Butyrylcholinesterase activity and molecular components in thymus of healthy and merosin-deficient Lama2dy mice

The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.     

The levels of both lipid rafts and raft-located acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency

Wild type and dystrophic (merosin-deficient) Lama2dy mice muscles were compared for their density of lipid rafts. The 5-fold higher level of caveolin-3 and the 2-3 times higher level of ecto-5’-nucleotidase activity in raft preparations (Triton X-100-resistant membranes) of dystrophic muscle supported expansion of caveolar and non-caveolar lipid rafts. The presence in rafts of glycosylphosphatidylinositol (GPI)-linked acetylcholinesterase (AChE) dimers, which did not arise from erythrocyte or nerve, not only revealed for the first time the capacity of the myofibre for translating the AChE-H mRNA but also an unrecognized pathway for targeting AChE-H to specialized membrane domains of the sarcolemma. Rafts of dystrophic muscle contained a 5-fold higher AChE activity/mg protein. RT-PCR for 3’-alternative mRNAs of AChE revealed AChE-T mRNA prevailing over AChE-R and AChE-H mRNAs in wild type mouse muscle. It also displayed principal 5’-alternative AChE mRNAs with exons E1c and E1e (the latter coding for N-terminally extended subunits) and fewer with E1d, E1a and E1b. The levels of AChE and butyrylcholinesterase mRNAs were unaffected by dystrophy. Finally, the decreased level of proline-rich membrane anchor (PRiMA) mRNA in Lama2dy muscle provided for a rational explanation to the loss of PRiMA-bearing AChE tetramers in dystrophic muscle.     

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis–Menten constant (K _m)for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the V _max was 0.547 mol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver–Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K_i(slope) and K _I(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. K _i was greater than K_i, indicating that CH has a greater binding affinity for the peripheral site than the active site.     

Dynamics of ribosomal activity and protein production in peripheral blood lymphocytes during an immune response

Microscopic spectrofluorimetry with acridine orange and 1-anilino-8-naphthalene sulfonate was used to assess the levels of translationally active rRNA (parameter α’, red/green emission ratio) and protein synthesis (parameter β, blue/green) in peripheral blood lymphocytes during rabbit immunization with ovalbumin. The averaged α’ and β changed synchronously and increased maximally after the third booster injection. At the height of the immune response, the distributions of lymphocytes in either parameter were broad and polymodal, suggesting differential activation of particular subpopulations. This two-dye, three-color assay holds much promise for monitoring the functional state of various cells.     

Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice

Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.     

TNF-alpha decreases hsp 27 in human blood mononuclear cells: involvement of protein kinase c

Treatment of PBMCs with TNF-alpha decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-alpha increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-alpha was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-alpha on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-alpha, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-alpha. These results indicate that TNF-alpha reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.     

Antileishmanial activity of a formulation of 2-n-propylquinoline by oral route in mice model

2-n-propylquinoline is presently a drug-candidate for the treatment of visceral leishmaniosis in pre-clinical development. As this compound is in an oily state, it needs to be formulated and the objectives of this study are: to prepare a formulation; to demonstrate that the new salted formulation did not alter the activity of the active ingredient; and finally, that this activity was quite good compared to the reference oral drug, miltefosine. Therefore, a 2-n-propylquinoline formulation, as camphorsulfonic salt, was prepared and characterised. On the Leishmania donovani / Balb/c mice model, a treatment by oral route at 60 μmoles/kg/day for ten consecutive days with this formulation was compared to 2-n-propylquinoline alone and to miltefosine, the oral reference drug. The salt formulation did not alter the activity of the 2-n-propylquinoline. The formulation reduced the parasite burden of 76% compared to 89% for miltefosine (not significant). The characteristics of this formulation results in a suitable drugability of 2-n-propylquinoline for further studies.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2)

The renin-angiotensin system (RAS), through angiotensin II and the angiotensin-converting enzyme (ACE), is involved in the genesis and progression of fibrotic diseases characterized by the replacement of normal tissue by an accumulation of an extracellular matrix (ECM). Duchenne muscular dystrophy (DMD) presents fibrosis and a decrease in muscle strength produced by chronic damage. The mdx mouse is a murine model of DMD and develops the same characteristics as dystrophic patients when subjected to chronic exercise. The connective tissue growth factor (CTGF/CCN2) and transforming growth factor type beta (TGF-β), which are overexpressed in muscular dystrophies, play a major role in many progressive scarring conditions. We have tested the hypothesis that ACE inhibition decreases fibrosis in dystrophic skeletal muscle by treatment of mdx mice with the ACE inhibitor enalapril. Both sedentary and exercised mdx mice treated with enalapril showed improvement in gastrocnemius muscle strength explained by a reduction in both muscle damage and ECM accumulation. ACE inhibition decreased CTGF expression in sedentary or exercised mdx mice and diminished CTGF-induced pro-fibrotic activity in a model of CTGF overexpression by adenoviral infection. Enalapril did not have an effect on TGF-β1 expression or its signaling activity in sedentary or exercised dystrophic mice. Thus, ACE inhibition might improve muscle strength and decrease fibrosis by diminishing specifically CTGF expression and activity without affecting TGF-β1 signaling. Our data provide insights into the pathogenic events in dystrophic muscle. We propose ACE as a target for developing therapies for DMD and related diseases.     

Chronic pharmacological activation of P2Y13 receptor in mice decreases HDL-cholesterol level by increasing hepatic HDL uptake and bile acid secretion

High level of high-density lipoprotein cholesterol (HDL-cholesterol) is inversely correlated to the risk of atherosclerotic cardiovascular disease. The protective effect of HDL is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and is later delivered to the liver for further metabolism and bile excretion. We have previously demonstrated that P2Y₁₃ receptor is critical for RCT and that intravenous bolus injection of cangrelor (AR-C69931MX), a partial agonist of P2Y₁₃ receptor, can stimulate hepatic HDL uptake and subsequent lipid biliary secretion without any change in plasma lipid levels. In the present study, we investigated the effect of longer-term treatment with cangrelor on lipoprotein metabolism in mice. We observed that continuous delivery of cangrelor at a rate of 35 μg/day/kg body weight for 3 days markedly decreased plasma HDL-cholesterol level, by increasing the clearance of HDL particles by the liver. These effects were correlated to an increase in the rate of biliary bile acid secretion. An increased expression of SREBP-regulated genes of cholesterol metabolism was also observed without any change of hepatic lipid levels as compared to non-treated mice. Thus, 3-day cangrelor treatment markedly increases the flux of HDL-cholesterol from the plasma to the liver for bile acid secretion. Taken together our results suggest that P2Y₁₃ appears a promising target for therapeutic intervention aimed at preventing or reducing cardiovascular risk.     

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A33:03 in peripheral blood mononuclear cells from patients and transgenic mice

Cytotoxic T lymphocyte (CTL) epitopes in the HBV protein of hepatitis B virus (HBV) may play a key role in viral control and liver damage. The aim of this study was to identify and study the function of HLA-A^∗33:03-restricted CTL epitopes in HBV protein of the HBV genotypes B and C, which are epidemic in China. Sixteen HBV peptides were predicated by computational analysis, and synthesized peptides were examined for their affinity to HLA-A^∗33:03 using a stable cell line. After being analyzed by enzyme-linked immunospot and cytolytic activity assays, as well as the tetramers staining method using peripheral blood mononuclear cells isolated from HBV-infected patients, five peptides (Hbs245–253, HBs335–343, HBc119–127, HBc104–112, and HBp391–399) were chosen to further confirm their HLA_A^∗33:03 restriction in transgenic mice.     

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24?weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF?+?F). Overweight/obese, T2DM patients on metformin therapy were given for 24?weeks corn oil (Placebo; 5?g/day) or n-3 PUFA concentrate as above (Omega-3; 5?g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF?+?F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF?+?F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.     

Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice

The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.     

Distorted leukocyte migration,angiogenesis, wound repair and metastasis in Tspan8 and Tspan8/CD151 double knockout mice indicate complementary activities of Tspan8 and CD51

The tetraspanin Tspan8 supports via associated integrins and proteases tumor progression and angiogenesis. To shed light on its activities in non-transformed cells, we generated a Tspan8 knockout (ko) mouse, comparing leukocyte migration, angiogenesis, wound healing and tumor growth with wild type, CD151ko and Tspan8/CD151ko (dbko) mice. CD151ko mice were included as CD151 activities resemble that of Tspan8, and dbko mice to exclude mutual substitution. Tspan8ko and dbko mice show no pathological phenotype. However, delayed type hypersensitivity reactions are mitigated in Tspan8ko mice, angiogenesis is severely impaired in Tspan8ko, CD151ko and dbko mice, with Tspan8 mostly affecting lymphangiogenesis. Distinct contributions of CD151 and Tspan8 to skin wound healing rely on preferentially CD151 anchoring basal keratinocytes and Tspan8 promoting motility. Proliferation of wounded skin keratinocytes is not affected. Metastasis formation of a melanoma and a Tspan8-expressing pancreatic cancer line was impaired in Tspan8ko and dbko mice, pointing towards a contribution of host Tspan8 to tumor progression. In line with the importance of tetraspanins in exosome-mediated intercellular communication, defects became mitigated by Tspan8/CD151-competent serum exosomes, which offers a most promising therapeutic option for chronic wounds and arteriosclerosis.