Reverse vaccinology approach to design a multi-epitope vaccine construct based on the Mycobacterium tuberculosis biomarker PE_PGRS17

Mycobacterium tuberculosis (Mtb) is responsible for high mortality rates in many low- and middle-income countries. This infectious disease remains accountable for around 1.4 million deaths yearly. Finding effective control measures against Mtb has become imperative. Vaccination has been regarded as the safe and lasting control measure to curtail the impact of Mtb. This study used the Mtb protein biomarker PE_PGRS17 to design a multi-epitope vaccine. A previous study predicted a strong antigenic property of PE_PGRS17. Immunogenic properties such as antigenicity, toxicity, and allergenicity were predicted for the PE_PGRS17 biomarker, specific B- and T-cell epitope sequences, and the final multiple epitope vaccine (MEV) construct. Algorithmic tools predicted the T- and B-cell epitopes and those that met the immunogenic properties were selected to construct the MEV candidate for predicted vaccine development. The epitopes were joined via linkers and an adjuvant was attached to the terminals of the entire vaccine construct. Immunogenic properties, and physicochemical and structural predictions gave insight into the MEV construct. The assembled vaccine candidate was docked with a receptor and validated using web-based tools. An immune simulation was performed to imitate the immune response after exposure to a dosed administrated predicted MEV subunit. An in silico cloning and codon optimisation gave insight into optimal expression conditions regarding the MEV candidate. In conclusion, the generated MEV construct may potentially emit both cellular and humoral responses which are vital in the development of a peptide-based vaccine against Mtb; nonetheless, further experimental validation is still required.

     

Multiple Sclerosis: Fas Signaling in Oligodendrocyte Cell Death

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas–ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase–mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 nonactivating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.     

Behavioral characterization of the novel GABAB receptor-positive modulator GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): anxiolytic-like activity without side effects associated with baclofen or benzodiazepines

The role of GABAB receptors in various behavioral processes has been largely defined using the prototypical GABAB receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABAB receptors in these disorders is largely unclear. We recently identified GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABAB receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital- and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABAB receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines.     

The Role of Gap Junction Channels During Physiologic and Pathologic Conditions of the Human Central Nervous System

Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.     

Importance of Extranuclear Estrogen Receptor-�� and Membrane G Protein�CCoupled Estrogen Receptor in Pancreatic Islet Survival

OBJECTIVE

We showed that 17β-estradiol (E₂) favors pancreatic β-cell survival via the estrogen receptor-α (ERα) in mice. E₂ activates nuclear estrogen receptors via an estrogen response element (ERE). E₂ also activates nongenomic signals via an extranuclear form of ERα and the G protein–coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival.

RESEARCH DESIGN AND METHODS

We used mice and islets deficient in estrogen receptor-α (αERKO^−/−), estrogen receptor-β (βERKO^−/−), estrogen receptor-α and estrogen receptor-β (αβERKO^−/−), and GPER (GPERKO^−/−); a mouse lacking ERα binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes.

RESULTS

We show that ERα protection of islet survival is ERE independent and that E₂ favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERβ plays a minor cytoprotective role compared to ERα. Accordingly, βERKO^−/− mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERα and ERβ in mice does not synergize to provoke islet apoptosis. In αβERKO^−/− mice and their islets, E₂ partially prevents apoptosis suggesting that an alternative pathway compensates for ERα/ERβ deficiency. We find that E₂ protection of islet survival is reproduced by a membrane-impermeant E₂ formulation and a selective GPER agonist. Accordingly, GPERKO^−/− mice are susceptible to streptozotocin-induced insulin deficiency.

CONCLUSIONS

E₂ protects β-cell survival through ERα and ERβ via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in β-cells and identifies GPER as a target to protect islet survival.Preserving insulin secretion by the pancreatic β-cells is critical in both type 1 and the late stages of type 2 diabetes. In type 1 diabetes, the death of insulin-producing β-cells of the pancreas by apoptosis leads to insulin dependence. Insulin replacement therapy by pancreatic islet transplantation is a treatment that most closely replicates normal physiological conditions for treatment of type 1 diabetes (1), but its effectiveness is reduced by the loss of functional islet mass from apoptosis, impairing the survival of islet grafts. Similarly, in the late stages of type 2 diabetes, evidence of β-cell apoptosis is documented in animal models (2,3) and in humans (4). Thus, in the absence of novel immunotherapy and antiapoptotic drugs, novel strategies to protect insulin-producing cells in vivo represent a major opportunity for therapeutic intervention. One promising approach to protect β-cells from apoptosis involves the cytoprotective actions of estrogens. In addition to its reproductive functions, the female sex steroid 17β-estradiol (E₂) is a neuroprotective hormone against multiple oxidative and proapoptotic insults in vivo and in vitro, acting via classic estrogen receptors (rev. in 5). Recently, we reported that E₂ protects β-cells from streptozotocin (STZ)-induced apoptosis in mice of both sexes via the estrogen receptor (ER)-α (6). In cultured mouse and human islets, E₂ has potent antiapoptotic properties against proinflammatory cytokines and reactive oxygen species (6,7). E₂ acts via classic estrogen receptors, ERα and ERβ (8). In ERα-deficient female mice, E₂ still partially protects β-cell survival via an alternative pathway (6), suggesting that ERβ may mediate the effects of E₂ in the absence of ERα.The G protein–coupled estrogen receptor (GPER), also known as GPR30, has been recognized as a membrane receptor for estrogens that mediates nongenomic signals (9). GPER is expressed in islets and has recently been suggested to mediate the estrogenic effect on islet insulin release (10). We analyzed the contribution of ERα, ERβ, and GPER to islet survival. We used mice individually deficient in ERα, ERβ, ERα and ERβ, and GPER; a mouse lacking ERα binding to the ERE; and human islets. These mutant mice and islets were exposed to oxidative stress using STZ or hydrogen peroxide, respectively, in combination with the use of specific pharmacological probes.     

Lung and respiratory muscle function in facioscapulohumeral muscular dystrophy

Pulmonary dysfunction is not a well‐recognized feature of facioscapulohumeral muscular dystrophy (FSHD). The aim of this study was to establish the prevalence and type of pulmonary and respiratory muscle dysfunction in FSHD. Sixteen patients with moderately advanced FSHD and 16 healthy controls were evaluated. Standard lung and respiratory muscle function tests were performed. Diaphragm muscle inspiratory action was evaluated with transdiaphragmatic pressure measurements. Lung function tests showed an increased residual volume in five patients. There was a significant difference in global respiratory muscle function in patients versus controls; weakness was mild, and it affected expiratory more than inspiratory muscles. There was no significant difference in the diaphragm inspiratory action of patients versus controls. The dystrophic process that underlies FSHD did not significantly involve the muscles of the diaphragm, but it caused mild global respiratory muscle weakness that affected expiratory more than inspiratory muscles. It is probably not necessary to routinely monitor respiratory muscle function in ambulant FSHD patients who lack symptoms or signs of respiratory impairment. Muscle Nerve, 2009