Measuring respiratory symptoms in clinical trials of COPD: reliability and validity of a daily diary.

BACKGROUND: Although respiratory symptoms are characteristic features of COPD, there is no standardised method for quantifying their severity in stable disease. OBJECTIVE: To evaluate the EXACT-Respiratory Symptom (E-RS) measure, a daily diary comprising 11 of the 14 items in the Exacerbations of Chronic Pulmonary Disease Tool (EXACT). METHODS: Qualitative: patient focus group and interviews to address content validity. Quantitative: secondary data analyses to test reliability and validity. RESULTS: Qualitative: n=84; mean (SD) age 65 (10) years, FEV1 1.2(0.4) L; 44% male. Subject descriptions of their respiratory symptoms were consistent with E-RS content and structure. Quantitative: n=188; mean (SD) age 66 (10) years, FEV1 1.2(0.5) L; 50% male. Factor analysis (FA) showed 3 subscales: RS-Breathlessness, RS-Cough & Sputum, and RS-Chest Symptoms; second-order FA supported a general factor and total score. Reliability (total and subscales): 0.88, 0.86, 0.73, 0.81; 2-day test-retest ICC: 0.90, 0.86, 0.87, 0.82, respectively. VALIDITY: Total scores correlated significantly (p < 0.0001) with SGRQ Total (r=0.75), Symptoms (r=0.66), Activity (r=0.57), Impact (r=0.70) scores; subscale correlations were also significant (r=0.26, p < 0.05 (RS-Chest Symptoms with Activity) to r=0.69, p < 0.0001 (RS-Cough & Sputum with Symptoms). RS-Breathlessness correlated with rescue medication use (r=0.32, p < 0.0001), clinician-reported mMRC (r=0.33, p < 0.0001), and FEV1% predicted (r=-0.17, p < 0.05). E-RS scores differentiated groups based on chronic bronchitis diagnosis (p < 0.01-0.001), smoking status (p < 0.05-0.001), and rescue medication use (p < 0.05-0.0001). CONCLUSIONS: Results suggest the RS-Total is a reliable and valid instrument for evaluating respiratory symptom severity in stable COPD. Further study of sensitivity to change is warranted.

The epidemiology of human T-cell lymphotropic virus types I and II in Canadian blood donors.

OBJECTIVES: Blood donors in Canada have been tested for Human T-Cell Lymphotropic Virus (HTLV) since 1990. We report the epidemiology, risk factors and lookback/traceback of HTLV-positive donors/recipients. METHODS: The annual HTLV rate was calculated from 1990 to 2010. Residual risk was estimated as the product of incidence and window period. Twenty-nine HTLV-positive donors and 116 matched controls (ratio 1 : 4) were interviewed about risk factors. For HTLV-positive donations, lookback investigations involved identification of all previous donations, and attempting to locate and test recipients. Traceback was initiated when transfusion transmission was queried for HTLV-positive blood recipients. All donors of products that the recipient received were identified, with an attempt to locate and test them. RESULTS: The HTLV rate decreased from 9.35 per 100,000 donations in 1990 to 1.11 in 2010. The residual risk of infection was 1 in 7.6 million donations. In logistic regression birth overseas (OR 18.7), history of sexually transmitted diseases (OR 32.9), sex with unknown background (OR 5.4) and blood transfusion (OR 8.9) were significant predictors. In the lookback study, of 109 HTLV-positive donors, 508 components were transfused, of whom 147 recipients were tested and 18 (12%) were positive. All were transfused prior to the implementation of donor testing. Twenty-three traceback investigations were requested involving 324 transfused untested products,of whom 219 (67.6%) of donors were tested and 13 (6%) were positive for HTLV. CONCLUSIONS: With testing of the blood supply, the risk from HTLV is very low and while most HTLV-positive donors have risk factors, deferrable risk is rare.

Cyclosporine A drives a Th17- and Th2-mediated posttransplant obliterative airway disease.

Calcineurin-inhibitor refractory bronchiolitis obliterans (BO) represents the leading cause of late graft failure after lung transplantation. T helper (Th)2 and Th17 lymphocytes have been associated with BO development. Taking advantage of a fully allogeneic trachea transplantation model in mice, we addressed the pathogenicity of Th cells in obliterative airway disease (OAD) occurring in cyclosporine A (CsA)-treated recipients. We found that CsA prevented CD8(+) T cell infiltration into the graft and downregulated the Th1 response but affected neither Th2 nor Th17 responses in vivo. In secondary mixed lymphocyte cultures, CsA dramatically decreased donor-specific IFN-γ production, enhanced IL-17 production and did not affect IL-13. As CD4(+) depletion efficiently prevented OAD in CsA-treated recipients, we further explored the role of Th2 and Th17 immunity in vivo. Although IL-4 and IL-17 deficient untreated mice developed an OAD comparable to wild-type recipients, a single cytokine deficiency afforded significant protection in CsA-treated recipients. In conclusion, CsA treatment unbalances T helper alloreactivity and favors Th2 and Th17 as coexisting pathways mediating chronic rejection of heterotopic tracheal allografts.

Jasmonates are phytohormones with multiple functions, including plant defense and reproduction.

The plant hormones jasmonic acid and methyl jasmonate, along with their intermediate compounds, produced in the octadecanoid pathway, are important signaling molecules that are collectively called jasmonates. These are widespread in the plant kingdom and play crucial roles in biotic/abiotic stress responses, as well as in processes related to plant growth and development. Recently, it has been shown that jasmonates are also involved in reproductive processes. We present the most recent findings related to the biosynthesis, regulation and signaling mechanisms of jasmonates. Additionally, we discuss the identification of [(+)-7-iso-JA-L-Ile] as the active biological hormonal form of jasmonate; this fills the greatest gap in our knowledge about the signaling mechanism that is responsible for the activation of downstream genes in the jasmonate-signaling cascade. The identification of several Arabidopsis thaliana mutants was crucial to the elucidation of the signaling mechanisms involved in jasmonate-mediated responses. Finally, the involvement of jasmonates in the reproductive process of Nicotiana tabacum L. is briefly discussed, since some of the main enzymes of the jasmonic acid biosynthesis pathway were identified in a stigma/style expressed sequence tag database (TOBEST) of this Solanaceae species.

Endotoxin-induced myeloid-derived suppressor cells inhibit alloimmune responses via heme oxygenase-1.

Inflammation and cancer are associated with impairment of T-cell responses by a heterogeneous population of myeloid-derived suppressor cells (MDSCs) coexpressing CD11b and GR-1 antigens. MDSCs have been recently implicated in costimulation blockade-induced transplantation tolerance in rats, which was under the control of inducible NO synthase (iNOS). Herein, we describe CD11b+GR-1+MDSC-compatible cells appearing after repetitive injections of lipopolysaccharide (LPS) using a unique mechanism of suppression. These cells suppressed T-cell proliferation and Th1 and Th2 cytokine production in both mixed lymphocyte reaction and polyclonal stimulation assays. Transfer of CD11b+ cells from LPS-treated mice in untreated recipients significantly prolonged skin allograft survival. They produced large amounts of IL-10 and expressed heme oxygenase-1 (HO-1), a stress-responsive enzyme endowed with immunoregulatory and cytoprotective properties not previously associated with MDSC activity. HO-1 inhibition by the specific inhibitor, SnPP, completely abolished T-cell suppression and IL-10 production. In contrast, neither iNOS nor arginase 1 inhibition did affect suppression. Importantly, HO-1 inhibition before CD11b+ cell transfer prevented the delay of allograft rejection revealing a new MDSC-associated suppressor mechanism relevant for transplantation.

Basophil activation tests for the diagnosis of food allergy in children.

BACKGROUND: Positive skin prick tests (SPT) for food allergens and specific IgE (sIgE) in serum indicate sensitization but do not enable distinction between sensitized but tolerant and clinically allergic patients. OBJECTIVE: Herein, we evaluate the clinical relevance of basophil activation tests (BATs) for peanut or egg allergy diagnosis. METHODS: Thirty-two peanut-allergic, 14 peanut-sensitized (sIgE(+) and/or SPT(+) to peanuts) but tolerant children and 29 controls with no history of an adverse reaction to peanuts were included. Similarly, 31 egg-allergic, 14 egg-sensitized children (sIgE(+) and/or SPT(+) to egg white) and 22 controls were studied. Flow cytometric analysis of CD63 expression or CD203c upregulation on basophils and the production of leukotrienes (LT) were performed in response to an in vitro crude peanut extract or ovalbumin (OVA) challenge. RESULTS: After in vitro peanut challenge, the basophils from peanut-allergic children showed significantly higher levels of activation than those from controls (P<0.001). After OVA challenge, a similar distinction (P<0.001) was observed between egg-allergics and controls. Interestingly, the majority of egg- or peanut-sensitized children failed to activate basophils, respectively, in response to OVA and peanut challenge. The sensitivity of the CD63, CD203c and LT assay was 86.7%, 89.5% and 76.0% with a specificity of 94.1%, 97.1% and 94.6% for peanut allergy diagnosis. The corresponding performances of BATs applied to egg allergy diagnosis were 88.9%, 62.5% and 77.8% for the sensitivity and 100%, 96.4% and 96.4% for the specificity. CONCLUSION: Neither conventional tests nor BATs are sensitive and specific enough to predict food allergy accurately. However, BATs may helpfully complete conventional tests, especially SPT, allowing improved discrimination between allergic and non-allergic individuals.

Quality aspects of the tissue microarray technique in a population-based cohort with ductal carcinoma in situ of the breast.

AIMS: Tissue microarray (TMA) is an efficient technique for analysis of molecular markers. Prospectively collected samples have been reported to give excellent concordance between TMA data and corresponding whole-sections. The aim was to evaluate the usefulness of TMA in a population-based cohort of 213 women with ductal carcinoma in situ of the breast (DCIS). METHODS AND RESULTS: We studied immunohistochemical HER2, oestrogen (ER) and progesterone (PR) receptor status. The prognostic impact was similar for all markers comparing whole sections and TMAs. The proportion of positive tumours was similar regarding HER2 and ER, whereas PR tumours were more frequently positive in the TMAs (P = 0.007). The concordance was 80% (kappa value 0.63) between original sections and TMAs. The proportion of successfully analysed tumours was 70%. Smaller tumours had a lower ratio (P < 0.0001) and a larger proportion of mismatched results (P = 0.05). CONCLUSIONS: Retrospective analyses of tumours from cohorts with long-term follow-up are indispensable. We have shown that the TMA technique is a useful tool for high-throughput analysis of DCIS. However, our study has pinpointed some technical hazards within a population-based cohort, including many small lesions and the poor condition of some donor blocks. Mismatched results may be due to tumour heterogeneity.

CD8+ T-Cell depletion and rapamycin synergize with combined coreceptor/stimulation blockade to induce robust limb allograft tolerance in mice.

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti-CD154 mAb, nondepleting anti-CD4 combined to either depleting or nondepleting anti-CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long-term allograft survival in both combinations. Nevertheless, robust donor-specific tolerance, defined by the acceptance of a fresh donor-type skin allograft and simultaneous rejection of third-party grafts, required initial CD8(+) T-cell depletion. Mixed donor-recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL-2, IFN-gamma and TNF-alpha in MLC with donor antigen while significant alloreactivity persisted against third- party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor-recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8(+) T-cell depletion and costimulation/coreceptor blockade.

Use of thoracic impedance sensors to screen for sleep-disordered breathing in patients with cardiovascular disease.

Screening patients for the possibility of sleep apnoea, one of the most common forms of sleep-disordered breathing, requires measurement of respiration. We propose a simple method to estimate the amplitude modulation of a respiratory tidal volume, using a semi-quantitative measure of respiration based on thoracic impedance (TI). Because respiratory volume changes may be accommodated by varying displacements of the rib cage (RC) and abdomen (AB), the latter produced by outward motion of the diaphragm, it is necessary for any useful measure of respiration to be closely related to both RC and AB displacements. Because the relative contributions of RC and AB displacements to respiratory tidal volume vary in different body positions, the present measurements were recorded from subjects in supine, and right and left lateral decubitus postures. We observed a clear linear relationship between TI and both RC and AB signals in all three body positions. There were no statistically significant differences between observed relationships between TI and AB and between TI and RC, and these relationships were independent of the body position. TI sensors appear to be a useful candidate for a simple method of screening for sleep apnoea, especially in a cardiology clinical setting. Further investigation is warranted for the refinement of algorithms to detect changes in amplitude modulation occurring with apnoeas and to remove artefacts due to gross body movements.

Skin biopsies in DC vaccines for stage III-IV melanoma patients: role of neutrophils?

Dendritic cell (DC) vaccines are used for the induction of anti-tumor T cell reaction in melanoma patients. DC are generated in vitro, pulsed with antigen and matured prior to injection. They are supposed to migrate to lymph nodes and to present the processed antigen to naive T cells allowing activation of tumor-specific lymphocytes. It has been suggested that intradermal injection allows a superior migration to the lymph node. Eight HLA-A2 positive patients with stage III or IV melanomas expressing NA 17 antigen were collected. They were included in a pilot trial of vaccination in which they received IL3/INFb DC presenting the NA17 A2 antigen. In each patient, a skin biopsy was performed at the injection site, 24 h after inoculation. The striking features of the biopsies were the presence of a perivascular CD3+/CD8+ T cell infiltrate with a slight population of CD4+ cells and the presence of a massive neutrophilic infiltrate associated with the injected DC still present, realizing a suppurative granuloma. The persistence of DC 24 h after the injection suggests that migration in the lymph node is not necessary for the induction of the immune response. The skin itself could be the location of a reaction starting with a massive recruitment of neutrophils.

A rapid test of alloreactivity based on interleukin-2 mRNA expression might identify liver transplant recipients with donor-specific hyporesponsiveness.

BACKGROUND: Immunosuppression withdrawal is feasible in some liver transplant (OLT) recipients but may lead to severe rejection in others, underlying the need for reliable biomarkers to identify patients with tolerant profile in whose weaning/withdrawal could be safely proposed. We evaluated the value of real-time polymerase chain reaction (PCR)-based measurement of interleukin (IL)-2 mRNA in mixed lymphocyte reaction (MLR) to monitor in vitro anti-donor reactivity in OLT patients. METHODS: MLR were performed in three patients undergoing living donor OLT using a tolerogenic protocol including donor stem cells. IL-2 mRNA production in MLR was measured by PCR at several intervals after OLT. RESULTS: In the early posttransplant period, three patients presented with global immunodeficiency, as indicated by low IL-2 mRNA production against both donor and third-party antigens. In the two patients who has immunosuppression successfully withdrawn, donor-specific hyporesponsiveness was observed thereafter: IL-2 mRNA production against donor cells remained low, while IL-2 mRNA production against a third-party antigen-presenting cells progressively recovered. No such modulation of the anti-donor response was observed in the patient in whom withdrawal led to rapid rejection. CONCLUSION: Measurement of IL-2 mRNA production in MLR might prefer a tool to monitor anti-donor reactivity after OLT for decisions to minimize or withdraw immunosuppression in patients displaying donor-specific hyporesponsiveness.

[From discovery to innovation: the example of hypereosinophilic syndrome]. / De la découverte a l'innovation: l'exemple du syndrome hyperéosinophilique.

Biomedical research is extremely productive so that each day new discoveries reveal novel (mechanisms and) molecular pathways of diseases. However, the number of innovative therapies approved each year by regulatory authorities is decreasing. The causes of this "innovation breakdown" are multiple and should be explored both in academic institutions and in the industrial world. New initiatives are underway in the Walloon Region to improve this situation, such as the setting up of the BioWin Health Cluster gathering pharmaceutical and biotechnology enterprises and university laboratories. This new vision of the partnership between academia and the private sector should be based on strong fundamental research activities. We illustrate this concept by discussing recent advances in the diagnosis and treatment of the hypereosinophilic syndrome.

N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells.

AIMS/HYPOTHESIS: The early loss of beta cells after islet cell transplantation has been attributed in part to blood coagulation at the implant site. Tissue factor expressed by beta cells and contaminating duct cells is considered to activate this process. Here, we investigated the ability of N-acetyl-L-cysteine to suppress the in vitro procoagulant activity of duct cells and human islet cell preparations. MATERIALS AND METHODS: The effects of Nacystelyn, a salt derivative of N-acetyl-L-cysteine, were first assessed on procoagulant activity induced in human plasma by recombinant tissue factor, human primary duct cells or human islet cell preparations. The influence of Nacystelyn on clot formation, platelet counts and D-dimers were measured in a whole blood tubing loop model. Human beta cell viability and insulin synthesis after Nacystelyn treatment were assessed to exclude cytotoxicity of Nacystelyn. RESULTS: Nacystelyn efficiently inhibited the procoagulant activity of human recombinant tissue factor, primary duct cells and human islet cell preparations at clinically relevant concentrations without cellular toxicity. CONCLUSIONS/INTERPRETATION: Nacystelyn is a pharmaceutical candidate to reduce early beta cell loss related to tissue factor-dependent coagulation after islet transplantation.

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles.

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Severe atherosclerosis of the aorta and development of peripheral T-cell lymphoma in an adolescent with angiolymphoid hyperplasia with eosinophilia.

We report an adolescent girl with a history of angiolymphoid hyperplasia with eosinophilia (ALHE) diagnosed at the age of 10 years. The patient also suffered from chronic persistent multiresistant herpes simplex virus infection. Atherosclerotic occlusive disease of the abdominal aorta and its major branches was observed at the age of 17 years, necessitating vascular surgical intervention 1 year later because of disease progression. Histological examination of the aorta disclosed widespread atherosclerosis and high levels of gene expression of both T-helper cell type (Th) 1- and Th2-derived cytokines. This suggests that a highly stimulated systemic immune response including increased production of both Th1- and Th2-derived cytokines such as interferon-gamma and interleukin-4 may result in severe atherosclerotic lesions at a very young age. In addition, the patient developed a peripheral T-cell lymphoma at the age of 18 years. Neither systemic atherosclerosis nor T-cell lymphoma has been reported in association with ALHE. It is suggested that a highly stimulated dysfunctional immune response may play a key role in persistent inflammatory disease and premature development of atherosclerosis as well as malignant transformation of T cells.

Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells.

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.

CD40 expression on human pancreatic duct cells: role in nuclear factor-kappa B activation and production of pro-inflammatory cytokines.

AIMS/HYPOTHESIS: Human pancreatic duct cells are closely associated with islet beta cells, and contaminate islet suspensions transplanted in Type 1 diabetes mellitus patients. Activated duct cells produce cytotoxic mediators and possibly contribute to the pathogenesis of Type 1 diabetes mellitus or islet graft rejection. As CD40 transduces activation signals involved in inflammatory and immune disorders, we investigated CD40 expression on duct cells and their response to CD40 engagement. METHODS: CD40 expression on human pancreatic duct cells was analysed by flow cytometry and immunohistochemical analyses. To assess the function of CD40 expression on duct cells, activation of the transcription factor nuclear factor-kappa B was determined using electrophoretic mobility shift assay and ELISA. Cytokine mRNA levels were quantified by real-time RT-PCR, and protein levels by Luminex technology. RESULTS: Isolated human pancreatic duct cells and Capan-2 cell lines were found to express constitutively CD40. The expression of CD40 on duct cells was confirmed in vivo on human normal and pathological pancreatic specimens. CD40 ligation on Capan-2 cells induced rapid nuclear factor-kappa B activation, and supershift assays demonstrated that p50/p65 heterodimers and p50/p50 homodimers were present in the activated complexes in the nucleus. This activation was accompanied by tumour necrosis factor-a and interleukin-1beta mRNA accumulation. Tumour necrosis factor-alpha protein secretion was confirmed in CD40-activated Capan-2 cells and in isolated human pancreatic duct cells. CONCLUSIONS/INTERPRETATION: Interaction between activated T lymphocytes expressing CD40 ligand and duct cells expressing CD40 may contribute to the immune responses involved in Type 1 diabetes mellitus and islet graft rejection. Interfering with CD40-mediated duct cell activation could alleviate beta cell damage of immune origin.