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Donor-derived infections (DDIs) caused by carbapenem-resistant gram-negative bacteria (CR-GNB) in solid organ transplant recipients are potentially life-threatening. In this prospective study, we evaluated the incidence, factors associated with transmission, and the outcome of recipients with unexpected CR-GNB DDIs after the implementation of our local active surveillance system (LASS). LASS provides for early detection of unexpected donor CR-GNB infections, prophylaxis of recipients at high risk, and early diagnosis and treatment of DDIs. Whole genome sequencing confirmed DDI. Among 791 recipients, 38 (4.8%) were at high risk of unexpected CR-GNB DDI: 25 for carbapenem-resistant Enterobacterales(CRE) and 13 for carbapenem-resistant Acinetobacter baumannii(CRAB). Transmission did not occur in 27 (71%) cases, whereas DDIs occurred in 9 of 25 of CRE and 2 of 13 of CRAB cases. Incidence of CR-GNB DDI was 1.4%. Recipients of organs with CR-GNB–positive preservation fluid and liver recipients from a donor with CRE infection were at the highest risk of DDI. There was no difference in length of hospital stay or survival in patients with and without CR-GNB DDI. Our LASS contains transmission and mitigates the negative impacts of CR-GNB DDI. Under well-defined conditions, organs from donors with CR-GNB may be considered after a thorough evaluation of the risk/benefit profile.

In this single center study, ex situ liver perfusates (LP) from deceased brain liver donors are collected and cytofluorimetric LP analysis is performed to obtain the lymphocyte phenotype and identify the potential clinical impact of natural killer cell fraction variety on the probability to experience an acute cellular rejection event.

We isolated a population of proliferating cells from cultured human fetal hepatocytes of 16–22 weeks gestational age. The cells shared a similar phenotype to that of mesenchymal stromal cells (MSCs) according to the International Society for Cellular Therapy (ISCT), including plastic adherence,antigen expression profile, and in vitromultilineage differentiation potential. Fetal liver (FL)-MSCs expressed the albumin gene, and harbored a subpopulation of CK18+cells (20–40%), which defined their hepatic origin. However, when subjected to in vitrohepatic differentiation, FL-MSCs did not acquire significant liver functions. Quantitative analysis of conditioned medium (CM) collected from cultured cells revealed the presence of growth factors and chemokines with potential liver regenerative properties, the most relevant of which (concentration ≥3000 pg/ml) were SDF-1 alpha, IL-6, MCP-1, IL-8, MIP-1 beta, VEGF-A, Gro-alpha, and HGF. Culturing of FL-MSCs as spheroids significantly enhanced the secretion of HGF and bFGF (approximately 5-fold) compared with culture monolayers. Moreover, CM assessed in vitroinduced capillary-like organization and migration of human umbilical vein endothelial cells (HUVECs) and fibroblasts as target cells. Interestingly, exosomes isolated from CM induced similar cellular responses in vitrowith high efficiency and in a dose-dependent manner.

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer.

Fetal hepatocytes have a high regenerative capacity. The aim of the study was to assess treatment safety and clinical efficacy of human fetal liver cell transplantation through splenic artery infusion. Patients with endstage chronic liver disease on the waiting list for liver transplantation were enrolled. A retrospectively selected contemporary matched-pair group served as control. Nonsorted raw fetal liver cell preparations were isolated from therapeutically aborted fetuses. The end points of the study were safety and improvement of the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores. Nine patients received a total of 13 intrasplenic infusions and were compared with 16 patients on standard therapy. There were no side effects related to the infusion procedure. At the end of follow-up, the MELD score (mean ± SD) in the treatment group remained stable from baseline (16.0 ± 2.9) to the last observation (15.7 ± 3.8), while it increased in the control group from 15.3 ± 2.5 to 19 ± 5.7 (p= 0.0437). The Child-Pugh score (mean ± SD) dropped from 10.1 ± 1.5 to 9.1 ± 1.4 in the treatment group and increased from 10.0 ± 1.2 to 11.1 ± 1.6 in the control group (p= 0.0076). All treated patients with history of recurrent portosystemic encephalopathy (PSE) had no further episodes during 1-year follow-up. No improvement was observed in the control group patients with PSE at study inclusion. Treatment was considered a failure in six of the nine patients (three deaths not liver related, one liver transplant, two MELD score increases) compared with 14 of the 16 patients in the control group (six deaths, five of which were caused by liver failure, four liver transplants, and four MELD score increases). Intrasplenic fetal liver cell infusion is a safe and well-tolerated procedure in patients with end-stage chronic liver disease. A positive effect on clinical scores and on encephalopathy emerged from this preliminary study.

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

We report that cells from human fetal dermis, termed here multipotent fetal dermal cells, can be isolated with high efficiency by using a nonenzymatic, cell outgrowth method. The resulting cell population was consistent with the definition of mesenchymal stromal cells by the International Society for Cellular Therapy. As multipotent fetal dermal cells proliferate extensively, with no loss of multilineage differentiation potential up to passage 25, they may be an ideal source for cell therapy to repair damaged tissues and organs. Multipotent fetal dermal cells were not recognized as targets by T lymphocytes in vitro, thus supporting their feasibility for allogenic transplantation. Moreover, the expansion protocol did not affect the normal phenotype and karyotype of cells. When compared with adult dermal cells, fetal cells displayed several advantages, including a greater cellular yield after isolation, the ability to proliferate longer, and the retention of differentiation potential. Interestingly, multipotent fetal dermal cells expressed the pluripotency marker SSEA4 (90.56 ± 3.15% fetal vs. 10.5 ± 8.5% adult) and coexpressed mesenchymal and epithelial markers (>80% CD90+/CK18+cells), coexpression lacking in the adult counterparts isolated under the same conditions. Multipotent fetal dermal cells were able to form capillary structures, as well as differentiate into a simple epithelium in vitro, indicating skin regeneration capabilities.

ABSTRACTWe describe two cases of bacteremic infections caused by a multidrug-resistant Klebsiella pneumoniaeisolate producing the OXA-48 carbapenemase that occurred in two solid organ transplant (liver and kidney) recipients, which was apparently transmitted with the allografts. This finding underscores the risk of donor-derived infections by multidrug-resistant Gram-negative pathogens in solid organ transplant recipients and emphasizes the need for rapid screening of organ donors for carriage of similar pathogens.

We examine the dynamics of patient-sharing relations within an Italian regional community of 35 hospitals serving approximately 1,300,000 people. We test whether interorganizational relations provide individual patients access to higher quality providers of care.

Innovation is crucial for competitive advantage of firms. The innovation model adopted by open source software projects has therefore attracted the attention of several managers. Consequently, private firms are increasingly supporting open source communities. However, the heterogeneous results obtained by open source software projects call for a deeper understanding of the organisational factors sustaining the innovativeness of these communities. We contribute to fill this gap through a case study that investigates which knowledge networks connecting developers inside open source communities support radical and incremental innovation. Our findings show that two distinguishable network configurations are associated with the two types of innovation. Particularly the presence of a single core of knowledge interaction involving the developers actually implementing the innovative code appears to be a requirement for radical innovation.

A discrepancy exists between the emphasis posed by practitioners on decentralized and non-hierarchical communication in Free Libre/Open Source Software (FLOSS) communities and empirical evidence of their hierarchical structure. To explain this paradox, it is hypothesized firstly that in FLOSS communities local sub-groups exist and are less hierarchical, more decentralized than the whole social network. Secondly, it is hypothesized that the bulk of communication exchanges taking place in the community happens inside local sub-groups formed by the most active community members. The recollection that practitioners have of FLOSS communities to which they participate would then be influenced by the position that they occupy inside those sub-groups. A measure of structural cohesion based on network node connectivity is proposed as an effective method to test whether FLOSS communication networks can be decomposed in nested hierarchies of progressively less centralized sub-groups. The recently introduced measure of weighted rich-club effect is adopted to test for the tendency of the most active community members to control communication by interacting more intensely with each other than with other members of the network. Results from a case study that are consistent with the hypotheses are presented and discussed.

Biomolecule-targeted imaging represents one of the most difficult challenges in medicine. Nanoerythrosomes (NERs) are nanovesicles obtained after lysis of red blood cells, and they are promising tools for drug delivery and imaging. In this work, a formulation based on NERs functionalized with 7-amino-3-methylcoumarin via cross-linking was tested on rat INS-1E and mouse MIN6 β-cells and endothelial MSI cell lines. First, the morphology, size, ζ-potentials, and spectroscopic properties of the aggregates were investigated, highlighting that the functionalization did not significantly affect the nanoparticles’ physicochemical features. In vitro, the nanoparticles did not significantly affect the proliferation and function of INS-1E and MIN6 β-cells at different concentrations. Only at the highest concentration tested on the MSI cell line, the formulation inhibited proliferation. Furthermore, NER aggregates were not internalized in both INS-1E and MIN6 cell lines, while a diffuse fluorescence was noticed in the cytosol of the MSI cell line at the highest concentrations. These findings proved that NER formulations might represent a new nanotool for β-cell imaging as a part of a strategy aimed to prevent any intracellular accumulation, thus reducing/avoiding side effects.

In experimental extracapillary glomerulonephritis (EG) podocytes migrate, proliferate and change phenotype, and play a pivotal role in crescent formation. Hepatocyte Growth Factor (HGF) is an injury-induced effector of tissue repair that causes cell migration, growth and transdifferentiation via its receptor Met.

Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with β-d-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN.

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitrowhether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG4, plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-α, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation which has been implicated in rejection.The interaction of anti--galactosyl natural antibodies (anti- gal Abs) with endothelial cells is the initial step for the development of xenograft rejection. In our study, we stimulated porcine aorthic endothelial cells (PAEC) with anti- gal IgG to investigate the synthesis of PAF from PAEC and its biological consequences.

We have reported that bcl-2 is expressed in normal human thyroid epithelium and that its expression is down-regulated in undifferentiated thyroid tumors. Production of IL-6 was concomitantly down-regulated in these forms. Based on these observations, we analyzed whether insertion of bcl-2 would reverse the highly malignant phenotype of a thyroid cell line (ARO) derived from an undifferentiated carcinoma. This cell line fails to produce Bcl-2 and IL-6. By infection with a bcl-2 retroviral vector, ARO cells expressing bcl-2 (ARObcl-2) were obtained. Compared with parental cells, expression of bcl-2 was associated with enhancement of growth potential (DNA synthesis, in vitro proliferation rate, anchorage-independent growth in semi-solid media). Chemotaxis and invasive potential in Boyden chambers were also increased. bcl-2-expressing cells showed a reduced response to apoptotic stimuli (low-serum conditions or anti-neoplastic drugs). Large branched colonies were formed in Matrigel from ARObcl-2 cells but not from parental cells. Finally, ARObcl-2 cells showed a decreased latency of tumor appearance when injected into immunodeficient mice. Potentiation of the malignant phenotype of ARO cells by bcl-2 was not ascribed to altered expression of (i) cytokine/growth factors (IL-4, IL-6, IL-8, IL-10, IL-12, TGF-α, TGF-β), (ii) thyroid-specific transcripts (TG, TPO, TSH-R, PIGF, PAX-8) or (iii) genes influencing tumor aggressiveness [VEGF, HMGI (Y), HMGI-C]. Our data indicate that bcl-2 potentiates the malignant phenotype of ARO cells not only by limiting the response to apoptotic stimuli but also by enhancing proliferation and tumor aggressiveness. Int. J. Cancer81:956–962, 1999. © 1999 Wiley-Liss, Inc.

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of β1 integrin and the down-regulation of αvβ3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells.

HIV-1 Tat protein has been shown to induce chemotaxis and recruitment of monocytes. In the present study, we evaluated whether HIV-1 Tat protein was able to induce the synthesis of platelet-activating factor (PAF), which is a potent mediator of cell motility, and whether the synthesis of PAF was instrumental in triggering Tat-induced monocyte chemotaxis. The results obtained indicate that Tat, but not gp120 and gp41, induced a time-dependent synthesis of PAF from monocytes at concentration as low as 0.1 ng/ml. As inferred by the inhibitory effect of anti-Flt-1 antibody and by the desensitization of monocytes following preincubation with vascular endothelial growth factor, the synthesis of PAF by monocytes stimulated with Tat was induced by activation of vascular endothelial growth factor receptor 1. Moreover, the Tat-induced chemotaxis of monocytes was abrogated both by WEB 2170 and by CV 3988, two chemically unrelated PAF receptor antagonists, suggesting that the synthesized PAF modulates the chemotactic response of monocytes to Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in the migratory response of monocytes.

We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379-1384, 1996), is presumably operational in patients during infections.

Fas–Fas ligand interaction is thought to be a crucial mechanism in controlling lymphocyte expansion by inducing lymphocyte apoptosis. However, Fas is also broadly expressed on nonlymphoid cells, where its function in vivo remains to be determined. In this study, we describe the development of inflammatory angiogenesis induced by agonistic anti-Fas mAb Jo2 in a murine model where Matrigel is used as a vehicle for the delivery of mediators. The subcutaneous implants in mice of Matrigel containing mAb Jo2 became rapidly infiltrated by endothelial cells and by scattered monocytes and macrophages. After formation and canalization of new vessels, marked intravascular accumulation and extravasation of neutrophils were observed. Several mast cells were also detected in the inflammatory infiltrate. The phenomenon was dose and time dependent and required the presence of heparin. The dependency on activation of Fas is suggested by the observation that the inflammatory angiogenesis was restricted to the agonistic anti-Fas mAb and it was absent in lpr Fas-mutant mice. Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement. These findings suggest a role for Fas–Fas ligand interaction in promoting local angiogenesis and inflammation.

In vitro experiments indicate that components of the host present in body fluids may prevent the attachment of human immunodeficiency virus type 1 (HIV‐1) to target cells. Fibronectin (Fn), a dimeric 440‐kDa extracellular matrix adhesion protein, is secreted by mesenchymal cells and assembled into insoluble matrices. Fn exerts important effects on cell growth and differentiation through a number of discrete functional domains. Several microorganisms are known to bind Fn. We show that, under physiological conditions, HIV‐1 gp120 and gp160 are capable of binding plasma and cellular Fn as well as laminin and vitronectin. Experiments were set up to analyze in detail the binding of HIV gp120 and gp160 to Fn. The gp120 and gp160 specifically recognize the C‐terminal heparin‐binding domain of Fn (Fn‐CTHBD) with a calculated KDof 2.8 × 10−7M for gp160. Binding of gp160 to Fn‐CTHBD is a saturable and specific process that is blocked by antibodies to Fn‐CTHBD and by heparin and is inhibited to a minor extent by heparan sulfate and dextran sulfate. These observations suggest that gp120/160 specifically recognize the III15repeat within Fn‐CTHBD. Intact Fn and Fn‐CTHBD strongly inhibit the interaction of gp120/160 with soluble CD4 and, under low serum conditions, are capable of neutralizing the infectivity of HIV‐1 for CD4‐positive T cells. Thus, Fn that is present in plasma and mucinous secretions may well affect HIV infectivity and virus distribution in vivo. J. Med. Virol. 54:44–53, 1998.© 1998 Wiley‐Liss, Inc.