and relationships that were with confidence 'experimentally observed' in leucocytes. The decidual and pbNK cells that were analysed here are a heterogeneous population and further study will help to disentangle whether there are differences in miRNA production by the different subsets of NK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study describing a different miRNA expression profile in dNK cells compared to matched pbNK cells during the first trimester of pregnancy. Our findings improved the body of knowledge on dNK cell biology and strongly suggest further investigation into the roles of miRNAs that are differentially expressed in human dNK compared to pbNK cells. Our results suggest that specific miRNAs can modulate dNK cell origin and functions, highlighting a potential role of this miRNA signature in human development and diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Pasteur, Fondazione Cenci Bolognetti, the European NoE EMBIC within FP6 (Contract number LSHN-CT-2004-512040), Istituto Italiano di Tecnologia, and Ministero dell'Istruzione, dell'Università e della Ricerca (Ricerche Universitarie), and from Università Politecnica delle Marche. There are no conflicts of interest to declare.

New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid.

Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.This result confirms the key role played by WNT and TGF-ß pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior.

Isolation and characterization of progenitor mesenchymal cells in human pituitary tumors.

The Cancer Stem Cells (CSCs) theory suggests that genetic alterations in stem cells are the direct cause for cancer. The evidence for a CSC population that results in pituitary tumors is poor. Some studies report the isolation of CSCs, but a deep characterization of the stemness of these cells is lacking. Here, we report the isolation and detailed characterization of progenitor mesenchymal cells (PMCs) from both growth hormone-secreting (GH(+)) and non-secreting (NS) pituitary adenomas, determining the immunophenotype, the expression of genes related to stemness or to pituitary hormone cell types, and the differentiative potential towards osteo-, chondro- and adipogenic lineages. Finally, the expression of CD133, known as a marker for CSCs in other tumors, was analyzed. Isolated cells, both from GH(+) and NS tumors, satisfy all the criteria for the identification of PMCs and express known stem cell markers (OCT4, SOX2, KLF4, NANOG), but do not express markers of pituitary hormone cell types (PITX2, PROP1, PIT1). Finally, PMCs express CD133. We demonstrated that pituitary tumors contain a stem cell population that can generate cell types characteristic of mesenchymal stem cells, and express CD133, which is associated with CSCs in other tumors.

Characterization and profiling of immunomodulatory genes in resident mesenchymal stem cells reflect the Th1-Th17/Th2 imbalance of psoriasis.

The expression of genes encoding for Th1, Th2 and Th17 cytokines has been extensively evaluated in differentiated skin cells of psoriatic patients. The microenvironment exerts a control on the phenotype of resident mesenchymal stem cells (MSCs) into the skin of psoriasis patients. Aim of the study was to extensively evaluate the relative expression of 43 genes encoding for Th1, Th2 and Th17 cytokines in MSCs isolated from skin of psoriasis patients. MSCs resident into psoriatic skin were isolated, characterized and profiled by PCR array for the relative expression of genes encoding for cytokines involved in Th1, Th2 and Th17 pathways. MSCs isolated from the skin of healthy subjects were used as control. The MSCs isolated from skin of psoriasis patients showed a greater relative expression of the most part of the analyzed genes encoding for Th1 and Th17 cytokines: INF-γ, CCR5, CXCL9, CXCL10, IL6, IL8, TNF-α, IL23A, CCL2, CCL20, CXCL2, CXCL5, IL17C, IL17F, IL17RA, IL21, TLR2 than healthy subjects. On the contrary, the relative expression of genes encoding for Th2 cytokines: CCL1, CCL22, CXCL12, IL2, IL3, IL4, IL13B, IL 22, IL 27, TGF-ß1, was similar between the MSCs isolated from psoriasis and healthy subjects. In conclusion, the MSCs isolated from psoriasis show an imbalance between the Th1-Th17 and Th2 pathways, which reflects the well-known abnormal balance observed in differentiated skin cells. This evidence could strengthen the hypothesis of an early involvement of resident MSCs in the pathogenesis of psoriasis.

Reinterpretation of mouse thyroid changes under space conditions: the contribution of confinement to damage.

During space missions, astronauts work in a state of separation from their daily social environment and in physical confinement. It has been shown that confinement influences mood and brain cortical activity, but no data has been obtained with regard to its effect on the thyroid gland, the structure and function of which change during spaceflights. Here, we report the results of a study on the effects of confinement on mouse thyroid, which was implemented with the Mice Drawer System Facility maintained on the ground, a system used for spaceflight experiments. The results show that confinement changes the microscopic structure of the thyroid gland and that it exhibits symptoms similar to those that result from physiological and/or pathological hyperfunction. What is left unchanged, however, is the sphingomyelinase-thyrotropin receptor relationship, which is important for thyrotropin response with a consequential production of hormones that act on the metabolism of almost all tissues and reduces the production of calcitonin, a hormone involved in bone metabolism. During space missions, the overexpression of pleiotrophin, a widespread cytokine up-regulated after tissue injury that acts on bone remodeling, attenuates changes to the thyroid that are spaceflight-dependent; therefore we studied the thyroids of pleiotrophin-transgenic mice in the Mice Drawer System Facility. In confinement, pleiotrophin overexpression does not protect from the loss of calcitonin. The contribution of confinement to thyroid damage during spaceflights is discussed.

Alterations of ROS pathways in scleroderma begin at stem cell level.

Scleroderma is a chronic systemic autoimmune disease (primarily of the skin) characterized by fibrosis (or hardening), vascular alterations and autoantibodies production.There are currently no effective therapies against this devastating and often lethal disorder. Despite the interest for the immunomodulatory effects of mesenchymal stem cells (MSCs) in autoimmune diseases, the role of MSCs in scleroderma is still unknown. A pivotal role in scleroderma onset is played by oxidative stress associated with the accumulation of great amounts of reactive oxygen species (ROS). This study depicts some phenotypic and functional features of MSCs isolated from the skin of healthy and scleroderma patients; the ROS production and accumulation, the expression of ERK1/2 and the effects of the stimulation with PDGF, were analyzed in MSCs; results were compared to those observed in primary fibroblasts (Fbs) isolated from the same subjects. We found that the pro-oxidant environment exerted by scleroderma affects MSCs, which are still able to counteract the ROS accumulation by improving the antioxidant defenses. On the contrary, scleroderma fibroblasts show a disruption of these mechanisms, with consequent ROS increase and the activation of the cascade triggered by scleroderma auto-antibodies against PDGFR.

Increased androgen receptor messenger RNA in frontal-parietal hair follicles of women with androgenetic alopecia.

Normal testosterone levels are frequently observed in women with androgenetic alopecia (AGA), suggesting the involvement of androgen sensitivity in this condition. Androgen sensitivity is related to androgen receptor (AR) messenger RNA (mRNA) production in hair follicles and is negatively related to the number of CAG repeats present in exon 1 of the AR gene. The aim of this study was to compare AR expression in AGA women with normal controls and to correlate this expression with the number of CAG repeats. Hair follicles were obtained from 27 women with AGA and 21 controls for AR gene expression analysis. AR expression was evaluated through AR mRNA quantification using real-time polymerase chain reaction and the number of CAG repeats in the AR gene was determined in complementary DNA samples obtained from hair follicles and analyzed with the Gene Scan software. AR mRNA in the frontal-parietal region was significantly higher than in the occipital region of AGA patients (paired t-test, P = 0.046). No significant difference was identified in controls (P = 0.67). Both regions in the same individual showed a significant positive correlation in AGA patients (r = 0.77; P < 0.05) and in controls (r = 0.91; P < 0.05). A negative correlation was identified between AR expression and the number of CAG repeats only in AGA patients (r = 0.510; P = 0.013). The identification of elevated AR mRNA quantitation in hair follicles is a useful tool for identifying potentially abnormal androgen sensitivity in AGA patients.

In vitro evaluation of mesenchymal stem cell isolation possibility from different intra-oral tissues.

Mesenchymal stem cells (MSCs) are of great interest for the regeneration of tissues and organs. Bone marrow is the first sources of MSCs, but in the recent years there has been interest in other tissues for the isolation of these pluripotent cells. In this study, we investigated the features of MSCs isolated from different oral regions in order to evaluate their potential application in the regeneration of damaged maxillofacial tissues. Sampling from human periodontal ligament, dental pulp, maxillary periosteum as well as bone marrow were collected in order to obtain different stem cell populations. Cells were morphologically and immunophenotipically characterized. Their proliferation potential and their ability to differentiate in osteoblasts were also assessed. All tested cell population showed a similar fibroblast-like morphology and superimposable immunophenotype. Slight differences were observed in proliferation and differentiation potential. Cells isolated from human periodontal ligament, dental pulp, maxillary periosteum had the characteristics of stem cells. Considering their peculiar feature they may alternatively represent interesting cell sources in stem cell-based bone/periodontal tissue regeneration approaches.

Observing the mouse thyroid sphingomyelin under space conditions: a case study from the MDS mission in comparison with hypergravity conditions.

This is a case report of apparent thyroid structural and functional alteration in a single mouse subjected to low Earth orbit spaceflight for 91 days. Histological examination of the thyroid gland revealed an increase in the average follicle size compared to that of three control animals and three animals exposed to hypergravity (2g) conditions. Immunoblotting analysis detected an increase in two thyroid gland enzymes, sphingomyelinase and sphingomyelin-synthase1. In addition, sphingomyelinase, an enzyme confined to the cell nucleus in the control animals, was found in the mouse exposed to hypogravity to be homogeneously distributed throughout the cell bodies. It represents the first animal observation of the influence of weightlessness on sphingomyelin metabolism.

Human periosteum-derived stem cells for tissue engineering applications: the role of VEGF.

Mesenchymal stem cells (MSCs) are promising tools for studying the mechanisms of development and for the regeneration of injured tissues. Correct selection of the MSCs source is crucial in order to obtain a more efficient treatment and, in this respect Periosteum-Derived Cells (PDPCs) may represent an interesting alternative to bone marrow MSCs for osteochondral tissue regeneration. In the present study we have isolated and characterized a MSCs population from the periosteum of human adult donors. PDPCs were expanded under specific culture conditions that prevent fibroblast contamination and support the maintenance of their undifferentiated phenotype. We show, for the first time, that PDPCs expresses VEGF receptor (Flt1 and KDR/Flk1) proteins and that they were similar to bone marrow Multipotent Adult Progenitor Cells (MAPCs). Since the latter are able to differentiate into endothelial cells, we tested the possible PDPCs commitment toward an endothelial phenotype in view of bone tissue engineering approaches that takes into account not only bone formation but also vascularization. PDPCs were treated with two different VEGF concentrations for 7 and 15 days and, alternatively, with the supernatant of human primary osteoblasts. Differently from MAPCs our PDPCs were unable to differentiate into endothelial cells after their in vitro VEGF treatment. On the contrary, growth factor stimulation induces PDPCs differentiation toward osteoblasts. We concluded that in PDPCs the presence of VEGF receptors is related to different cross-talk between osteogenesis and angiogenesis that could involve in situ PDPCs recruitment.

The thyroid lobes: the different twins.

Although differences in size of the right and left thyroid lobes are well defined, differences in morphology, follicles structure, cAMP production, thyrotropin receptor, and protein involved in cell signalling have not previously been reported. This study provides morpho-functional data of right and left thyroid lobes by biochemical, immunohistochemistry, immunoblotting and immunofluorescence analysis. We demonstrate that, in comparison with the left lobe, the right lobe has a higher activation index, is more sensitive to thyrotropin treatment, is rich in thyrotropin receptor and caveolin 1 involved in thyroid hormone synthesis as well as in epithelial thyroid cell homeostasis, is characterised by a high content of molecules involved in cell signalling such as stat3, raf1, sphingomyelinase and sphingomyelin-synthase whose activity ratio is necessary for epithelial cell activity and finally has more areas calcitonin-dependent. The relation between structure/function of right lobe and its susceptibility to the higher risk of pathological modifications with respect the left lobe is discussed.

Thyrotropin receptor and membrane interactions in FRTL-5 thyroid cell strain in microgravity.

The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264 km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.

An embedded portable biosensor system for bacterial concentration detection.

Microbial screening is a primary concern for many products. Traditional techniques based on standard plate count (SPC) are accurate, but time consuming. Furthermore, they require a laboratory environment and qualified personnel. The impedance technique (IT) looking for changes in the electrical characteristics of the sample under test (SUT) induced by bacterial metabolism represents an interesting alternative to SPC since it is faster (3-12h vs. 24-72 h for SPC) and can be easily implemented in automatic form. With this approach, the essential parameter is the time for bacteria concentration to reach a critical threshold value (about 10(7) cfu mL(-1)) capable of inducing significant variations in the SUT impedance, measured by applying a 100 mV peak-to-peak 200 Hz sinusoidal test signal at time intervals of 5 min. The results of this work show good correlation between data obtained with the SPC approach and with impedance measurements lasting only 3h, in the case of highly contaminated samples (10(6) cfu mL(-1)). Furthermore, this work introduces a portable system for impedance measurements composed of an incubation chamber containing the SUT, a thermoregulation board to control the target temperature and an impedance measurement board. The mix of cheap electronics and fast detection time provides a useful tool for microbial screening in industrial and commercial environments.

Diazepam decreases leukocyte-endothelium interactions in situ.

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

The effects of insulin and insulin-like growth factors on tumor vascularization: new insights of insulin-like growth factor family in cancer.

It is currently believed that the development of a clinically relevant tumor needs new vessel formation provided by both angiogenesis (primary involving endothelial cells) and postnatal vasculogenesis (primary involving bone marrow-derived cells). Clearly, it is important to identify factors that help to enhance the growth and "health" of tumors, as well as their further vascularization. The Insulin and Insulin-like Growth Factors (IGFs) systems play a key role in cellular metabolism, differentiation, proliferation, transformation and apoptosis, during normal and malignant growth. Moreover, these molecules seem essential in promoting tumor vascularization. Due to the complexity of these systems, the review has been focused on the role of insulin and IGFs signaling in the regulation of tumor angiogenesis and postnatal vasculogenesis. Since targeting on IGF for cancer therapy is rapidly becoming a clinical reality, a better understanding of IGFs-mediated pathways has a relevant impact, providing new insights to help the design of newly developed drugs.

Decitabine, differently from DNMT1 silencing, exerts its antiproliferative activity through p21 upregulation in malignant pleural mesothelioma (MPM) cells.

Malignant pleural mesothelioma (MPM) is a locally aggressive neoplasm, principally linked to asbestos fibres exposure. Strong evidences associate this pollutant with induction of DNA breaks, aberrant chromosomes segregation and important chromosomal rearrangements, considered crucial events in malignant transformation. A considerable contribution to cellular transformation in MPM is also given by the presence of high genomic instability, as well as by the increased DNA methylation, and consequent decreased expression, of tumor-suppressor genes. In this study we first demonstrated that MPM cells are characterized by a decreased methylation level of pericentromeric DNA sequences which can justify, at least in part, the genomic instability observed in this neoplasia. Concomitantly, we found a paradoxical increased expression of DNMT1, the most expressed DNA methyltransferases in MPM cells, DNMT3a and all five isoforms of DNMT3b. Thus, we compared two experimental strategies, DNMT1 silencing and usage of a demethylating agent (5-aza-2'-deoxycytidine or Decitabine), both theoretically able to revert the locally hypermethylated phenotype and considered potential future therapeutic approaches for MPM. Interestingly, both strategies substantially decrease cell survival of MPM cells but the antitumor activity of Decitabine, differently from DNMT1 silencing, is mediated, at least in part, by a p53-independent p21 upregulation, and is characterized by the arrest of MPM cells at the G2/M phase of the cell cycle. These results indicate that the two approaches act probably through different mechanisms and, thus, that DNMT1 silencing can be considered an effective alternative to Decitabine for cancer treatment.

Effects of single or repeated amphetamine treatment and withdrawal on lung allergic inflammation in rats.

The effects of single or repeated amphetamine (AMPH) treatment and those of AMPH withdrawals on immune-mediated lung inflammatory response were studied in rats. Two experiments were done. In the first, rats egg-albumin (OVA) sensitized were singularly or repeatedly (21 days, once daily) treated with AMPH (1.0 mg/kg) or with a similar number and volume of 0.9% NaCl. The OVA aerosol challenge was performed 12 h after the single or last repeated AMPH treatment and also 72 and 120 h after AMPH withdrawal. In the second experiment, the effects of reserpine (1.0 mg/kg/day for 5 consecutive days) on single AMPH actions on lung allergic response of rats were analyzed. Single and repeated AMPH treatment induced opposite actions on Bronchoalveolar lavage fluid (BAL) cellularity of allergic rats: single treatment decreased and repeated treatment increased the total number of cells as well as those of macrophages, neutrophils and eosinophils. Our data also showed that single but not repeated AMPH treatment decreased the number of neutrophils, monocytes and lymphocytes in the peripheral blood, and increased the total number of bone marrow cells in rats sensitized and challenged with OVA. Furthermore, it was shown that reserpine treatment precluded the effects of single AMPH treatment on cellular migration to the lung of OVA-sensitized and challenged rats. It was concluded that AMPH effects on lung inflammatory response and cell recruitment to the lung in allergic rats rely at least partially on corticosterone serum levels. The possible involvement of vesicular monoamine transporter type 2 (VMAT2) with these observed effects was discussed.

Detection of microbial concentration in ice-cream using the impedance technique.

The detection of microbial concentration, essential for safe and high quality food products, is traditionally made with the plate count technique, that is reliable, but also slow and not easily realized in the automatic form, as required for direct use in industrial machines. To this purpose, the method based on impedance measurements represents an attractive alternative since it can produce results in about 10h, instead of the 24-48h needed by standard plate counts and can be easily realized in automatic form. In this paper such a method has been experimentally studied in the case of ice-cream products. In particular, all main ice-cream compositions of real interest have been considered and no nutrient media has been used to dilute the samples. A measurement set-up has been realized using benchtop instruments for impedance measurements on samples whose bacteria concentration was independently measured by means of standard plate counts. The obtained results clearly indicate that impedance measurement represents a feasible and reliable technique to detect total microbial concentration in ice-cream, suitable to be implemented as an embedded system for industrial machines.

Antidepressants for patients with tinnitus.

BACKGROUND: Tinnitus is described as the perception of sound or noise in the absence of real acoustic stimulation. It has been compared with chronic pain, and may be associated with depression or depressive symptoms which can affect quality of life and the ability to work. Antidepressant drugs have been used to treat tinnitus in patients with and without depressive symptoms. OBJECTIVES: To assess the effectiveness of antidepressants in the treatment of tinnitus and to ascertain whether any benefit was due to a direct tinnitus effect or a secondary effect due to treatment of concomitant depressive states. SEARCH STRATEGY: We searched the Cochrane Ear, Nose and Throat Disorders Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) The Cochrane Library Issue 1, 2006); MEDLINE (January 1951 to 2006); EMBASE (1974 to 2006), CINAHL (to 2006), PSYCINFO (to 2006), LILACS (to 2006), and Cambridge Scientific Abstracts. The date of the most recent search was March 2006. SELECTION CRITERIA: Randomised controlled clinical studies of antidepressant drugs versus placebo in patients with tinnitus. DATA COLLECTION AND ANALYSIS: The studies retrieved were critically appraised and data extracted independently by two authors. Where necessary study authors were contacted for further information. MAIN RESULTS: Five trials involving 525 patients were included. Four of these trials looked at the effect of tricyclic antidepressants on tinnitus, investigating 405 patients. One trial investigated the effect of a selective serotonin reuptake inhibitor (SSRI) in a group of 120 patients. No trials involving other antidepressant agents met the inclusion criteria. Only the trial using the SSRI drug met the highest quality standard. None of the other included trials met the highest quality standard, due to use of inadequate outcome measures, large drop out rates or failure to separate the effects on tinnitus from the effects on symptoms of anxiety and depression. All the trials assessing tricyclic antidepressants suggested that there was a slight improvement in tinnitus but these effects may have been attributable to methodological bias. The trial that investigated the SSRI drug found no overall improvement in any of the validated outcome measures that were used in the study although there was possible benefit for a subgroup that received higher doses of the drug. This observation merits further investigation. Reports of side effects including sedation, sexual dysfunction and dry mouth were common. AUTHORS' CONCLUSIONS: There is insufficient evidence to say that antidepressant drug therapy improves tinnitus.