Multiple sclerosis (MS) is a chronic inflammatory dis- ease of the central nervous system (CNS) that is histo- logically characterized by focal mononuclear cell infil- tration with a varying extent of demyelination (1, 2). It is postulated that autoimmune T cells recognizing myelin autoantigens such as myelin basic protein (MBP) or proteolipid protein (PLP) play a central role in the pathogenesis of MS. This postulate is based on sub- stantial evidence such as increased frequency of activat- ed MBP- or PLP-specific T cells in peripheral blood and cerebrospinal fluid of MS patients (3–6), and significant complementarity-determining region 3 (CDR3) homol- ogy between T-cell infiltrates within MS plaques and MBP- or PLP-specific T-cell clones generated from MS patients (7, 8). Moreover, previous studies have identi- fied the peptides MBP84-102 and PLP95-116 as immunodominant and possible encephalitogenic epi- topes in MS patients with the HLA-DR2 haplotype (9,
Oral administration of antigen is a long recognized method of inducing systemic immune tolerance. In animals with ex- perimental autoimmune disease, a major mechanism of oral tolerance triggered by oral administration of antigen in- volves the induction of regulatory T cells that mediate ac- tive suppression by secreting the cytokine TGF- b 1. Multiple sclerosis (MS) is a presumed T cell–mediated Th1 type au- toimmune disease. Here, we investigated whether in MS pa- tients oral myelin treatment, containing both myelin basic protein (MBP) and proteolipid protein (PLP), induced anti- gen specific MBP or PLP reactive T cells that either secreted IL-4, TGF- b 1, or alternatively did Th1 type sensitization occur as measured by IFN- g secretion. Specifically, 4,860 short-term T cell lines were generated to either MBP, PLP, or tetanus toxoid (TT) from 34 relapsing-remitting MS pa- tients: 17 orally treated with bovine myelin daily for a mini- mum of 2 yr as compared to 17 nontreated patients. We found a marked increase in the relative frequencies of both MBP and PLP specific TGF- b 1–secreting T cell lines in the myelin treated MS patients as compared to non-treated MS patients (MBP P , 0.001, PLP P , 0.003). In contrast, no change in the frequency of MBP or PLP specific IFN- g or TT specific TGF- b 1 secreting T cells were observed. These results suggest that the oral administration of antigens gen- erates antigen specific TGF- b 1 secreting Th3 cells of pre- sumed mucosal origin that represent a distinct lineage of T cells. Since antigen-specific TGF- b 1 secreting cells localize to the target organ and then suppress inflammation in the local microenvironment, oral tolerization with self antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not depend upon knowledge of the antigen specificity of the original T cell clone triggering the autoimmune cascade. ( J. Clin. In- vest. 1996. 98:70–77.) Key words: demyelination • oral toler- ance • autoantigen • regulatory cytokines
It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen- specific immunotherapies for T cell–mediated autoimmune diseases. In patients with the demyelinating disease multi- ple sclerosis (MS), multiple potentially autoantigenic epi- topes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fu- sion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP ( D PLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating dis- ease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of ac- tivated T cells completely prevented subsequent clinical pa- ralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Preven- tion of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived func- tional peptide–MHC complexes in vivo, as well as by a sig- nificant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resis- tant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 ad- ministration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyeli- nating disease. ( J. Clin. Invest. 1996. 98:1602–1612). Key words: autoimmunity • apoptosis • T cells • tolerance • im-
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METHODS: We induced EAE by subcutaneous inoculation in two marmosets by human white matter (HWM) and in seven marmosets by MP4 (a chimeric recombinant fusion protein of myelin- basic and proteolipid protein) in adjuvant along with intravenous inactivated pertussis vaccine to facilitate the disease process. The HWM-inoculated animals were induced with Freund’s adjuvant as the established model of marmoset EAE. The MP4-inoculated animals were induced with either Freund’s incomplete adjuvant or TiterMax as part of a preclinical treatment trial. MR imaging was performed at 1.5 T at baseline, and repeated at 1- to 2-week intervals for a period of up to 16 weeks in six EAE-induced marmosets, and intermittently for up to 70 weeks in three EAE- induced and two control marmosets. Proton density– (PD-) and T2-weighted, pre- and postgado- pentetate dimeglumine enhancement, T1-weighted, and magnetization transfer (MT) images were obtained. The brains were prepared for histologic evaluation of lesion distribution and counts, characterization of lesions as demyelinating or inflammatory, and histopathologic scoring. The clinical, MR, and pathologic scoring were done on grading systems, and correlated for evaluation. RESULTS: White matter (WM) changes after EAE induction were observed first at 9 days in the HWM-induced animals and at 2.5 weeks in the MP4-induced animals, with subsequent week- to-week fluctuations on PD- and T2-weighted images. Contrast-enhancing lesions were not ob- served in all animals. MR-revealed WM lesions correlated to histopathologic analysis of EAE lesions, measuring from 0.5 mm to 1.5 mm. The lesion count and extent of demyelination was greater in the HWM-induced animals than in the MP4-induced animals. Some MR-revealed lesions correlated directly to clinical symptoms, but the majority of lesions were clinically silent. CONCLUSION: On MR images, lesions in the EAE marmoset model were confined to the WM, and their development, resolution, distribution, and enhancing characteristics fluctuated over the duration of the study. The dynamic presentation of MR-revealed lesions confirms the parallels between EAE in the marmoset and relapsing-remitting MS. Clinical symptoms alone were not representative of ongoing pathologic brain lesions. Therefore, serial MR imaging serves as a very important adjunct to clinical and histologic surveillance of the development of new and the persistence of existing brain lesions in this animal model of MS.
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Multiple sclerosis (MS) is a disease of the human central nervous system (CNS), characterized by perivascular inflammation, accompanied by primary demyelination and axonal damage. It is believed to result from autoim- mune mechanisms leading to destruction of myelin, pre- sumably initiated by abnormally activated T cells that recognize CNS components in MS patients. The patho- genic autoimmunity in MS appears to be associated with complex immune reactivity directed against several CNS-specific and non CNS-specific components [1,2]. Many of the primary target antigens detected in T cell responses of MS patients share identity with those CNS antigens demonstrated to cause overt, clinical EAE in laboratory animals. Thus far, several myelin proteins, myelin basic protein (MBP), proteolipid protein (PLP), and more recently, myelin oligodendrocyte glycoprotein (MOG), myelin-oligodendrocytic basic protein (MOBP), oligodendrocye specific protein (OSP) [1,2], and the neuronal components [(b-synuclein (bSyn), neurofila- ment light (NF-L)] [3,4] fulfill these criteria. In attempts to establish a molecular etiology of MS that both explains the genetic associations and potentiates specific therapeutic interventions, defining the potentially patho- genic epitopes of major MS-related CNS target antigens, in the context of their HLA restricting genes/alleles, and characterization of the corresponding responder T cells will be essential.
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PLP is the major myelin protein of the CNS. Point muta- tions and deletions/duplications of the PLP1 gene are as- sociated with X-linked dysmyelinating leukodystrophies, PMD and SPG2 . Duplication of the normal PLP1 gene is associated with approximately 70% of PMD cases [44,45]. Point mutations in the PLP1 gene are a minority of the PMD and SPG2 cases, but can cause the entire spectrum of the PMD and SPG2 clinical manifestations . Recently, we have identified in a group of PMD patients that a deletion in the PLP1 intron 3 splicing enhancer caused a mild form of PMD . The PLP- ISEdel mouse was generated to investigate the mechan- ism by which loss of the intron 3 splicing enhancer leads to the progressive neurological disability, demyelination, and axonal loss in the PMD patients with this PLP1 gene mutation . As we previously reported, by electron microscopy analysis of the optic nerve, myelin in the PLP-ISEdel mouse forms normally although there are re- dundant loops of myelin at one month. At three and six months, myelin compaction is progressively abnormal suggesting myelin instability; however, changes in the levels of myelin basic protein and CNP in the optic nerve were not found . We report here the temporal and spatial profile of microglia and astrocyte activation Table 1 Summary of quantitative neuropathological analysis of glial fibrillary acidic protein immunohistochemistry (GFAP IHC)
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While T cells are thought to be the primary effectors of CNS damage in autoimmune disease, they interact and communicate with various cell types, including DC and B cells (Stern et al., 2014). Shortly after injection with myelin protein fragments and adjuvant to induce EAE, an influx of DC into the spinal cord and surround- ing regions can be observed (Izikson et al., 2000; Greter et al., 2005; McMenamin, 1999; Bailey et al., 2007; Lande et al., 2008). This increase in cell numbers—which has been proposed to be due to in situ differentiation (Gott- fried-Blackmore et al., 2009)—is thought to indicate an ongoing immune response targeting the myelin sheath of neurons.
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9. Feldmann A, Amphornrat J, Schönherr M, Winterstein C, Möbius W, Ruhwedel T, et al. Transport of the major myelin proteolipid protein is directed by VAMP3 and VAMP7. J Neurosci. 2011; 31(15): 5659-5672. 10. Jung J, Jo HW, Kwon H, Jeong NY. However, ATP Release through Lysosomal Exocytosis from Peripheral Nerves: The Effect of Lysosomal Exocytosis on Peripheral Nerve Degeneration and Regeneration after Nerve Injury. Biomed Research International. 2014; (3): 936891- 936891.
Infection of mice with a sublethal neurotropic JHM strain of mouse hepatitis virus (JHMV) results in an acute encephalo- myelitis accompanied by demyelination that progresses to a chronic infection (3). Oligodendrocytes are a major target of infection during acute replication and a reservoir during viral persistence, which is characterized by viral RNA, viral antigen, and ongoing demyelination (3). Infection of gamma interferon (IFN- ␥ )-deficient mice and immunodeficient recipients of memory T-cell subsets suggests a critical requirement for IFN- ␥ in controlling virus replication in oligodendrocytes but not in astrocytes or microglia/macrophages (5, 19). Infection of C57BL/6 transgenic (TG) mice expressing a dominant negative (dn) IFN- ␥ receptor 1 (IFN- ␥ R1) specifically in oligodendro- cytes under the control of the proteolipid protein promoter (10) demonstrated increased numbers of infected oligodendro- cytes without altering the extent of demyelination or tissue destruction (11). These data revealed that IFN- ␥ signaling to oligodendrocytes is critical for control of virus replication but that the extent of tissue destruction and morbidity is indepen- dent of the viral load. This notion is supported by recent studies suggesting that immune modulatory effects of IFN- ␥ are more critical to pathogenesis than virus replication per se (5, 12, 20, 24). As BALB/c mice mount increased Th1 re- sponses to mouse hepatitis virus infection (13) and H-2 d -
Chronic relapsing-remitting experimental allergic encephalomyelitis (EAE) was induced in cynomolgus monkeys by a single immunization with a homogenate of human brain white matter (BH) in adjuvant. Proliferative T lymphocyte responses to BH, to myelin basic protein (MBP), but not to proteolipid protein, were detected in peripheral blood mononuclear cells (PBMC) of all animals and persisted until their death or, in surviving animals, for greater than 10 mo postimmunization. Responses of higher magnitude tended to be associated with fatal, compared with nonfatal, episodes of clinical EAE. The frequency of MBP-reactive T cells in PBMC of animals with acute EAE was quantitated with a soft agar colony system; the ratio of T cells that proliferated specifically to MBP was estimated at between 5 and 20 per 10(6) PBMC. A similar frequency of peptide-specific T cells was estimated from PBMC of monkeys immunized with a synthetic 14-mer peptide corresponding to a region near the carboxy terminus of MBP. Thus, autoantigen-reactive T cells can be detected in the
Although T cell responses to the quantitatively major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), are likely to be of importance in the course of multiple sclerosis (MS), cell-mediated autoimmune responses to other myelin antigens, in particular quantitatively minor myelin antigens, such as myelin-associated glycoprotein (MAG) and the central nervous system-specific myelin oligodendrocyte glycoprotein (MOG), could also play a prevalent role in disease initiation or progression. Highly purified myelin antigens were used in this study to assess cell-mediated immune response to MOG in MS patients, in the context of the reactivity to other myelin antigens, MBP, PLP, and MAG. The greatest
One major distinction is between obligate complexes, which, by definition, do not form their characteris- tic structure in vivo unless bound, and non-obligate complexes, which can exist as stable monomers [47,48]; complexes are also divided along a continuum between transient and permanent interactions , based on temporal length or energetic strength [48,50-52]. Many methods are designed to predict transient interfaces (TIs) [8,17,22,28,35-37,53,54], as they have greater phar- macological relevance, particularly for signal trans- duction cascades [50,52]. However, TIs tend to be more difficult to predict than permanent interfaces [18,20,23,27,33,39,52,55], possibly resulting from the weaker nature of the interaction manifesting itself as a weaker signal in the properties defining the interacting residues [27,33,52]. However, the existence of fewer train- ing examples due to data gathering difficulties may also play a role [47,49,56-58]. In general, TIs are less evolu- tionarily conserved than permanent interfaces [50,59-61], but more conserved than the rest of the protein sur- face . Further, TIs tend to be more compact  and richer in water (i.e. more prone to water-mediated bind- ing) [51,62,63]. They also differ in residue propensities , including fewer hydrophobic  and more polar residues . Thus, unsurprisingly, training on one inter- face type to predict on the other tends to decrease scores [18,33], though this is sometimes not the case . Gen- erally, analysis of transient versus permanent complexes uses predefined sets [13,60,66] or programs designed to separate them [52,67-69].
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RdRp always switched from pT7Rep3-L in either the small remaining region of PV3 VP1 (of which only 84 nts were present) or the 3’ terminus of luciferase, but never from the 2A region. These subgenomic replicons were constructed in such a way that a small portion of VP1 remained after the reporter gene and before the 2A region. This was to ensure that the appropriate virus protease cleavage site was retained. Even though the number of recombinant viruses studied was small, no clones were recovered which showed the RdRp leaving the replicon in the 5’ portion of the 2A coding region. Although template switching may have been random at the nucleotide level, it was possible that viruses with complete virus proteins and not those with chimeric CVA21/PV proteins were detected because of viable protein function. Once again, the recombination events adhered to some general rules already observed; the crossover sites were unlikely to be precise, the inserted sequence was no more than 513 nts in length, and chimeric 2A or 2B coding regions were not observed.
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The docking of one protein with another is a complex process that involves two basic types of complementarity; geometric and chemical. The majority of docking algorithms are based upon methods of evaluating and matching the geometric shape of the potential interacting surfaces. This type of matching requires a molecular surface representation and in many cases this is achieved by depicting the molecular envelope as a Connolly surface (Connolly 1983a; Connolly 1983b). This method represents a molecular surface by a dense population of dots. Many of these can be excluded, leaving only the critical points, which describe local knobs and holes on the protein surface. Connolly (1986) developed this representation of a molecular surface into a docking algorithm, matching quartets of knobs and holes on each surface, and then screening for overlaps between the two molecules. This method proved successful for the docking of a and p subunits of haemoglobin, but failed for the trypsin-trypsin inhibitor complex. Recently Norel et al. (1994) improved this method, reducing the complexity of the docking method, and successfully docked 14 out of 16 protein complexes. Many other algorithms involve similar matching of knobs and holes (Greer and Bush 1978; Kuntz et a i, 1982; Lee and Rose, 1985; Jaing and Kim, 1991), and many more match geometric criteria using similar simplified interpretations of the molecular surface (e.g. Wodak and Janin, 1978; Helmer-Citterich and Tramontane, 1994).
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Results: YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of − 0.132 and − 0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. Conclusion: This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of candidate genes and their important interacting residues likely to be provide a gateway for developing computer aided drug designing.
Central to the function of proteins is the concept of molecular recognition. Protein–ligand and protein–protein interactions make up the bulk of the chemical processes that give rise to living things. Realizing the full potential of protein design technology will therefore require an increased understanding of the design principles of molecular recognition. We have tackled problems involving molecular recognition by using computational methods to design novel protein-ligand and protein-protein interactions. Firstly, we set out to design a protein capable of recognizing lanthanide metal ions. Protein-lanthanide systems are of interest for their potential to serve as purification agents for use under biological conditions. We have designed a highly dense 6-coordinate lanthanide binding at the core of a de novo protein, and used the dynamical aspects of the protein to achieve a degree of differentiation between elements in the lanthanide series. Secondly, we investigated systems of homo-oligomeric protein complexes that self-assemble into hollow cages. We have studied the structural determinants of naturally occurring self-assembling ferritin cages and identified a single mutation that greatly increased the stability of the ferritin cage, as well as dramatically altered the overall structure of the assembly. We have also used the formulation of probabilistic protein design to arrive at novel sequences for α-helical peptides that can adjust their surfaces in accordance to different local environments. This formulation was used to identify a sequence for a peptide designed to self-assemble into a spherical particle with broken symmetry. Taken together, these efforts will lead to an increased understanding of the role of kinetics and structural plasticity in protein-ligand and protein-protein interactions.
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The inset highlights the pattern of hydrogen bonds that stabilizes alpha helices. Many functional proteins fold into a compact globular shape, with many carbon-rich amino acids sheltered inside away from the surrounding water. The folded structure of hemoglobin includes a pocket to hold heme, which is the molecule that carries oxygen as it is transported throughout the body. The folding pattern of a polypeptide chain can be described in terms of the angles of rotation around the main chain bond. Chemical bonds have characteristic lengths, the peptide bond has partial double- bond character, meaning it is shorter, and rigid. Two or more polypeptide chains can come together to form one functional molecule with several subunits. The four subunits of hemoglobin cooperate so that the complex picks up and delivers more oxygen than is possible with single subunits. Protein function can be thought of on different interdependent levels and may be divided into three major categories: molecular function, biological process and cellular component (David Lee et al. 2007). Molecular function describes activity on the molecular level, such as catalysis, whereas biological process describes broader functions that are carried out by assemblies of molecular functions, such as a particular metabolic pathway. Cellular component describes the compartment or compartments of a cell in which the protein performs its function
Protein–protein interactions are central to many processes within cells and organisms, ranging from immune defense to cellular communication. For biological regulation, it is necessary to recognize their targets, and the networks responsible for interactions in macromolecular complexes 1 . Tools to alter and interfere with protein interactions offer great promise to help understand and delineate these networks. So, it is important to know the three dimensional structure of the protein molecules as well as the protein-protein interface. But, the limited nature of Protein Data Bank, and further limited number of X-ray crystallographic structures of high resolution has been a major constraint in the previous studies. Recently, however, there has been a large increase in the number of known three-dimensional structures that contain protein-protein recognition sites and more high-resolution structures have been solved. These structures cover a much broader range of activities than the earlier ones, which were almost exclusively protease inhibitor and antibody antigen complexes. The knowledge of those few structures guided us to determine the rules for general structural study.
There are many reports on the encephalitogenic po tential of PLP.3,4.12 So we extracted this protein for in ducing EAE, The typical PLP recovery from 50 g of bovine brain ranged from 40-80 mg depending upon the amount of myelin in the portion of brain utilized. PLP prepared in this study is 20 mg from 50 gr bovine brain, A minor contaminant having slightly higher electro phoretic mobility (around 20 kd) than PLP, suggested previously to be DM-20, has been detected in some of our preparations. 1
The understanding of myelin autoimmunity at the mo- lecular level is poor, also taking into account the fact that myelin protein epitopes in general are buried within the myelin sheath, a tightly packed multilayered membrane. On the other hand, these epitopes are hid- den from the immune system in a healthy myelin en- vironment, and might only be exposed in case of prior insult to the myelin membrane. All our experiments point towards highly similar properties for all the stud- ied peptides. In this respect, it is of interest to note the possible potential of myelin protein-derived pep- tides in MS therapy; these include both cyclic and mu- tated variants of myelin antigen peptides, especially MBP85-99 [59, 94–98]; the design of such peptides could also take into account our observations on simi- lar overall structural and membrane interaction prop- erties of peptides with limited sequence similarity per se. While the link between membrane binding, membrane- induced folding, and autoimmune response is unclear, molecular mimicry possibly taking place in autoimmune demyelination may be based on 3D similarity of host and pathogen epitopes. In the case of MBP85-99 mimics, for example, while the peptides fold into amphipathic helices on lipid membranes, the extended conformation bound to the MHC shows the con- served residues being presented outwards, being im- portant for immune response.
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