The biomarkers of immune dysregulation and inflammation response in Parkinson disease

Parkinson’s disease (PD) is referring to the multi-systemic α-synucleinopathy with Lewy bodies deposited in midbrain. In ageing, the environmental and genetic factors work together and overactive major histocompatibility complex pathway to regulate immune reactions in central nerve system which resulting in neural degeneration, especially in dopaminergic neurons. As a series of biomarkers, the human leukocyte antigen genes with its related proteomics play cortical roles on the antigen presentation of major histocompatibility complex molecules to stimulate the differentiation of T lymphocytes and i-proteasome activities under their immune response to the PD-related environmental alteration and genetic variation. Furthermore, dopaminergic drugs change the biological characteristic of T lymphatic cells, affect the α-synuclein presentation pathway, and inhibit T lymphatic cells to release cytotoxicity in PD development. Taking together, the serum inflammatory factors and blood T cells are involved in the immune dysregulation of PD and inspected as the potential clinic biomarkers for PD prediction

Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Paired Cysteine Mutagenesis

BACKGROUND: GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt(Ph), it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)(3) (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1. METHODOLOGY/PRINCIPAL FINDINGS: To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)(3) is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd(2+). CONCLUSIONS/SIGNIFICANCE: Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle

Probiotic Bifidobacterium lactis HN019 enhances the resistance and immunity against enteric pathogens : a thesis presented in partial fulfilment of the requirements for master [sic] degree in nutritional science at Massey University

Probiotics are defined as a group of live microorganisms, including some microbial stimulants that exert health promoting effects, such as the maintenance of a normal intestinal microbiotia, increased nutritional value of foods, anticarcinogenic activity, reduction of serum cholesterol levels, alleviation of lactose intolerance and stimulation of the immune system. Some of strains of lactic acid bacteria (LAB) are representative probiotics. The objective of this study was to examine the immunomodulatory and antiinfection properties of a new identified LAB strain- Bifidobacterium lactis using two animal models. Two experiments were conducted and reported in this thesis. In the first experiment, a piglet weaning diarrhoea model was used to test the efficacy of Bifidobacterium lactis HN019 protecting against diarrhoea associated with Rotavirus and E. coli. 17 three-week-old piglets were allocated into two groups balanced for liveweight and litter of origin. The first group (n=8) was orally administered B. lactis HN019 (109 cfu/piglet/day) through the experiment; the second group (n=9) was not given B. lactis HN019 (control). After one week, the animals were penned individually and weaned onto a weaner diet. Blood samples were taken to measure the antibody responses, cell proliferation, and phagocytic activity of leukocytes (monocytes and neutrocytes). Also the effect of B. lactis HN019 on weaning diarrhoea was assessed by monitoring the severity of diarrhoea, feed intake and liveweight gain of the piglets on the weaner diet. Compared to the controls, piglets receiving B. lactis HN019 had lower severity of weaning diarrhoea, higher survival rate and feed conversion efficiency (or liveweight gain). The protection was associated with lower levels of faecal rotavirus and E. coli shedding, higher phagocytie activities and cell proliferative response to mitogens, and higher specific antibody titers. These results suggest that dietary B. lactis can reduce the severity of weaning diarrhoea associated with rotavirus and E. coli, and the probiotic is associated with enhanced immune responsiveness. In the second experiment, the protective effects of Bifidobacterium lactis HN019 against E. coli O157:H7 and associated immunological parameters were investigated using murine models. After one week acclimatisation on a skim milk powder (SMP)-based diet, eighty-six BALB/c and C57 mice were selected and randomised to two treatment groups. One group was fed on the SMP-based diet until the end of the experiment, while the other group was fed the SMP-based diet supplemented with B. lactis HN019 (3 x 108 cfu/g). After one week on these diets, mice were intragastrically inoculated with 0.1 ml E. coli O157:H7 suspension (109 cfu/ml). Protection against E. coli O157:H7 infection was assessed by monitoring the morbidity, feed intake, bacterial translocation to visceral tissues (spleen and liver) and immune responsiveness. Phagocytic activities of blood and peritoneal cells, and antibody titres against E. coli O157:H7 in intestinal content were also measured. The results showed that B. lactis HN019-fed mice conferred a significant degree of protection against E. coli O157:H7 challenge in comparison to the control mice that did not receive B. lactis HN019. Protection included lower morbidity and higher post-challenge feed intake, reduced pathogen translocation to blood, spleen and liver, as well as significantly higher phagocytic activities of blood and peritoneal cells and anti-E. coli IgA level in gut content. These results suggest that B. lactis HN019 can enhance the host resistance to E. coli O157:H7 and that the protection is associated with enhanced immune functions. In summary, potential immune enhancing effects of B. lactis HN019 were investigated in one pig trial and one mice trial. The results showed that supplement of B. lactis HN019 relieved diarrhoea associated with rotavirus and E. coli infection in piglets and enhance the host resistance to E. coli Ol57:117 challenge in mice. Immunological measurements indicated B. lactis HN019 fed groups had significant higher phagocytosis and anti-E. coli IgA levels. And the pathogen shedding was also reduced in B. lactis HN019 fed groups. As concluded, B. lactis HN019 can provide a protective role against special enteric pathogen infection by its immunomodulatory effects

The Accessibility in the External Part of the TM5 of the Glutamate Transporter EAAT1 Is Conformationally Sensitive during the Transport Cycle. PLoS One 2012

Background: Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet. Methodology/Principal Findings: We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. Vmax of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA. Conclusions/Significance: All these facts suggest that the accessibility of some positions of the external part of the TM5 i

Topology model and transport cycle of GLT-1.

<p>(<b><i>A</i></b>) Topology model of GLT-1 by analogy to that of Glt_Ph shown with <i>black dots</i> denotes the approximate locations of the following three cysteine substitutions: I295C, G297C, and I463C. TM helices 1– 8 and hairpins (HP1 and HP2) are labeled. (<b><i>B</i></b>) Transport cycle of GLT-1. After binding of sodium, glutamate, and a proton from the extracellular medium (<i>up</i>), the outward-facing substrate-loaded translocation complex is formed. After the external gate closes, the internal gate opens and the substrate and cotransported ions dissociate into the cytoplasm (<i>bottom</i>). Subsequently, intracellular potassium enters the binding pocket. After the internal gate closes, the external gate opens and potassium is released into the extracellular medium.</p

Effect of the composition of the external medium on the inhibition of double cysteine mutants by CuPh.

<p>HeLa cells expressing double cysteine mutants were preincubated for 5 min in the presence and absence of 200 µM CuPh. The indicated preincubation solutions contained NaCl, NaCl +1 mM L-glutamate, ChCl +1 mM L-glutamate, NaCl +1 mM GABA, NaCl +1 mM glycine, NaCl +20 µM TBOA, KCl, choline chloride. Values are given as percent of control (preincubation without CuPh) and represent the mean ± S.E. of at least three different experiments done in triplicate. (<b><i>A</i></b>) I295C/I463C double cysteine mutants. (<b><i>B</i></b>) G297C/I463C double cysteine mutants.</p

Effect of CuPh on the activity of cysteine mutants.

<p>HeLa cells expressing double cysteine mutants or the indicated control mutants, all in the background of CL-GLT-1, were preincubated in NaCl-containing medium with 200 µM CuPh for 5 min at room temperature, washed twice with choline chloride-containing solution, and subsequently D-[³H]aspartate transport was assayed. Co-expression of two single cysteine mutants in HeLa cells is marked by “co”. The values shown represent the percentage of activity after treatment with 200 µM CuPh relative to that obtained after preincubation in the absence of CuPh. Values represent the mean ± S.E. of at least three separate experiments each done in triplicate. (<b><i>A</i></b>) I295C/I463C double cysteine mutants and its control mutants. (<b><i>B</i></b>) G297C/I463C double cysteine mutants and its control mutants.</p

Effect of the composition of the external medium on the inhibition of single cysteine mutants by MTSET.

<p>HeLa cells expressing the single cysteine mutants L296C (<b><i>A</i></b>), I297C (<b><i>B</i></b>), G299C (<b><i>C</i></b>) or K300C (<b><i>D</i></b>), were preincubated for 5 min in the presence or absence of 0.2 (<b><i>A</i></b>), 1.0 (<b><i>B</i></b>), 0.6 (<b><i>C</i></b>) or 0.01 (<b><i>D</i></b>) mM MTSET. The indicated preincubation solutions contained NaCl, NaCl+1mM L-glutamate, NaCl+20 µM TBOA, NaCl+1mM GABA, KCl, choline chloride. Subsequently the cells were washed and D-[³H]-aspartate transport was measured as described under “Materials and methods”. Values are given as a percentage of control (preincubation without MTSET) and represent the mean ± S.E. of at least three different experiments done in triplicate.</p