Micronucleus test on Triturus carnifex as a tool for environmental biomonitoring.

The amphibian micronucleus test has been widely used during the last 30 years to test the genotoxic properties of several chemicals and as a tool for ecogenotoxic monitoring. The vast majority of these studies were performed on peripheral blood of urodelan larvae and anuran tadpoles and to a lesser extent adults were also used. In this study, we developed protocols for measuring micronuclei in adult shed skin cells and larval gill cells of the Italian crested newt (Triturus carnifex). Amphibians were collected from ponds in two protected areas in Italy that differed in their radon content. Twenty-three adult newts and 31 larvae were captured from the radon-rich pond, while 20 adults and 27 larvae were taken from the radon-free site. The animals were brought to the laboratory and the micronucleus test was performed on peripheral blood and shed skins taken from the adults and on larval gills. Samples from the radon-rich site showed micronucleus frequencies higher than those from the radon-free site and the difference was statistically significant in gill cells (P < 0.00001). Moreover, the larval gills seem to be more sensitive than the adult tissues. This method represents an easy (and noninvasive in the case of the shed skin) application of the micronucleus assay that can be useful for environmental studies in situ. Environ. Mol. Mutagen. 56:412-417, 2015. © 2014 Wiley Periodicals, Inc.

Female major histocompatibility complex type affects male testosterone levels and sperm number in the horse (Equus caballus).

Odours of vertebrates often contain information about the major histocompatibility complex (MHC), and are used in kin recognition, mate choice or female investment in pregnancy. It is, however, still unclear whether MHC-linked signals can also affect male reproductive strategies. We used horses (Equus caballus) to study this question under experimental conditions. Twelve stallions were individually exposed either to an unfamiliar MHC-similar mare and then to an unfamiliar MHC-dissimilar mare, or vice versa. Each exposure lasted over a period of four weeks. Peripheral blood testosterone levels were determined weekly. Three ejaculates each were collected in the week after exposure to both mares (i.e. in the ninth week) to determine mean sperm number and sperm velocity. We found high testosterone levels when stallions were kept close to MHC-dissimilar mares and significantly lower ones when kept close to MHC-similar mares. Mean sperm number per ejaculate (but not sperm velocity) was positively correlated to mean testosterone levels and also affected by the order of presentation of mares: sperm numbers were higher if MHC-dissimilar mares were presented last than if MHC-similar mares were presented last. We conclude that MHC-linked signals influence testosterone secretion and semen characteristics, two indicators of male reproductive strategies.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4-specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14(++)CD16(-) monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68(+)/CD163(+) macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti-CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.

Postmortem angiography using femoral cannulation and postmortem microbiology.

Despite the undeniable advantages of postmortem angiography, numerous questions have arisen concerning the influence that the injected contrast media may exercise on biological fluids and tissues collected for toxicological and biochemical investigations. Moreover, cardiac blood for microbiological investigations cannot be obtained post-angiography. In this study, we examined whether the peripheral blood collected prior to postmortem angiography, using percutaneous access to femoral vessels after skin surface disinfection, could be suitable for microbiological investigations when postmortem angiography with femoral vessel cannulation is also performed. A total of 66 cases were included in the study and were divided into two subgroups (angiography and bacteriology group, 33 cases and control group, 33 cases). Autopsies, histology, toxicology, bacteriology, and biochemical investigations (procalcitonin, C-reactive protein, interleukin-6, and soluble triggering receptors expressed on myeloid cells type 1) were performed in all cases. No statistically significant differences between the two groups were noted, and identified category distribution (death unrelated to infection, true infection, false positive, and undetermined) was rather similar in both studied populations. These preliminary results suggest that postmortem angiography using a femoral approach does not constitute an impediment to the collection of peripheral blood for microbiology and vice versa. Moreover, the use of femoral blood for microbiology does not lead to an increased risk of doubtful results.

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28-Costimulated T Cells Prepared for Adoptive Therapy.

PURPOSE: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)-vaccine primed T cells in the context of cell-based immunotherapy. EXPERIMENTAL DESIGN: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. RESULTS: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. CONCLUSIONS: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840-50. ©2015 AACR.

Circulating FABP4 Is a prognostic biomarker in patients with acute coronary syndrome but not in asymptomatic individuals.

OBJECTIVE: Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS: Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS: Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS.

Assessment of Immature Platelet Fraction in the Diagnosis of Wiskott-Aldrich Syndrome.

Children with Wiskott-Aldrich syndrome (WAS) are often first diagnosed with immune thrombocytopenia (ITP), potentially leading to both inappropriate treatment and the delay of life-saving definitive therapy. WAS is traditionally differentiated from ITP based on the small size of WAS platelets. In practice, microthrombocytopenia is often not present or not appreciated in children with WAS. To develop an alternative method of differentiating WAS from ITP, we retrospectively reviewed all complete blood counts and measurements of immature platelet fraction (IPF) in 18 subjects with WAS and 38 subjects with a diagnosis of ITP treated at our hospital. Examination of peripheral blood smears revealed a wide range of platelet sizes in subjects with WAS. Mean platelet volume (MPV) was not reported in 26% of subjects, and subjects in whom MPV was not reported had lower platelet counts than did subjects in whom MPV was reported. Subjects with WAS had a lower IPF than would be expected for their level of thrombocytopenia, and the IPF in subjects with WAS was significantly lower than in subjects with a diagnosis of ITP. Using logistic regression, we developed and validated a rule based on platelet count and IPF that was more sensitive for the diagnosis of WAS than was the MPV, and was applicable regardless of the level of platelets or the availability of the MPV. Our observations demonstrate that MPV is often not available in severely thrombocytopenic subjects, which may hinder the diagnosis of WAS. In addition, subjects with WAS have a low IPF, which is consistent with the notion that a platelet production defect contributes to the thrombocytopenia of WAS. Knowledge of this detail of WAS pathophysiology allows to differentiate WAS from ITP with increased sensitivity, thereby allowing a physician to spare children with WAS from inappropriate treatment, and make definitive therapy available in a timely manner.

Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes.

BACKGROUND: Increasing evidences link T helper 17 (Th17) cells with multiple sclerosis (MS). In this context, interleukin-22 (IL-22), a Th17-linked cytokine, has been implicated in blood brain barrier breakdown and lymphocyte infiltration. Furthermore, polymorphism between MS patients and controls has been recently described in the gene coding for IL-22 binding protein (IL-22BP). Here, we aimed to better characterize IL-22 in the context of MS. METHODS: IL-22 and IL-22BP expressions were assessed by ELISA and qPCR in the following compartments of MS patients and control subjects: (1) the serum, (2) the cerebrospinal fluid, and (3) immune cells of peripheral blood. Identification of the IL-22 receptor subunit, IL-22R1, was performed by immunohistochemistry and immunofluorescence in human brain tissues and human primary astrocytes. The role of IL-22 on human primary astrocytes was evaluated using 7-AAD and annexin V, markers of cell viability and apoptosis, respectively. RESULTS: In a cohort of 141 MS patients and healthy control (HC) subjects, we found that serum levels of IL-22 were significantly higher in relapsing MS patients than in HC but also remitting and progressive MS patients. Monocytes and monocyte-derived dendritic cells contained an enhanced expression of mRNA coding for IL-22BP as compared to HC. Using immunohistochemistry and confocal microscopy, we found that IL-22 and its receptor were detected on astrocytes of brain tissues from both control subjects and MS patients, although in the latter, the expression was higher around blood vessels and in MS plaques. Cytometry-based functional assays revealed that addition of IL-22 improved the survival of human primary astrocytes. Furthermore, tumor necrosis factor α-treated astrocytes had a better long-term survival capacity upon IL-22 co-treatment. This protective effect of IL-22 seemed to be conferred, at least partially, by a decreased apoptosis. CONCLUSIONS: We show that (1) there is a dysregulation in the expression of IL-22 and its antagonist, IL-22BP, in MS patients, (2) IL-22 targets specifically astrocytes in the human brain, and (3) this cytokine confers an increased survival of the latter cells.

Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Probiotic Sonicates Selectively Induce Mucosal Immune Cells Apoptosis Through Ceramide Generation Via Neutral Sphingolyelinase.

Background: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect. Methodology/Principal Findings: Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L.brevis (LB)and S. thermophilus (ST) and the non-probiotic E. coli EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn"s disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB -induced poptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase. Conclusions: These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Enhancement of viability of radiosensitive (PBMC) and resistant (MDA-MB-231) clones in low-dose-rate cobalt-60 radiation therapy

AbstractObjective:In the present study, the authors investigated the in vitrobehavior of radio-resistant breast adenocarcinoma (MDA-MB-231) cells line and radiosensitive peripheral blood mononuclear cells (PBMC), as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy.Materials and Methods:The cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed.Results:Radiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation.Conclusion:Low dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer.

Valeur diagnostique du rapport CD103+CD4+/CD4+ pour différencier la sarcoïdose d'autres causes de lymphocytose alvéolaire.

Introduction: CD103 is a specific integrin present on some CD4+ lymphocytes of the mucosal immune system. It has been hypothesized that most CD4+ lymphocytes in pulmonary sarcoidosis do not originate from mucosal sites but from redistribution from the peripheral blood, and therefore do not bear the CD103 integrin. Several studies have suggested that a low CD103+ percentage among bronchoalveolar lavage (BAL) CD4+ lymphocytes discriminates between sarcoidosis and other causes of lymphocytic alveolitis, but contradictory data exist. Methods: We reviewed 1151 consecutive patients with BAL lymphocytosis >10% and flow cytometry performed between 2006 and 2014. 944 cases were excluded due to poor BAL quality (n= 97), unavailable clinical data (n= 760), or unclear diagnosis (n= 87). The remaining 207 patients were grouped into 9 diagnostic categories. To assess the discriminative value of the CD103+CD4+/CD4+ ratio to distinguish sarcoidosis from the other entities, area under ROC curves (AUC) were determined. Results: Sarcoidosis patients (n=53) had a lower CD103+CD4+/CD4+ ratio than the other diagnostic categories. AUC was 62% for sarcoidosis compared to all other patients and 69% for sarcoidosis compared to other interstitial lung diseases. When combining CD103+CD4+/CD4+ and CD4+/CD8+ ratios, AUC increased to 76% and 78% respectively. When applying published cut-offs from 4 previous studies to our population, AUC varied between 54 and 73%. Conclusions: The CD103+CD4+/CD4+ ratio does not accurately discriminate between sarcoidosis and other causes of lymphocytic alveolitis, neither alone nor in combination with CD4+/CD8+ ratio, and is not a relevant marker for the diagnosis of sarcoidosis.

Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.