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.

The mouse mutation muscle deficient (mdf) is characterized by a progressive motoneuron disease.

Muscle deficient (mdf) is an autosomal-recessive mutation mapped to mouse chromosome 19. The clinical phenotype and the muscle histopathology, briefly described in 1980, and the nervous system histopathology are detailed in the present study. Homozygotes develop a posterior waddle at 4 to 8 weeks of age. Soon thereafter, the hindlimbs become paralyzed and weakness appears in forelimbs, leading to a serious disability. The disease progresses slowly and the mean lifespan is reduced to 8 months. Skeletal muscles exhibit a neurogenic atrophy with signs of reinnervation. Peripheral nerves display axonal degeneration. Neurons within the spinal cord ventral horn, and some motor nuclei of the brain stem, are affected by a cytoplasmic vacuolar degeneration. Ascending and descending spinal cord tracts appear normal. An astrogliosis, restricted to the ventral horn of the spinal cord, occurs in mdf/mdf mice of 10 weeks of age. These clinical and histological features are indicative of a progressive motor neuronopathy. Among the murine spinal muscular atrophies, the programmed cell death of the mdf motoneurons is morphologically similar to wobbler. Because of the long time course, the mdf mutation may represent a valuable tool for understanding juvenile motoneuron diseases with chronic evolution, even though the murine locus is not syntenic with the human ones.

Transforming growth factor alpha expression as a response of murine motor neurons to axonal injury and mutation-induced degeneration.

We previously showed that degenerating adult motor neurons of the murine mutant wobbler, a model of spinal muscular atrophy, express Transforming Growth Factor alpha (TGF alpha), a growth factor endowed with glio- and neurotrophic activities. Here, we evaluated whether TGF alpha expression is a general response of adult motor neurons to injury. Synthesis of its precursor (pro-TGF alpha) was investigated in another model of motoneuronal degeneration, the murine mutant muscle deficient, and in hypoglossal motor neurons following axonal crush and cut. In control conditions, motor neurons were devoid of pro-TGF alpha immunoreactivity. In the mutant lumbar spinal cord, pro-TGF alpha immunoreactive motor neurons appeared as soon as the disease developed and pro-TGF alpha expression persisted until the latest stages of degeneration. Motor neurons and astrocytes of the white matter weakly immunoreactive for the TGF alpha receptor were also present in both control and mutant lumbar spinal cords. Following hypoglossal nerve crush and cut, motoneuronal pro-TGF alpha expression was precocious and transient, visible at one day post-injury and lasting for only 3 days, during which time astrocyte-like cells immunoreactive for both TGF alpha and its receptor appeared within the injured nucleus. Enhanced TGF alpha mRNA levels following nerve crush showed that activation occurred at the transcriptional level. These results show that upregulation of TGF alpha is an early and common response of adult murine motor neurons to injury, regardless of its experimental or genetic origin.

The effect of high doses of vitamin B6 on autistic children: a double-blind crossover study.

The authors used data from an earlier nonblind study to identify 16 autistic-type child outpatients who had apparently improved when given vitamin B6 (pyridoxine). In a double-blind study each child's B6 supplement was replaced during two separate experimental trial periods with either a B6 supplement or a matched placebo. Behavior was rated as deteriorating significantly during the B6 withdrawal.

Posttraumatic dysautonomic cephalalgia. Clinical observations and treatment.

Five patients developed posttraumatic vascular headaches associated with autonomic dysfunction. The precipitating injury affected the anterior triangle of the neck, presumably involving the region of the carotid artery sheath. Disturbance of sympathetic function, characterized by excessive sweating and pupillary dilation associated with headache, was noted. Appropriate pharmacologic studies revealed evidence of partial sympathetic devervation. While headache was resistant to ergotamine preparations, prompt relief was obtained with propranolol hydrochloride, and adrenergic beta-receptor blocking agent.

Perinatal neuropathy as an early manifestation of Krabbe's disease.

An asymptomatic child with antenatally diagnosed globoid cell leukodystrophy was found to have electrodiagnostic evidence of peripheral neuropathy at 7 weeks of age. The earliest prior such evidence in the literature was in a 6-month-old-child. Thus, peripheral neuropathy can be both the initial manifestation and a very early finding in Krabbe's disease.

Paroxysmal genital pain: an unusual manifestation of epilepsy.

A male patient who is now 15 years old has experienced a seizure disorder since age 9 years. The seizures were expressed as episodes of excruciating pain localized to the genital region. Appropriate anticonvulsant medication has controlled both pain and seizures.

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.

Short increase of BDNF messenger RNA triggers kainic acid-induced neuronal hypertrophy in adult mice.

Neurotrophin gene expression in adult brain varies according to physiological activity and following brain injury, suggesting a role in neuronal maintenance and plasticity. However, the exact roles and mechanisms of action of neurotrophins in the adult brain are still poorly understood. We have recently demonstrated that neurons of the adult mouse dentate gyrus can develop a conspicuous morphogenetic response to intrahippocampal injection of kainic acid. This response is correlated with long-lasting overexpression of the brain-derived neurotrophic factor gene, suggesting a causal relationship between molecular and structural changes. To test this hypothesis, brain-derived neurotrophic factor messenger RNA were sequestered in vivo by administration of antisense oligodeoxynucleotides. When administered before 20 h post-kainate, antisense oligodeoxynucleotides totally prevented the kainate-induced neuronal hypertrophy, while sense or missense sequences had no effect. On the other hand, the hypertrophic response was observed when antisense administration was begun 24 h post-kainate, indicating an involvement of brain-derived neurotrophic factor messenger RNA in the initiation of structural changes, but not in their evolution. The hypertrophy was blocked by inhibition of tyrosine kinase activities by K252a, suggesting an involvement of Trk high affinity receptors. Administration of human recombinant brain-derived neurotrophic factor without previous treatment by kainate failed to induce any morphogenetic response. These results show that a short activation of the brain-derived neurotrophic factor gene can, in association with neuronal activation by kainate, trigger dramatic and long-lasting morphological changes in adult neurons. A physiological role of brain-derived neurotrophic factor in adult brain could therefore be to link, by autocrine/paracrine action, activation of glutamate receptors and neuronal morphological adaptive responses.

Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy.

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.

Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors in human dilated cardiomyopathy: potential role of MMP-9 in myosin-heavy chain degradation.

BACKGROUND: End-stage of human dilated cardiomyopathy (DCM) is characterized by myocyte loss and fibrosis, and associated with ventricular dilatation and reduced cardiac function. Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) have been involved in the myocardial remodeling. AIMS: To evaluate the potential role of matrix gelatinases (MMP-2 and MMP-9) in DCM, the balance between gelatinases and TIMPs and the gelatinase localization were investigated in left free wall ventricles from six normal donors and six patients with DCM at the transplantation time. METHODS: TIMP-(1, 2, 3 and 4) mRNAs were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). TIMP-1 and -2 protein content was assessed by ELISA. MMP-2 and MMP-9 expression were examined by zymography and immunological techniques. RESULTS: All TIMPs were down-regulated in DCM hearts, especially TIMP-1 (reduced by 80%). Gel zymography revealed similar activity of MMP-2 and MMP-9 in both tissues. By in situ zymography and immunohistochemistry, active and immunoreactive gelatinases were pericardiomyocyte in control hearts and intracardiomyocyte in DCM hearts. Intracellular MMPs were associated with sarcomeric structure in DCM. To estimate a putative role of these gelatinases, several sarcomeric contractile proteins were digested in vitro by purified active MMP-9. Only myosin-heavy chain was cleaved in vitro giving 180-, 120-, 80- and 20-kDa proteolytic fragments. In vivo, two major myosin-heavy chain proteolytic fragments (80 and 20 kDa) were detected by specific monoclonal antibody against myosin-heavy chain in DCM left ventricular homogenates, only. CONCLUSIONS: Taken together, these data highly suggest that MMP-2 and MMP-9 may be involved in the disorganization of the contractile apparatus in DCM hearts.

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ.

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl(2) but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.

Generalized molecular defects of the neuromuscular junction in skeletal muscle of the wobbler mutant mouse.

Available models of motorneuron disease that occur naturally in animals provide a useful approach to study human motorneuron disease. The wobbler mutant mouse displays a hereditary lower motorneuron disease which leads to progressive partial denervation of skeletal muscle and, at the same time, axonal regeneration with attempted reinnervation. In order to determine the consequences of these processes at the neuromuscular level, we undertook a study of key molecular components of the neuromuscular junction in wobbler mice. Increased levels of acetylcholine-receptor (AChR) and neural cell adhesion molecule (N-CAM) in wobbler muscle, together with an intense axon sprouting, suggest a complex denervation-reinnervation phenomenon. Furthermore, the appearance of ectopic clusters of AChR, spatially related with regrowth of axons, suggests ectopic formation of new synaptic areas, while, at the same time, some old synaptic sites fail to be reinnervated. Finally, 90% of wobbler neuromuscular contacts present a reduced acetylcholinesterase activity and a lack of N-CAM, which suggests a generalized defect of the mutant neuromuscular junction. These observed abnormalities may well be the consequence of a specific motorneuron defect.

Hydrophilic and hydrophobic attachment of both globular and asymmetric acetylcholinesterase to frog muscle basal lamina sheaths.

We prepared myofiber basal lamina sheaths (BLs) using the in vivo experimental procedure of Sanes et al. (J. Cell Biol.78, 176-198, 1978) on frog cutaneus pectoris muscle. On the 15 days post-operatively, acetylcholinesterase (AChE) is still found concentrated in native BLs and purified BLs preparations and both globular and asymmetric molecular forms coexist (Nicolet et al., J. Cell Biol., 107, 762-768, 1986). We describe here at least two distinct AChE pools, according to their differential solubility in non-ionic detergent and high-salt media. One is detergent-extracted (DE) and the other is detergent-insoluble, high-salt extracted (HSS). In the BLs preparation as well as in control motor end-plate rich regions (MEP-r) of muscle, both globular and asymmetric forms of AChE are found as DE and HSS variants. These observations suggest that all AChE forms are present in the extracellular muscle basal lamina and are bound through not only hydrophilic but also hydrophobic bonds, to probably distinct structural domains of the muscle basal lamina.

A dimeric form of acetylcholinesterase anchored through a glycolipid in mouse skeletal muscle.

A glycolipid anchorage for acetylcholinesterase (AChE) has been found in some tissues. In this paper, the possibility of such an anchorage has been explored in mammalian muscle membranes. We report that a phosphatidylinositol-specific phospholipase C (PIPLC) solubilizes AChE from microsomal membranes of mouse intercostal muscle. Among the several molecular forms of AChE, PIPLC specifically releases in a dose dependent manner one molecular form which migrates on linear sucrose gradients as a single peak of sedimentation coefficient 6.3 s. In other subcellular membrane fractions, including motor endplate enriched fraction, PIPLC fails to solubilize AChE. This type of membrane glycolipid mediated anchorage for AChE is then only detectable in a precise region of skeletal muscle.

Association of tailed acetylcholinesterase to lipidic membranes in mammalian skeletal muscle.

Tailed acetylcholinesterase (AChE) was studied in three subcellular membrane fractions of mouse skeletal muscle: a fraction enriched in isolated motor endplates (C), an extrasynaptic membrane fraction (A) and a microsomal fraction (S). In the (C) fraction, tailed asymmetric 16S AChE required high salt conditions to be extracted, while in (A) and (S) microsomal membranes, a collagenase sensitive 16S form, was extracted by detergent alone. This apparent "hydrophobic" property suggests that there is a pool of 16S AChE which is probably bound to lipidic membranes. The detergent extractable (DE) 16S AChE was not concentrated in motor endplate-rich regions and differential inhibition of external and internal AChE demonstrated that it could have both intra- and extracellular locations in the adult differentiated muscle fibres.

Early detection of mouse wobbler mutation: a model of pathological motoneurone death.

The mouse recessive mutation wobbler, carried by the C57BL/6J strain, is a naturally occurring model of motoneurone death. The gene is unknown and in the absence of predictive markers, mutants have to be diagnosed by phenotypic criteria at 4 weeks after birth. We localized the wobbler gene to chromosome 11 at 0.98 +/- 1.1 cM from the glutamine synthetase (Glns) gene. A polymorphic allele of the Glns gene was then introduced into the congenic wobbler strain by intraspecific crossing. One-quarter of the offspring expressed the same phenotypic mutation as true wobbler and were detectable by PCR, as they are homozygous for the wobbler-linked Glns allele. The new mutants exhibit motoneurone degeneration despite the new genetic background.

Peripheral injections of Freund's adjuvant in mice provoke leakage of serum proteins through the blood-brain barrier without inducing reactive gliosis.

Breakdown of the blood-brain barrier (BBB) and ensuing gliosis are common events following physical trauma to the central nervous system (CNS) or during autoimmune diseases such as experimental allergic encephalomyelitis (EAE). Some studies of EAE in rodents report that peripheral injections of complete Freund's adjuvant (CFA), which contains heat-inactivated Mycobacterium to provoke peripheral inflammation without adversely affecting the CNS, can itself lead to increased BBB permeability to small tracer molecules and certain serum proteins. To study the equivocal relationship between serum protein extravasation and reactive gliosis, we injected C57BL/6 mice with CFA and histologically assessed the permeability of various serum proteins and the reactivity of proximal microglia and astrocytes in the uninjured brainstem and spinal cord enlargements after 1-4 weeks. Our results confirm that CFA injections induce progressive increases in the perivascular extravasation of serum IgG, albumin, IgM, and exogenous horseradish peroxidase, all to varying degrees, most prominently in the brainstem and cervical spinal cord after 2-3 weeks. More importantly, neither microglial cells nor astrocytes in regions of focal serum protein leakage appeared morphologically reactive based on immunoreactivity for CR3 receptors (Mac-1) or glial fibrillary acidic protein (GFAP), respectively. Because we found no evidence of T cell infiltration accompanying the exudates, our results indicate that in the absence of physical trauma or inflammatory cells resident CNS neuroglia remain quiescent upon exposure to extravasated serum proteins.