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There are different BCR-ABL1 fusion genes that are translated into proteins that are different from each other, yet all leukemogenic, causing chronic myeloid leukemia (CML) or acute lymphoblastic leukemia. Their frequency has never been systematically investigated. In a series of 45503 newly diagnosed CML patients reported from 45 countries, it was found that the proportion of e13a2 (also known as b2a2) and of e14a2 (also known as b3a2), including the cases co-expressing e14a2 and e13a2, was 37.9% and 62.1%, respectively. The proportion of these two transcripts was correlated with gender, e13a2 being more frequent in males (39.2%) than in females (36.2%), was correlated with age, decreasing from 39.6% in children and adolescents down to 31.6% in patients ≥ 80 years old, and was not constant worldwide. Other, rare transcripts were reported in 666/34561 patients (1.93%). The proportion of rare transcripts was associatedwith gender (2.27% in females and 1.69% in males) and with age (from 1.79% in children and adolescents up to 3.84% in patients ≥ 80 years old). These data show that the differences in proportion are not by chance. This is important, as the transcript type is a variable that is suspected to be of prognostic importance for response to treatment, outcome of treatment, and rate of treatment-free remission.

Solid pseudopapillary neoplasms of the pancreas are rare neoplasms that have been shown to harbor recurrent somatic pathogenic variants in the beta-catenin gene, CTNNB1. Here, we used targeted next generation sequencing to analyze these tumors for other associated mutations. Six cases of solid pseudopapillary neoplasms were studied. DNA extracted from formalin-fixed paraffin embedded tissue blocks was analyzed using the Ion Torrent platform, with the 50-gene Ampliseq Cancer Hotspot Panel v2 (CHPv2), with further variant validation performed by Sanger sequencing. Four tumors (67%) were confirmed to harbor mutations within CTNNB1, two with c.109T > G p.(Ser37Ala) and two with c.94G > A p.(Asp32Asn). One case showed a frameshift deletion in the Adenomatous Polyposis Coli gene, APC c.3964delG p.(Glu1322Lysfs*93) with a variant allele frequency of 42.6%. Sanger sequencing on non-tumoral tissue confirmed the variant was somatic. The patient with the APC mutation developed metastasis and died. In addition to the four cases harboring CTNNB1 variants, we found a case characterized by poor outcome, showing a rare frameshift deletion in the APC gene. Since the APC product interacts with beta-catenin, APC variants may, in addition to CTNNB1, contribute to the pathogenesis of solid pseudopapillary neoplasms via the Wnt signaling pathway.

Imatinib mesylate is considered standard of care for first-line treatment of chronic phase chronic myeloid leukemia (CML-CP). In the phase III, randomized, open-label International Randomized Study of Interferon vs STI571 (IRIS) trial, previously untreated CML-CP patients were randomized to imatinib (n=553) or interferon-alpha (IFN) plus cytarabine (n=553). This 6-year update focuses on patients randomized to receive imatinib as first-line therapy for newly diagnosed CML-CP. During the sixth year of study treatment, there were no reports of disease progression to accelerated phase (AP) or blast crisis (BC). The toxicity profile was unchanged. The cumulative best complete cytogenetic response (CCyR) rate was 82%; 63% of all patients randomized to receive imatinib and still on study treatment showed CCyR at last assessment. The estimated event-free survival at 6 years was 83%, and the estimated rate of freedom from progression to AP and BC was 93%. The estimated overall survival was 88% -- or 95% when only CML-related deaths were considered. This 6-year update of IRIS underscores the efficacy and safety of imatinib as first-line therapy for patients with CML.

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5–10% of AML and 2–3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5–9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse. © 1999 Cancer Research Campaign

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

OBJECTIVE: We estimated the adequacy of antibiotic therapy to guidelines for nosocomial and community-acquired urinary tract infections in hospital. DESIGN: For 4 weeks, all adult patients hospitalized with positive bacteriuria were included in our retrospective study. Data was collected from urine culture results and from patient medical files. Adequacy to guidelines was analyzed by two infectious disease specialists, focusing on the indication, antibiotic choice, dosage, route of administration, and duration of treatment. RESULTS: Overall 202 patients were enrolled in the study (63.9% women). The decision of initiating or not antibiotic therapy was appropriate in 66.8% of cases. Antibiotherapy indication and antibiotic choice were adequate in 94 cases in empiric prescription (50.8%) and in 123 cases (60.9%) after receiving culture antibiogram results. Route of administration was adequate in 94.4% and dosage in 70.8% of prescriptions. This poor compliance with guidelines was mainly due to unnecessary prescriptions in asymptomatic bacteriuria, unnecessary biotherapies and spectrum errors. CONCLUSIONS: It seems important to remind prescribers of recommendations for urinary tract infections.

Three cases of acute lymphoblastic leukemia (ALL) with the rare t(17;19)(q22;p13) translocation were investigated for E2A/HLF fusion genes using reverse transcription coupled with polymerase chain reaction (RT-PCR). The patients had C-ALL, F/17 years (case 1) or pre-B ALL, M/11 years (case 2) and M/13 years (case 3). Case 1 had an event-free survival (EFS) of 42 months. Case 2 was ultimately refractory to treatment. Case 3 presented following EFS of 16 months in morphological remission (1% blasts), but with immunological and cytogenetic evidence of active disease, then relapsed, remitted and relapsed. Type II E2A/HLF fusion cDNA was found at diagnosis (cases 1, 2), at presentation (case 3) and in all samples tested, whether with active disease or in complete remission (CR). Case 3 showed, in addition, type I fusion E2A/HLF cDNA at presentation, through induction therapy when there was evidence of active disease, but not in CR. Cases 1 and 3 had bone marrow transplantation while in CR but with residual disease detectable by RT-PCR. All patients have died of ALL. Two cases (2 and 3) had hypercalcemia with bone lesions. No case had any evidence of disseminated intravascular coagulation. This is the first demonstration of the value of RT-PCR for the detection of minimal residual disease in t(17;19) ALL.

We have studied the capacity of peripheral blood cells from 26 chronic B cell leukaemias to proliferate continuously in culture; 72 attempts to establish cell lines were made. The cells were treated in vitro with or without stimulating agents: Epstein-Barr virus (EBV) and/or phorbol-ester (TPA) were the most frequently used. Fourteen cell lines of continuous growth were established from cells from 11 patients, but only four of these were proven to be derived from the original leukaemic cells. Only in the latter four lines were the karyotypic abnormalities and the patterns of immunoglobulin (Ig)-gene rearrangements identical to those found in the patients' leukaemic cells. On the other 10 lines, five had both kappa- and lambda-producing cells, and the remaining five, despite showing light-chain restriction, were proved to be non-leukaemic clones by comparing the Ig-gene rearrangement patterns before and after culture. Three of the four leukaemic cell lines (JVM-2, JVM-3 and JVM-13) were induced by EBV + TPA and derived from prolymphocytic leukaemia (PLL) cases; the fourth (JVM-14) originated from a case of chronic lymphocytic leukaemia (CLL) with increased percentage of prolymphocytes whose cells were stimulated in vitro with EBV. The immunophenotype of the three PLL lines is more mature than that of the original prolymphocytes, as shown by a reduction in surface-Ig and FMC7 expression, enhancement of cytoplasmic-Ig and increase in CD38- and transferrin receptor-positive cells. The cells from line JVM-14 retained the CD5-antigen, a marker of CLL. This study suggests that PLL and some CLL clones are arrested at a stage of maturation ideally suited to be triggered to continuous proliferation in culture. The presence of consistent chromosomal abnormalities in PLL may offer an alternative explanation for the greater proliferative potential of these cells in vitro.

beta and gamma T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL-leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR beta was found in germline configuration. In one of them rearrangement of T cell-rearranging gene gamma (TRG gamma) and a gamma mRNA were demonstrated. We suggest that in this patient the leukemic T cells arose from one of the rare T cells bearing a gamma-delta rather than an alpha-beta TCR heterodimeric molecule. In the other patient several discrete TRG gamma rearrangements were detected. Because her leukemic cells were shown to be monoclonal on the grounds of their karyotype, we suggest that her leukemia originated before any rearrangement had taken place. The combined use of TCR beta and TRG gamma probes provides new information on the origin and clonal expansion of lymphoid cells in LGL-leukemia.

We have investigated the configuration of immunoglobulin (Ig) genes in leukaemic cells in 17 patients with B-cell leukaemias (11 chronic lymphocytic leukaemias (B-CLL); 4 prolymphocytic leukaemias (B-PLL), and two hairy cell leukaemias (HCL)). In addition we studied four patients with T chronic lymphocytic leukaemia (T-CLL); four patients with acute leukaemia (3 acute lymphoblastic leukaemias (ALL), and 1 mixed acute leukaemia (M.AL)); and six patients with chronic granulocytic leukaemias in blastic crisis (CGL.BC). The heavy chain genes (H) were analysed by using probes for the constant region of the mu chains (C mu) and for the joining region (JH). The light chain genes were analysed by using probes for the constant region of the kappa (C kappa) and lambda (C lambda) chains. We have found rearranged Ig genes in all cases of B-CLL, B-PLL and HCL, but in none of the patients with T-CLL. In one case of HCL, both mu genes were deleted, indicating that in this case the class switch has taken place. In four out of six cases with either ALL or lymphoid CGL.BC and in one case of M.AL, an Ig gene rearrangement was also found. No rearrangement was detected in two cases of myeloid CGL.BC. When the combination of rearrangement versus germ-line configuration was considered, a variety of patterns emerge, but in no case did we find a L chain gene rearranged without at least one H chain gene being rearranged as well. Whereas in the majority of cases of B-CLL only one H chain gene is rearranged, in nearly all cases of B-PLL both H chain genes are rearranged. By systematic analysis of restriction fragment sizes of rearranged genes, we have established that a large number of different variable regions for the H chain (VH) are involved in Ig gene rearrangement in B-cell malignancies. Our data confirm that testing for Ig gene rearrangement may be the most sensitive and specific test for identifying leukaemic cells of B lineage.

Antigen receptor stereotypy across B-cell lymphoproliferations: the case of IGHV4-59/IGKV3-20 receptors with rheumatoid factor activity

Background aims. The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. Methods. Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitr o study of the stemness features and multipotency of these multidistrict MSC populations. Results. Notch-1 � , Stro-1 � , Sca-1 � and Oct-4 � cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothe- lial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high effl ux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. Conclusions. Overall, these fi ndings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) are currently under evaluation in phase III clinical trials for inflammatory bowel disease and other intestinal disease manifestations. The therapeutic efficacy of these treatments may derive from a combination of the differentiation, trophic and immunomodulatory abilities of the transplanted cells. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specific functions and hold advantages for engraftment and contribution in the gastrointestinal environment.Intestinal specimens were collected, and the mucosa and submucosa mechanically separated and enzymatically digested. Mesenchymal stromal populations were isolated, expanded and characterized under conditions commonly used for MSC. The differentiation potential, trophic effect and immunomodulatory ability were investigated. Results We successfully isolated and extensively expanded populations showing the typical MSC profile: CD29+, CD44+, CD73+, CD105+ and CD166+, and CD14(-), CD34(-) and CD45(-). Intestinal mucosal (IM) MSC were also CD117+, while submucosal cultures (ISM MSC) showed CD34+ subsets. The cells differentiated toward osteogenic, adipogenic and angiogenic commitments. Intestinal-derived MSC were able to induce differentiation and organization of intestinal epithelial cells (Caco-2) in three-dimensional collagen cultures. Immunomodulatory activity was evidenced in co-cultures with normal heterologous phytohemagglutinin-stimulated peripheral blood mononuclear cells. Conclusions Multipotent MSC can be isolated from intestinal mucosal and submucosal tissues. IM MSC and ISM MSC are able to perform trophic and immunomodulatory functions. These findings could open a pathway for novel approaches to intestinal disease treatment.

Cooperation between research communities and software-development teams has led to the creation of novel software. The purpose of this paper is to show an alternative work method based on the usage of ImageJ (http://rsb.info.nih.gov/ij/), which can be effectively employed in solving common microscopic and ultrastructural image analysis tasks. As an open-source software, ImageJ provides the possibility to work in a free-development/sharing world. Its very "friendly" graphical user interface helps users to manage and edit biomedical images. The on-line material such as handbooks, wikis, and plugins leads users through various functions, giving clues about potential new applications. ImageJ is not only a morphometric analysis software, it is sufficiently flexible to be adapted to the numerous requirements tasked in the laboratories as routine as well as research demands. Examples include area measurements on selectively stained tissue components, cell count and area measurements at single cell level, immunohistochemical antigen quantification, and immunoelectron microscopy gold particle count.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10 6, 1.08±0.11 × 10 5, 1.03±0.10 × 10 4, 1.02±0.09 × 10 3, 1.04±0.10 × 10 2 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.Leukemia advance online publication, 27 February 2015; doi:10.1038/leu.2015.29.

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me 2 SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 °C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49 ± 16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of “CLL-biased” features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

Objectives and design To establish whether in diabetic patients with peripheral artery obstructive disease (PAOD) vasa vasorum (vv) neoangiogenesis is altered with increased arterial damage. Materials Thirty-three patients with PAOD and critical lower limb ischaemia, 22 with type II diabetes. Methods Immunohistochemistry for endothelial cell markers (CD34 and von Willebrand Factor); real-time reverse transcription polymerase chain reaction (RT-PCR) to quantify arterial wall expression of vascular endothelial growth factor (VEGF); enzyme-linked immunosorbent assay (ELISA) to assess blood VEGF; flow cytometry to detect circulating endothelial cells (CECs). Results Patients with PAOD and diabetes have a higher frequency (60% vs. 45%) of advanced atherosclerotic lesions and a significant reduction ( p = 0.0003) in CD34 + capillaries in the arterial media. Adventitial neoangiogenesis was increased equally (CD34 + and vWF + ) in all patients. Likewise, all patients have increased CEC and VEGF concentration in the blood as well as in-situ VEGF transcript expression. Conclusions Patients with PAOD have remarkable arterial damage despite increased in-situ and circulating expression of the pro-angiogenic VEGF; a dysfunctional vv angiogenesis was seen in diabetics which also showed a higher frequency of parietal damage; it is suggested that in diabetic arterial wall, injury is worsened by vv inability to finalise an effective VEGF-driven arterial wall neoangiogenesis.

Morphological and immunophenotypic characterization of cells grown on poly(l-lactic acid) (PLLA) electrospun scaffolds is usually performed using immunofluorescence and cryosections. However, these methods present practical limits; histological processing, on the other hand, is believed to lead to artifactual changes in the scaffold structure. Here the formalin-fixed paraffin-embedding (FFPE) procedure was tailored to process PLLA electrospun scaffolds grown with human umbilical vein endothelial cells. After 1 to 7 days of culture, the scaffolds were processed with the FFPE procedure. Using this protocol, not only cross sections but also "en face" sections were obtained. This made possible to perform the effective light microscopy analysis of cell morphology and to assess cell adhesion and penetration without considerable scaffold damage. The method was also suitable for immunohistochemical assays, such as proliferation (Ki67), extracellular matrix production (type IV collagen), survival (cleaved caspase-3), and immunophenotyping (KDR, CD44, vimentin, CD45); results were compared with those obtained using complementary techniques (scanning electron microscopy, Alamar Blue assay, and cryosections). The FFPE protocol can be safely applied to PLLA scaffolds and provides information that are essential to study the mechanisms of interaction between cells and PLLA fibers before their potential implantation in vivo.

Infection by human parvovirus B19 is widespread and can be associated with a wide range of different pathologies and clinical manifestations. We provide the first evidence of localization of an active parvovirus B19 infection in the intestinal mucosa and its association with a severe inflammatory bowel disease, characterized by duodenal villous atrophy with increased intraepithelial lymphocytes and inflammatory infiltrates in the colonic mucosa. Virus in the intestinal mucosa was detected in cells of the inflammatory infiltrate, identified as T lymphocytes and selectively localized in sites of active tissue degeneration.

Neovascularization can be improved using polymer scaffolds supporting endothelial progenitor cells (EPCs). The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured in a hyaluronan-based non-woven mesh (HYAFF-11). eEPCs were seeded on HYAFF-11 at the density of 1 x 10(6)/cm(2) and cultured with endothelial differentiating factors for 3 weeks. After 24 h, nearly 90% of EPCs were adherent. Cell viability, evaluated by methyltetrazolium test, was greater in HYAFF-11 than on the most commonly used fibronectin-coated dishes, even if a progressive decline in viability was observed starting from approximately the second week of culture. eEPCs easily migrated to and aggregated on the scaffold. Evidence of active protein synthesis and features of endothelial differentiation, including cellular transcytotic channels and micropinocytotic vesicles, was revealed using electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction analysis. eEPCs cultured in the scaffold also showed a certain angiogenic activity, as demonstrated by hepatocyte growth factor transcription and vascular endothelial growth factor secretion. In conclusion, eEPCs can migrate and adhere inside HYAFF-11, maintain their pre-endothelial phenotype, and express angiogenic factors, especially within the first week of growth. These results indicate that non-woven HYAFF-11 could be a promising candidate as a vehicle for eEPCs for regenerative medicine applications.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Human mesenchymal stromal (stem) cells (hMSCs) isolated from adult bone marrow (BM-hMSCs) as well as amnion (AM-hMSCs) and chorion (CM-hMSCs) term placenta leaves were studied by transmission electron microscopy (TEM) to investigate their ultrastructural basic phenotype. At flow cytometry, the isolated cells showed a homogeneous expression of markers commonly used to identify hMSCs, i.e., CD105, CD44, CD90, CD166, HLA-ABC positivities, and CD45, AC133, and HLA-DR negativities. However, TEM revealed subtle yet significant differences. BM-hMSCs had mesenchymal features with dilated cisternae of rough endoplasmic reticulum (rER) and peripheral collections of multiloculated clear blisters; this latter finding mostly representing complex foldings of the plasma membrane could be revelatory of the in situ cell arrangement in the niche microenvironment. Unlike BM-hMSCs, CM-hMSCs were more primitive and metabolically quiescent, their major features being the presence of rER stacks and large peripheral collections of unbound glycogen. AM-hMSCs showed a hybrid epithelial-mesenchymal ultrastructural phenotype; epithelial characters included non-intestinal-type surface microvilli, intracytoplasmic lumina lined with microvilli, and intercellular junctions; mesenchymal features included rER profiles, lipid droplets, and well-developed foci of contractile filaments with dense bodies. These features are consistent with the view that AM-hMSCs have a pluripotent potential. In conclusion, this study documents that ultrastructural differences exist among phenotypically similar hMSCs derived from human bone marrow and term placenta leaves; such differences could be revelatory of the hMSCs in vitro differentiation potential and may provide useful clues to attempt their in situ identification.

The clinical use of endothelial progenitor cells is hampered by difficulties in obtaining an adequate number of functional progenitors. This study aimed to establish whether human thoracic aortas harvested from healthy multiorgan donors can be a valuable source of angiogenic progenitors. Immunohistochemical tissue studies showed that two distinct cell populations with putative stem cell capabilities, one composed of CD34+ cells and the other of c-kit+ cells, are present in between the media and adventitia of human thoracic aortas. Ki-67+ cells with high growth potential were located in an area corresponding to the site of CD34+ and c-kit+ cell residence. We thus isolated cells (0.5 approximately 2.0 x 10(4) aortic progenitors per 25 cm2) which, upon culturing, coexpressed molecules of mesenchymal stromal cells (i.e., CD44+, CD90+, CD105+) and showed a transcript expression of stem cell markers (e.g., OCT4, c-kit, BCRP-1, Interleukin-6) and BMI-1. Cell expansion was adequate for use in a clinical setting. A subset of cultured cells acquired the phenotype of endothelial cells in the presence of vascular endothelial growth factor (e.g., increased expression of KDR and von Willebrand factor positivity), as documented by flow cytometry, immunofluorescence, electron microscopy, and reverse transcription-polymerase chain reaction assays. An in vitro angiogenesis test kit revealed that cells were able to form capillary-like structures within 6 hours of seeding. This study demonstrates that thoracic aortas from multiorgan donors yield mesenchymal stromal cells with the ability to differentiate in vitro into endothelial cells. These cells can be used for the creation of an allogenic bank of angiogenic progenitors, thus providing new options for restoring vascularization at ischemic sites. Disclosure of potential conflicts of interest is found at the end of this article.

We have developed a mixed ester of hyaluronan with butyric and retinoic acid (HBR) that acted as a novel cardiogenic/vasculogenic agent in human mesenchymal stem cells isolated from bone marrow, dental pulp, and fetal membranes of term placenta (FMhMSCs). HBR remarkably enhanced vascular endothelial growth factor (VEGF), KDR, and hepatocyte growth factor (HGF) gene expression and the secretion of the angiogenic, mitogenic, and antiapoptotic factors VEGF and HGF, priming stem cell differentiation into endothelial cells. HBR also increased the transcription of the cardiac lineage-promoting genes GATA-4 and Nkx-2.5 and the yield of cardiac markerexpressing cells. These responses were notably more pronounced in FMhMSCs. FMhMSC transplantation into infarcted rat hearts was associated with increased capillary density, normalization of left ventricular function, and significant decrease in scar tissue. Transplantation of HBR-preconditioned FMhM-SCs further enhanced capillary density and the yield of human vWF-expressing cells, additionally decreasing the infarct size. Some engrafted, HBR-pretreated FMhMSCs were also positive for connexin 43 and cardiac troponin I. Thus, the beneficial effects of HBR-exposed FMhMSCs may be mediated by a large supply of angiogenic and antiapoptotic factors, and FMhMSC differentiation into vascular cells. These findings may contribute to further development in cell therapy of heart failure.

This study was aimed to establish whether the cryopreservation procedure we currently use in clinics can modify arterial homograft antigenicity. To this purpose, we performed an immunohistochemical study on fresh and cryopreserved human arterial homografts to visualize the expression of HLA class I heavy and light chains "in situ" by using the HC-10 and Namb-1 monoclonal antibodies. Human femoral arteries and thoracic aortas were harvested from 18 heart-beating donors and sampled before and after cryopreservation. Arterial segments were frozen in liquid nitrogen vapors in a controlled rate freezing system. After thawing, samples were processed for routine immunohistochemistry. To standardize immunostaining, flow-cytometry indirect immunofluorescence analysis was performed on HUVEC; immunohistochemistry of human ovarian cortical vessels was performed as an additional positive control. Negative controls were performed by omitting tissue incubation with primary antibodies. HLA-class I antigens were markedly expressed by endothelial cells lining surface intima and adventitial vasa vasorum; a moderate expression was found in medial smooth muscle cells. Except for the surface unreactivity caused by loss of endothelium, results from cryopreserved arterial allografts were strictly comparable to those observed in fresh, unfrozen tissues. These results support the view that cryopreserved arterial allografts are immunogenic as their fresh counterparts; apart from smooth muscle cells which retained a moderate expression of HLA class I antigens following cryopreservation, our study suggests that the highly HC-10 positive endothelial cells we found to line the rich adventitial network of vasa vasorum are expected to be one of the major targets of the serological response in the recipient.