Neuromyelitis optica is a terrible demyelinating autoimmune disease. The recent find of NMO-IgG positive serum on most patients with the disease, has given new impulse to the survey of how the disease develops and the exact mechanisms of development. A important characteristic of NMO is the activation of complement which causes loss of AQP4 and the formation of lesions. This experiment injects NMO-IgG into mouse encephalon tissue showing AQP4 or AQP1 along with human complement. The consequences showed that when NMO-IgG was injected onto AQP4 showing cells with human complement a greater figure of cell decease was recorded. Conversely there was no addition in cell decease when no human complement was added. As a control the experiment was besides carried out with AQP1 cells which showed no marks of increased cell decease in either conditions.
Background of Neuromyelitis optica Page 2-3
Background of Aquaporins Page 3-4
Production of antibodies Page 4-5
Activation of complement Page 5
Methods Page 5-7
Hypothesis Page 5
Results Page 7-8
Discussion Page 8-
Keywords: Devics ; Neurolmyelitis ; Complement ; Aquaporin ; Immunoglobulin.
NMO- Neuromyelitis optica
MRI- Magnetic resonance imagination
FAB-Fragment antigen binding
Fc Region- Fragment Crystalizable part
Background on NMO
Neuromyelitis optica ( NMO ) is a progressive autoimmune upset. It is an inflammatory demylinating disease of the spinal cord and ocular nervus called myelitis and ocular neuritis severally. ( 1 ) They can happen together or individually. Ocular neuritis is known to do complete ocular damage with a low incidence of recovery. ( 2 ) Myelitis is more terrible as it affects the spinal cord and depending on the location of the lesions can impact a big portion of the lower organic structure doing hurting in the limbs and vesica palsy. In terrible instance if the lesions travel upwards they can damage the encephalon root and cause neurogenic respiratory failure. ( 2 ) It has besides got a really high rate of backsliding, 85 % , particularly when compared to Multiple Sclerosis. ( 4 )
T2-weighted ( left ) and T1-weighted ( right ) sagittal MRI of the cervical spinal and upper thoracic cord in a patient with
NMO, demoing longitudinal extended transverse myelitis with swelling, mortification and additive Gd
sweetening. ( 1 )
NMO was foremost discovered in the late nineteenth century by Eugene Devic. It was after sing several patients all whom suffered from loss of vision, palsies and loss of vesica control. The most dramatic features observed by Eugene Devic and where the same patients developed non organ specific autoantibody conditions. ( 3 ) The conditions were found to be a consequence of redness in the spinal cord and ocular nervus. This was discovered during post-mortems which showed immunoglobulin deposition and complement activation. ( 3 ) For many old ages after Eugene Devic recorded his findings, NMO was still erroneously classed as a subtype of multiple induration ( 1 ) . It was non until Clifford Allbutt in the late nineteenth century, studied Eugene ‘s work and associated the symptoms with a separate upset ( 12 ) . In the past the diagnosing was based on clinical features such as ; ocular neuritis, bilateral motor shortages, pain cross myelitis and many other symptoms ( 12 ) . Recently nevertheless an auto-antibody for AQP4 has been discovered and called NMO-IgG ( 6 ) . It is significantly accurate and is now considered an accurate marker for NMO ( 12 ) . It is 73 % sensitive and can separate NMO from classical MS with an truth of 91 % ( 13 ) . This has lead to the belief the oncoming of disease may be caused or exacerbated by the immune system ( 1 ) . Furthermore the development of MRI has allowed the lesions to be viewed in vivo on life patients.
Treatment of NMO
Presently there are two long term interventions widely used. These are intervention with interferon beta and immunosuppressive therapy ( 15 ) . Current research suggests that interferon intervention is significantly less effectual than immunosuppressive intervention ( 16 ) . Both interventions nevertheless still fail to hold disease development ( 15 ) . A new drug being trialed, Rituximab, is a monoclonal antibody which works against CD20+ cells and has shown promising consequences ( 17 ) .
Of late there has been increased involvement in the function of aquaporins within the encephalon and the difference between the different isotopes. It has become progressively of import to to the full understand the maps and functions H2O channels have within the encephalon as strong grounds is emerging proposing the AQP4 molecule is targeted in NMO. ( 6 ) Aquaporins ( AQPs ) are a household of transmembrane molecules with 6 membrane spheres organizing a ring with a pore in the center with which H2O can be transported in both waies ( 5 ) . AQPs were ab initio discovered by Petre Agre in 1988 ( 7 ) who showed that toad oocytes which expressed AQPs, where far more susceptible to H2O lysis than thoses non showing AQPs, therefore turn outing improved H2O conveyance ( 8 ) . Furthermore it has been shown that where encephalon hydrops has occurred the astrocytes associated with that country will demo increased look of AQPs on the cell surface ( 11 ) . Therefore far over 10 different subtypes of AQP have been isolated in mammals ( 5 ) .The chief AQP in footings of copiousness in the human encephalon and importance in NMO is AQP4 ( 11 ) . Brain AQP4 is chiefly found in astrocyte pes procedures, encephalon parenchyma and major fluid compartments ( 9 ) . AQP4 is farther subdivided into an isoform of M1 and M23 ( 10 ) . The two isoforms, M1 and M23, are due to translation induction differences in the N end point of the first and 2nd methionine amino acid severally ( 18 ) . M23 is besides 3 times more abundant in the human encephalon than M1 ( 19 ) . Functionally no differences have been noted between the 2 isoforms, nevertheless they do look to change the organisation of AQP4 on the intramembrane ( 10 ) . This is thought to be due to the M23 isoform organizing big square arrays with abundant cross Bridgess whereas the M1 isoform appears to curtail square array assembly ( 10 ) .
Production of antibodies.
The human organic structure ‘s defence mechanisms are grouped into two chief classs, the innate and the adaptative immune system. Whilst they are grouped they work together and rely on one another to work in unison and supply maximal protection for the human organic structure. ( 14 ) One mechanism of the innate immune system is the usage of antibodies. These belong to the Ig ace household. ( 14 ) They consist of 2 upper visible radiation ironss and 2 heavy ironss. Depending on their isoform they can be found on the surface of cells or go arounding freely within the blood vass. Immunoglobulins are produced from mature B cells and come in the undermentioned isoforms, IgA, IgD, IgE, IgG and IgM and each isotope is farther divided into subclasses. They are distinguished by differences in their changeless heavy concatenation parts. This is thought to give each isoform its different biological map, with fluctuation in the lighter upper ironss ensuing in different antigenic determinant specificity. When a B cell matures it will get down to bring forth the different antibodies depending on the stimulation which caused it to maturate.
The chief Ig isotype we shall discussed is IgG. It is secreted out of the cell and every bit present in intra and excess vascular surfaces. ( 14 ) It has the highest half life of the Ig molecules with an mean life span of 23 yearss. Furthermore IgG is the lone antibody which can go through through the placenta to the foetus giving unsusceptibility for first hebdomads of life. ( 14 ) The chief manner in which IgG works is via opsonisation and activation of the complement system.
When an antibody detects a pathogen of right specificity it bind to it with the FAB parts of the visible radiation ironss. This leave the lower heavy Fc part lodging out. Macrophages have receptors which can observe the Fc part and will steep anything attached to that antibody. This is called opsonisation and comes from the Greek opsonin which means to fix for feeding. ( 14 ) Furthermore one time an antibody is bound both to an antigen and a scavenger cell it activates the scavenger cells and causes release of Interleukins and cytokines, which mediate an inflammatory response. This is thought to be the cause of redness seen around the site of lesions.
Another manner in which antibodies protect the human organic structure from disease is by triping complement. The complement system is a set of 20 or so proteins go arounding in the blood. It can be activated via three tracts, the surrogate tract, the lectin tract and the classical tract. Finally all the tracts converge but the induction is different. Immunoglobulins are first-class triggers of the classical tract. It was named so as it was the first of the 3 tracts to be discovered. ( 14 ) The chief proteins are called C1 through to C9, the Numberss do non denote order of activation instead the order in which they were discovered. When two IgG molecules are bound to an antigen they cause C1 to split C3 and therefore get down the cascade. The following composite will ensue in the cleavage of the following composite with most adhering to the pathogens surface. Once C9 is reached a membrane onslaught composite ( MAC ) is formed. This literally punctures the surface of the pathogen and causes lysis. ( 14 ) Again it is this lytic map which is thought to be the cause of lesions and demyelination.
What is non clear is whether the presence of NMO-IgG is adequate to do demyelination or whether it requires other factors present in NMO typical patients, but which we have non discovered yet. The purposes of this survey is hence to find whether NMO-IgG can do lesions and redness in mouse tissue with or without human complement.
The hypothesis for this experiment is that there will be a statistically significance in the addition of cell decease when NMO-IgG is injected into cells showing AQP4 along with human complement.
Isolating IgG from patient serum: Serum was obtained from five patients ( P1 – P5 ) with an established diagnosing of NMO and strong AQP4-Ab serum positiveness. We besides used three different pooled non-NMO control sera ( C1 – C3 ) . Clinical inside informations of the NMO patients are shown in Supplementary Table 1. Five milliliter of each serum ( P1 – P5 ) or pooled serum ( C1 – C3 ) was diluted 1 in 4 in PBS and loaded onto a Protein-A column ( Sigma, Poole, UK ) . After rinsing with PBS the edge IgG was eluted with glycine pH 2.3 and instantly neutralised in 1 M Tris pH 8.0. The positive fractions were pooled and dialysed against Hartmann ‘s solution. They were concentrated by dialysis against polythene ethanediol, dialysed once more against Hartmann ‘s solution and stored at 4oC. IgG concentration in the samples were 6 – 38 mg/mL. We term IgGNMO the entire IgG isolated from the serum NMO patients ( that contains AQP4-Ab ) and IgGCON the entire IgG isolated from the serum of non-NMO topics. The presence or absence of AQP4-Ab in each sample was confirmed by immunocytochemistry on civilized Chinese Hamster Ovary ( CHO ) cells showing AQP4 or AQP1 ( as control ) . AQP4-Ab titers were independently measured by fluoroimmunoprecipitation and cell-based checks ( Supplementary Table 2 ) . Each mouse was injected with IgG from a individual sample ( P1 – P5 or C1 – C3 ) . In experiments affecting several mice, we used at least three different IgGNMO samples ( from P1 – P5 ) and at least two different IgGCON samples ( from C1 – C3 ) .
Human and mouse complement: Non-haemolysed blood was collected from human voluntaries or CD1 mice in a field glass tubings and allowed to coagulate at room temperature for 30 min. The samples were centrifuged at 1,000 r.p.m. and the serum supernatant was collected, aliquoted and stored at -80 0C. Serum collected in this manner preserves complement activity and is termed human ( hC ) or mouse ( megahertz ) complement in the manuscript.
Cell civilization: CHO K1 cells stably transfected with plasmids encoding M23 AQP4 ( CHO-AQP4 ) or AQP1 ( CHO-AQP1 ) were grown on coverslips in F12 medium with 10 % FBS ( Invitrogen, Paisley, UK ) . More than 95 % of cells expressed the several proteins in their plasma membranes ( Saadoun et al. , 2005 ) .
Immunocytochemistry: For AQP1, AQP4 and C5b-9 immunostaining, CHO cells were washed in PBS, fixed in 4 % impersonal buffered methanal ( Sigma, Poole, UK ) for 5 min followed by coney anti-AQP1 ( 1:200, Chemicon-Millipore, Livingstone, UK ) or rabbit anti-AQP4 ( 1:200, Chemicon-Millipore, Livingstone, UK ) or rabbit anti-C5b-9 ( 1:100, Abcam, Cambridge, UK ) primary antibody for 1 H at 25 0C. Cells were washed with PBS and incubated with AlexaFluor-linked caprine animal anti-rabbit secondary antibody ( 1:200, Invitrogen, Paisley, UK ) . For AQP4 immunostaining with IgGNMO, unrecorded cells were washed with PBS, exposed to IgGNMO ( 1:200,15 min, 4 0C in PBS + 5 millimeter dextroglucose ) , washed with PBS, post-fixed in 4 % impersonal buffered methanal ( Sigma, Poole, UK ) , washed with PBS and incubated with Texas-red-linked anti-human IgG secondary antibody ( Vector Laboratories, Peterborough, UK ) . After secondary antibody incubation, the coverslips were washed with PBS and mounted in Aquamount medium with DAPI ( Vector Laboratories, Peterborough, UK ) . Coverslips were examined utilizing a BX-51 Olympus epifluorescence microscope.
Complement activation and cell viability assays: CHO cells on coverslips were exposed at 37 0C to F12 medium without serum containing ( by vol. ) 5 % IgGNMO or IgGCON and 5 % hC or megahertz, and where stated 0.25 mg/mL C1inh ( Biopur, Bubendorf, Switzerland ) concluding concentration. After 1 H, some coverslips were immunostained for C5b-9. After 2 h the cells were stained with a LIVE/DEAD® cell viability kit ( Molecular Probes – Invitrogen, Paisley, UK ) harmonizing to the maker ‘s instructions. Live cells stain fluorescent green and dead cells with damaged plasma membranes stain f
uorescent ruddy. Coverslips were examined utilizing a BX-51 Olympus epifluorescence microscope and the figure of ruddy and green cells were counted.
Fig 2: AQP4 staining with Dapi and Red
Fig 1: AQP1 staining with Dapi and Red
Fig3: Control IgG + AQP1 Fig 4: Control IgG + AQP4
Fig 5: NMO- IgG + AQP1 Fig 6: NMO-IgG + AQP4