Towards ethical drug pricing: the European Orphan Genomic Therapies Fund.

An increasing number of novel genomic therapies are expected to become available for patients with rare or ultra-rare diseases. However, the primary obstacle to equal patient access to these orphan genomic therapies are currently very high prices charged by manufacturers in the context of limited healthcare budgets. Taking into account ethical pricing theories, the paper proposes the implementation of a pricing infrastructure covering all European member states, which has the potential to promote distributive justice while maintaining the attractiveness of genomic therapy development.

Human germline editing in the era of CRISPR-Cas: risk and uncertainty, inter-generational responsibility, therapeutic legitimacy.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) technology may allow for efficient and highly targeted gene editing in single-cell embryos. This possibility brings human germline editing into the focus of ethical and legal debates again. MAIN BODY: Against this background, we explore essential ethical and legal questions of interventions into the human germline by means of CRISPR-Cas: How should issues of risk and uncertainty be handled? What responsibilities arise regarding future generations? Under which conditions can germline editing measures be therapeutically legitimized? For this purpose, we refer to a scenario anticipating potential further development in CRISPR-Cas technology implying improved accuracy and exclusion of germline transmission to future generations. We show that, if certain concepts regarding germline editing are clarified, under such conditions a categorical prohibition of one-generation germline editing of single-cell embryos appears not to be ethically or legally justifiable. CONCLUSION: These findings are important prerequisites for the international debate on the ethical and legal justification of germline interventions in the human embryo as well as for the harmonization of international legal standards.

Coarse-graining strategy for modeling effective, highly diffusive fluids with reduced polydispersity: A dynamical study.

We present a coarse-graining strategy for reducing the number of particle species in mixtures to achieve a simpler system with higher diffusion while preserving the total particle number and characteristic dynamic features. As a system of application, we chose the bidisperse Lennard-Jones-like mixture, discovered by Kob and Andersen [Phys. Rev. Lett. 73, 1376 (1994)], possessing a slow dynamics due to the fluid's multi-component character with its apparently unconventional choice for the pair potential of the type-A-type-B arrangement. We further established in a so-formed coarse-grained and temperature-independent monodisperse system an equilibrium structure with a radial distribution function resembling its mixture counterpart. This one-component system further possesses similar dynamic features such as glass transition temperature and critical exponents while subjected to Newtonian mechanics. This strategy may finally lead to the manufacturing of new nanoparticle/colloidal fluids by experimentally modeling only the outcoming effective pair potential(s) and no other macroscopic quantity.

Framing the ethical and legal issues of human artificial gametes in research, therapy, and assisted reproduction: A German perspective.

Recent results from studies on animals suggest that functional germ cells may be generated from human pluripotent stem cells, giving rise to three possibilities: research with these so-called artificial gametes, including fertilization experiments in vitro; their use in vivo for therapy for the treatment of human infertility; and their use in assisted reproductive technologies in vitro. While the legal, philosophical, and ethical questions associated with these possibilities have been already discussed intensively in other countries, the debate in Germany is still at its beginning. A systematic and detailed analysis of the legal framework in Germany is provided with regard to the three possibilities, including the applicable statutory laws as well as the constitutional law. The question emerges as to whether the statutory laws as well as the constitution justify a distinction to be made between embryos of artificial and natural origin. This question is subject to philosophical analysis, discussing the distinction between person and thing, dignity and price, personality and property, and nature and technique. As a result, the criterion of naturalness alone may not be sufficient to differentiate between embryos of natural and artificial origin, and other criteria need to be identified.

Coarse-graining strategy for molecular pair interactions: A reaction coordinate study for two- and three-dimensional systems.

We investigate and provide optimal sets of reaction coordinates for mixed pairs of molecules displaying polar, uniaxial, or spherical symmetry in two and three dimensions. These coordinates are non-redundant, i.e., they implicitly involve the molecules' symmetries. By tabulating pair interactions in these coordinates, resulting tables are thus minimal in length and require a minimal memory space. The intended fields of application are computer simulations of large ensembles of molecules or colloids with rather complex interactions in a fluid or liquid crystalline phase at low densities. Using effective interactions directly in the form of tables can help bridging the time and length scales without introducing errors stemming from any modeling procedure. Finally, we outline an exemplary computational methodology for gaining an effective pair potential in these coordinates, based on the Boltzmann inversion principle, by providing a step-by-step recipe.

Equilibrium structures of anisometric, quadrupolar particles confined to a monolayer.

We investigate the structural properties of a two-dimensional system of ellipsoidal particles carrying a linear quadrupole moment in their center. These particles represent a simple model for a variety of uncharged, non-polar conjugated organic molecules. Using optimization tools based on ideas of evolutionary algorithms, we first examine the ground state structures as we vary the aspect ratio of the particles and the pressure. Interestingly, we find, besides the intuitively expected T-like configurations, a variety of complex structures, characterized with up to three different particle orientations. In an effort to explore the impact of thermal fluctuations, we perform constant-pressure molecular dynamics simulations within a range of rather low temperatures. We observe that ground state structures formed by particles with a large aspect ratio are in particular suited to withstand fluctuations up to rather high temperatures. Our comprehensive investigations allow for a deeper understanding of molecular or colloidal monolayer arrangements under the influence of a typical electrostatic interaction on a coarse-grained level.

Coarse-grained electrostatic interactions of coronene: Towards the crystalline phase.

In this article, we present and compare two different, coarse-grained approaches to model electrostatic interactions of disc-shaped aromatic molecules, specifically coronene. Our study builds on our previous work [T. Heinemann et al., J. Chem. Phys. 141, 214110 (2014)], where we proposed, based on a systematic coarse-graining procedure starting from the atomistic level, an anisotropic effective (Gay-Berne-like) potential capable of describing van der Waals contributions to the interaction energy. To take into account electrostatics, we introduce, first, a linear quadrupole moment along the symmetry axis of the coronene disc. The second approach takes into account the fact that the partial charges within the molecules are distributed in a ring-like fashion. We then reparametrize the effective Gay-Berne-like potential such that it matches, at short distances, the ring-ring potential. To investigate the validity of these two approaches, we perform many-particle molecular dynamics simulations, focusing on the crystalline phase (karpatite) where electrostatic interaction effects are expected to be particularly relevant for the formation of tilted stacked columns. Specifically, we investigate various structural parameters as well as the melting transition. We find that the second approach yields consistent results with those from experiments despite the fact that the underlying potential decays with the wrong distance dependence at large molecule separations. Our strategy can be transferred to a broader class of molecules, such as benzene or hexabenzocoronene.

Angle-resolved effective potentials for disk-shaped molecules.

We present an approach for calculating coarse-grained angle-resolved effective pair potentials for uniaxial molecules. For integrating out the intramolecular degrees of freedom we apply umbrella sampling and steered dynamics techniques in atomistically-resolved molecular dynamics (MD) computer simulations. Throughout this study we focus on disk-like molecules such as coronene. To develop the methods we focus on integrating out the van der Waals and intramolecular interactions, while electrostatic charge contributions are neglected. The resulting coarse-grained pair potential reveals a strong temperature and angle dependence. In the next step we fit the numerical data with various Gay-Berne-like potentials to be used in more efficient simulations on larger scales. The quality of the resulting coarse-grained results is evaluated by comparing their pair and many-body structure as well as some thermodynamic quantities self-consistently to the outcome of atomistic MD simulations of many-particle systems. We find that angle-resolved potentials are essential not only to accurately describe crystal structures but also for fluid systems where simple isotropic potentials start to fail already for low to moderate packing fractions. Further, in describing these states it is crucial to take into account the pronounced temperature dependence arising in selected pair configurations due to bending fluctuations.

HLA-DR alpha 2 mediates negative signalling via binding to Tirc7 leading to anti-inflammatory and apoptotic effects in lymphocytes in vitro and in vivo.

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRalpha2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-zeta chain & ZAP70, and inhibition of IFN-gamma and FasL expression. HLA-DRalpha2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRalpha2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.

T-cell immune response cDNA 7 in allograft rejection and inflammation.

The membrane protein T-cell immune response cDNA 7 (TIRC7) is transiently expressed in subsets of lymphocytes following antigen stimulation. The importance of TIRC7 in immune activation is demonstrated by the effect of antibodies directed against extracellular domains of TIRC7. In vitro targeting of TIRC7 inhibits proliferation and cytokine expression in human, mouse and rat lymphocytes, and these inhibitory effects have been associated with induction of cytotoxic T-lymphocyte antigen 4 mRNA and protein in the presence of TIRC7 antibodies. In vivo, anti-TIRC7 antibodies prevent kidney transplant rejection in rats and heart allograft rejection in mice. Treatment with an anti-TIRC7 antibody as monotherapy or in combination with TNFalpha blockade inhibits disease progression in collagen-induced arthritis. TIRC7 expression decreases in the peripheral blood of humans who have undergone cardiac transplant prior to clinical rejection, and is therefore a promising noninvasive tool for the prediction of rejection. Thus, targeting of TIRC7 may lead to the development of specific and effective therapeutic and diagnostic approaches by unifying relevant cellular and molecular responses in T- and B-cell subsets, and represents a promising new pathway for immune regulation in transplantation and autoimmune disease.

TIRC7 inhibits T cell proliferation by modulation of CTLA-4 expression.

Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.

TIRC7 deficiency causes in vitro and in vivo augmentation of T and B cell activation and cytokine response.

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7(-/-) mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7(-/-) mice exhibited significantly increased proliferation and expression of IL-2, IFN-gamma, and IL-4 in response to different stimuli. Resting T cells from TIRC7(-/-) mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7(-/-) mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.

Monoclonal antibody specific for TIRC7 induces donor-specific anergy and prevents rejection of cardiac allografts in mice.

T cell immune response c-DNA (TIRC7) is up-regulated during the early stages of T-cell activation in response to alloantigens. In this study, we analyzed the effects of newly developed monoclonal antibodies (mAb) against TIRC7 in acute cardiac allograft rejection. Fully vascularized heterotopic allogeneic heart transplantation was performed in mice across a full-mismatch barrier (C57Bl/10 into CBA). Recipients received seven injections (day 0-7) of a novel anti-TIRC7 mAb or remained untreated. Graft survival, histology and ex vivo lymphocyte functions were tested. Targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into grafts resulting in delay of morphological graft damage and prolongation of allograft survival. The lymphocytes from anti-TIRC7 mAb-treated animals exhibit hypo-responsiveness without evidence of lymphocyte depletion against the donor allo-antigens. Proliferation and expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were down-regulated while interleukin-4 (IL-4) and IL-10 expression were spared. Moreover, anti-TIRC7 mAb enhanced up-regulation of CTLA-4 expression but suppressed up-regulation of CD25 on stimulated lymphocytes in vitro and in vivo. Ligation of TIRC7 has important effects on the regulation of co-stimulatory signaling pathways associated with suppressing of T-cell activation. Targeting of TIRC7 may therefore provide a novel therapeutic approach for modulating T cell immune responses during organ transplantation.

Principles of ethical decision making regarding embryonic stem cell research in Germany.

The availability of embryonic stem (ES) cells isolated from human blastocysts may open novel avenues for medical treatment of otherwise incurable diseases. Yet the generation of human ES cells requires the destruction of early human embryos. This confronts us with the moral problem of whether it is justifiable to sacrifice human life in order to treat other human life. This article outlines the development of the German debate about research with ES cells and explicates the arguments that are central to that debate with respect to the aims and means of research with ES cells. With regard to the means, the isolation of ES cells from human embryos raises the question of the moral status of the human embryo. A restrictive position acknowledges the human dignity of the embryo in its very early stage of development and claims that the embryo's life must be protected accordingly. In contrast, a gradualist position acknowledges human dignity, and therefore the full level of protection, only when the embryo has reached a certain stage of development. In addition, the intentions behind the generation of human embryos, i.e. exclusively for research purposes, and the mode of generating them, i.e. by nuclear transfer technology, have strong ethical relevance in the German debate. Based on these results, the ethical reasoning underlying the draft of a Stem Cell Act recently passed by the German Parliament is outlined.

Cis-4-methylsphingosine phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK mitogen-activated protein kinases.

Intracellular phosphorylation of cis-4-methylsphingosine was previously shown to result in a metabolically stable compound that accumulates in Swiss 3T3 fibroblasts and mimics the mitogenic effect induced by the short-lived sphingosine metabolite, sphingosine-1-phosphate. In the present study incubation of neuroblastoma B104 cells with cis-4-methylsphingosine (10 microM) also resulted in an intracellular accumulation of its phosphorylated derivative that was, however, associated with the concentration-dependent induction of apoptosis, not observed after treatment with 10 microM of sphingosine-1-phosphate or sphingosine, respectively. In B104 cells, cis-4-methylsphingosine stimulated p38 mitogen-activated protein kinase (p38 MAPK) and simultaneously inhibited extracellular signal-regulated kinase (ERK), whereas sphingosine and sphingosine-1-phosphate only stimulated p38 MAPK without suppression of ERK. Inhibition of cis-4-methylsphingosine phosphorylation reduced both, apoptosis and concurrent regulation of mitogen-activated protein kinases (MAPKs), suggesting that the unusual accumulation of the phosphorylated sphingoid base was responsible for the biological effects. Furthermore, inhibition of p38 MAPK prevented cis-4-methylsphingosine-induced apoptosis, while suppression of the ERK pathway in the presence of sphingosine or sphingosine-1-phosphate resulted in apoptosis, indicating that the simultaneous opposite regulation of the two MAPKs was required for the induction of apoptosis.