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BACKGROUND: In neonates, antibody-mediated destruction of neutrophils may occur as a consequence of auto- or isoimmune disorders. There are few studies on this topic, and particularly on neonatal alloimmune neutropenia (NAN). MATERIALS AND METHODS: We retrospectively evaluated the clinical and molecular/serological findings of 83 neutropenic infants referred to our Reference Laboratory for diagnostic evaluation of NAN, from 2008 to 2021. We also genotyped 260 Italian healthy subjects for the four principal human neutrophil antigens (HNA). RESULTS: The diagnosis of NAN was confirmed in 31 cases. The other 52 cases were autoimmune neutropenia (n=21), neutropenia caused by maternal neutrophil autoantibodies (n=8), neutropenia of non-immune origin (n=17), and cases in which a diagnosis could not be reached (n=6). The median age at neutropenia onset and absolute neutrophil count (ANC) were significantly lower in NAN than in non-NAN cases (1 vs 30 days, p

Objetivo: Expor os conceitos básicos sobre a depressão pós-parto e psicose puerperal, ressaltando a história natural da doença e possíveis consequências para o binômio mãe-criança. Revisão bibliográfica: Existem vários fatores de risco para a depressão pós-parto: história pessoal de depressão pré-natal, renda familiar baixa e má relação conjugal. No que se refere à psicose puerperal, tem-se como principais fatores de risco: história pessoal ou familiar de transtorno bipolar e psicose pós-parto em uma gestação anterior. O diagnóstico de depressão pós-parto pode acarretar algumas consequências para o bebê, como baixo peso, diarreia, cólicas, dor durante a rotina de vacinação e doença febril na criança. A psicose puerperal pode afetar diretamente na segurança da mãe e do bebê, visto que a mulher diagnosticada tem tendência ao suicídio, comportamento hostil e prejudicial com a criança. A abordagem acerca destas patologias precisa ser multidisciplinar. A intervenção farmacológica está reservada aos casos graves. Considerações finais: A depressão pós-parto e a psicose puerperal são patologias psiquiátricas que podem acometer mulheres no período do puerpério. Nesse período, ocorrem alterações tanto de caráter emocional como hormonal, potencializadas por diversos fatores. Resultados indicam que essas patologias podem levar a um padrão negativo de interação com o bebê.

Berzuini et al report the observation that nearly half of patients with COVID-19 tested at their blood center had a positive direct antiglobulin test (DAT). However, eluates did not react with any test cells but did react with red cells from other patients with COVID-19 that were DAT negative. This suggests that COVID-19 may modulate the red cell membrane and present novel antigenic epitopes.

Porta Valbona in Urbino: its representation between history and restorationThe Porta Valbona study is part of a complex project of conservation and valorisation of the defensive walls of Urbino that the research group, of the School of Conservation and Restoration of the University of Urbino Carlo Bo, has developed in recent years. Built in 1621 it is the most important gate of the city both because it is connected to Via Mazzini, one of the main streets of the historic centre, and for its spectacular architectural appearance created for the wedding of Prince Federico Ubaldo della Rovere with Princess Claudia de Medici. The two eagles, in limestone, placed at the sides of the door, date back to the mid-eighteenth century and are the work of the Rimini architect Giovan Francesco Buonamici. It is also the only Gate of Urbino which has a monumental facing facing outwards, or towards Piazza del Mercatale. Despite having undergone several restorations and consolidations over the centuries, it has not been modified in its original appearance. Porta Valbona, together with the city walls, represents a real urban palimpsest, an exceptional case of sedimentation and stratification which, despite the events, still allows us to reconstruct its historical events. The applied design method was based on the following analyzes: a) urban analysis: knowledge of the characteristics and urban potential of the door; b) historical analysis: knowledge of the historical evolution and of the specific qualities of the door; c) geometric analysis: metric and architectural survey; d) material analysis, study of materials and forms of deterioration; e) structural analysis: identification of the morphological and constructive organization from the structural point of view. All the large amount of information obtained from the analysis was managed thanks to the use of GIS systems. Thus it was possible to identify the shape and character of the monument and its testimonial, constructive and architectural values ​​were recognized. On the basis of an internal analysis of the cultural asset and an external analysis of the context in which it is located, it was possible to define the strengths, weaknesses, opportunities and threats.

BackgroundSevere acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown.MethodsWe evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis and the presence of association with inflammatory biomarkers and 28-days mortality.ResultsLymphocytopenia was present in 51 of 63 (80.9%) patients. This reduction was mirrored also on CD8+ lymphocytes (128 cells/μL), natural killer cells (67 cells/μL) and natural killer T cells (31 cells/μL). Monocytes were preserved in total number but displayed a subpopulation composed mainly of cells with a reduced expression of both CD14 and HLA-DR. A direct correlation was found between serum values of IL-6 and the frequency of Th2 lymphocytes (R=0.17; p=0.04) but not with the monocytes count (R=0.01; p=0.60). Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (p=0.028) and CD4+ cells (p=0.042) and higher mean percentages of CD8+/CD38+/HLA-DR+ lymphocytes (p=0.026).ConclusionsThe early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2 lymphocytes and less immunocompetent monocytes, which include atypical mononuclear cells.eTOC-At diagnosis patients with COVID-19 have lymphocytopenia-Monocytes with both normal or altered scatter properties display a reduced expression of CD14 and HLA-DR in most of COVID-19 patients-Patients who die in the 28 days from admission have lower values of CD3+ and CD4+ cells and higher percentages of activated CTL cells compared to those who survive

The castle Colonna of Arnara: from the restoration of the monument to the enhancement of the ancient village and its territoryThe castle of Arnara, is located in Ciociaria in southern Lazio, in what was once the feud of the Conti of Ceccano. For the territory it is the most important monumental complex of medieval architecture. In fact, despite its small size, it is one of the few examples of fortified architecture still original in its essential lines. This castle represents an exceptional case of sedimentation and stratification, which even today, from its reading, allows us to reconstruct both its historical events and those related to the urban development of the adjacent historic village. Moreover it can be considered of priority importance also from the urbanistic point of view because it represents the first historical nucleus of Arnara. Unfortunately, today, the castle is in a bad state of conservation. With the passing of time its conditions have become more and more aggravated also because of the complex events linked to the various passages of property, of private and public nature, which have strongly influenced the management of the monument. The study of the castle of Arnara is part of the national and international debate that considers participation a key factor for the sustainable enhancement of the cultural heritage, because it promotes greater awareness of its social and economic value. The objective of the research work is to develop, taking the castle as a case study, a new approach that sees cultural heritage (tangible and intangible) as an irreplaceable heritage of knowledge and as a precious resource for economic growth, employment and social cohesion; therefore a cultural heritage seen as a driving force. In the specific case of Colonna Castle, attention has been focused on the importance of being able to activate virtuous circles around this very important place of culture, enhancing its role as a centre of knowledge and incubator of creativity and social innovation.

The presented study describes how to document in a dynamic way a complex and full of historical information architecture with a flexible system. Thanks to the collaboration between various professional figures and the integration of various digital systems, it has been possible to create an open documentary model able to satisfy the need to manage complex information.Our case study is the Valbona gate, the main entrance of the city of Urbino. The project presents two different phases: a first study is characterised by the setting up of a Datawarehouse concerning state of conservation and restoration managed with a GIS system; the second part is focused on a new type of documentation, developed with an online software, CDR - Conservation Digital Report.Thanks to the capacity of organising all data into a single Geodatabase system, the information relating to the conservation status and to deterioration, restoration interventions carried out.The result is a geodatabase linked with conservative sheets expressly scheduled. In this way the documentation is completed by a system which allows a simple updating of information and its implementation, if necessary.

BACKGROUND Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. STUDY DESIGN AND METHODS We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). RESULTS Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45dimGly-AdimCD71bright cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. CONCLUSION MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Small extracellular vesicles (EVs) are able to pass from the central nervous system (CNS) into peripheral blood and contain molecule markers of their parental origin. The aim of our study was to isolate and characterize total and neural-derived small EVs (NDEVs) and their micro RNA (miRNA) cargo in Alzheimer&rsquo, s disease (AD) patients. Small NDEVs were isolated from plasma in a population consisting of 40 AD patients and 40 healthy subjects (CTRLs) using high throughput Advanced TaqMan miRNA OpenArrays&reg, which enables the simultaneous determination of 754 miRNAs. MiR-23a-3p, miR-223-3p, miR-100-3p and miR-190-5p showed a significant dysregulation in small NDEVs from AD patients as compared with controls (1.16 &plusmn, 0.49 versus 7.54 &plusmn, 2.5, p = 0.026, 9.32 &plusmn, 2.27 versus 0.66 &plusmn, 0.18, p &lt, 0.0001, 0.069 &plusmn, 0.01 versus 0.5 &plusmn, 0.1, p &lt, 0.0001 and 2.9 &plusmn, 1.2 versus 1.93 &plusmn, 0.9, p &lt, 0.05, respectively). A further validation analysis confirmed that miR-23a-3p, miR-223-3p and miR-190a-5p levels in small NDEVs from AD patients were significantly upregulated as compared with controls (p = 0.008, p = 0.016, p = 0.003, respectively) whereas miR-100-3p levels were significantly downregulated (p = 0.008). This is the first study that carries out the comparison between total plasma small EV population and NDEVs, demonstrating the presence of a specific AD NDEV miRNA signature.

BACKGROUND Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS RBC antibody–bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p

Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial.We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts).Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly.MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Ex vivo expansion of hematopoietic stem cells has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. Recently, we demonstrated the existence of a circulating myogenic progenitor expressing the CD133 antigen. The relative inability of circulating CD133+ stem cells to reproduce themselves ex vivo imposes substantial limitations on their use for clinical applications in muscular dystrophies. Here we report that the use of cluster-assembled nanostructured titanium dioxide (ns-TiO 2 ) substrates, in combination with cytokine enriched medium, enables high-level expansion of circulating CD133+ stem cells in vitro. Furthermore, we demonstrate that expanded circulating CD133+ stem cells retain their in vitro capacity to differentiate into myogenic cells. The exploitation of cluster-assembled ns-TiO 2 substrates for the expansion of CD133+ stem cells in vitro could therefore make the clinical application of these stem cells for the treatment of muscle diseases practical.

To evaluate the fully automated FACSCanto software, we compared lymphocyte subpopulation counts obtained using three-color FACSCalibur-CELLQuest and six-color FACSCanto-FACSCanto software techniques. High correlations were observed between data obtained with these techniques. Our study indicated that FACSCanto clinical software is accurate and sensitive in single-platform lymphocyte immunophenotyping.

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS.RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect.A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and β-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency.RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.

Accurate estimation of haematopoietic stem cell (HSC) counts by flow cytometry may be difficult in laboratories in which sophisticated equipment and staff with specific expertise are not available. Affordable flow cytometers that can perform basic functions may help to overcome these difficulties. In this study we compared HSC and leucocyte counts determined by volumetric and bead-based protocols performed with the small, low-cost Accuri(®) C6, with those obtained with two gold-standard instruments, the four-colour FACSCalibur(®) and the eight-colour FACSCantoII(®), our reference flow cytometers.With the three cytometers we tested, in parallel, 111 consecutive samples from cord blood, peripheral blood from patients with myelofibrosis and myeloproliferative syndromes, fresh and thawed HSC collected by apheresis and bone marrow products. The findings were compared with one-way ANOVA, Bland-Altman analysis and linear regression.The results of HSC and leucocyte enumeration by the three devices were strongly correlated (r(2)0.99; p0.0001). ANOVA performed on different subgroups of samples did not reveal significant differences between HSC count determined by the C6 bead-based and reference flow cytometers in any of the subgroups. Regarding the C6 volumetric protocol, a statistically significant difference was observed only in the cord blood subgroup. Time for instrument set-up, calibration and analysis was slightly longer with Accuri(®) C6 (40 min) than with FACSCantoII(®) (30 min).Accuri(®) C6 is a reliable instrument for HSC enumeration in fresh samples, using both volumetric and bead-based approaches, although the volumetric protocol on cord blood samples needs to be improved. The Accuri(®) C6 is easy to use, does not require profound knowledge of flow cytometry and could be employed in an urgent setting. Its performance may be improved by more efficient calibration and shorter analysis time.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality. Antibodies against human leucocyte antigens (HLA) and human neutrophil antigens (HNA) are often detected in the implicated donors. We investigated the incidence and aetiology of TRALI in Lombardy. Moreover, we determined the rate of HLA and HNA alloimmunisation and the HNA genotype in a cohort of local blood donors.During a 2-year observational study in eight blood transfusion services, suspected TRALI cases were collected and characterised by means of HLA and HNA antibody screening of implicated donors, donor/recipient cross-matching and HLA/HNA molecular typing. In addition, 406 Italian donors were evaluated for alloimmunisation and in 102 of them HNA gene frequencies were determined.Eleven cases were referred to the central laboratory, of whom three were diagnosed as having TRALI, seven as having possible TRALI and one as having transfusion-associated circulatory overload. Seven TRALI cases were immune-mediated whereas in three we did not find either alloantibodies in implicated donors or a positive reaction in the cross-match. The most frequently implicated blood component was red blood cells (in 5 males and in 1 female), whereas four cases of TRALI were associated with transfusion of fresh-frozen plasma (in 3 females and in 1 male). The frequency of reported TRALI/possible TRALI cases was 1:82,000 for red blood cells and 1:22,500 for fresh-frozen plasma. No cases were observed for platelets. Overall, the frequency of HLA or HNA alloimmunisation in blood donors was 29% for females and 7% for males. The latter could be related, at least in part, to natural antibodies. HNA gene frequencies showed that HNA-1b is more frequent than HNA-1a in our sample of donors.The recently adopted national policy to prevent TRALI, i.e. using only plasma donated by males, would have had a positive impact in our setting.

Background and Aims: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC). Methods: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ-/- mice. Results: The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. Conclusions: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution. © 2011 Colombo et al.

The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.

s / Digestive and Liver Disease 47S (2015) e19–e42 e37 Conclusions NAFLD-HCC rises in the absence of cirrhosis in about 50% of the cases, and it is detected at a later tumor stage than in HCV-infected patients. Future research should identify patients with NAFLD who require surveillance in order to offer them the most timely and effective treatment. http://dx.doi.org/10.1016/j.dld.2015.01.081

Background and aim Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. Methods Hepatocytes were isolated from the liver within 15 min after death (t = 15 min) and after 4, 8 and 12 h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. Results The number of cells per gram of tissue was similar at 15 min, 4 and 8 h, while it was significantly decreased at 12 h. About 0.2 × 106 viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8 h of ischaemia per gram of tissue. Conclusions Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.

The antiviral drug ribavirin (RBV) is widely used in combination with pegylated interferon (PegIFN) for the treatment of hepatitis C virus (HCV) infection. A major side effect of RBV is a reversible anemia. The mechanism underlying anemia in RBV treated patients remains speculative although hemolysis was suggested. To get further insights, we evaluated in vitro the effect of RBV and PegIFN on proliferation and erythroid differentiation of CD34 pheripheral blood cells. The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. RBV (0.5mM and 0.1mM) and PegIFN (100U and 1000U) were added alone or in combination at different days of culture: precisely at day 0 corresponding to CD34 stage and at day 7 corresponding to proerythroblast stage. Cells growth and viability were evaluated by tripan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glicophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific (γ-globin and GATA2) gene expression analysis with real-time PCR. RBV added at day 0 of culture drastically inhibited cell growth and differentiation (RBV 0.1mM: 8.5-fold reduction in cells growth and 1.6-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 12-fold reduction in cells growth and 30-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of RBV at day 7 of culture had a milder inhibitory effect (RBV 0.1mM: 2.4-fold reduction in cells growth and 0.7-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 3.6-fold reduction in cells growth and 2.3-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of PegIFN both at day 0 and day 7 of culture delayed erythroid differentiation, with reduction of GPA+ cells, decrease of orthocromatic erythroblasts and increase of more undifferentiated polychromatophilic erythroblasts, and increased expression of primitive erythropoietic genes (tab 1). The combined addition of PegIFN and RBV both at day 0 and day 7 of culture affected cell growth and erythroid differentiation less than RBV alone, with increase of GPA+ cells, decrease of basophilic erythroblasts and increase of more differentiated orthocromatic erythroblasts, and decreased expression of γ-globin gene. However cells treated with the combination of PegIFN and RBV were more undifferentiated than cells treated with PegIFN alone. All these observations confirmed the inhibitory effect of PegIFN on erythroid differentiation and strongly suggested an inhibitory action of RBV on cell growth, particularly on undifferentiated stem cells. These observations provide a model for the pathophysiology of anemia observed with ribavirin treatment. Tab. 1 % GPA+ cells % polychromatophilic e. % orthocromatic e. γ-gene GATA2 gene Untreated cells 61 17 55 1 1 PegIFN 100U 50 37 56 1,75 1,16 PegIFN 1000U 47 57 23 2,39 1,7

Background Various prognostic serum and cellular markers have been identified for many diseases, such as cardiovascular diseases and tumor pathologies. Here we assessed whether the levels of certain stem cells may predict the progression of Duchenne muscular dystrophy (DMD). Methods and Findings The levels of several subpopulations of circulating stem cells expressing the CD133 antigen were determined by flow cytometry in 70 DMD patients. The correlation between the levels and clinical status was assessed by statistical analysis. The median (±SD) age of the population was 10.66±3.81 (range 3 to 20 years). The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD patients compared to healthy controls (mean±standard deviation: 17.38±1.38 vs. 11.0±1.70; P = 0.03) with a tendency towards decreased levels in older patients. Moreover, the levels of this subpopulation of cells correlated with the clinical condition. In a subgroup of 19 DMD patients after 24 months of follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be associated with a phenotype characterised by slower disease progression. The circulating CD133+CXCR4+CD34- cells in patients from different ages did not exhibit significant differences in their myogenic and endothelial in vitro differentiation capacity. Conclusions Our results suggest that levels of CD133+CXCR4+CD34- could function as a new prognostic clinical marker for the progression of DMD.

Chronic neutropenia (CN) is defined as a blood absolute neutrophil count (NC) below 1500/ml, lasting for at least 6 months. We report the results of serological screening for anti-neutrophil antibodies, performed in our laboratory during 2002–2005, in 100 consecutive children affected from CN and followed in 4 Italian pediatric centers. The patients, 65 males (m) and 35 females (f), had a median age of 18 months (range: 1–180). Both IgG bound to patient neutrophils (CD16+ cells) and the patient serum reactivity against healthy donor granulocytes (Direct and Indirect Granulocyte-Immunofluorescence Test, D-GIFT and I-GIFT) were assessed by flow-cytometry. The results are shown in the table, grouped according to the pattern of reactivity observed with D- and I-GIFT: A NC above 1500/ μl on the day of serological screening was found in 5.5%, 22.4%, 0% and 35% of patients in groups 1, 2, 3 and 4, respectively. Secondary CN, usually in association with other autoimmune disorders or malignant diseases, was reported in 5.5%, 10.3%, 0% and 50% of patients in groups 1, 2, 3 and 4, respectively. Infections were present in 72%, 79%, 25% and 8% of patients in groups 1, 2, 3 and 4, respectively. In group 2, 50 of the 58 patients (86%), who didn’t show anti-neutrophil antibodies in their sera, presented neutropenia with concomitant infections. In these patients only D-GIFT was able to detect neutrophil antibodies. Patients testing positive for I-GIFT had more severe neutropenia. Patients negative by both techniques were mostly found to have non-immunomediated neutropenia at clinical follow up. Although many laboratories perform only an indirect test to detect antineutrophil antibodies, our results suggest that direct test is more sensitive in identifying immunomediated chronic neutropenia. table group GIFT no. of patients m/f NC (cells/μl)* WBC (x102/μl)* * Mean ± SD 1 D+ I+ 18 15/3 757 ± 520 7.58 ± 3.1 2 D+ I− 58 36/22 968 ± 818 7.29 ± 5.3 3 D– I+ 4 3/1 525 ± 369 6.71 ± 2.3 4 D- I− 20 11/9 1693 ± 1510 8.28 ± 3.2

Replication of mature hepatocytes can ensure hepatic tissue renewal in normal liver. However, in certain physiopathologic conditions, cells with stem-like properties can proliferate and/or be recruited from extra-hepatic compartments. For a better understanding of the mechanisms related to liver regeneration, more data on the morphological and differentiative profiles of liver stem cells are needed. This study was aimed at the immunophenotypic characterization of circulating and intrahepatic stem cells in organ donors and in patients with chronic liver disease. We performed a four-colors flow cytometric evaluation of liver mononuclear cell suspensions (L-MNC) obtained after enzymatic digestion of liver biopsy specimens from 8 patients and 6 multi-organ donors. Paired peripheral blood mononuclear cells (PB-MNC) from the same subjects were also examined. L-MNC and PB-MNC were incubated with antibodies to the following markers: CD34, CD133, CD45, CD117 (c-kit) and CXCR4 (Stromal derived factor-1 receptor). Cell viability was performed using 7-aminoActinomycin D to exclude dead cells. In patients, the stem cell marker CD34 was detected on the surface of 0.6±0.9% of the L-MNC, a proportion similar to that observed in donors (0.6±0.5%). In both patients and donors, the majority of CD34+ cells co-expressed CD45 (86±9% and 60±31% respectively). The subset of CD34+/CD45- cells expressing c-kit was increased in L-MNC from patients as compared to donors (13.8±12% vs.1.5±1% of the total CD34+ cells; p Flow cytometry analysis of intrahepatic CD34+ stem cells allows the identification of at least two phenotipically distinct populations. The first population co-expresses c-kit, is increased in the presence of chronic hepatocellular damage and is likely resident in the liver, being rarely detectable in peripheral blood. The second population resembles the classical hematopoietic stem cell and harbors CD133 and the chemokine receptor CXCR4 for trafficking to the liver in response to liver injury.