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The conversion of chemical structures into computer-readable descriptors, able to capture key structural aspects, is of pivotal importance in the field of cheminformatics and computer-aided drug design. Molecular fingerprints represent a widely employed class of descriptors; however, their generation process is time-consuming for large databases and is susceptible to bias. Therefore, descriptors able to accurately detect predefined structural fragments and devoid of lengthy generation procedures would be highly desirable. To meet additional needs, such descriptors should also be interpretable by medicinal chemists, and suitable for indexing databases with trillions of compounds. To this end, we developed—as integral part of EXSCALATE, Dompé's end-to-end drug discovery platform—the DompeKeys (DK), a new substructure-based descriptor set, which encodes the chemical features that characterize compounds of pharmaceutical interest. DK represent an exhaustive collection of curated SMARTS strings, defining chemical features at different levels of complexity, from specific functional groups and structural patterns to simpler pharmacophoric points, corresponding to a network of hierarchically interconnected substructures. Because of their extended and hierarchical structure, DK can be used, with good performance, in different kinds of applications. In particular, we demonstrate how they are very well suited for effective mapping of chemical space, as well as substructure search and virtual screening. Notably, the incorporation of DK yields highly performing machine learning models for the prediction of both compounds' activity and metabolic reaction occurrence. The protocol to generate the DK is freely available at https://dompekeys.exscalate.eu and is fully integrated with the Molecular Anatomy protocol for the generation and analysis of hierarchically interconnected molecular scaffolds and frameworks, thus providing a comprehensive and flexible tool for drug design applications. [ABSTRACT FROM AUTHOR]

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle's principle, "The whole is greater than the sum of its parts", multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available. [ABSTRACT FROM AUTHOR]

The 1.33-mm survey of protoplanetary discs in the Taurus molecular cloud found annular gaps and rings to be common in extended sources (≳ 55 au), when their 1D visibility distributions were fit parametrically. We first demonstrate the advantages and limitations of non-parametric visibility fits for data at the survey's 0.12-arcsec resolution. Then we use the non-parametric model in Frankenstein (frank) to identify new substructure in three compact and seven extended sources. Among the new features, we identify three trends: a higher occurrence rate of substructure in the survey's compact discs than previously seen, underresolved (potentially azimuthally asymmetric) substructure in the innermost disc of extended sources, and a 'shoulder' on the trailing edge of a ring in discs with strong depletion at small radii. Noting the shoulder morphology is present in multiple discs observed at higher resolution, we postulate it is tracing a common physical mechanism. We further demonstrate how a superresolution frank brightness profile is useful in motivating an accurate parametric model, using the highly structured source DL Tau in which frank finds two new rings. Finally, we show that sparse (u, v) plane sampling may be masking the presence of substructure in several additional compact survey sources. [ABSTRACT FROM AUTHOR]

We present the first ALMA survey of protoplanetary discs at 3 mm, targeting 36 young stellar objects in the Lupus star-forming region with deep observations (sensitivity 20–50  |$\mu$| Jy beam−1) at ∼0.35 arcsec resolution (∼50 au). Building on previous ALMA surveys at 0.89 and 1.3 mm that observed the complete sample of Class II discs in Lupus at a comparable resolution, we aim to assess the level of grain growth in the relatively young Lupus region. We measure 3 mm integrated fluxes, from which we derive disc-averaged 1–3 mm spectral indices. We find that the mean spectral index of the observed Lupus discs is |$\alpha _\mathrm{1-3\, mm}=2.23\pm 0.06$|⁠ , in all cases |$\alpha _\mathrm{1-3\, mm}\lt 3.0$|⁠ , with a tendency for larger spectral indices in the brightest discs and in transition discs. Furthermore, we find that the distribution of spectral indices in Lupus discs is statistically indistinguishable from that of the Taurus and Ophiuchus star-forming regions. Assuming the emission is optically thin, the low values |$\alpha _\mathrm{1-3\, mm}\le 2.5$| measured for most discs can be interpreted with the presence of grains larger than 1 mm. The observations of the faint discs in the sample can be explained without invoking the presence of large grains, namely through a mixture of optically thin and optically thick emission from small grains. However, the bright (and typically large) discs do inescapably require the presence of millimetre-sized grains in order to have realistic masses. Based on a disc mass argument, our results challenge previous claims that the presence of optically thick substructures may be a universal explanation for the empirical millimetre size-luminosity correlation observed at 0.89 mm. [ABSTRACT FROM AUTHOR]

We analyse spatially resolved ALMA observations at 0.9, 1.3, and 3.1 mm for the 26 brightest protoplanetary discs in the Lupus star-forming region. We characterize the discs multiwavelength brightness profiles by fitting the interferometric visibilities in a homogeneous way, obtaining effective disc sizes at the three wavelengths, spectral index profiles, and optical depth estimates. We report three fundamental discoveries: first, the millimetre continuum size–luminosity relation already observed at 0.9 mm is also present at 1.3 mm with an identical slope, and at 3.1 mm with a steeper slope, confirming that emission at longer wavelengths becomes increasingly optically thin. Second, when observed at 3.1 mm the discs appear to be only 9 per cent smaller than when observed at 0.9 mm, in tension with models of dust evolution that predict a starker difference. Third, by forward modelling the sample of measurements with a simple parametric disc model, we find that the presence of large grains (⁠|$a_\mathrm{max}\gt 1\,$| mm) throughout the discs is the most favoured explanation for all discs as it reproduces simultaneously their spectral indices, optical depth, luminosity, and radial extent in the 0.9–1.3 mm wavelength range. We also find that the observations can be alternatively interpreted with the discs being dominated by optically thick, unresolved, substructures made of mm-sized grains with a high scattering albedo. [ABSTRACT FROM AUTHOR]

Within the circular economy framework, our study aims to assess the rhamnolipid production from winery and olive oil residues as low-cost carbon sources by nonpathogenic strains. After evaluating various agricultural residues from those two sectors, Burkholderia thailandensis E264 was found to use the raw soluble fraction of nonfermented (white) grape marcs (NF), as the sole carbon and energy source, and simultaneously, reducing the surface tension to around 35 mN/m. Interestingly, this strain showed a rhamnolipid production up to 1070 mg/L (13.37 mg/g of NF), with a higher purity, on those grape marcs, predominately Rha-Rha C14-C14, in MSM medium. On olive oil residues, the rhamnolipid yield of using olive mill pomace (OMP) at 2% (w/v) was around 300 mg/L (15 mg/g of OMP) with a similar CMC of 500 mg/L. To the best of our knowledge, our study indicated for the first time that a nonpathogenic bacterium is able to produce long-chain rhamnolipids in MSM medium supplemented with winery residues, as sole carbon and energy source. Key points: • Winery and olive oil residues are used for producing long-chain rhamnolipids (RLs). • Both higher RL yields and purity were obtained on nonfermented grape marcs as substrates. • Long-chain RLs revealed stabilities over a wide range of pH, temperatures, and salinities [ABSTRACT FROM AUTHOR]

The synthesis of a new dithiolethione-cysteine ethyl ester hybrid, ACS94, its metabolites, and its effect on GSH levels in rat tissues and on the concentration of circulating H2S is described. ACS94 rapidly enters the cells, where it is metabolised to cysteine and the dithiolethione moiety ACS48. Experiments performed through the oral administration of ACS94 to healthy rats showed that it is capable of increasing the GSH levels in most of the analysed organs and the concentration of circulating H2S. Although the increase in GSH concentration was similar to that obtained by ACS48 and N-acetylcysteine ethyl ester, the H2S increase was long-lasting and more evident with respect to the parent molecules. Moreover, a decrease of homocysteine in several rat organs and in plasma was noted. This effect may represent a potential therapeutic use of ACS94, as hyperhomocysteinaemia is considered a risk factor for cardiovascular diseases. Lastly, ACS94 was more efficient than N-acetylcysteine in protecting the liver and kidneys against acute acetaminophen toxicity. [ABSTRACT FROM AUTHOR]

Malignant peritoneal mesothelioma (MpM), arising in the setting of local inflammation, is a rare aggressive tumour with a poor prognosis and limited therapeutic options. The three major MpM histological variants, epithelioid (E-MpMs), biphasic, and sarcomatoid MpMs (S-MpMs), are characterised by an increased aggressiveness and enhanced levels of EZH2 expression. To investigate the MpM immune contexture along the spectrum of MpM histotypes, an extended in situ analysis was performed on a series of 14 cases. Tumour-infiltrating immune cells and their functionality were assessed by immunohistochemistry, immunofluorescence, qRT-PCR, and flow cytometry analysis. MpMs are featured by a complex immune landscape modulated along the spectrum of MpM variants. Tumour-infiltrating T cells and evidence for pre-existing antitumour immunity are mainly confined to E-MpMs. However, Th1-related immunological features are progressively impaired in the more aggressive forms of E-MpMs and completely lost in S-MpM. Concomitantly, E-MpMs show also signs of active immune suppression, such as the occurrence of Tregs and Bregs and the expression of the immune checkpoint inhibitory molecules PD1 and PDL1. This study enriches the rising rationale for immunotherapy in MpM and points to the E-MpMs as the most immune-sensitive MpM histotypes, but it also suggests that synergistic interventions aimed at modifying the tumour microenvironment (TME) should be considered to make immunotherapy beneficial for these patients. [ABSTRACT FROM AUTHOR]

We present GALARIO, a computational library that exploits the power of modern graphical processing units (GPUs) to accelerate the analysis of observations from radio interferometers like Atacama Large Millimeter and sub-millimeter Array or the Karl G. Jansky Very Large Array. GALARIO speeds up the computation of synthetic visibilities from a generic 2D model image or a radial brightness profile (for axisymmetric sources). On a GPU, GALARIO is 150 faster than standard PYTHON and 10 times faster than serial C++ code on a CPU. Highly modular, easy to use, and to adopt in existing code,GALARIOcomes as two compiled libraries, one for Nvidia GPUs and one for multicore CPUs, where both have the same functions with identical interfaces. GALARIO comes with PYTHON bindings but can also be directly used in C or C++. The versatility and the speed of GALARIO open new analysis pathways that otherwise would be prohibitively time consuming, e.g. fitting high-resolution observations of large number of objects, or entire spectral cubes of molecular gas emission. It is a general tool that can be applied to any field that uses radio interferometer observations. The source code is available online at http://github.com/mtazzari/galario under the open source GNU Lesser General Public License v3. [ABSTRACT FROM AUTHOR]

Background: The presence of antibodies to CTLA-4, a negative regulator of T-cell activation, was investigated in multiply transfused patients with malignant and non- malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA-4 antibodies in the patients analyzed.Study Design and Methods: Patient sera were tested for binding to the recombinant full-length CTLA-4 beta-galactosidase fusion protein by an ELISA. Immuno-fluorescence stainings were performed to analyze the CTLA-4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units.Results: CTLA-4 antibodies were found in multiply transfused patients with beta-thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA-4 antibodies was found in patients receiving non-WBC-reduced blood (88%) than in those receiving WBC-reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA-4 epitopes.Conclusions: The apoptotic WBCs present in the RBC units, after cold storage, express CTLA-4 epitopes. These epitopes can be released and induce formation of CTLA-4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis. [ABSTRACT FROM AUTHOR]

In this paper, we revisit the issue of estimating the 'fossil' disc mass in the circumprimary disc, during the merger of a supermassive black hole binary. As the binary orbital decay speeds up due to the emission of gravitational waves, the gas in the circumprimary disc might be forced to accrete rapidly and could in principle provide a significant electromagnetic counterpart to the gravitational wave emission. Since the luminosity of such flare is proportional to the gaseous mass in the circumprimary disc, estimating such mass accurately is important. Previous investigations of this issue have produced contradictory results, with some authors estimating super-Eddington flares and large disc mass, while others suggesting that the 'fossil' disc mass is very low, even less than a Jupiter mass. Here, we perform simple 1D calculations to show that such very low estimates of the disc mass are an artefact of the specific implementation of the tidal torque in 1D models. In particular, for moderate mass ratios of the binary, the usual formula for the torque used in 1D models significantly overestimates the width of the gap induced by the secondary and this artificially leads to a very small leftover circumprimary disc. Using a modified torque, calibrated to reproduce the correct gap width as estimated by 3D models, leads to fossil disc masses of the order of one solar mass. The rapid accretion of the whole circumprimary disc would produce peak luminosities of the order of 1-20 times the Eddington luminosity. Even if a significant fraction of the gas escapes accretion by flowing out the secondary orbit during the merger (an effect not included in our calculations), we would still predict close to Eddington luminosities that might be easily detected. [ABSTRACT FROM AUTHOR]

Background: Clear cell sarcoma (CCS), initially named malignant melanoma of soft parts, is an aggressive soft tissue sarcoma (STS) that, due to MITF activation, shares with melanoma the expression of melanocyte differentiation antigens. CCS is poorly sensitive to chemotherapy. Multi-kinase inhibitors have been used as therapeutic agents. In the case we report here, treatment with sunitinib induced a long-lasting clinical response that was associated with an immune activation directed against Melan-A/MART-1 antigen. Case presentation: A 28 years old female patient with an advanced molecularly confirmed CCS resistant to conventional chemotherapy was started in January 2012 on sunitinib, 37.5 mg/day, with evidence of radiologic and metabolic response at the primary and metastatic sites of disease. Pathologic response and loss of the Melan-A/MART-1 antigen were evidenced on residual tumor removed in April 2012. Immunological monitoring performed on patient's blood during pharmacological treatment revealed a systemic, Melan-A/MART-1 specific immunity and a low frequency of immunosuppressive cells. Sunitinib was restarted in May 2012, with a new response, and continued for 11 months although with repeatedly interruptions due to toxicity. Disease progression and new responses were documented at each treatment interruption and restart. Sunitinib was definitively interrupted in April 2013 for disease progression. Conclusion: The analysis of this case proves that antigens expressed by CCS, as for melanoma, can be immunogenic in vivo and that tumor-antigen specific T cells may exert anti-tumor activity in CCS patient. Thus, manipulation of the immune response may have therapeutic potential for this STS subtype and immunotherapy approaches, can be promising therapeutic options for these patients. Keywords: Sarcoma, Sunitinib, Clear cell sarcoma, Tumor- [ABSTRACT FROM AUTHOR]

Background:Host immunity is emerging as a key player in the prognosis and response to treatment of cancer patients. However, the impact of the immune system and its modulation by therapies are unknown in rare soft tissue sarcomas such as solitary fibrous tumours (SFTs), whose management in the advanced forms includes anti-angiogenic therapy. Here, we studied the in situ and systemic immune status of advanced SFT patients and the effects of sunitinib malate (SM) in association with the clinical efficacy.Methods:Immune contexture of SFTs was assessed by immunohistochemistry in lesions from untreated or SM-treated patients. Frequency of circulating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and T-cell functions was assessed ex vivo in SFT patients prior and during anti-angiogenic therapy. Patients with long-term tumour control were included to correlate immune profiles and clinical responses.Results:Anti-angiogenic naïve SFT lesions were heavily infiltrated by CD163+CD14+CD68− and CD163+CD14−CD68− myeloid cells but devoid of T cells. Conversely, post-SM tumours acquired a new subset of CD68+CD14+ myeloid cells and displayed traits of an on-going adaptive immunity, strongly enriched in activated CD8+ and CD4+ T cells. These changes at the tumour site paralleled the alleviation of systemic immunosuppression and the drop in the frequency of circulating monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs). Rebound in the number of mMDSCs, but not of gMDSCs occurred at disease progression, and a reduced percentages of mMDSCs, comparable to those found in healthy donors (HDs), endured only in the SM-responsive patients.Conclusions:The immune contexture of SFT patients is heavily involved in anti-angiogenic therapy and it could be exploited to achieve more durable disease control through immune-based combination strategies. [ABSTRACT FROM AUTHOR]

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma and the clinical management of patients with unresectable, metastatic disease is still challenging. ASPS expresses an array of potentially therapeutically targetable, angiogenesis-related molecules and, importantly, it has a distinctive angiogenic phenotype marked by a peculiar tumor-associated vasculature. Several studies, conducted in transgenic mouse models and in a large variety of human tumors of different histotype, clearly proved the substantial contribution of tumor-infiltrating myeloid cells, such as myeloid derived suppressor cells, monocytes and macrophages, in the formation and maintenance of abnormal blood vessels in tumors. By immunohistochemistry we thus explored the presence and the distribution of cells expressing myeloid markers in the inflammatory infiltrate of surgical treated metastatic ASPS. Indeed, we found that myeloid cells expressing CD14 and CD163 markers constitute the prominent cells in the inflammatory infiltrate of ASPS. These macrophage-like cells form a network surrounding the endothelial cells, or, interspersed in the tumor nest, they keep deep contact with tumor cells. In this commentary, we discussed our findings in relation to the recently published paper by Kummar and colleagues reporting the clinical and molecular results of a phase II clinical trial in patients with unresectable, metastatic ASPS treated with the anti-angiogenic drug cediranib, targeting the VEGFR-1,-2,-3 tyrosine kinases. [ABSTRACT FROM AUTHOR]

Abstract: A series of dithiolethione derivatives was synthesized and the in vitro HDAC inhibitory activity was tested. The most active compounds, 1 and 2, exhibited an IC50 in nM range with a strong hyperacetylation of histone H4 in A549 cells. The HDAC inhibitory activity comparable to that of SAHA and the inhibition of A549 cell proliferation suggest that these compounds are worthy of further studies as potential anticancer agents. [Copyright &y& Elsevier]

The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (L-DOPA} is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to the continuing neuronal loss are oxidative stress, including oxidation of L-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors is to produce L-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such L-DOPA hybrids based on coupling L-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to he capable of conversion by isolated mitochondria to H2S or equivalent SH- ions. This capability was confirmed by in vivo results, showing a large increase in intra-cerebral dopamine and glutathione after iv administration in rats. When human microglia, astrocytes, and SH-SY5Y neuro-blastoma cells were treated with these donating agents, they all accumulated H2S intracellularly as did their derivatives coupled to L-DOPA. The donating agents and the L-DOPA hybrids reduced the release of tumor necrosis factor-α, interleukin-6, and nitric oxide from stimulated microglia, astrocytes as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants from these stimulated cells to SH-SY5Y cells. L-DOPA itself was without effect in any of these assays. The H2S-releasing L-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant anti-inflammatory, antioxidant, and neuroprotective properties. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4–11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34+ cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K–Akt survival pathway.Leukemia (2008) 22, 147–160; doi:10.1038/sj.leu.2404980; published online 11 October 2007 [ABSTRACT FROM AUTHOR]

The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG-dependent events. It has been demonstrated that nuclear DGK-ζ interferes with cell cycle progression. We previously reported that nuclear DGK-ζ expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK-ζ in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-ζ, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-l6-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK-ζ by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK-ζ was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-ζ, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK-ζ as a key determinant of cell cycle progression and differentiation of C2C12 cells. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34+ cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.Leukemia (2007) 21, 886–896. doi:10.1038/sj.leu.2404643; published online 15 March 2007 [ABSTRACT FROM AUTHOR]

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression. [ABSTRACT FROM AUTHOR]

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

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myeloid leukemia (AML). The progression of myelodysplastic syndromes (MDSs) to AML is thought to be associated with abrogation of apoptotic control mechanisms. However, little is known about signal transduction pathways which may be involved in enhanced survival of MDS cells. In this report, we have performed immunocytochemical and flow cytometric analysis to evaluate the levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt) staining in 90% of the cases (n=22) diagnosed as high-risk MDS, whereas mononuclear cells from normal bone marrow or low-risk MDS patients showed low or absent Ser473 p-Akt staining. Furthermore, all high-risk MDS patients also demonstrated high expression of the Class I PI3K p110delta catalytic subunit and a decreased expression of PTEN. Taken together, our results suggest that Akt activation might be one of the factors contributing to the decreased apoptosis rate observed in patients with high-risk MDS. [ABSTRACT FROM AUTHOR]

Recent studies suggest that the prosurvival signal transduction pathway involving phosphoinositide 3-kinase (PI3K)/Akt can confer an aggressive, apoptosis-resistant phenotype to acute leukaemia cells. We have investigated the effect of modulating this signalling pathway on the sensitivity of leukaemic cell lines (NB-4, CEM, Jurkat, MOLT-4) and acute promyelocytic primary blasts to apoptosis induced by 1 μmol/l As2O3. Whereas parental NB-4 cells did not display any phosphorylated (active) Akt, CEM, Jurkat and MOLT-4 cells exhibited high levels of Akt activation. Consistently, treatment of NB-4 cells with pharmacological inhibitors of the PI3K/Akt pathway ( , wortmannin) did not increase sensitivity of these cells to arsenic trioxide (As2O3), whereas siRNA knock-down of Akt enhanced As2O3-induced apoptosis of CEM, Jurkat and MOLT-4 cells. Overexpression of a constitutively active Akt cDNA rendered NB-4 cells less susceptible to As2O3. Upon prolonged exposure to As2O3, we isolated a NB-4 cell clone that was resistant to As2O3 and displayed high levels of active Akt. treatment of acute promyelocytic primary blasts with elevated Akt phosphorylation levels resulted in an increased sensitivity to As2O3. These results may provide a rationale for the development of combined or sequential treatment with PI3K/Akt inhibitors to improve the efficacy of As2O3 on acute leukaemias and also to overcome As2O3 resistance. [ABSTRACT FROM AUTHOR]

Summary:Healthy allogeneic donors, who were treated with G-CSF and underwent peripheral blood haematopoietic precursor collection at our Institution, were enrolled in a short- and long-term haematological surveillance protocol for a 5–7-year period. To date, 94 donors have been assessed with a mean follow-up of 30 months (4–84); for 30 subjects, the follow-up is 48 months. During G-CSF administration, 23/94 donors showed a significant platelet count decrease from the baseline. Pre-apheresis platelet decrement correlated with the total G-CSF dose administered, baseline platelet level and donor age. Normal platelet counts returned within 4–8 months. PMN and/or lymphocyte lower values were observed in 55/94 donors 2 weeks after G-CSF administration, with mean drops from the baseline of 40 and 36% for PMN and lymphocytes, respectively. The PMN decrease correlated inversely with donor age, as younger donors were more affected than older ones, whereas the lymphocyte decrease correlated directly with the total blood volumes processed in the apheresis courses, in particular for donors subjected to large volume leukaphereses. Long-term observation showed moderate neutrophil reduction (25% count drop from the baseline) in four of the 30 donors observed for four years or more. 14 donors showed persistent, slight lymphocytopenia (mean drop of 13%) until the third year, with recovery in the fourth year of follow-up.Bone Marrow Transplantation (2005) 36, 289–294. doi:10.1038/sj.bmt.1705066; published online 20 June 2005 [ABSTRACT FROM AUTHOR]

Activation of the phosphoinositide 3 kinase (PI3K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its downregulation, by means of PI3K inhibitors, lowers resistance to various types of therapy in tumour cell lines. Recently, it has been reported that deguelin, a naturally occurring rotenoid, is a powerful inhibitor of PI3K. We investigated whether or not deguelin could enhance the sensitivity to chemotherapeutic drugs of human U937 leukaemia cells and acute myeloid leukaemia (AML) blasts with an activated PI3K/Akt network. Deguelin (10 nmol/l) induced S phase arrest with interference of progression to G2/M, and at 100 nmol/l significantly increased apoptotic cell death of U937. At 10–100 nmol/l concentrations, deguelin downregulated Akt phosphorylation of leukaemia cells and markedly increased sensitivity of U937 cells to etoposide or cytarabine. A 10 nmol/l concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells, whereas it increased sensitivity of AML blasts to cytarabine. Deguelin was less toxic than wortmannin on erythropoietin- and stem cell factor-induced erythropoiesis from CD34+ progenitor cells. Overall, our results indicate that deguelin might be used in the future for increasing sensitivity to therapeutic treatments of leukaemia cells with an active PI3K/Akt signalling network. [ABSTRACT FROM AUTHOR]

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts. [ABSTRACT FROM AUTHOR]

Activation of the phosphoinositide 3-kinase (PI3-K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its down-regulation, by means of pharmacological inhibitors of PI3-K, considerably lowers resistance to various types of therapy in cell lines derived from solid tumours. Recently, a new class of Akt inhibitors, referred to as phosphatidylinositol ether lipids (PIAs), have been synthesized. We tested whether two new PIAs could lower the sensitivity threshold to chemotherapeutic drugs of human leukaemia cell lines with an activated PI3-K/Akt network. We used HL60AR (for apoptosis resistant), K562 and U937 cells. The two pharmacological inhibitors, used at 5 μmol/l, down-regulated Akt kinase activity and phosphorylation. Neither of the two chemicals affected the activity of other signalling proteins in the Akt pathway, such as phosphoinositide-dependent protein kinase-1 or PTEN. When employed at 5 μmol/l, the Akt inhibitors markedly reduced the resistance of the leukaemic cell lines to etoposide or cytarabine. Remarkably, a 5 μmol/l concentration of the inhibitors did not negatively affect the survival rate of human cord blood CD34+ cells. Overall, our results indicate that new selective Akt pharmacological inhibitors might be used in the future for overcoming Akt-mediated resistance to therapeutic treatments of acute leukaemia cells. [ABSTRACT FROM AUTHOR]

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting. [ABSTRACT FROM AUTHOR]

It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIPL, and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-? and it was less effective in downregulating p70S6 kinase (p70S6K) activity. The Akt inhibitor increased sensitivity to apoptotic inducers of K562 and U937, but not of MOLT-4, leukemia cells. Overall, our results indicate that selective Akt pharmacological inhibitors might be used in the future for enhancing the sensitivity of leukemia cells to therapeutic treatments that induce apoptosis or for overcoming resistance to these treatments.Leukemia (2003) 17, 1794-1805. doi:10.1038/sj.leu.2403044 [ABSTRACT FROM AUTHOR]

Background and Objectives Apoptosis affects white blood cells (WBCs) contained in packed red blood cell (RBC) units. This phenomenon was recently described also in residual WBCs after filtration. The aim of this study was to better characterize the residual WBCs postfiltration by using apoptosis markers and morphology. Materials and Methods Immunofluorescence, flow cytometry and cell-sorting techniques were utilized. Results Residual leucocytes of leucodepleted packed RBC units showed increasing values of apoptotic elements in a time-course experiment. We also demonstrated that these elements are positive for APO 2·7 monoclonal antibody (mAb), poly ADP-ribose polymerase (PARP) cleavage and fluorescein isothiocyanate (FITC)-conjugated N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), all of which indicate that programmed death is a feature of this population of cells. Phenotypic analysis with CD45 side-scatter gating demonstrated also that CD15 and CD16 granulocyte-associated antigens are present on a subset of postfiltration leucocytes. Moreover, the expression of human leucocyte antigen (HLA) class I antigens is maintained. Sorting of CD45-positive cells and morphological analysis of these samples confirmed that leucocytes in postfiltration units have morphological characteristics of dying cells. Conclusions Our study extends previous observations regarding the morphology and function of apoptotic cells in leucodepleted blood units, which suggested the presence of apoptotic cells in postfiltration leucocytes. Cleaved PARP, APO 2·7 mAb and positivity for the FITC-conjugated Z-VAD-analogous reagent strongly suggest the activation of programmed death pathways. In addition, the maintained granulocyte-associated and HLA class I antigens might recall an immune response in multitransfused patients. [ABSTRACT FROM AUTHOR]

TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis. [ABSTRACT FROM AUTHOR]

Background and Objectives Packed red blood cells (pRBCs) contain apoptotic white cells. We studied apoptotic cells in pRBCs after filtration and at various time-points during storage. Materials and Methods To maintain the same subset of cells, seven pRBC units were pooled in a single bag and divided equally into seven aliquots. Two series of five experiments were performed: in the first we utilized the Biofil RO1 Max filter, and in the second the Pall BPF4 filter was used. One aliquot was immediately leucodepleted while the others were stored at 4°C and filtered on days 3, 7, 10, 14, 21 and 42 of storage. The postfiltration leucocyte counts and apoptotic evaluations were performed by using the Nageotte chamber and flow cytometry. Results The absolute number of residual leucocytes was always less than 0.5 x 10[sup 6] in each experiment. Nageotte chamber counts showed a greater number of white blood cells than flow cytometry during the 42 days of storage. On day O, the percentage of apoptotic cells in non-leucodepleted pRBCs was 1.1 ±0.4 and 1.2 ±0.4, while in filtered pRBCs it was high from day O, at 53.5 ±16.3 and 52 ±18.5, respectively, with Biofil and Pall filters. On day 10 of storage, apoptotic cells reached a percentage of 42.5 ±15.8 and 41.6 ±18.6 in non-leucodepleted pRBCs, while in filtered units an average value of ≈ 90% was found with both filters. Conclusions The percentage of apoptotic cells was higher in leucodepleted than in non-leucodepleted pRBCs. After filtration, the degree of apoptosis was already high on day O, and reached a mean of ≈ 90% by day 10. The difference in residual WBC counts between the Nageotte chamber and flow cytometry could be related to the presence of a high percentage of apoptotic cells in filtered blood components, and to the method used to distinguish viable from apoptotic cells. [ABSTRACT FROM AUTHOR]

Summary A total of 70 serum samples from heparin-induced thrombocytopenia (HIT II) patients, non-HIT patients and healthy subjects, respectively, were studied for the presence of antiheparin/PF4 antibodies. Two enzyme-linked immunosorbent assay (ELISA) assays were compared with the particle gel immunoassay (PaGIA). Beads of the PaGIA kit were also used to evaluate the feasibility of flow cytometric detection of antiheparin/PF4 antibodies in patient samples. Experiments have shown that all samples found positive by ELISA and PaGIA, were also positive when analysed by flow cytometry by an indirect test using the high-density particles coated with heparin/PF4 complexes and a second step fluorescein isothiocyanate (FITC) antihuman immunoglobulin (Ig)G reagent. The procedure was easy to perform, repetitive and beads were promptly visualized by physical parameters, with a very low background. In conclusion, the results of this study suggest that flow cytometry is a reliable method for the detection of antiheparin/PF4 antibodies. [ABSTRACT FROM AUTHOR]

Recombinant human monoclonal antibodies against CD152 have been generated by selecting a synthetic phage scFv library with purified CD152-Ig fusion protein. Sixteen scFv fragments were isolated which specifically react with CD152 by enzyme-linked immunoabsorbent assay (ELISA) and Western blot resulting in their clustering into two groups recognizing different antigenic determinants. One group of scFvs (number3, number13, number40, number44, number47, number51, number57, number80 number83) recognized an epitope on CD152 dimer whereas another group (number15, number18, number31, number35, number54, number72, number81) recognized an epitope on both dimeric and monomeric CD152 molecule suggesting their possible use in understanding the subunit structure of CD152 which is still controversial. Sequencing of the VH genes revealed that all the scFvs belonged to the VH3 gene family but they were different in CDR3 length and composition. It was possible to correlate specific CDR3 sequences with reactivity of the two groups of scFvs. Four scFvs, number3, number40, number81 and number83, each representative of one specific CDR3, were selected for further analysis. Competition ELISA experiments showed that they recognize CD152 in its native configuration and bound to different epitopes from the CD80/CD86 interaction site. The scFvs were able to stain human T lymphocytes stimulated either with anti-CD3 and CD28 antibodies or PHA, PMA and ionomycin by cytofluorimetry suggesting that they can be useful reagents for monitoring the kinetics of surface-bound and intracellular CD152. [ABSTRACT FROM AUTHOR]

Soft tissue sarcomas (STSs) are a family of rare malignant tumors encompassing more than 80 histologies. Current therapies for metastatic STS, a condition that affects roughly half of patients, have limited efficacy, making innovative therapeutic strategies urgently needed. From a molecular point of view, STSs can be classified as translocation-related and those with a heavily rearranged genotype. Although only the latter display an increased mutational burden, molecular profiles suggestive of an "immune hot" tumor microenvironment are observed across STS histologies, and response to immunotherapy has been reported in both translocation-related and genetic complex STSs. These data reinforce the notion that immunity in STSs is multifaceted and influenced by both genetic and epigenetic determinants. Cumulative evidence indicates that a fine characterization of STSs at different levels is required to identify biomarkers predictive of immunotherapy response and to discover targetable pathways to switch on the immune sensitivity of "immune cold" tumors. In this review, we will summarize recent findings on the interplay between genetic landscape, molecular profiling and immunity in STSs. Immunological and molecular features will be discussed for their prognostic value in selected STS histologies. Finally, the local and systemic immunomodulatory effects of the targeted drugs imatinib and sunitinib will be discussed. [ABSTRACT FROM AUTHOR]

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimuIated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9x10-13 to 5.7 x 10-11M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10-8M (immunotoxin containing gelonin) and 5x10-9M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S ≤ 10-11M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes. [ABSTRACT FROM AUTHOR]

Three monoclonal antibodies (MoAb), named 8A. 8F6 and 62B1. reacting with plasma cell-associated antigens. were characterized. 8A was found to he positive throughout the B cell lineage maturation steps from the immature B-committed CD10+ cell to the plasma cells. 8F6 and 62B1 reactivity is restricted to more mature cells and related lymphoid malignancies. In particular 62B1 appears to be limited to hairy cells and plasma cells. The results show that it is possible to obtain reagents reacting with plasma cells by immunizing mice with cells derived from human multiple myclomas. Furthermore, the obtained results suggest that it is possible to elicit antibodies against antigens which are present throughout all the differentiation steps of the B cell lineage. These new MoAb could help in elucidating the phenotype of the plasma cells and the relationships of multiple myclomas with other B cell proliferative disorders. [ABSTRACT FROM AUTHOR]

Immunotoxins were prepared with three ribosome-inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6, linked to the anti-CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22-expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC50 (concentration causing 50% inhibition) ranging from < 5 × 10-15 to 7.6 × 10-11 M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5 × 10-14 to 5 × 10-8 M, with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 × 10-8 M). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10-9 M. Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3 × 10-6), when 60% of the animals remained tumour-free. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are professional antigen-presenting cells of the mammalian immune system. Ex vivo differentiated DCs represent a unique Advanced Therapy Medicinal Product (ATMP), used in several clinical trials as personalized cancer immunotherapy. The therapy's reliability depends on its capacity to produce high-quality mature DCs (mDCs) in compliance with Good Manufacturing Practices. From March 2010 to December 2023, 103 patients were enrolled in multiple clinical trials at the Immuno-Gene Therapy Factory at IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori". Six hundred forty-two doses were produced, and the manufacturing process was implemented to optimize production. Our study is a retrospective analysis focusing on the quality control results. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating viability, purity and phenotype of mDCs and their quality in terms of microbiological safety. The data obtained are given with median and interquartile range. The batches were found to be microbiologically safe in terms of sterility, mycoplasma, and endotoxins. An increase in DC maturation markers was found. The release criteria checks showed a high percentage of viability and purity was maintained during the production process. Our findings have confirmed that the measures implemented have ensured the safety of the products and have contributed to the establishing a robust "Pharmaceutical Quality System." This has enabled many safe mDCs to be produced for clinical trials. [ABSTRACT FROM AUTHOR]

We analyze the dust morphology of 29 transition disks (TDs) observed with Atacama Large (sub-)Millimeter Array (ALMA) at (sub-)millimeter emission. We perform the analysis in the visibility plane to characterize the total flux, cavity size, and shape of the ring-like structure. First, we found that the Mdust–M⋆ relation is much flatter for TDs than the observed trends from samples of class II sources in different star-forming regions. This relation demonstrates that cavities open in high (dust) mass disks, independent of the stellar mass. The flatness of this relation contradicts the idea that TDs are a more evolved set of disks. Two potential reasons (not mutually exclusive) may explain this flat relation: the emission is optically thick or/and millimeter-sized particles are trapped in a pressure bump. Second, we discuss our results of the cavity size and ring width in the context of different physical processes for cavity formation. Photoevaporation is an unlikely leading mechanism for the origin of the cavity of any of the targets in the sample. Embedded giant planets or dead zones remain as potential explanations. Although both models predict correlations between the cavity size and the ring shape for different stellar and disk properties, we demonstrate that with the current resolution of the observations, it is difficult to obtain these correlations. Future observations with higher angular resolution observations of TDs with ALMA will help discern between different potential origins of cavities in TDs. [ABSTRACT FROM AUTHOR]

The Sunyaev–Zel'dovich (SZ) effect can potentially be used to investigate the heating of the circumgalactic medium and subsequent suppression of cold gas accretion on to the host galaxy caused by quasar feedback. We use a deep ALMA observation of HE0515-4414 in band 4, the most luminous quasar known at the peak of cosmic star formation (z  = 1.7), to search for the SZ signal tracing the heating of the galaxy's halo. ALMA's sensitivity to a broad range of spatial scales enables us to disentangle emitting compact sources from the negative, extended SZ signal. We obtain a marginal SZ detection (∼3.3σ) on scales of about 300 kpc (30–40 arcsec), at the 0.2 mJy level, 0.5 mJy after applying a correction factor for primary beam attenuation and flux that is resolved out by the array. We show that our result is consistent with a simulated ALMA observation of a similar quasar in the fable cosmological simulations. We emphasize that detecting an SZ signal is more easily achieved in the visibility plane than in the (inferred) images. We also confirm a marginal detection (3.2σ) of a potential SZ dip on smaller scales (<100 kpc) already claimed by other authors, possibly highlighting the complex structure of the halo heating. Finally, we use SZ maps from the fable cosmological simulations, convolved with ALMA simulations, to illustrate that band 3 observations are much more effective in detecting the SZ signal with higher significance, and discuss the optimal observing strategy. [ABSTRACT FROM AUTHOR]

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients' blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients' sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors. [ABSTRACT FROM AUTHOR]

A correlation between proto-planetary disc radii and sub-mm fluxes has been recently reported. In this letter, we show that the correlation is a sensitive probe of grain growth processes. Using models of grain growth and drift, we have shown in a companion paper that the observed disc radii trace where the dust grains are large enough to have a significant sub-mm opacity. We show that the observed correlation emerges naturally if the maximum grain size is set by radial drift, implying relatively low values of the viscous α parameter ≲0.001. In this case, the relation has an almost universal normalization, while if the grain size is set by fragmentation the flux at a given radius depends on the dust-to-gas ratio. We highlight two observational consequences of the fact that radial drift limits the grain size. The first is that the dust masses measured from the sub-mm could be overestimated by a factor of a few. The second is that the correlation should be present also at longer wavelengths (e.g. 3mm), with a normalization factor that scales as the square of the observing frequency as in the optically thick case. [ABSTRACT FROM AUTHOR]

Anti-human platelet antigens (HPA) alloantibodies are seldom involved in febrile nonhaemolytic reactions (FNHTRs). We describe a case in which anti-HPA-5a alloantibodies are related to an FNHTR. We studied the specificity of the alloantibodies by flow cytometry, ELISA and MACE. Typing of donors and the patient was performed by sequence-specific polymerase chain reaction. The alloantibodies were found reactive with HPA-5a antigens. The patient was HPA-5b/b, whereas the donor of the platelet apheresis involved in the FNHTR was HPA-5a/a. Despite the low frequency of anti-HPA-5a antibodies, they might be responsible for FNHTR. [ABSTRACT FROM AUTHOR]

Background: Clear cell sarcoma (CCS), initially named malignant melanoma of soft parts, is an aggressive soft tissue sarcoma (STS) that, due to MITF activation, shares with melanoma the expression of melanocyte differentiation antigens. CCS is poorly sensitive to chemotherapy. Multi-kinase inhibitors have been used as therapeutic agents. In the case we report here, treatment with sunitinib induced a long-lasting clinical response that was associated with an immune activation directed against Melan-A/MART-1 antigen.Case Presentation: A 28 years old female patient with an advanced molecularly confirmed CCS resistant to conventional chemotherapy was started in January 2012 on sunitinib, 37.5 mg/day, with evidence of radiologic and metabolic response at the primary and metastatic sites of disease. Pathologic response and loss of the Melan-A/MART-1 antigen were evidenced on residual tumor removed in April 2012. Immunological monitoring performed on patient's blood during pharmacological treatment revealed a systemic, Melan-A/MART-1 specific immunity and a low frequency of immunosuppressive cells. Sunitinib was restarted in May 2012, with a new response, and continued for 11 months although with repeatedly interruptions due to toxicity. Disease progression and new responses were documented at each treatment interruption and restart. Sunitinib was definitively interrupted in April 2013 for disease progression.Conclusion: The analysis of this case proves that antigens expressed by CCS, as for melanoma, can be immunogenic in vivo and that tumor-antigen specific T cells may exert anti-tumor activity in CCS patient. Thus, manipulation of the immune response may have therapeutic potential for this STS subtype and immunotherapy approaches, can be promising therapeutic options for these patients. [ABSTRACT FROM AUTHOR]

Protoplanetary disks emit radiation across a broad range of wavelengths, requiring a multiwavelength approach to fully understand their physical mechanisms and how they form planets. Observations at submillimeter to centimeter wavelengths can provide insights into the thermal emission from dust, free-free emission from ionized gas, and possible gyro-synchrotron emission from the stellar magnetosphere. This work is focused on CX Tau, a ~0.4 M⊙ star with an extended gas emission and a compact and apparently structureless dust disk, with an average millimeter flux compared to Class II sources in Taurus. We present observations from the Karl G. Jansky Very Large Array across four bands (between 9.0 mm and 6.0 cm) and combine them with archival data from the Atacama Large Millimeter/submillimeter Array, the Submillimeter Array, and the Plateau de Bure Interferometer. This multiwavelength approach allows us to separate the dust continuum from other emissions. After isolating the dust thermal emission, we derived an upper limit of the dust disk extent at 1.3 cm, which is consistent with theoretical predictions of a radial drift-dominated disk. The centimeter data show a peculiar behavior: deep observations at 6.0 cm did not detect the source, while at 1.3 cm, the flux density is anomalously higher than adjacent bands. Intraband spectral indices suggest a dominant contribution from free-free emission, whereas gyro-synchrotron emission is excluded. To explain these observations, we propose a strong variability among the free-free emission with timescales shorter than a month. Another possible interpretation is the presence of anomalous microwave emission from spinning dust grains. [ABSTRACT FROM AUTHOR]

Introduction: Three-dimensional (3D) multicellular spheroids are fundamental in vitro tools for studying in vivo tissues. Volume is the main feature used for evaluating the drug/treatment effects, but several other features can be estimated even from a simple 2D image. For high-content screening analysis, the bottleneck is the segmentation stage, which is essential for detecting the spheroids in the images and then proceeding to the feature extraction stage for performing morphotypic analysis. Problem: Today, several tools are available for extracting morphological features from spheroid images, but all of them have pros and cons and there is no general validated solution. Thanks to new deep learning models, it is possible to standardize the process and adapt the analysis to big data. Novelty: Starting from the first version of AnaSP, an open-source software suitable for estimating several morphological features of 3D spheroids, we implemented a new module for automatically segmenting 2D brightfield images of spheroids by exploiting convolutional neural networks. Results: Several deep learning segmentation models (i.e., VVG16, VGG19, ResNet18, ResNet50) have been trained and compared. All of them obtained very interesting results and ResNet18 ranked as the best-performing. Conclusions: A network based on an 18-layer deep residual architecture (ResNet-18) has been integrated into AnaSP, releasing AnaSP 2.0, a version of the tool optimized for high-content screening analysis. The source code, standalone versions, user manual, sample images, video tutorial, and further documentation are freely available at: https://sourceforge.net/p/anasp. [ABSTRACT FROM AUTHOR]

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma and the clinical management of patients with unresectable, metastatic disease is still challenging. ASPS expresses an array of potentially therapeutically targetable, angiogenesis-related molecules and, importantly, it has a distinctive angiogenic phenotype marked by a peculiar tumor-associated vasculature. Several studies, conducted in transgenic mouse models and in a large variety of human tumors of different histotype, clearly proved the substantial contribution of tumor-infiltrating myeloid cells, such as myeloid derived suppressor cells, monocytes and macrophages, in the formation and maintenance of abnormal blood vessels in tumors. By immunohistochemistry we thus explored the presence and the distribution of cells expressing myeloid markers in the inflammatory infiltrate of surgical treated metastatic ASPS. Indeed, we found that myeloid cells expressing CD14 and CD163 markers constitute the prominent cells in the inflammatory infiltrate of ASPS. These macrophage-like cells form a network surrounding the endothelial cells, or, interspersed in the tumor nest, they keep deep contact with tumor cells. In this commentary, we discussed our findings in relation to the recently published paper by Kummar and colleagues reporting the clinical and molecular results of a phase II clinical trial in patients with unresectable, metastatic ASPS treated with the anti-angiogenic drug cediranib, targeting the VEGFR-1,-2,-3 tyrosine kinases. [ABSTRACT FROM AUTHOR]