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.

Nrf2-dependent persistent oxidative stress results in stress-induced vulnerability to depression.

Stressful life events produce a state of vulnerability to depression in some individuals. The mechanisms that contribute to vulnerability to depression remain poorly understood. A rat model of intense stress (social defeat (SD), first hit) produced vulnerability to depression in 40% of animals. Only vulnerable animals developed a depression-like phenotype after a second stressful hit (chronic mild stress). We found that this vulnerability to depression resulted from a persistent state of oxidative stress, which was reversed by treatment with antioxidants. This persistent state of oxidative stress was due to low brain-derived neurotrophic factor (BDNF) levels, which characterized the vulnerable animals. We found that BDNF constitutively controlled the nuclear translocation of the master redox-sensitive transcription factor Nrf2, which activates antioxidant defenses. Low BDNF levels in vulnerable animals prevented Nrf2 translocation and consequently prevented the activation of detoxifying/antioxidant enzymes, ultimately resulting in the generation of sustained oxidative stress. Activating Nrf2 translocation restored redox homeostasis and reversed vulnerability to depression. This mechanism was confirmed in Nrf2-null mice. The mice displayed high levels of oxidative stress and were inherently vulnerable to depression, but this phenotype was reversed by treatment with antioxidants. Our data reveal a novel role for BDNF in controlling redox homeostasis and provide a mechanistic explanation for post-stress vulnerability to depression while suggesting ways to reverse it. Because numerous enzymatic reactions produce reactive oxygen species that must then be cleared, the finding that BDNF controls endogenous redox homeostasis opens new avenues for investigation.

Nrf2-dependent persistent oxidative stress results in stress-induced vulnerability to depression.

This corrects the article DOI: 10.1038/mp.2016.144.

Repeated social defeat-induced depression-like behavioral and biological alterations in rats: involvement of cholecystokinin.

Cholecystokinin (CCK) involvement in depression-like disorders is poorly documented. Here, we investigated whether CCKergic neurotransmission is relevant to depressive-like symptoms and antidepressant therapy using a novel preclinical model based on repeated social defeat over 4 weeks in rats. Repeated social defeat triggers changes that could be considered as behavioral and biological correlates of depressive symptoms in humans, such as a hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis (increase of serum corticosterone levels and of adrenal gland weight), increased immobility time in the forced swimming test (FST), decrease of body weight and of sweet water consumption and reduction of hippocampal volume associated with a decreased cell proliferation in the dentate gyrus. In addition, in vivo microdialysis showed that cortical CCK release was tonically increased in defeated rats. Chronic imipramine treatment (16 mg kg(-1) per day for 25 days) prevented both the repeated social defeat-induced alterations of biological and behavioral parameters and the associated increase of cortical CCK release. Chronic blockade of CCK2 receptors by the specific antagonist CI-988 (1 mg kg(-1) per day for 25 days) also normalized immobility time in the FST and prevented HPA axis hyperactivity, reduction of hippocampal volume and cell proliferation and decreased sweet water intake normally evoked by repeated social defeat. These data showed that the repeated social-defeat paradigm can be considered as a suitable model of 'depression' in rats. The causal link between social defeat-evoked (1) increase in cortical CCKergic neurotransmission and (2) depression-like symptoms that we highlighted here strongly suggests that CCKergic systems may be a relevant target for novel antidepressant therapy.

[Low doses ketamine: antihyperalgesic drug, non-analgesic]. / Kétamine à faibles doses: antihyperalgésique, non analgésique.

Recent data in animal experiments as in clinical trials have clearly reported that pain modulation is related to an equilibrium between antinociceptive and pronociceptive systems. Therefore, the apparent pain level could not only be a consequence of a nociceptive input increase but could also result from a pain sensitization process. Glutamate, via NMDA receptors, plays a major role in the development of such a neuronal plasticity in the central nervous system, leading to a pain hypersensitivity that could facilitate chronic pain development. By an action on NMDA receptors opioids also induce, in a dose dependent manner, an enhancement of this postoperative hypersensitivity. "Antihyperalgesic" doses of ketamine, an NMDA receptor antagonist, were able to decrease this central sensitization not only in painful animal but also in human volunteers exposed to different pain models, or in the postoperative period. Many studies have reported that ketamine effects are elicited when this drug is administered the following manner: peroperative bolus (0.1 to 0.5 mg/kg), followed by a constant infusion rate (1 to 2 microg/kg per min) during the peroperative period and for 48 to 72 hours after anaesthesia. Those ketamine doses improved postoperative pain management by reducing hyperalgesia due to both surgical trauma and high peroperative opioid doses. This antihyperalgesic action of ketamine also limited the postoperative morphine tolerance leading to a decrease in analgesic consumption and an increase in the analgesia quality.

Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine.

BACKGROUND: It has been reported that mu-opioid receptor activation leads to a sustained increase in glutamate synaptic effectiveness at the N-methyl-D-aspartate (NMDA) receptor level, a system associated with central hypersensitivity to pain. One hypothesis is that postoperative pain may result partly from the activation of NMDA pain facilitatory processes induced by opiate treatment per se. The authors tested here the effectiveness of the opiate analgesic fentanyl for eliciting a delayed enhancement in pain sensitivity. METHODS: The consequences of four bolus injections (every 15 min) of fentanyl (20-100 microg/kg per injection, subcutaneously) on immediate (for several hours) and long-term (for several days) sensitivity to nociceptive stimuli in the rat (paw-pressure vocalization test) were evaluated. The effects of the combination of the NMDA-receptor antagonist ketamine (10 mg/kg, subcutaneously) with fentanyl also were assessed. RESULTS: Fentanyl administration exhibited a biphasic time-dependent effect: first, an early response (for 2-5 h) associated with a marked increase in nociceptive threshold (analgesia), and second, a later response associated with sustained lowering of the nociceptive threshold (5 days for the longest effect) below the basal value (30% of decrease for the maximal effect) indicative of hyperalgesia. The higher the fentanyl dose used, the more pronounced was the fentanyl-induced hyperalgesia. Ketamine pretreatment, which had no analgesic effect on its own, enhanced the earlier response (analgesia) and prevented the development of long-lasting hyperalgesia. CONCLUSIONS: Fentanyl activates NMDA pain facilitatory processes, which oppose analgesia and lead to long-lasting enhancement in pain sensitivity.

Microporous microparticles designed as stable immunoadsorbents.

We have developed a solid-phase immunoadsorbent based on encapsulated goat anti-apolipoprotein B polyclonal antibodies previously crosslinked with a 0.25% glutaraldehyde solution, and designed to remove by immunoaffinity the excess of apolipoproteins B from the plasma of patients affected by familial hypercholesterolemia. Compared to a classical immunoadsorbent prepared by activation of Sepharose CL-4B with cyanogen bromide, the resulting immunoadsorbent exhibits both optimal adsorption capacity and stability over the entire range of chemical and biochemical conditions during its practical handling. This approach will serve as a model system to demonstrate the applicability of microparticles as immunoadsorbents, which can be achieved for other encapsulated crosslinked proteins.

Selective in vitro removal of anti-A antibodies by adsorption on encapsulated erythrocyte-ghosts.

Large volume plasma exchanges are used for the removal of anti-A or anti-B antibodies from the plasma of patients undergoing transplantation from donors with major ABO incompatibility. Previous works suggest that solid-phase immunoadsorption can be substituted for plasma exchange in situations where antigens can be purified and immobilized on columns through which plasma is percolated. However, the preparation of purified antigens of the ABO system is large quantities is laborious and requires the use of considerable blood volumes. Studies were therefore undertaken to determine the feasibility of an original immunoadsorbent based on porous microparticles prepared by a water/oil/water emulsification-solvent evaporation method, within which erythrocytes-ghosts carrying blood group antigens were entrapped. The decrease of the antibody hemagglutinating titre after adsorption onto encapsulated ghosts suggests that antibodies can cross the polymeric membrane and bind to the antigens. This original approach of using encapsulated antigens for the batchwise removal of antibodies could be extended to affinity chromatography, and immunoadsorption therapy with a chromatographic column linked to an extracorporeal circulation could be considered.

Regenerated cellulose-based hemodialyzers with immobilized proteins as potential devices for extracorporeal immunoadsorption procedures: an assessment of protein coupling capacity and in vitro dialysis performances.

The development of immunoadsorbents usable with whole blood should offer the potential for making significant improvements in extracorporeal immunoadsorption procedures. In contrast to traditional chromatographic media, these hemocompatible matrices could be used without requiring the previous step of the separation of blood cells and plasma. Conventional hemodialyzers seem to be particularly appropriate for such a purpose. This paper describes a feasibility study of the preparation of immunoaffinity supports, from regenerated cellulose (Cuprophan)-based dialyzers by cyanogen bromide activation and coupling of bovine serum albumin or human immunoglobin G, used as models for immunochemical ligands. Several parameters of the activation and coupling steps were studied in order to define the optimum preparation conditions. In addition, the preservation of the transport properties (clearance and ultrafiltration) of the modified hemodialyzers was evaluated in vitro to ensure that the device potentially could be used in future human therapeutic applications with no risk of massive removal of solutes and fluid from the blood. Results indicate that 150-300 mg of immunoglobulins can be immobilized per square meter of Cuprophan. Modified hemodialyzers show a slight decrease of their clearance values and a slight increase of their ultrafiltration coefficients, and thus they can be proposed as reliable carrier material for extracorporeal cleansing systems.

Antibodies released from immunoadsorbents: effect of support, activation and elution conditions.

Immunoadsorption is an application of affinity chromatography, as a therapeutic method to specifically deplete biological fluids such as blood plasma from proteins in excess, or to extract a biomolecule from a complex mixture. However, the leakage of small amounts of antibodies covalently immobilized on the support hampers the practical use of this method. In fact, these released antibodies contaminate the purified proteins or depleted media and, when they are of animal nature, they may lead to immunization of patients, or cause an anaphylactic shock when a clinical use is concerned. It is therefore of prime importance that the immunoadsorbents exhibit a satisfactory stability over the whole range of chemical and biochemical conditions involved during their clinical handling. To determine optimal conditions for the preparation of stable immunoadsorbents designed to remove selectively Low Density Lipoproteins (LDLs) from the plasma of patients affected by familial hypercholesterolemia, various immunoadsorbents were prepared by covalent immobilization of goat anti-apolipoprotein B polyclonal antibodies on different supports (Sepharose CL-4B, Sepharose 6 Fast Flow, Sphérodex and Fractogel) previously activated by various chemical reagents (cyanogen bromide, divinyl sulphone, tresyl chloride and trichloro-s-triazine). Their adsorption capacity, specificity, stability and the amount of immobilized antibodies were compared in terms of the activation method and the support used. It turns out that the immunoadsorbents prepared with Sepharose 6 Fast Flow lead to optimal yield of coupling, adsorption capacity, and an excellent stability at neutral pH. TC-activated-Fractogel turns out as well to afford an excellent coupling yield, a good adsorption capacity and an optimal stability in the whole pH range tested.

Immunoadsorption procedure as a potential method for the specific beta 2-microglobulin removal from plasma of patients with chronic renal failure.

beta 2-Microglobulin (beta 2-M), which accumulates in the plasma of patients undergoing long-term dialysis, has been identified as the principal precursor protein of amyloid fibrils in dialysis-related amyloidosis. As no specific treatment for this affection has been yet established, an extracorporeal immunoadsorption procedure appears to be an attractive therapeutic approach to remove beta 2-M. Several murine monoclonal antibodies to human beta 2-M were developed and compared as affinity ligands. One of them was selected on the basis of its specificity and adsorption capacity. In order to achieve maximum efficiency in protein removal, different parameters of the procedure were studied and optimized: effect of antibody coupling density, determination of maximum adsorption capacity of the immunoadsorbents and influence of antigen concentration and of flow-rate on antigen capture efficiency. The conditions of regeneration of immunoaffinity sorbents were also investigated to allow their multiple use without loss of adsorption capacity. The results show the validity of the proposed technique in removing beta-M from plasma of patients with chronic renal failure.

Importance of the matrix and its chemical activation on the stability of immuno-adsorbents.

Various matrices were reacted with four different activation reagents, in order to prepare immuno-adsorbents for the selective removal of low-density lipoproteins from blood plasma of patients affected by hypercholesterolaemia. The resulting immuno-adsorbents were compared to that obtained earlier with cyanogen bromide activation, in terms of coupling yield, adsorption capacity and moreover stability towards the various chemical and biochemical conditions to which they are submitted during their handling. Tresyl chloride-activated Sepharose 6 Fast Flow turns out to afford optimal stability in the whole pH range tested.

[Validation of the virus inactivation capacity of a procedure of human plasma albumin purification by chromatography]. / Validation des capacités d'inactivation des virus d'un procédé de purification de l'albumine plasmatique humaine par chromatographie.

Almost the whole of the human plasma albumin preparations intended for clinical or biological uses is at present fractionated by cold ethanol precipitation technics based on the Cohn method. However, ion-exchange chromatographic processes have been recently developed. The aim of this work was the evaluation of the viral inactivation efficacy of an automated industrial chromatographic process allowing fractionation of 350 to 400 l of plasma per cycle (one precipitation step, three ion-exchange chromatography steps using the Spherodex-Spherosil gels--Sepracor-IBF, Villeneuve la Garenne, France--and one pasteurization step. Three relevant viruses were selected for this validation study: the hepatitis B virus (HBV), the poliomyelitis virus and the human immunodeficiency virus (HIV). In order to comply with EEC and FDA regulatory documents, significant amounts of the tested viruses were spiked into the different fractions obtained during the various purification steps and their removal or inactivation during the subsequent step were determined. The validation study was performed under conditions which mimic the manufacturing process using fractions obtained during a semi-industrial fractionation. Moreover, residual viral infectivity was checked on after elution and washing of the columns for each chromatographic step. Results have pointed out: a) an overall reduction of 4.4 log 10 for HBV. Infectivity is judged by a combination of several markers and the DNA polymerase activity is the most affected particularly during the three ending purification steps; b) an overall reduction in virus titer > 10 log 10 for the poliomyelitis virus; c) an overall reduction in virus titer > 10 log 10 for HIV (four of the five steps have an important potential to inactivate this virus increasing the safety of the process). Moreover, no residual viral infectivities were detected after washing of the columns. In conclusion, this study showed the viral safety of human albumin purified using the chromatographic Spherodex-Spherosil process. As had been observed for fractionation by means of ethanol, the pasteurization step is necessary to ensure inactivation of two of the three viruses tested (HBV and poliomyelitis virus). This validation study allowed the preparation of a manufacturing and controls document for albumin and a marketing authorization has been issued by the "Laboratoire National de la Santé" (LNS, France).

Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

Single-step method for purification of human transferrin from a by-product of chromatographic fractionation of plasma.

A rapid, simple and convenient method is described for the isolation, on a pilot scale, of pure and functional human transferrin from an unexploited by-product of chromatographic fractionation of plasma. In a single chromatographic step on DEAE-Spherodex, 97% pure transferrin was obtained in 75% yield. A virus inactivation treatment was included in the preparative process in order to guarantee the safety of the final product, which could be used in culture media.

Dye-affinity purification of transthyretin from an unexploited by-product of human plasma chromatographic fractionation.

Dye-affinity chromatography of human plasma transthyretin on Remazol Yellow GGL-Sepharose from an unexploited by-product of chromatographic fractionation of plasma was optimized for large-scale preparation of a therapeutic product. With this system, transthyretin is only weakly bound to the gel. The residence time on gel and the transthyretin level in the by-product were observed to have no influence on the binding capacity of gel, and the optimum amount of transthyretin to be applied to the gel was found to be 1 g/l of gel. The adsorbent can be used more than ten times. The procedure resulted in the isolation, with a 30% yield with respect to plasma, of an 80% pure protein, which retained its thyroxine-binding capacity. Although the purity is acceptable for substitutive therapy, it can be improved further with a second chromatography on Cibacron Blue-Sepharose.

Specific removal of transthyretin from plasma of patients with familial amyloidotic polyneuropathy: optimization of an immunoadsorption procedure.

Familial amyloidotic polyneuropathy is characterized by the presence in patients plasma of a genetic variant of transthyretin. No specific treatment has been found and extracorporeal immunoadsorption on immobilized anti-transthyretin antibodies appears as a potentially attractive procedure. Parameters involved in specific immunoadsorption of transthyretin were studied and optimized. Several monoclonal anti-TTR antibodies were compared as affinity ligands and one of them was found to be suitable for such purposes. Optimum quantities of antibodies to be immobilized on the gel were determined. Three desorption agents were tested for regenerating immunoadsorbents and best results were obtained with basic variation of pH, allowing total desorption of TTR and possibility of multiple use without loss of adsorption capacity. Simulation of an immunoadsorption procedure in well-defined conditions showed efficiency and specificity of adsorption to remove TTR and the system thus should be subjected to clinical trials.