Targeting the splicing of mRNA in autoimmune diseases: BAFF inhibition in Sjögren's syndrome as a proof of concept.

BAFF (B-cell-activating factor of the tumor necrosis factor family), a pivotal cytokine for B-cell activation, is overexpressed by salivary gland (SG) epithelial cells in primary Sjogren's syndrome (pSS). ΔBAFF, a physiological inhibitor of BAFF, is a minor alternative splice variant of BAFF. A U7 RNA was reengineered to deliver antisense sequences targeting BAFF splice regions. A major decrease of BAFF messenger RNA (mRNA) and protein secretion, concomitantly with the increase of ΔBAFF mRNA, was observed in vitro. In vivo, SG retrograd instillation of nonobese diabetic mice by the modified U7 cloned into an adeno-associated virus vector significantly decreased BAFF protein expression and lymphocytic infiltrates and improved salivary flow. This study offers a rationale for localized therapeutic BAFF inhibition in pSS and represents a proof of concept of the interest of exon skipping in autoimmune diseases.

Muco-ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro.

BACKGROUND: Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. METHODS: Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. RESULTS: In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. CONCLUSION: After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

Muscle precursor cell autografting in a murine model of urethral sphincter injury.

OBJECTIVE: To determine whether muscle precursor cells (MPCs) harvested from limb skeletal muscle can enhance the regeneration process of the striated urethral sphincter after injury. MATERIAL AND METHODS: Striated urethral sphincters of male mice were injured by an injection of a myotoxic substance (notexin). In the experimental group, 2 days after injury, MPCs were enzymatically harvested from striated muscles of the lower limbs and labelled with PKH 26, then immediately re-injected into the injured urethral sphincter of the same animal. In the control group, saline buffer was injected instead of MPCs. Animals were killed 7 days or 1 month after injury and the sphincters removed for histological study (the presence of PKH 26-labelled myofibres, measurement of myofibre diameter and total number of myofibres). RESULTS: MPC autografting accelerated sphincter muscle repair, as shown by a higher myofibre diameter (P = 0.03) and number (P = 0.01) in the experimental group than in the controls at 7 days. One month after their injection MPCs were still detectable in the regenerating sphincters and participated in the formation of new myofibres. CONCLUSION: This study provides the experimental basis for a new therapeutic approach to urethral sphincter insufficiency after surgical or obstetrical injury, based on MPC autografting.

Oligoclonal T-cells in blood and target tissues of patients with anti-Hu syndrome.

T-cell clones of unknown significance (TCUS), assessed by monoclonal or oligoclonal T-cell patterns in PCR-DGGE, were detected in blood of 7/9 patients with anti-Hu syndrome. Clonal patterns were also detected in 2/2 neoplastic lymph nodes, and in 2/2 inflamed dorsal root ganglia from three patients. Only some T-cell clones found in target tissues were also detected in blood or non-target tissues, and likely corresponded to TCUS. In one patient, an identical T-cell clone was found in both neoplastic lymph node tissue and dorsal root ganglia, but not in blood. Dorsal root-infiltrating lymphocytes were cytotoxic CD8(+) TIA-1(+) T-cells. They were often found in close contact to sensory neurons, most of which expressed MHC-1. Taken together, these data support a direct effector role of cytotoxic CD8(+) T-cells, the same clones being likely operative in sensory neuron damage and immune-mediated tumor growth control.

Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle.

Macrophagic myofasciitis (MMF) is an emerging condition of unknown cause, detected in patients with diffuse arthromyalgias and fatigue, and characterized by muscle infiltration by granular periodic acid-Schiff's reagent-positive macrophages and lymphocytes. Intracytoplasmic inclusions have been observed in macrophages of some patients. To assess their significance, electron microscopy was performed in 40 consecutive cases and chemical analysis was done by microanalysis and atomic absorption spectrometry. Inclusions were constantly detected and corresponded to aluminium hydroxide, an immunostimulatory compound frequently used as a vaccine adjuvant. A lymphocytic component was constantly observed in MMF lesions. Serological tests were compatible with exposure to aluminium hydroxide-containing vaccines. History analysis revealed that 50 out of 50 patients had received vaccines against hepatitis B virus (86%), hepatitis A virus (19%) or tetanus toxoid (58%), 3-96 months (median 36 months) before biopsy. Diffuse myalgias were more frequent in patients with than without an MMF lesion at deltoid muscle biopsy (P < 0.0001). Myalgia onset was subsequent to the vaccination (median 11 months) in 94% of patients. MMF lesion was experimentally reproduced in rats. We conclude that the MMF lesion is secondary to intramuscular injection of aluminium hydroxide-containing vaccines, shows both long-term persistence of aluminium hydroxide and an ongoing local immune reaction, and is detected in patients with systemic symptoms which appeared subsequently to vaccination.

Ciliary neurotrophic factor may activate mature astrocytes via binding with the leukemia inhibitory factor receptor.

Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTFalpha receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFRalpha-/- cells; (iv) it is potentiated by soluble CNTFRalpha added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFRalpha.

Angiotensin II induces nuclear factor- kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway.

Rat neonatal ventricular cardiomyocytes (RNVM) possess G protein-coupled AT(1)receptors for angiotensin II (AngII) that activate multiple intracellular pathways. To elucidate potential signaling mechanisms involved, we focussed on the nuclear transcription factor-kappa B (NF- kappa B) in RNVM culture. Using specific antibody to NF- kappa Bp65, immunolocalization of NF- kappa B was cytoplasmic in unstimulated cardiomyocytes, whereas NF- kappa B was translocated into the RNVM nucleus in response to AngII. This translocation was inhibited in the presence of calphostin C, a specific inhibitor of protein kinase C (PKC). Western blot analysis showed an increase of NF- kappa B in AngII-stimulated cardiomyocyte nuclear extracts as compared to controls. Biomolecular interaction analysis (BIA analysis) of NF- kappa B activation showed that only AngII-nuclear extracts bound to NF- kappa B consensus sequence with a high degree of affinity. This DNA-binding capacity was completely lost in calphostin C-treated cells. At transcriptional level in RNVM, AngII mediates the upregulation of matrix gelatinase (MMP-9), which is totally inhibited by calphostin C treatment. In conclusion, cardiomyocyte nuclear NF- kappa B translocation in response to Ang II via PKC pathway activates cardiomyocyte-specific transcription of MMP-9 and may activate transcription from responsive genes which are involved in cardiac hypertrophy process and/or cardiac remodeling.

Heart and lung VEGF mRNA expression in rats with monocrotaline- or hypoxia-induced pulmonary hypertension.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is upregulated during exposure to hypoxia. In this study, we analyzed heart and lung VEGF mRNA expression and examined pulmonary vascular remodeling as well as myocardial capillary density in two rat models of pulmonary hypertension involving exposure to chronic hypoxia (CH) and treatment with monocrotaline (MCT), respectively. The rats were studied after 0.5, 1, 3, 15, and 30 days of exposure to 10% O2 or 1, 6, and 30 days after a subcutaneous MCT injection (60 mg/kg). Both CH and MCT induced pulmonary hypertension and hypertrophy of the right ventricle (RV) with increased RV weight and atrial natriuretic peptide mRNA expression. VEGF mRNA expression as assessed by Northern blot analysis was potently induced after 12 h of hypoxia in both the right and left ventricles. After prolonged exposure to hypoxia, VEGF mRNA returned to baseline in the left ventricle (LV) but remained increased in the RV, where it peaked after 30 days. In MCT rats, VEGF mRNA was unchanged in the LV but decreased by 50% in the RV and by 90% in the lungs after 30 days. VEGF mRNA remained unchanged in the lungs from CH rats. Pulmonary vascular remodeling was more pronounced in MCT than in CH rats. The number of capillaries per RV myocyte was increased in rats exposed to 30 days of hypoxia, whereas it remained unchanged in MCT rats despite a similar degree of RV hypertrophy. Our results suggest that the sustained increase in VEGF expression in the hypertrophied RV during CH may account for the increased number of capillaries per myocyte. In contrast, reduced VEGF expression in the lungs and RV of MCT rats may aggravate pulmonary vascular remodeling and compromise RV myocardial perfusion.

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Bcl-2 sensitivity differentiates two pathways for motoneuronal death in the wobbler mutant mouse.

The molecular events leading to motoneuronal death are still poorly understood. In mammals, the bcl-2 proto-oncogene, which encodes a membrane-associated protein, has been shown to suppress both developmental motoneuronal death and experimental axotomy-induced motoneuronal death. We assessed a potential protective effect of Bcl-2 on pathological motoneuronal death processes in adult rodents. We took advantage of the murine mutant wobbler, which undergoes progressive degeneration of the spinal and brainstem motoneurons. A hybrid carrying both the wobbler mutation and the human bcl-2 transgene under the control of the neuron-specific enolase promoter was produced. Although Bcl-2 protected spinal and brainstem motoneurons from developmental death and the postnatal motoneurons of the facial nucleus from axotomy-induced death, the pathological motoneuronal death was not altered in the adult hybrid. These results demonstrate that Bcl-2 sensitivity distinguishes at least two different motoneuronal death pathways in the wobbler mutant. They support the hypothesis that experimental and pathological motoneuronal death are dependent on different cellular mechanisms.

Targeting widespread sites of damage in dystrophic muscle: engrafted macrophages as potential shuttles.

Inherited muscle diseases are characterized by widespread muscle damage in the body. This limits the clinical relevance of cell or gene therapy based upon direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells, capable of homing naturally to the sites of lesion, to deliver therapeutic substances. Certain muscular dystrophies present successive cycles of degeneration-regeneration. These sporadic necrotic lesions trigger local inflammations with subsequent infiltration of blood-borne mononuclear cells. We have, therefore, tested the possibility that homing monocytes and macrophages could be appropriate shuttles for delivering a therapeutic agent to disseminated pathogenic sites, their targeting being triggered by the pathogeny itself. First, fluorescently labeled immortalized monocytes were intravenously injected into mice which had previously undergone freeze-damaging of individual muscles. In agreement with our hypothesis, intense labelling was observed in the muscle, specifically in damaged regions. Second, the technique was adapted to meet the needs of chronic diseases with characteristic continuous, widespread degeneration of muscle fibers, by creating a reservoir of genetically engineered monocytes, via bone marrow transplantation. Mdx mice received bone marrow from transgenic mice expressing the lacZ reporter gene, under the control of the vimentin promoter, which is active in monocytes and macrophages. Histological and molecular analyses demonstrated the homing of engineered macrophages at the sites of muscle damage, for periods as long as 2 months. Bone marrow progenitor cells, appropriately engineered to elicit the synthesis, in macrophages, of therapeutically relevant substances, may be of clinical value in various pathologies involving an inflammatory phase.

Transforming growth factor alpha (TGF alpha) expression in degenerating motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?

The enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity.

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes.

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.

De novo amplification within a "silent" human cholinesterase gene in a family subjected to prolonged exposure to organophosphorous insecticides.

A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing the "silent" CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorous insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the "onion skin" model, based on amplification of genes in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where we previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. Our findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor. Its occurrence in two generations from a family under prolonged exposure to parathion indicates that organophosphorous poisons may be implicated in previously unforeseen long-term ecological effects.

Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides.

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.

[Rheumatological manifestations of adrenal insufficiencies]. / Manifestations rhumatologiques des insuffisances surrénales.

Muscular, articular and periarticular symptoms were reviewed in 19 patients with adrenal failure and compared with previously published data. Symptoms are variable and non-specific; muscular cramps and periarticular calcification were common. Hypercalcaemia was uncommon but its incidence was probably underestimated. Calcification of the ear cartilage was rare but characteristic.