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The spread of multidrug-resistant Gram-negative bacterial infections is a significant issue for worldwide public health. Gram-negative organisms regularly develop resistance to antibiotics, especially to β-lactam antimicrobials, which can drastically restrict the number of therapies. A third-generation cephalosporin and the non-β-lactam β-lactamase inhibitor avibactam, which exhibits broad-spectrum β-lactamase inhibition in vitro , are combined to form ceftazidime-avibactam (CAZ-AVI). In this narrative review, we summarize data on pharmacokinetic (PK) parameters for CAZ-AVI in both animal and human models of pneumonia, as well as in healthy individuals. We assessed current literature performing an extensive search of the literature, using as search words 'CAZ-AVI', 'pharmacokinetics', 'pneumonia', 'lung', and 'epithelial lining fluid'. Overall, lung exposure studies of CAZ-AVI revealed that the epithelial lining fluid penetration ranges between 30% and 35% of plasma concentration. Despite the fair lung penetration of CAZ-AVI, this antimicrobial agent has a pivotal role in managing patients with multi-drug resistant Gram-negative pneumonia, however further studies are needed to better assess its PK profile.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue., (Copyright © 2024. Published by Elsevier Inc.)

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging., (Copyright © 2024. Published by Elsevier Inc.)

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Single-cell (SC) gene expression analysis is crucial to dissect the complex cellular heterogeneity of solid tumors, which is one of the main obstacles for the development of effective cancer treatments. Such tumors typically contain a mixture of cells with aberrant genomic and transcriptomic profiles affecting specific sub-populations that might have a pivotal role in cancer progression, whose identification eludes bulk RNA-sequencing approaches. We present scMuffin, an R package that enables the characterization of cell identity in solid tumors on the basis of a various and complementary analyses on SC gene expression data., Results: scMuffin provides a series of functions to calculate qualitative and quantitative scores, such as: expression of marker sets for normal and tumor conditions, pathway activity, cell state trajectories, Copy Number Variations, transcriptional complexity and proliferation state. Thus, scMuffin facilitates the combination of various evidences that can be used to distinguish normal and tumoral cells, define cell identities, cluster cells in different ways, link genomic aberrations to phenotypes and identify subtle differences between cell subtypes or cell states. We analysed public SC expression datasets of human high-grade gliomas as a proof-of-concept to show the value of scMuffin and illustrate its user interface. Nevertheless, these analyses lead to interesting findings, which suggest that some chromosomal amplifications might underlie the invasive tumor phenotype and the presence of cells that possess tumor initiating cells characteristics., Conclusions: The analyses offered by scMuffin and the results achieved in the case study show that our tool helps addressing the main challenges in the bioinformatics analysis of SC expression data from solid tumors., (© 2023. The Author(s).)

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis., Competing Interests: IZ and RR have a patent accepted concerning the use of Agents that modulate TMEM230 in tumor associated angiogenesis. Patent Application International Publication number: 20200247882. Agents that modulate TMEM230 as angiogenesis regulators and that detect TMEM230 AS markers of metastasis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cocola, Magnaghi, Abeni, Pelucchi, Martino, Vilardo, Piscitelli, Consiglio, Grillo, Mosca, Gualtierotti, Mazzaccaro, La Sala, Di Pietro, Palizban, Liuni, DePedro, Morara, Nano, Kehler, Greve, Noghero, Marazziti, Bussolino, Bellipanni, D’Agnano, Götte, Zucchi and Reinbold.)

Objectives: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired peripheral neuropathy of immunological origin with a clinical presentation and course that are extremely variable. The therapeutic approach generally includes corticosteroid drugs, intravenous immunoglobulins (IVIGs) or plasmapheresis alone or in combination as first line therapy, and immunosuppressants. In 2014 the Italian regulatory agency included subcutaneous immunoglobulins (SCIGs) in the list of off-label drugs reimbursed by the national health service. Our aim is to compare costs and outcomes of IVIG versus SCIG therapy., Methods: Patients medical records and therapeutic plans were retrospectively analysed to collect data on IVIG treatments 1 year before the switch to SCIG, and after 1 year of treatment with SCIG. A budget impact analysis was conducted through resource identification and quantification, and healthcare and non-health care costs evaluation., Results: 13 of 34 patients affected by CIDP who were referred to our neurophysiopathological unit and treated with IVIG were switched to home-based SCIG. After 1 year of receiving SCIG, 12 patients remained neurologically stable and reported good outcomes. Considering the cost of IVIG (€30.97/g) and adding to this the direct and indirect healthcare costs, the total cost of IVIG treatment for the 12 patients in a year was €371 417.06, compared with the cost of SCIG (€51.57/g) for a total annual cost of €631 745.16, not including indirect costs., Conclusions: We observe a higher cost for SCIG treatment versus IVIG, which is not in line with data in the literature. However, SCIGs offer some important safety benefits and improvements in patient quality of life., Competing Interests: Competing interests: None declared., (© European Association of Hospital Pharmacists 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Patients with neuromuscular diseases, during their illness are more susceptible to respiratory infections due to predisposing factors. Ineffective cough and the presence of atelectasis and hypoventilation, dysphagia and drooling can represent risk factors for the development of respiratory infection and fatal respiratory failure. Infections of respiratory tract with acute respiratory failure are the most common reason for hospitalizations, and pneumonia is among the leading causes of morbidity and mortality worldwide. The setting in which pneumonia is acquired heavily influences diagnostic and therapeutic choices. We will focus on aetiopathogenesis, diagnosis and treatment of pneumonia in these subjects, particularly considering the disease severity, rates of antibiotic resistance and the possible complications. In this case consultations with specialized physicians are strongly recommended., (©2021 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.)

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Background: In this article, we present a clinical case of refractory septic shock resulting from intestinal perforation treated with high doses of vasopressin and hydrocortisone during emergency surgery. The use of such high doses of vasopressin for this type of shock is not described in the literature., Case Presentation: A 49-year-old white woman with grade III obesity, Crohn's disease, and an intestinal perforation presented with refractory septic shock. Initially, a low dose of vasopressin was used. Then, the dosage was increased to 0.4 U/minute; in the literature, this is defined as "salvage therapy." This therapy consists of an initial load followed by a continuous infusion of hydrocortisone., Conclusions: The significant increase in her cardiac index and stroke volume index resulted in an improvement in peripheral resistance, gas exchange, and urine output and a decrease in her heart rate, interleukin-6 level, and tumor necrosis factor-α level. The administration of high doses of vasopressin and corticosteroids was demonstrated to be safe for the immune system, to reduce the systemic inflammatory response, and to have direct cardiovascular effects. Further studies are required to examine the use of vasopressin as an initial vasopressor as well as its use in high dosages and in combination with corticosteroids.

Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation.

Background: The identification of the organisation and dynamics of molecular pathways is crucial for the understanding of cell function. In order to reconstruct the molecular pathways in which a gene of interest is involved in regulating a cell, it is important to identify the set of genes to which it interacts with to determine cell function. In this context, the mining and the integration of a large amount of publicly available data, regarding the transcriptome and the proteome states of a cell, are a useful resource to complement biological research., Results: We describe an approach for the identification of genes that interact with each other to regulate cell function. The strategy relies on the analysis of gene expression profile similarity, considering large datasets of expression data. During the similarity evaluation, the methodology determines the most significant subset of samples in which the evaluated genes are highly correlated. Hence, the strategy enables the exclusion of samples that are not relevant for each gene pair analysed. This feature is important when considering a large set of samples characterised by heterogeneous experimental conditions where different pools of biological processes can be active across the samples. The putative partners of the studied gene are then further characterised, analysing the distribution of the Gene Ontology terms and integrating the protein-protein interaction (PPI) data. The strategy was applied for the analysis of the functional relationships of a gene of known function, Pyruvate Kinase, and for the prediction of functional partners of the human transcription factor TBX3. In both cases the analysis was done on a dataset composed by breast primary tumour expression data derived from the literature. Integration and analysis of PPI data confirmed the prediction of the methodology, since the genes identified to be functionally related were associated to proteins close in the PPI network. Two genes among the predicted putative partners of TBX3 (GLI3 and GATA3) were confirmed by in vivo binding assays (crosslinking immunoprecipitation, X-ChIP) in which the putative DNA enhancer sequence sites of GATA3 and GLI3 were found to be bound by the Tbx3 protein., Conclusion: The presented strategy is demonstrated to be an effective approach to identify genes that establish functional relationships. The methodology identifies and characterises genes with a similar expression profile, through data mining and integrating data from publicly available resources, to contribute to a better understanding of gene regulation and cell function. The prediction of the TBX3 target genes GLI3 and GATA3 was experimentally confirmed.

Context: The von Hippel-Lindau (VHL) syndrome is an inherited multitumour disorder characterized by clinical heterogeneity and high penetrance. Pheochromocytoma (Pheo) is present in 10%-15% of cases and can be isolated or associated with other lesions such as haemangioblastomas, kidney cysts or cancer and pancreatic lesions. In VHL patients, Pheos generally secrete norepinephrine and are located in the adrenals. Extra-adrenal Pheos (paragangliomas, PGLs) are rare., Objective: While performing genetic testing in patients affected by apparently sporadic Pheos or PGLs, we found two novel different VHL germline mutations in two females who presented with two distinct very uncommon clinical pictures. One patient was studied for the presence of an adrenal incidentaloma and the other for the presence of a neck tumour., Methods and Results: Patients coding regions and exon-intron boundaries of RET (exons 10, 11, 13-15), VHL, SDHD, SDHB and SDHC genes were amplified and sequenced. We identified two novel VHL point mutations: a L198V missense mutation in a 32-year-old female affected by a right adrenal compound and mixed tumour constituted by an epinephrine secreting Pheo, a ganglioneuroma and an adrenocortical adenoma, and a T152I missense mutation in a 24-year-old female affected by a left carotid body tumour. No other lesions were found in the patients or in the VHL mutation positive relatives., Conclusions: These cases enlarge the list of VHL mutations and add new insights in the clinical variability of VHL disease, thus confirming the importance of genetic testing in patients affected by apparently sporadic Pheos or PGLs.

Objective: Microvascular endothelium is one of the main targets of the inflammatory response. On specific activation, endothelial cells recruit Th1-lymphocytes at the inflammatory site. We investigated the intracellular signaling mediating tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma inflammatory response in human microvascular endothelial cells (HMEC-1) and the interfering effects of the peroxisome-proliferator-activated-receptor (PPARgamma) agonist, rosiglitazone (RGZ)., Methods and Results: TNFalpha and IFNgamma, mainly when combined, stimulate IFNgamma-inducible protein of 10 kDa (IP10) and fractalkine production evaluated by ELISA and TaqMan analyses. This effect is not only mediated by activation of the NFkB and Stat1 classic pathways, but also involves a rapid increase in phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) as measured by Western blot. RGZ interferes with TNFalpha and IFNgamma stimulation of IP10, fractalkine, and adhesion molecule through a novel rapid mechanism which involves the blocking of ERK activation., Conclusions: Our findings shed new light on the mechanisms underlying the inflammatory response of microvascular endothelium and on the possible therapeutic use of RGZ in vasculopathies involving Th1-responses.

Rosiglitazone (RGZ), a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR)-gamma, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R) of human adrenocortical carcinoma (ACC) and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR). We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K)-Akt, and extracellular signal-regulated kinase (ERK1/2) cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The KmPRPP (18.4 +/- 3.5 microM) and kcat (2.7 +/- 0.3 s-1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 +/- 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (Ki = 81.1 microM) and competitive (Ki = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5'-phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2'-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.

Objective: Children and adolescents with type 1 (insulin-dependent) diabetes mellitus (T1DM) show several impairment of bone metabolism and structure, resulting in a higher risk of decreased bone mass and its related complications later in life. Alterations of the nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) system have been implicated in several metabolic bone diseases characterized by increased osteoclast differentiation and activation and enhanced bone resorption., Design: We aimed to assess OPG levels and to investigate the possible relation between OPG levels, bone status and glycemic control in a group of prepubertal children with T1DM without microvascular complications., Methods: Twenty-six prepubertal T1DM children (median age 9.9 years, range 4.1-13.1 years) were studied. In all patients, serum OPG, hemoglobin (Hb)A1c, parathyroid hormone (PTH) and 25-dihy-droxyvitamin D (25-D) levels were evaluated. Bone quality was determined by measuring the attenuation of ultrasound waves by bone (broadband ultrasound attenuation (BUA)) at the calcaneal site. The data were compared with those of a group of 45 age-, sex-and body-size-matched healthy children., Results: Children with T1DM showed a reduced Z-score BUA in comparison with the control group (Student's t-test, P < 0.0001). Plasma OPG levels were significantly higher in diabetic children than in controls (Student's t-test, P < 0.0001). In T1DM children, Z-score BUA values displayed a significant correlation with OPG (Student's t-test, r = -0.62; P = 0.001), and HbA1c (r = -0.59; P = 0.007). OPG levels were significantly correlated with HbA1c (r = 0.56; P = 0.008). In a multiple regression analysis including age, duration of diabetes, physical activity, calcium intake, mean HbA1c and Z-score BUA, only HbA1c significantly predicted serum OPG levels (beta 0.67; P = 0.003)., Conclusions: Prepubertal children with T1DM have a significant increase of OPG levels. OPG serum concentrations are correlated to calcaneal BUA and HbA1c values. OPG could be a new marker of reduced bone mass in children with T1DM.

Objective: Emerging evidence from in vitro studies and mouse genetics attributes to osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, an important role in vascular biology. We evaluated serum levels of OPG in a group of children with Kawasaki disease (KD), before immunoglobulin (IVIG) infusion and at 3-month followup., Methods: Fifty patients (38 boys, 20 girls, median age 3.6 yrs, range 4 mo-7.4 yrs) fulfilling criteria for the diagnosis of KD, 30 febrile controls with infectious diseases, 18 patients with juvenile systemic lupus erythematosus (JSLE), and 40 healthy controls were enrolled. All KD patients received IVIG treatment within the first 10 days of illness, and aspirin. Coronary artery abnormalities (CAA) were reported in 6 out of 58 patients; all were male and younger than 5 years. Serum OPG was measured by ELISA in patients with KD before IVIG and at 3-month followup (median time 3.2 mo, range 3-3.5)., Results: At baseline and at the 3-month followup, KD patients had significantly higher OPG serum levels than febrile controls (p < 0.001 and p < 0.004, respectively), JSLE patients (p < 0.0001), and healthy controls (p < 0.0001). At baseline, KD patients who developed CAA had higher OPG serum levels than those without CAA (p = 0.0001); this difference was not present at 3-month followup. The optimal OPG cutoff value of 123.2 pg/ml was a significant predictor for CAA, with a sensitivity of 100% (6/6), a specificity of 96% (50/52), and a positive predictive value of 75% (6/8)., Conclusion: High OPG levels might be the result of compensatory production during acute and subacute phases of KD. OPG assay might be an additional clinically useful marker to monitor and differentiate patients who develop, from those who do not develop, such coronary artery abnormalities.