Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Measurement of the Vector and Tensor Asymmetries at Large Missing Momentum in Quasielastic (e[over →],e

We report the measurement of the beam-vector and tensor asymmetries A_{ed}^{V} and A_{d}^{T} in quasielastic (e[over →],e^{'}p) electrodisintegration of the deuteron at the MIT-Bates Linear Accelerator Center up to missing momentum of 500 MeV/c. Data were collected simultaneously over a momentum transfer range 0.1

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages.

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.

Precise measurement of deuteron tensor analyzing powers with BLAST.

We report a precision measurement of the deuteron tensor analyzing powers T(20) and T(21) at the MIT-Bates Linear Accelerator Center. Data were collected simultaneously over a momentum transfer range Q=2.15-4.50 fm(-1) with the Bates Large Acceptance Spectrometer Toroid using a highly polarized deuterium internal gas target. The data are in excellent agreement with calculations in a framework of effective field theory. The deuteron charge monopole and quadrupole form factors G(C) and G(Q) were separated with improved precision, and the location of the first node of G(C) was confirmed at Q=4.19±0.05 fm(-1). The new data provide a strong constraint on theoretical models in a momentum transfer range covering the minimum of T(20) and the first node of G(C).

Charge form factor of the neutron at low momentum transfer from the 2H-->(e-->,e'n)1H reaction.

We report new measurements of the neutron charge form factor at low momentum transfer using quasielastic electrodisintegration of the deuteron. Longitudinally polarized electrons at an energy of 850 MeV were scattered from an isotopically pure, highly polarized deuterium gas target. The scattered electrons and coincident neutrons were measured by the Bates Large Acceptance Spectrometer Toroid (BLAST) detector. The neutron form factor ratio GEn/GMn was extracted from the beam-target vector asymmetry AedV at four-momentum transfers Q2=0.14, 0.20, 0.29, and 0.42 (GeV/c)2.

-Measurement of the proton's electric to magnetic form factor ratio from 1H(over -->)(e(over -->),e'p).

We report the first precision measurement of the proton electric to magnetic form factor ratio from spin-dependent elastic scattering of longitudinally polarized electrons from a polarized hydrogen internal gas target. The measurement was performed at the MIT-Bates South Hall Ring over a range of four-momentum transfer squared Q2 from 0.15 to 0.65 (GeV/c)(2). Significantly improved results on the proton electric and magnetic form factors are obtained in combination with existing cross-section data on elastic electron-proton scattering in the same Q2 region.

Measurements of the generalized electric and magnetic polarizabilities of the proton at low Q2 using the virtual-compton-scattering reaction.

The mean square polarizability radii of the proton have been measured for the first time in a virtual-Compton-scattering experiment performed at the MIT-Bates out-of-plane scattering facility. Response functions and polarizabilities obtained from a dispersion analysis of the data at Q2 = 0.057 GeV2/c2 are in agreement with O(p3) heavy baryon chiral perturbation theory. The data support the dominance of mesonic effects in the polarizabilities.

The influence of refractory ceramic fibres on pulmonary morphology, redox and immune system in rats.

Refractory ceramic fibres (RCF) were studied in male SPRD rats by both in vivo long term sequential and in vitro methods. RCF was administered by single intratracheal instillation and the lungs were examined at the end of months 1, 3 and 6 after exposure. In addition, the direct toxicity of the fibres was examined in a primary culture of alveolar macrophages (AM) and in pneumocytes type II (T2). Pulmonary morphological changes, a number of parameters of the redox system, such as activity of extracellular Cu,Zn/superoxide dismutase (EC-SOD), total glutathione content of the lungs (GSH) and immunoglobulins in bronchoalveolar lavage (IgA, IgG, IgM) and in the blood were measured. The composition of the original RCF and the elemental content of the lung tissue were compared by energy dispersive x-ray analysis (EDXA) before and after exposure. Macrophage alveolitis became confluent and moderate fibrosis developed by the end of month 3, and after 6 months of exposure the intensity decreased to the level of the first month. The RCF did not significantly influence the activity of EC-SOD or the total glutathione content of the lungs. Although aluminium and silicon could be demonstrated by EDXA in the lung tissue at the end of month 3, these elements were no longer detectable by the end of month 6. The RCF decreased IgA significantly in bronchoalveolar lavage (BAL). The main components of RCF induced pulmonary alterations, whereas no significant change could be detected in EC-SOD and GSH. Injuries caused by direct toxicity could be observed in the cell membranes only at the highest concentration. On the basis of these results RCF can be determined as moderately toxic fibres.

Investigation of the conjectured nucleon deformation at low momentum transfer.

We report new precise H(e,e(')p)pi(0) measurements at the Delta(1232) resonance at Q(2)=0.127 (GeV/c)(2) obtained at the MIT-Bates out-of-plane scattering facility which are particularly sensitive to the transverse electric amplitude (E2) of the gamma(*)N-->Delta transition. The new data have been analyzed together with those of earlier measurements to yield precise quadrupole to dipole amplitude ratios: Re(E(3/2)(1+)/M(3/2)(1+))=(-2.3+/-0.3(stat+syst)+/-0.6(model))% and Re(S(3/2)(1+)/M(3/2)(1+))=(-6.1+/-0.2(stat+syst)+/-0.5(model))% for M(3/2)(1+)=(41.4+/-0.3(stat+syst)+/-0.4(model))(10(-3)/m(pi(+))). The derived amplitudes give credence to the conjecture of deformation in hadrons favoring, at low Q2, the dominance of mesonic effects.

Pulmonary toxicity of wollastonite in vivo and in vitro.

Pulmonary toxicity of wollastonite has been studied in both in vivo long-term sequential and in vitro methods in Sprague-Dawley rats. Wollastonite was administered by intratracheal instillation and the lungs were examined after 1, 3 and 6 months by morphological methods. UICC crocidolite was applied as the positive control. In addition, the effects of both fibres were examined in primary cultures of pulmonary alveolar macrophages and type II pneumocytes to determine the effects of the fibres on the membranes of these cells, the activity of Cu,Zn/superoxide dismutase and the redox system and the release of proinflammatory peptides: macrophage chemoattractant protein-1 (MCP-1) and macrophage inhibitory protein-1alpha (MIP-1alpha). By the end of six months wollastonite had induced mild pulmonary interstitial fibrosis, whereas crocidolite induced progressive interstitial fibrosis as a function of time. The membranes of macrophages and pneumocytes were disrupted at the lowest concentration of crocidolite. The activity of enzymes of the redox system and cytoplasmic superoxide dismutase significantly decreased with crocidolite. Wollastonite decreased only the activity of gamma-glutamyl transpeptidase and glutathione peroxidase. Crocidolite induced expression of the proinflammatory peptides at the lowest concentration (1 micro g ml(-1)) but wollastonite increased production of these peptides only at medium and high concentrations (5 and 10 micro g ml(-1)). These results underline the importance of further human epidemiological studies and the need for the determination of a hygienic standard.

Spin-dependent electron-proton scattering in the Delta-excitation region.

We report on measurements of the cross section and provide first data on spin correlation parameters A(TT') and A(TL') in inclusive scattering of longitudinally polarized electrons from nuclear-polarized hydrogen. Polarized electrons were injected into an electron storage ring operated at a beam energy of 720 MeV. Polarized hydrogen was produced by an atomic beam source and injected into an open-ended cylindrical cell, located in the electron storage ring. The four-momentum transfer squared ranged from Q2 = 0.2 GeV(2)/c(2) at the elastic scattering peak to Q2 = 0.11 GeV(2)/c(2) at the Delta(1232) resonance. The data provide a stringent test of pion electroproduction models.

Spin-momentum correlations in quasielastic electron scattering from deuterium.

The spin-momentum correlation parameter A(V)(ed) was measured for the 2H-->(e-->,e'p)n reaction for missing momenta up to 350 MeV/c at Q2 = 0.21 (GeV/c)(2) for quasielastic scattering of polarized electrons from vector-polarized deuterium. The data give detailed information about the deuteron spin structure and are in good agreement with the results of microscopic calculations based on realistic nucleon-nucleon potentials and including various spin-dependent reaction mechanism effects. The experiment reveals in a most direct manner the effects of the D state in the deuteron ground-state wave function and shows the importance of isobar configurations for this reaction.

Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor.

The Notch signaling pathway is essential in many cell fate decisions in invertebrates as well as in vertebrates. After ligand binding, a two-step proteolytic cleavage releases the intracellular part of the receptor which translocates to the nucleus and acts as a transcriptional activator. Although Notch-induced transcription of genes has been reported extensively, its endogenous nuclear form has been seldom visualized. We report that the nuclear intracellular domain of Notch1 is stabilized by proteasome inhibitors and is a substrate for polyubiquitination in vitro. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Lin12/Notch-negative regulators in Caenorhabditis elegans. We isolated human and murine counterparts of SEL-10 and investigated the role of a dominant-negative form of this protein, deleted of the F-box, on Notch1 stability and activity. This molecule could stabilize intracellular Notch1 and enhance its transcriptional activity but had no effect on inactive membrane-anchored forms of the receptor. We then demonstrated that SEL-10 specifically interacts with nuclear forms of Notch1 and that this interaction requires a phosphorylation event. Taken together, these data suggest that SEL-10 is involved in shutting off Notch signaling by ubiquitin-proteasome-mediated degradation of the active transcriptional factor after a nuclear phosphorylation event.

Capsid size determination in the P2-P4 bacteriophage system: suppression of sir mutations in P2's capsid gene N by supersid mutations in P4's external scaffold gene sid.

The sid gene of the P2-dependent phage P4 provides an external scaffold so P2 N gene encoded protomers assemble as T = 4 capsids rather than as P2's T = 7 capsids. Mutations (sir) in the middle of N interfere with Sid's function. We describe a new P4 mutant class, nms ("supersid") mutations, which direct also P2 sir to provide small capsids. Three different nms mutations were located near the sid end, commingled with sid(-) mutations. Suppression of sir by nms is not allele-specific. Our results favor this interpretation of capsid size control: (i) sir mutations reduce pN protomer flexibility and thereby interfere with the generation of T = 4 compatible hexons; (ii) the C-termini of Sid molecules link up when forming the scaffold; nms mutations strengthen these Sid-Sid contacts and thus allow the scaffold to force even sir-type protomers to form T = 4 compatible hexons. Some related findings concern suppression of N ts mutations by P4.

Experiments with longitudinally polarized electrons in a storage ring using a siberian snake

We report on first measurements with polarized electrons stored in a medium-energy ring and with a polarized internal target. Polarized electrons were injected at 442 MeV (653 MeV), and a partial (full) Siberian snake was employed to preserve the polarization. Longitudinal polarization at the interaction point and polarization lifetime of the stored electrons were determined with laser backscattering. Spin observables were measured for electrodisintegration of polarized 3He, with simultaneous detection of scattered electrons, protons, neutrons, deuterons, and 3He nuclei, over a large phase space.

The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L).

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.

In vitro and in vivo tests for determination of the pathogenicity of quartz, diatomaceous earth, mordenite and clinoptilolite.

The effects of samples of crystalline quartz, diatomaceous earth, mordenite and clinoptilolite were investigated in vitro (as concerns erythrocyte haemolysis and lactate dehydrogenase (LDH) release from peritoneal macrophages) and in vivo (on LDH, protein and phospholipids in rat bronchoalveolar lavage (BAL), and phospholipids in rat lung tissue). The respirable mineral samples were instilled intratracheally. Determinations in the BAL were carried out after 15, 60 and 180 days, and in the lung tissue after 90, 180 and 360 days. Quartz DQ and quartz FQ induced acute, subacute and chronic inflammation and progressive fibrosis. However, due to the Al2O3 contamination on the surface of the particles quartz FQ caused a delayed response in vivo. Diatomaceous earth produced acute/subacute inflammation that gradually became more moderate after 60 days. Clinoptilolite was inert, whereas the other zeolite sample, mordenite, was cytotoxic in vivo. The reason for this was presumably the needle and rod-shaped particles in the mordenite samples. The investigation revealed that different in vitro and in vivo methods canprovide valuable data concerning the pulmonary toxicity of minerals.

Translation of two nested genes in bacteriophage P4 controls immunity-specific transcription termination.

In phage P4, transcription of the left operon may occur from both the constitutive PLE promoter and the regulated PLL promoter, about 400 nucleotides upstream of PLE. A strong Rho-dependent termination site, timm, is located downstream of both promoters. When P4 immunity is expressed, transcription starting at PLE is efficiently terminated at timm, whereas transcription from PLL is immunity insensitive and reads through timm. We report the identification of two nested genes, kil and eta, located in the P4 left operon. The P4 kil gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the PLE promoter, and its expression is controlled by P4 immunity. Overexpression of kil causes cell killing. This gene is the terminal part of a longer open reading frame, eta, which begins upstream of PLE. The eta gene is expressed when transcription starts from the PLL promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both kil and eta overlap the timm site. By cloning kil upstream of a tRNA reporter gene, we demonstrated that translation of the kil region prevents premature transcription termination at timm. This suggests that P4 immunity might negatively control kil translation, thus enabling transcription termination at timm. Transcription starting from PL proceeds through timm. Mutations that create nonsense codons in eta caused premature termination of transcription starting from PLL. Suppression of the nonsense mutation restored transcription readthrough at timm. Thus, termination of transcription from PLL is prevented by translation of eta.