Haploidentical HSCT with post transplantation cyclophosphamide versus unrelated donor HSCT in pediatric patients affected by acute leukemia.

T-cell replete hematopoietic stem cell transplantation (HSCT) from a haploidentical donor followed by high doses of cyclophosphamide has been demonstrated to provide the best chances of a cure for many children in need of an allograft but who lack both a sibling and an unrelated donor. In this study we retrospectively compared the outcome of pediatric patients undergoing T-replete haploidentical HSCT (Haplo) for acute leukemia with those undergoing transplantation from unrelated HLA-matched donor (MUD) and HLA mismatched unrelated donor (MMUD) from 2012 to 2017 at our Center. Both univariable and multivariable analyses showed similar 5-year overall survival rates for MUD, MMUD, and Haplo patients: 71% (95% CI 56-86), 72% (95% CI 55-90), and 75% (95% CI 54-94), respectively (p = 0.97). Haplo patients showed reduced event-free survival rates compared to MUD and MMUD patients: 30% (95% CI 12-49) versus 70% (95% CI 55-84) versus 53% (95% CI 35-73), respectively (p = 0.007), but these data were not confirmed by a multivariable analysis. Non-relapse mortality (NRM) and relapse incidence (RI) were similar for the three groups. Therefore, our data confirm that Haplo is a suitable clinical option for pediatric patients needing HSCT when lacking both an MUD and an MMUD donor.

Phase dependent encapsulation and release profile of ZIF-based biocomposites.

Biocomposites composed of Zeolitic Imidazolate Frameworks (ZIFs) are generating significant interest due to their facile synthesis, and capacity to protect proteins from harsh environments. Here we systematically varied the composition (i.e. relative amounts of ligand (2-methylimidazole), metal precursor (Zn(OAc)2·2H2O), and protein) and post synthetic treatments (i.e. washes with water or water/ethanol) to prepare a series of protein@ZIF biocomposites. These data were used to construct two ternary phase diagrams that showed the synthesis conditions employed gave rise to five different phases including, for the first time, biocomposites based on ZIF-CO3-1. We examined the influence of the different phases on two properties relevant to drug delivery applications: encapsulation efficiency and release profile. The encapsulation efficiencies of bovine serum albumin and insulin were phase dependent and ranged from 75% to 100%. In addition, release profiles showed that 100% protein release varied between 40 and 300 minutes depending on the phase. This study provides a detailed compositional map for the targeted preparation of ZIF-based biocomposites of specific phases and a tool for the straightforward analysis of the crystalline phases of ZIF based materials (web application named "ZIF phase analysis"). These data will facilitate the progress of ZIF bio-composites in the fields of biomedicine and biotechnology.

Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancer cells.

Aberrant activity of the hedgehog (Hh) pathway is prevalent in pathologies such as cancer. Improved understanding of Hh activity in the aggressive tumor cell phenotype is being pursued for development of targeted therapies. Recently, we described a link between Hh activity and carbonic anhydrase XII (CAXII) expression. Extracellular facing CAs (IX/XII) are highly expressed in hypoxia, contribute to tumor pH regulation and are thus of clinical interest. Here we have extended the investigation of potential interactions between Hh activity and CAXII utilizing genomic disruption/knockout of either GLI1 (the main transcriptional factor induced with Hh activity) or CAXII in the triple negative breast cancer cell lines MDA-MB-231 and BT-549. Knockout of GLI1 and CAXII significantly decreased hallmarks of tumor aggressiveness including proliferation and migration. Most intriguingly, CAXII knockout caused a massive induction of the Sonic hedgehog (Shh) ligand expression (gene and protein). This novel finding indicates that CAXII plays a potential role in suppression of Shh and may act in a feedback loop to regulate overall Hh activity. Enhanced knowledge of these CA-Hh interactions in future studies may be of value in understanding this currently 'incurable' subclass of breast cancer.

Olivine in an unexpected location on Vesta's surface.

Olivine is a major component of the mantle of differentiated bodies, including Earth. Howardite, eucrite and diogenite (HED) meteorites represent regolith, basaltic-crust, lower-crust and possibly ultramafic-mantle samples of asteroid Vesta, which is the lone surviving, large, differentiated, basaltic rocky protoplanet in the Solar System. Only a few of these meteorites, the orthopyroxene-rich diogenites, contain olivine, typically with a concentration of less than 25 per cent by volume. Olivine was tentatively identified on Vesta, on the basis of spectral and colour data, but other observations did not confirm its presence. Here we report that olivine is indeed present locally on Vesta's surface but that, unexpectedly, it has not been found within the deep, south-pole basins, which are thought to be excavated mantle rocks. Instead, it occurs as near-surface materials in the northern hemisphere. Unlike the meteorites, the olivine-rich (more than 50 per cent by volume) material is not associated with diogenite but seems to be mixed with howardite, the most common surface material. Olivine is exposed in crater walls and in ejecta scattered diffusely over a broad area. The size of the olivine exposures and the absence of associated diogenite favour a mantle source, but the exposures are located far from the deep impact basins. The amount and distribution of observed olivine-rich material suggest a complex evolutionary history for Vesta.

Guidelines for the use of long-term central venous catheter in children with hemato-oncological disorders. On behalf of supportive therapy working group of Italian Association of Pediatric Hematology and Oncology (AIEOP).

In the last 30 years, the use of long-term central venous catheters (CVC) is increased especially for children with hemato-oncological disorders. However, the use of CVC is associated to complications, as mechanical accidents, thrombosis, and infections that can determine a prolongation of hospital stay, an increase of costs, and sometimes life-threatening conditions that require urgent systemic treatment or CVC removal. CVC removal may be troublesome especially in neonates, infants, or any other "highly needed CVC patients"; in these selected cases, the prevention and treatment of CVC-related complications play a pivotal role and specific surveillance programs are crucial. While extensive literature is focused on CVC management in adults, no guidelines are available for children. To this aim, the first recommendations for the management of CVC infectious complication in pediatric age have been written after pediatric and adult literature review and collegial discussion among members of Supportive Therapy working group of Italian Association of Pediatric Hematology and Oncology. Compared to the adult age, the necessity of peripheral vein cultures for the diagnosis of CVC-related infection remains controversial in children because of the poorer venous asset and a conservative, pharmacologically focused management through CVC remains mandatory, with CVC removal to be performed only in selected cases.

Spectroscopic characterization of mineralogy and its diversity across Vesta.

The mineralogy of Vesta, based on data obtained by the Dawn spacecraft's visible and infrared spectrometer, is consistent with howardite-eucrite-diogenite meteorites. There are considerable regional and local variations across the asteroid: Spectrally distinct regions include the south-polar Rheasilvia basin, which displays a higher diogenitic component, and equatorial regions, which show a higher eucritic component. The lithologic distribution indicates a deeper diogenitic crust, exposed after excavation by the impact that formed Rheasilvia, and an upper eucritic crust. Evidence for mineralogical stratigraphic layering is observed on crater walls and in ejecta. This is broadly consistent with magma-ocean models, but spectral variability highlights local variations, which suggests that the crust can be a complex assemblage of eucritic basalts and pyroxene cumulates. Overall, Vesta mineralogy indicates a complex magmatic evolution that led to a differentiated crust and mantle.

The surface composition and temperature of asteroid 21 Lutetia as observed by Rosetta/VIRTIS.

The Visible, InfraRed, and Thermal Imaging Spectrometer (VIRTIS) on Rosetta obtained hyperspectral images, spectral reflectance maps, and temperature maps of the asteroid 21 Lutetia. No absorption features, of either silicates or hydrated minerals, have been detected across the observed area in the spectral range from 0.4 to 3.5 micrometers. The surface temperature reaches a maximum value of 245 kelvin and correlates well with topographic features. The thermal inertia is in the range from 20 to 30 joules meter(-2) kelvin(-1) second(-0.5), comparable to a lunarlike powdery regolith. Spectral signatures of surface alteration, resulting from space weathering, seem to be missing. Lutetia is likely a remnant of the primordial planetesimal population, unaltered by differentiation processes and composed of chondritic materials of enstatitic or carbonaceous origin, dominated by iron-poor minerals that have not suffered aqueous alteration.

Role of the hypoxic microenvironment in the antitumor activity of tyrosine kinase inhibitors.

The tumor microenvironment is characterized, not only by marked gradients in drug concentration, but also by gradients in the rate of cell proliferation and by regions of hypoxia and acidity, all of which can influence tumor cell sensitivity to drug treatment. Hypoxia is also an important environmental factor in chronic myeloid leukemia (CML), because bone marrow is intrinsically hypoxic in nature. Systems-wide analyses of tumors have recently identified receptor tyrosine kinase coactivation as an important mechanism by which cancer cells achieve chemoresistance. Recent work suggests that Src activation might play a prominent role in the response to hypoxia to promote cell survival, progression, and metastasis of a variety of human cancer. Other studies also established a functional link between Bcr-Abl and the Src family tyrosine kinases. It is well known that mutations can also cause some tyrosine kinases to become constitutively active, a nonstop functional state that may contribute to initiation or progression of cancer as in CML. Leukemic cells carrying chromosomal alteration, are sensitive to imatinib that induces complete remission in most patients. This inhibitor is a highly selective Bcr-Abl tyrosine kinase inhibitor (TKI). There is a considerable interest in understanding how activated signaling pathways enhance tumor cell survival under hypoxia, because this might lead to the introduction of more effective treatments to target these resistant subpopulations. For all these reasons it is important to identify new TKIs which are also active in hypoxia, the real tumor microenvironment, as possible alternative therapy.

Adenosine kinase gene expression in human colorectal cancer.

Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate gene expression of adenosine kinase, a key enzyme in adenosine metabolism, in human intestinal biopsy specimens of 10 colorectal cancer patients. Quantitative mRNA expression levels were normalized against the reference gene beta-actin. The results showed that adenosine kinase gene expression was significantly higher in cancer than in normal-appearing tissue, in line with our previous measurements of adenosine kinase enzyme activities in colorectal tumor samples.

Analysis of protease-activated receptor-1 and -2 in human scar formation.

Protease-activated receptor (PAR)-1 and PAR-2 are reported to contribute to the fibrotic process in a number of organs, including lung, liver, pancreas, and kidney. The aim of this study was to localize expression and biological activity of PAR-1 and PAR-2 in normal and pathological cutaneous scars. First, we investigated the immunohistochemical expression of PAR-1 and PAR-2 proteins in a series of human normal scars (NS, n = 10), hypertrophic scars (HS, n = 10), and keloids (K, n = 10). Expression of PAR-1 and PAR-2 was observed in all types of scar. Specifically, in HS and K, diffuse PAR-1 and PAR-2 positivity was found in dermal cellular areas composed of myofibroblasts, while no or minor staining was observed in the scattered fibroblasts embedded in abundant extracellular matrix in the context of the more collagenous nodules, irrespective of the type of scar. The hyperplastic epidermis overlying K was also found to be strongly PAR-1 and PAR-2 positive, whilst in most NS and HS the epidermis was faintly to moderately stained. Second, ribonuclease protection assay on paraffin-embedded specimens showed overexpression of PAR-1 and PAR-2 mRNA in K compared to NS and HS. Third, cultured human fibroblasts exposed to TGF-beta1 expressed a myofibroblast phenotype associated with overexpression of PAR-2, while PAR-1 expression was unaffected. Intracellular Ca(2+) mobilization by PAR-2 agonists in myofibroblasts was increased as compared to fibroblasts, whereas the effect of PAR-1 agonists was unchanged. Our in vivo study indicates that PAR-1 and PAR-2 are expressed in cells involved in physiological and pathological scar formation and suggests that in vitro overexpression and exaggerated functional response of PAR-2 may play a role in the function of myofibroblasts in scar evolution from a physiological repair process to a pathological tissue response.

Osteosarcoma cell line growth inhibition by zoledronate-stimulated effector cells.

Bisphosphonates have a profound effect on bone resorption and are widely used in the treatment of osteoclast-mediated bone diseases. Zoledronic acid (ZA), a third-generation biphosphonate, has a potent antitumor activity and expands gammadelta (gammadelta) T cells endowed of major histocompatibility complex-unrestricted lytic activity. Many solid tumors express tumor-specific antigens on their surface, representing targets for immune effector T cells. Nevertheless, the immune surveillance against clinically manifested tumors is relatively inefficient. Therefore, we investigated the hitherto unknown effects of ZA activated gammadelta T cells of normal donors on osteosarcoma cell lines. gammadelta T cells were stimulated with ZA and low doses of interleukin-2, and then analyzed for proliferation and generation of effector activity against osteosarcoma cell lines. Our results show the potent anti-tumor activity of ZA-stimulated gammadelta T cells and the enhanced immunosensitivity of osteosarcoma cell lines to gammadelta T cells suggesting that osteosarcoma is another gammadelta T cell susceptible tumor type.

New pyrazolo[3,4-d]pyrimidines endowed with A431 antiproliferative activity and inhibitory properties of Src phosphorylation.

New 4-aminopyrazolo[3,4-d]pyrimidines bearing various substituents at the position 1 and 6, were synthesized. The new compounds showed antiproliferative activity toward A431 cells, were found to be inhibitors of Src phosphorylation, and induced apoptotic cell death. In particular, 2h was a better inhibitor of Src phosphorylation than the reference compound PP2.

Inhibition of interleukin-12 expression by alpha-thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a powerful regulator of inflammatory responses and is known to affect cell-mediated immunity. Interleukin (IL)-12 is a strong promoter of the development of Th1-type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL-6, a Th2-related cytokine, in human peripheral blood mononuclear cells (PBMC). Here we show that thrombin downregulates IL-12 production at both protein and mRNA levels in human PBMC. The inhibition of IL-12 production was accompanied by an enhanced release of IL-10, which inhibits Th1-related processes and promotes Th2-type responses. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin-specific and requires thrombin proteolytic activity. In addition, activation of coagulation inhibits IL-12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation. Decreased IL-12 production appears to be related also to IL-10 production, since the addition of an anti-IL-10 monoclonal antibody to thrombin-treated PBMC resulted in a partial restoration of IL-12 production. In conclusion, the observation that thrombin significantly affects the production of IL-12, as well as of IL-10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.

Regulation of angiogenesis by Th1- and Th2-type cytokines.

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.

Hypoxia induces the expression and release of interleukin 1 receptor antagonist in mitogen-activated mononuclear cells.

Hypoxia modulates the expression of inflammatory mediators in a variety of cell types. Since interleukin (IL-)1 receptor antagonist (Ra) is a cytokine widely associated with an inflammatory state and is expressed by activated mononuclear cells, we investigated whether hypoxia induces IL-1Ra expression in human peripheral blood mononuclear cells (PBMC) activated by phytohaemagglutinin (PHA). RNase protection assay, conducted on PHA-activated PBMC cultured under hypoxic conditions (2% O(2)) for 16-40 h, revealed that hypoxia enhances IL-1Ra mRNA expression. Further, IL-1Ra release was significantly affected by hypoxia, as determined by ELISA. Concomitantly, hypoxia enhanced, even though at a lesser extent, both IL-1alpha and IL-1beta mRNA expression and release, as determined by RPA and ELISA. However, at 40 h of treatment, hypoxia did not affect cell viability and DNA fragmentation, but caused an inhibition of the proliferation index after PHA stimulation, obtained by MTT assay. These results suggest that activated mononuclear cells tend to respond to hypoxic stress by modulating the expression of IL-1Ra and IL-1-related molecules and their release in the surrounding microenvironment.

[The domestic processing of the common bean resulted in a reduction in the phytates and tannins antinutritional factors, in the starch content and in the raffinose, stachiose and verbascose flatulence factors]. / O processamento doméstico do feijão-comum ocasionou uma redução nos fatores antinutricionais fitatos e taninos, no teor de amido e em fatores de flatulência rafinose, estaquiose e verbascose.

The objective of this study was to evaluate the effect of the soaking step and the domestic processing of the common bean, on the chemical composition, the levels of phytate, tannin, starch and flatulence factors by utilizing the follows treatments: raw bean (FC), freeze-dried cooked unsoaked bean (FCSM), freeze-dried cooked bean without the non-absorbed soaking water (FCSAM), freeze-dried cooked bean with the non-absorbed soaking water (FCCAM) and the soaking water (AM). The beans were soaking for a period for 16 hours in the proportion 3:1 (water:beans) at room temperature. The effect of the phytates and tannins on the net protein efficiency ratio (NPR) and protein digestibility using male Wistar rats were studied. A decrease in the phytate content of the beans (85%) with use of soaking was observed. In the case of the tannin content, only the cooking of the beans promoted high decomposition (84%). In the (FCSAM) treatment a decrease in the raffinose (25.0%), stachiose (24.8%), verbascose (41.7%) and starch (26.8%) contents was observed. Diets containing casein (control), casein plus the soluble solids obtain from the soaking water showed no significant difference (p > 0.05) for the NPR, as well as for the different bean treatments, although these showing lower values. The treatment (FCSM) showed the higher digestibility (74.3 +/- 5.8%) of the bean treatments, the casein diets showing 94.6 +/- 0.9%. The reduction of the phytates, tannin, starch contents and flatulence factors in the common bean was most effective when the soaking water not absorbed was discarded (FCSAM).

Thrombin regulates the expression of proangiogenic cytokines via proteolytic activation of protease-activated receptor-1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types, including monocytes and endothelial cells, involved in the control of angiogenesis. Different cytokines produced by mononuclear cells have been implicated in angiogenic processes associated with tissue repair and certain human malignancies. We have previously shown that thrombin enhances proliferative responses in T lymphocytes. More recently, we reported that interferon-gamma-differentiated monocytes have increased expression of protease-activated receptor-1 (PAR-1) and increased thrombin binding. Since cytokines may be involved directly and indirectly in angiogenesis, we initiated studies to determine thrombin effects on the induction of cytokines, such as interleukin (IL)-1 and IL-6, in human mononuclear cells. IL-1 and IL-6 protein expression was significantly enhanced by thrombin (P<.05), as determined by enzyme-linked immunosorbent assay (ELISA). Treating mononuclear cells with the PAR-1 peptide, SFLLRN, has effects similar to those of thrombin. Thus, it appears that these thrombin effects are mediated through activation of PAR-1. These results confirm that thrombin is a strong activator of monocytes and could be involved in angiogenesis by inducing cytokines that could enhance the angiogenic process in tissue repair.

Hypoxia modulates cyclin and cytokine expression and inhibits peripheral mononuclear cell proliferation.

Previously, we found that hypoxia can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC). Here, we demonstrated that the cycle progression of hypoxic PBMC, cultured in the presence or not of a specific T cell activator such as phytohaemagglutinin (PHA), was delayed when compared with aerobic cultures. This delay was accompanied by a decrease of the expression of specific cyclins associated to cell cycle progression phases. Ribonuclease Protection Assay (RPA) studies reveal a decrease in the expression of cyclin A and B in PHA-stimulated PBMC kept for 40 hr under hypoxic condition (2% O(2)), when compared with aerobic cultures (20% O(2)). In concomitance, a decrease of cyclin D2 expression was present after 16 hr of hypoxic treatment. However, the decrease was transient and disappeared after 40 hr of hypoxic treatment. Furthermore, cyclin C expression was not affected by hypoxia. Hypoxia-induced cyclin modulation was accompanied by an increased synthesis of interleukin (IL)-2 and IL-4, analyzed by ELISA. By evaluating these results, it appears that hypoxia induces a growth suppressive state in mitogen-activated PBMC by inhibiting the synthesis of mitotic cyclins A and B. However hypoxic PBMC maintain their viability and capability of producing stimulatory cytokines, after mitogen treatment. This should be important in local hypoxia, usually associated with necrotic areas, in inflammation, and infections, where T lymphocyte capability of producing stimulatory cytokines is desirable.

Interleukin 10 production in patients undergoing cardiopulmonary bypass: evidence of inhibition of Th-1-type responses.

The cardiopulmonary bypass (CPB) procedure has long been associated with a generalized immunosuppression. To understand further the cytokine-mediated regulation of the complex physiological and immunological changes induced by CPB, the authors decided to investigate whether CPB affects the release of interleukin (IL)-10, as well as other cytokines, in correlation to the inhibition of T cell responses. Using phytohaemagglutinin (PHA) as mitogen and peripheral blood mononuclear cells (PBMC) isolated from patients undergoing CPB, we investigated whether this procedure has an effect on the secretion of different patterns of cytokines (Th1- and Th2-type) and PBMC proliferation. In all patients, CPB significantly enhances IL-10 and IL-6 production in resting and PHA-stimulated PBMC. On the other hand, IL-2 production, in response to PHA, was significantly diminished. Reduced IL-2 and enhanced IL-10 production were associated with a significant decrease in PBMC proliferation. Immunosuppression was also associated to lymphopenia, while neutrophil counts were significantly enhanced. These results show that after CPB there is a transient but clear unbalanced immune response demonstrated by a differentiated production of Th1- and Th2-type cytokines. The release of different patterns of cytokines observed after CPB may be helpful in understanding and preventing the development of infectious and immune complications in surgical procedure employing CPB.

Hypoxia affects cytokine production and proliferative responses by human peripheral mononuclear cells.

We have shown that hypoxia (2% O2 approximately pO2 14 mmHg) as opposed to O2 atmospheric pressure (20.9% O2 approximately pO2 140 mmHg) can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC) in the presence or absence of a specific T-cell activator such as phytohemagglutinin (PHA). In hypoxia, interleukin (IL)-2, IL-4, and interferon (IFN)-gamma production increased by 110, 70, and 50% over that of controls, respectively, in PHA-stimulated PBMC (P < 0.05). Moreover, in hypoxia, IL-6 production was significantly enhanced in both resting and PHA-stimulated PBMC by 36 and 37%, respectively (P < 0.05). However, in hypoxia, IL-10 production decreased in both resting and stimulated PBMC, being 80 and 67% of controls, respectively (P < 0.05). PBMC proliferation was not significantly affected by hypoxia, although PBMC susceptibility to PHA was about 80% of that of the control (P < 0.05) after 40 hr of treatment, whereas the cycle progression of hypoxic PBMC was delayed. From an evaluation of these results, hypoxia apparently modifies the production of cytokines by PBMC. These results have both theoretical and practical interest because local hypoxia is very common in several conditions, such as inflammation and local ischemia, and is a host-nonspecific defense against infection. Furthermore, these results suggest a differential pattern of cytokine production in vivo in hypoxic tissues.