Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

© 2016, Springer Science+Business Media Dordrecht.Objective: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. Results: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. Conclusion: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

© 2016 Springer Science+Business Media DordrechtObjective: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. Results: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. Conclusion: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

OBJECTIVE: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.RESULTS: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT.CONCLUSION: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

MscS-like mechanosensitive channels in plants and microbes

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity

Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein’s directionality and unusual stepping behaviour

Uveal effusion syndrome (clinical case)

The purpose — to evaluate the effectiveness of the posterior sclerectomy with the corneal trepan in a uveal effusion syndrome.Patients and methods. Patient 1. The man, 61 years, complained about a blindness of the right eye and the progressing decrease in vision of the left eye. According to data of examinations the diagnosis was: uveal effusion syndrome, detachment of the choroid, exudative retinal detachment, the complicated cataract of both eyes. Patient 2. The man, 62 years, with complaints to low vision of the right eye and a blindness of the left-hand eye. Diagnosis: uveal effusion syndrome, detachment of the choroid, exudative retinal detachment, the initial complicated cataract of the right eye. Subatrophy operated retinal detachment, complicated cataract, silicone into the vitreal cavity of the left eye. Both patients underwent trepanation posterior sclera. Results. The patient 1noted significant improvement of vision in both eyes at the last examination. Мisual acuity with correction was OD — 0,2, OS — 0.3. Intraocular pressure was normal, improvement of electrical sensitivity and lability was diagnosed. On ultrasound examination of the retina belonged, moderate swelling of the choroid remained. Patient 2. Visual acuity was 0,1 + 3,0 D at the time of the last inspection. According to the ultrasonic B-scan of the right eye retinal detachment decreased to 3,8 mm, moderate swelling of the choroid remained. The OСT has showed detachment of the neuroepithelium in the macula to 60 μm. In ultrasonic biomicroscopy circular, ciliochoroidal detachment to 0,15 mm was diagnised.Conclusion. Described clinical cases confirmed the almost complete recovery of patients with the uveal effusion syndrome. Sclerectomy with the use of corneal trepan is a technically simple procedure and helps to define the path of the outflow suprachoroidal fluid subtenon space

The modern view on the concept of treatment of active stages of retinopathy of prematurity

Purpose: To show the basic regulations of the complex retinopathy of prematurity (ROP) treatment with use of retinal pattern scanning laser coagulation and early vitrectomy.Methods: Pascal pattern scanning lasercoagulation is performed for stage 2 ROP type 2, stage 3 ROP type 2 and subclinical stageand early clinical stage of aggressive posterior ROP (S. N. Fyodorov Federal State Institution «IRTC «Eye Microsurgery» Kaluga Branchclassification). Early 3‑ports 25G vitrectomy is performed if ROP progressing 2‑4 weeks after the laser treatment.Results: In 2003‑2011 823 different interventions for infants with active ROP was performed: 737 retinal lasercoagulations, 3‑ports vitrectomy — 72, 3‑ports lensvitrectomy — 14. Treatment efficacy was 98.6 % for stage 2 and stage 3 ROP (regress of the disease occurred in 557 of 565 eyes), and 74.4 % for aggressive posterior ROP (regress of the disease occurred in 128 of 172 eyes). The total efficacy of the complex treatment was 92.9 % (regress of the disease occurred in 685 of 737 eyes).Conclusion: The basic regulations of the complex ROP treatment are early, within first 6 weeks of chronologic age, photocoagulationto delay progression of the retinal detachment and to stabilize vascularity, and early vitrectomy if ROP progressing after the lasertreatment

AGGRESSIVE POSTERIOR RETINOPATHY OF PREMATURITY CLASSIFICATION

Based on dynamic monitoring of 163 premature infants (326 eyes) with aggressive posterior retinopathy of prematurity (ROP), digital retinoscopy and computer morphometry the disease clinical and morphometric features were revealed and systematized, and their consecutive replacement was fixed. As a result the separate classification of aggressive posterior disease was worked up. In aggressive posterior ROP course the next consecutive stages were marked out: subclinical, early clinical appearances stage, manifestation stage, advanced, far-advanced and terminal stages. The peculiarity of early clinical appearances stage and manifestation stage is the presence of such course types: favorable and unfavorable

STAGES OF PROVIDING COMPREHENSIVE EYE CARE FOR PREMATURE CHILDREN WITH PREMATURE RETINOPATHY

Purpose. Functional results analysis of eye care system for premature infants including a complete cycle of measures in an early detection, treatment and regular medical check-up activities for patients with retinopathy of prematurity (ROP) in the Central Region of Russia. Material and methods. Visits of the clinical specialists were carried out to the Departments for Nursing of Premature Infants for screening examinations in groups of ROP development risk. Infants with the revealed ROP were referred to the Kaluga Branch of the S. Fyodorov Eye Microsurgery State Institution for a detailed diagnostic examination and a subsequent treatment and monitoring. Results. There were made 454 visits to Kaluga, Tula, Bryansk, and Orel regions in the period between 2003 and 2011 and 8861 infants were examined. ROP was found in 1834 infants (20.7%). There were performed 823 different interventions for infants with active ROP: 737 retinal laser coagulations, 3-port lens preserving vitrectomy – 72, lens vitrectomy – 14. The total efficacy of the treatment was 92.9%. Conclusion.  The proposed eye care system for premature infants in the Central Region of Russia combines all trends: from a detailed precise diagnosis to a high-technologic treatment. It allows to reproduce it all over the Russian Federation territory