CD3/CD19 Depletion for T-cell Reduction of Allogeneic Transplants: Mostly Efficient, but not Robust

Aggressive T-cell depletion, in vitro or in vivo, is a prerequisite for survival of haplo-identical stem cell transplantation. The classical T-cell-depleted transplant, immunomagnetically enriched CD34+ cells, is very safe with respect to graft-versus-host reactivity, but associated with very high transplant-related and relapse mortality with an overall probability of survival of only 20%. Protocols for T- and B-cell depletion were therefore developed, reasoning that transplantation of the majority of Natural Killer (NK) cells and the substantial dose of residual T-cells might improve survival, which was, in principle, confirmed. Anecdotal reports of frequent failure to achieve adequate T-cell depletion prompted review of the aggregate data for transplant quality at our center. The first observation is the relative paucity of combined CD3/CD19 depletion processes as PTCy protocols have made inroads, 13 depletions in 8 years. Median T- and B-cell log-depletion were −3.89 and −1.92, respectively; instead of, CD34+ cell recovery was generally high (median 92%), as was NK-cell recovery (median 52%). However, the process failed to yield satisfactory T- and B-cell depletion in two out of 13 preparations, of which one product could be rescued by a second round of depletion, at the expense of CD34+ cell recovery. In our hands, the process is thus insufficiently robust for routine clinical use. Assuming similar observations in other centers, this may explain implementation of alternative protocols, such as TCRαβ/CD19 depletion or transplantation of unmanipulated grafts with subsequent in vivo depletion

Редкие металлы в космической технике

В данной статье рассматривается применение редких металлов в космической технологии. Такжев статье описана классификация редких металлов, уникальные свойства и место каждой группыэлементов в конструировании комических аппаратов.Im vorliegenden Artikel wird die Verwendung von seltenen Metallen in der Raumfahrttechnik betrachtet.Der Artikel beschreibt auch die Klassifizierung von seltenen Metallen, die unikalen Eigenschaften jeder Gruppe undderen Funktion bei der Konstruktion von kosmischen Geräten

Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts

<p>Abstract</p> <p>Background</p> <p>In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.</p> <p>Methods</p> <p>We made use of an <it>in vitro </it>transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.</p> <p>Results</p> <p>Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors <it>in vivo</it>. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.</p> <p>Conclusions</p> <p>Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.</p

Mobilization of hematopoietic stem cells with the novel CXCR4 antagonist POL6326 (balixafortide) in healthy volunteers—Results of a dose escalation trial

Background: Certain disadvantages of the standard hematopoietic stem and progenitor cell (HSPC) mobilizing agent G-CSF fuel the quest for alternatives. We herein report results of a Phase I dose escalation trial comparing mobilization with a peptidic CXCR4 antagonist POL6326 (balixafortide) vs. G-CSF. Methods: Healthy male volunteer donors with a documented average mobilization response to G-CSF received, following ≥6 weeks wash-out, a 1–2 h infusion of 500–2500 µg/kg of balixafortide. Safety, tolerability, pharmacokinetics and pharmacodynamics were assessed. Results: Balixafortide was well tolerated and rated favorably over G-CSF by subjects. At all doses tested balixafortide mobilized HSPC. In the dose range between 1500 and 2500 µg/kg mobilization was similar, reaching 38.2 ± 2.8 CD34 + cells/µL (mean ± SEM). Balixafortide caused mixed leukocytosis in the mid-20 K/µL range. B-lymphocytosis was more pronounced, whereas neutrophilia and monocytosis were markedly less accentuated with balixafortide compared to G-CSF. At the 24 h time point, leukocytes had largely normalized. Conclusions: Balixafortide is safe, well tolerated, and induces efficient mobilization of HSPCs in healthy male volunteers. Based on experience with current apheresis technology, the observed mobilization at doses ≥1500 µg/kg of balixafortide is predicted to yield in a single apheresis a standard dose of 4× 10E6 CD34+ cells/kg from most individuals donating for an approximately weight-matched recipient. Exploration of alternative dosing regimens may provide even higher mobilization responses. Trial Registration European Medicines Agency (EudraCT-Nr. 2011-003316-23) and clinicaltrials.gov (NCT01841476

BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β3 expression

BACKGROUND The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells. METHODS In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay. RESULTS BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion. CONCLUSION Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).Prostatic carcinom

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Transforming growth factor-beta (TGF-beta) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-beta induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-beta-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-beta-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-beta receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-beta or TGF-beta-like activity. TGF-beta-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-beta-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-beta was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-beta/Smad- and MMP2- and MMP9-dependent breast cancer invasion.Urolog

TGF-beta,Smad signaling in hepatic stellate cells and during liver fibrogenesis

Transforming growth factor (TGF)-beta is a key mediator of hepatic stellate cell (HSC) activation and extracellular matrix accumulation leading to fibrosis. Aim of the present study was to perturb the TGF-beta pathway in this process by overexpression of Smad7, an intracellular antagonist of TGF-beta signaling. Moreover, a new TGF-beta response gene was identified, which participates in the control of profibrogenic transdifferentiation to myofibroblast-like cells. Ligation of the common bile duct was used to induce liver fibrosis in rats. Animals received injections of an adenovirus overexpressing Smad7. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary cultured cells. AdSmad7 infected rats displayed reduced collagen deposition and hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed, when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary cultured HSC. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSC was performed, resulting in identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression reduced Id1 expression. Conversely, Id1 overexpression in HSC enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSC interfered with the transdifferentiation process, indicating a pivotal role of Id1 for fibrogenesis. Additionally, treatment of HSC with TGF-beta led to increased Id1 protein expression, which was dependent on activation of the novel ALK1/Smad1/5/8 pathway. Knocking-down expression of ALK1 in HSC interfered with TGF-beta dependent Smad1 phosphorylation and Id1 upregulation, but did not block the activation of the Smad2/3 pathway. In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation. Moreover, a novel TGF-beta pathway involving ALK1 and Smad1 was shown to mediate HSC activation via Id1 upregulation. These new findings represent valuable information for the development of more efficient antifibrotic strategies to treat liver damage in the future

Spheroid Assay to Measure TGF-β-induced Invasion

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage(1,2). Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes(5). In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways(6). To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy