Karyotype studies on populations of two Hypochaeris species (H. catharinensis and H. lutea), Asteraceae, endemics to southern Brazil.

Hypochaeris is an excellent system for studying different modes of chromosome evolution in plants. We carried out a cytogenetic analysis on populations of 2 Hypochaeris species, comprising 10 populations of H. catharinensis and 5 of H. lutea, to assess possible changes on chromosome organization in this interesting genus. Conventional Feulgen staining and fluorescent banding revealed that the general aspects of chromosome morphology for all populations of both species were similar, evidence of the typical bimodal karyotypes with 2n = 8 chromosomes that characterize the South American Hypochaeris. Comparative analysis of the karyotypes identified minor variations in the absolute size and arm ratio of corresponding chromosome pairs. One population of H. lutea was entirely polyploid adding a novel cytotype to this species. Fluorescent banding revealed strong chromomycin A3 (CMA3)-positive signals on both arms of chromosomes 3 and 4 of H. catharinensis, revealing a new pattern for the distribution of GC-rich heterochromatin in Hypochaeris. A strong CMA-positive signal was observed on the short arm of chromosome 3 in one population of H. lutea, while the other populations validated the CMA3 pattern already described for this species. While the overall karyotype similarities of the 2 species are in compass with all South American Hypochaeris, the presence of unusual large blocks of GC-rich heterochromatin suggests that chromosome rearrangements, related to dispersion of heterochromatin, are taking place in the karyotype of H. catharinensis. The novel polyploid cytotype identified in H. lutea provides support that polyploidization is an active process in the mode of chromosome evolution in Hypochaeris.

Clinical and virological findings in the ongoing outbreak of West Nile virus Livenza strain in northern Italy, July to September 2012.

In July-September 2012, one month earlier than in previous years, 13 confirmed human cases of West Nile virus infection were diagnosed in northern Italy, including five with neuroinvasive disease, three with West Nile fever, and five West Nile virus (WNV)-positive blood donors. In nine cases, the presence of the WNV lineage 1a Livenza strain, characterised in 2011, was ascertained. Symptomatic patients had prolonged viruria with high viral load.

New endemic West Nile virus lineage 1a in northern Italy, July 2012.

We report here the first blood donation positive for West Nile virus (WNV) by nucleic acid amplification testing collected in north-eastern Italy in July 2012.Partial sequencing of the WNV RNA demonstrated identity with a WNV lineage 1a genome identified in the same area in 2011 and divergence from the strain responsible for the outbreak in northern Italy in 2008­09. These data indicate that WNV activity in northern Italy is occurring earlier than expected and that different WNV strains are circulating.

Coexistence of erythrocyte agglutination and EDTA-dependent platelet clumping in a patient with thymoma and plasmocytoma.

For 8 years, EDTA-dependent pseudothrombocytopenia was observed in a 55-year-old woman with a history of rheumatoid arthritis who had undergone surgery for lymphoepithelial thymoma 11 years earlier. The clinical picture was characterized by the presence of platelet clumps and antiplatelet antibodies of the IgM class. With the recent appearance of a solitary extramedullary plasmocytoma in the right retrobulbar region and the detection of an IgGlambda monoclonal gammopathy, blood examination also revealed erythrocyte agglutinates alongside the platelet clumps and the presence of a cold IgG antibody with antiI specificity. Both phenomena were observed in vitro when the sample temperature declined to 20 degrees C to 25 degrees C, but not at 37 degrees C. While the EDTA-dependent antiplatelet antibodies did not appear to be chronologically correlated with the patient's diseases, the cold antierythrocyte autoantibodies were strictly related to the plasmocytoma and the IgGlambda monoclonal component in serum. To our knowledge, this is the first description of an association between EDTA-dependent platelet and erythrocyte agglutinates, with a clinical picture of pseudothrombocytopenia and pseudoerythrocytopenia due to cold agglutinins.

IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques.

When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group O-test red cell panels at 37 degrees C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.

Abnormalities of membrane protein composition in patients with autoimmune haemolytic anaemia.

Acquired abnormalities of red cell membrane protein composition in 37 patients with a positive direct antiglobulin test have been studied: 17 patients had true autoimmune haemolytic anaemia and 20 were HIV-infected subjects with a positive direct antiglobulin test but without signs of haemolysis. The study was carried out by performing sodium dodecyl sulphate polyacrylamide gel electrophoresis of ghost proteins followed by densitometric evaluation of the areas under the peaks, normalized by the total (alpha + beta) spectrin content. Results show a significant decrease of bands 3, 4.1 and 4.2 over spectrin in patients with autoimmune haemolysis as compared to controls; at least in a small subset of patients, different specificities recognized by autoantibodies do not seem to account for these abnormalities which are reproducible independently from the molecular size of bands immunoprecipitated by autoantibodies. A similar decrease of protein 4.2 but not of band 3 staining intensity is also noticeable in HIV patients with a positive direct antiglobulin test. These results are consistent with the hypothesis that, following interactions between autoantibodies and autoantigens, modifications occur on membrane proteins resembling a variety of quantitative defects described in inherited haemolytic anaemias, and mainly the "vertical interaction defects' of hereditary spherocytosis. Moreover, the decrease of band 3 staining intensity seems to represent a feature of patients with immune mediated haemolysis and not only with autoantibody binding.