Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.     

Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients'' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.Autoantibodies specific for proteins or nonprotein antigens (dsDNA, snRNP, carbohydrates) are often the serological hallmark of autoimmune diseases. Autoantibodies can be simply an epiphenomenon secondary to a chronic inflammatory milieu (1), but they can also play a direct pathogenetic role, as antithyroglobulin antibodies do in Hashimoto''s thyroiditis (2).Autoimmune hepatitis (AIH)¹ is a chronic necro-inflammatory disease of unknown etiology that affects predominantly women with an incidence of 1 to 2 per 100,000 per year and a prevalence of 10 to 20 out of 100,000 (3, 4). AIH is subdivided into two major types on the basis of autoantibody reactivity (5). Antibodies to nuclei and/or to smooth muscle characterize type 1 AIH, whereas antibodies to a liver-kidney microsomal constituent define patients with type 2 AIH. Because the detection of these autoantibodies is done by means of immunofluorescence on rodent multi-organ sections (liver, kidney, stomach), there are problems with the standardization and interpretation of the immunostaining patterns (6). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an international committee to define guidelines and develop procedures and reference standards for more reliable testing (7, 8). Although ELISA and bead assays with purified or recombinant autoantigens are under development (9), they actually represent a complementary, rather than alternative, approach to traditional immunofluorescence. Moreover, serological overlap is frequently observed between AIH and other non-autoimmune liver diseases such as chronic viral hepatitis (10). Therefore, new, highly specific markers represent an unmet medical need for the more accurate diagnosis and classification of AIH.Besides the potential diagnostic application, the discovery of novel AIH autoantigens could provide insights into the disease pathogenicity mechanism. Although some AIH target-autoantigens have been identified and characterized, little is known about their pathogenetic role, and other autoantigens are probably still unknown. Autoantibodies, to be considered pathogenetic, must have at least two features: (i) the target-autoantigen should be either expressed on the plasma membrane of target cells or secreted by cells (i.e. should be exposed to autoantibodies), and (ii) binding of the autoantibodies to the target antigen should disturb a cellular function directly or indirectly. A possible pathogenetic role in AIH has been put forward for autoantibodies specific for cytochrome P450 2D6 (CYP2D6) or Asialoglycoprotein receptor 1 (AGPR-1), which are both present on the hepatocyte cell membrane (10).Protein microarrays are a powerful technology, as they allow the simultaneous screening of thousands of analytes (11). In the present study, to identify new autoantigens with potential diagnostic and/or pathogenetic roles in AIH, we printed a microarray with 1626 human proteins whose main feature was being either secreted or membrane associated (i.e. potentially exposed to autoantibody recognition). We used this microarray to screen panels of sera from patients with AIH and identified six new protein antigens that are recognized with high sensitivity and specificity. One of these six autoantigens is the interleukin-4 (IL4) receptor fibronectin type III (FNIII) domain of the IL4 receptor (CD124), and, interestingly, patients'' autoantibodies specific for CD124 neutralize IL4 signaling, suggesting a possible pathogenetic role for IL4 neutralization in AIH.     

Determination of the biological diatom index (IBD NF T 90–354): results of an intercomparison exercise

A diatom intercomparison exercise on sampling, slide preparation, countsand identification has been carried out in September 1999 with 24French-speaking diatomists on the River Loup (Alpes Maritimes, France). Fewoperators strictly respected the standard, especially for the number ofindividuals to be counted. It has however been demonstrated this had a very lowimpact on the index value and that old counts can be used for indexcalculation,if there are at least 300 frustules or valves. Counts higher than 400 did notlead to a better precision. The total variability largely depended on theoperator and was distributed unevenly between sampling, which appears to be acritical stage, slide preparation and counting. For those who strictlyrespectedthe standard procedure, the natural variability can be estimated to about oneunit value of the Biological Diatom Index. This intra-operator variability canbe considered as very low, as the IBD scale is from 0 to 20, and notsignificantin assessing water quality. Despite this, the inter-operator variability isgreater. When comparing operators' results, after excluding those who did notrespect the standard requirements, the main source of variability is due tomisidentifications, especially for small Achnanthes(A. minutissima and A. biasolettiana)and Cocconeis (C. placentula var.placentula, var. euglypta and var.lineata). Intercomparison exercises, internet exchangesforchecking and reference material collections should be encouraged both tosatisfythe internal and external quality controls and to acquaint biologists with theimportance of field and laboratory protocols, as well as to allow progress indiatom identification.     

Blockade of the nociceptin/orphanin FQ/NOP receptor system in the rat ventrolateral periaqueductal gray potentiates DAMGO analgesia

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG.     

Anti-tumoral Effects of miR-3189-3p in Glioblastoma

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.     

Hedgehog signaling: From the cuirass to the heart of pancreatic cancer

Exocrine pancreatic cancer is the fifth cause of cancer-related death in Europe and carries a very poor prognosis for all disease stages. To date no medical treatment has significantly increased patients' survival. One of the reasons for pancreatic cancer's chemoresistence is the complex tumor architecture: cancer cells are surrounded by a dense desmoplastic stroma that blocks drug delivery. Moreover, pancreatic cancer is characterized by a marked heterogeneity of cells, including cancer stem cells (CSCs) that act as tumor-initiating cells and hierarchically control the differentiated cancer cells. In particular, this subpopulation is resistant to classic cytotoxic therapies, and seems to be responsible for disease renewal. Hedgehog signaling (HH) is implicated in pancreatic gland development during embryogenesis and is reactivated during tumorigenesis and the maintenance of pancreatic cancer. Some studies demonstrated that the Hedgehog-secreted signaling proteins are overexpressed in both the stromal and CSCs pools, implying an abnormal activation of HH in the main compartment of pancreatic cancer. For this reason, the Hedgehog pathway could be an interesting target for clinical trials to increase drug concentration in neoplastic cells and hence deplete the stroma and directly kill tumor-initiating cells.     

Supraspinal injection of Substance P attenuates allodynia and hyperalgesia in a rat model of inflammatory pain

The neuropeptide Substance P (SP), that has a high affinity for the neurokinin 1 (NK1) receptor, is involved in modulation of pain transmission. Although SP is thought to have excitatory actions and promote nociception in the spinal cord, the peptide induces analgesia at the supraspinal level. The aim of this study was to evaluate the role of supraspinal SP and the NK1 receptor in inflammatory pain induced by injection of carrageenan in the hind paw of the rat. There are two nociceptive behavioral responses associated with this pain state: mechanical allodynia and heat hyperalgesia. Because the NK1 receptor colocalizes with the MOP receptor in supraspinal sites involved in pain modulation, we also decided to study the possible involvement of the opioid system on SP-induced analgesia. We found that treatment with SP, at doses of 3.5, 5 and 7 μg/5 μl/rat i.c.v., clearly showed inhibition of allodynia and hyperalgesia. Pretreatment with the selective NK1 antagonist L-733,060 (10mg/kg i.p.) blocked the SP-induced analgesia, suggesting the involvement of the NK1 receptor. This SP-induced analgesia was significantly reduced by administration of the opioid antagonist naloxone (3mg/kg s.c.). This reduction occurred when SP was administered either before or after the carrageenan injection. These results suggest a significant antinociceptive role for SP and the NK1 receptor in inflammatory pain at the supraspinal level, possibly through the release of endogenous opioids.     

1H-NMR-Based Metabolomic Study for Identifying Serum Profiles Associated with the Response to Etanercept in Patients with Rheumatoid Arthritis

Objective

A considerable proportion of patients with rheumatoid arthritis (RA) do not have a satisfactory response to biological therapies. We investigated the use of metabolomics approach to identify biomarkers able to anticipate the response to biologics in RA patients.

Methods

Due to gender differences in metabolomic profiling, the analysis was restricted to female patients starting etanercept as the first biological treatment and having a minimum of six months’ follow-up. Each patient was evaluated by the same rheumatologist before and after six months of treatment. At this time, the clinical response (good, moderate, none) was determined according to the EUropean League Against Rheumatism (EULAR) criteria, based on both erythrocyte sedimentation rate (EULAR-ESR) and C-reactive protein (EULAR-CRP). Sera collected prior and after six months of etanercept were analyzed by ¹H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate data analysis.

Results

Twenty-seven patients were enrolled: 18 had a good/moderate response and 9 were non responders according to both EULAR-ESR and EULAR-CRP after six months of etanercept. Metabolomic analysis at baseline was able to discriminate good, moderate, and non-responders with a very good predictivity (Q² = 0.68) and an excellent sensitivity, specificity, and accuracy (100%). In good responders, we found an increase in isoleucine, leucine, valine, alanine, glutamine, tyrosine, and glucose levels and a decrease in 3-hydroxybutyrate levels after six months of treatment with etanercept with respect to baseline.

Conclusion

Our study confirms the potential of metabolomic analysis to predict the response to biological agents. Changes in metabolic profiles during treatment may help elucidate their mechanism of action.     

Comparative monitoring by means of diatoms,macroinvertebrates and chemical parameters of an Apennine watercourse of central Italy: The river Tenna

The WFD/60/2000/EC establishes the use of different biological indicators to assess the ecological status of rivers, in addition to chemical, physical and bacteriological parameters. At this purpose, the present study concerning the river Tenna, a central Apennine watercourse (Italy), aimed to analyse and discuss the information given by two biological indices: the Extended Biotic Index or E.B.I. that uses benthic macroinvertebrates, in the version adapted to the Italian rivers (I.B.E.), and the diatom-based Eutrophication and/or Pollution Index (EPI-D). Sampling has been carried out twice, in May (deep water conditions) and October 2006 (low water conditions), in ten stations along the hydrographic basin of the river. Chemical parameters have been determined monthly during a year because they present a higher variability than biotic indicators. The correlations between the biological and abiotic data obtained have been statistically analysed by means of the Spearman's test. The statistical data, in general, showed a direct correlation between the two biotic indices, and an inverse correlation between these and the chemical-physical and bacteriological parameters. Some of the differences of judgement emerging from the application of the two biotic indices showed a complementary response of the diatoms and macroinvertebrates to various environmental stresses. These results suggest the usefulness of an integrated multidisciplinary approach, in order to obtain a more accurate diagnosis of the health status of the watercourses.