Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study.

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

ARC Syndrome-Linked Vps33B Protein Is Required for Inflammatory Endosomal Maturation and Signal Termination.

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. Here, we report that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of ARC syndrome, a disease linked to loss of Vps33B.

Two novel mutations in myosin binding protein C slow causing distal arthrogryposis type 2 in two large Han Chinese families may suggest important functional role of immunoglobulin domain C2.

Distal arthrogryposes (DAs) are a group of disorders that mainly involve the distal parts of the limbs and at least ten different DAs have been described to date. DAs are mostly described as autosomal dominant disorders with variable expressivity and incomplete penetrance, but recently autosomal recessive pattern was reported in distal arthrogryposis type 5D. Mutations in the contractile genes are found in about 50% of all DA patients. Of these genes, mutations in the gene encoding myosin binding protein C slow MYBPC1 were recently identified in two families with distal arthrogryposis type 1B. Here, we described two large Chinese families with autosomal dominant distal arthrogryposis type 2(DA2) with incomplete penetrance and variable expressivity. Some unique overextension contractures of the lower limbs and some distinctive facial features were present in our DA2 pedigrees. We performed follow-up DNA sequencing after linkage mapping and first identified two novel MYBPC1 mutations (c.1075G>A [p.E359K] and c.956C>T [p.P319L]) responsible for these Chinese DA2 families of which one introduced by germline mosacism. Each mutation was found to cosegregate with the DA2 phenotype in each family but not in population controls. Both substitutions occur within C2 immunoglobulin domain, which together with C1 and the M motif constitute the binding site for the S2 subfragment of myosin. Our results expand the phenotypic spectrum of MYBPC1-related arthrogryposis multiplex congenita (AMC). We also proposed the possible molecular mechanisms that may underlie the pathogenesis of DA2 myopathy associated with these two substitutions in MYBPC1.

Antibodies identified by cell-based assays in myasthenia gravis and associated diseases.

We have established cell-based assays for the improved detection of acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) antibodies in myasthenia gravis. This approach has enabled us to demonstrate antibodies to "clustered" AChRs in patients who were previously AChR antibody negative and can also be used to distinguish between adult and fetal AChR antibodies in mothers of babies with arthrogryposis multiplex congenita. We summarize our recent evidence for the pathogenicity of MuSK and clustered AChR antibodies using in vivo models. Cell-based assays are now also being used for the detection of other antibodies, such as those directed to components of the VGKC/CASPR2/LGI1 complex in Morvan's syndrome, and to AQP4 antibodies in neuromyelitis optica; both of these diseases can be associated with MG and sometimes thymoma. The cell-based method is time consuming but has many advantages and may provide a gold standard for the future in the detection of pathogenic autoantibodies in patients with immune-mediated diseases.

Fetal arthrogryposis and maternal serum antibodies.

Arthrogryposis multiplex congenital (AMC) describes multiple joint contractures resulting from lack of movement in utero. Antibodies directed at the fetal isoform of the muscle acetylcholine receptor (AChR) have been reported in a small number of asymptomatic mothers of AMC babies. We examined sera from 179 mothers of AMC babies and 20 parous and non-parous controls to look for antibodies to AChR or undefined muscle or neuronal proteins. We found positive AChR antibodies in only three sera (1.5%) from asymptomatic AMC mothers. However, there was reactivity with muscle or with neuronal antigens in 33% of the sera, and reactivity to undefined neuronal antigens was more common in sera from mothers of AMC babies with CNS involvement (p=0.001) than those without. The offspring of mothers with AChR antibodies may benefit from treatment during pregnancy. Other maternal antibodies require further study, but these observations add to the emerging literature on maternal antibodies associated with developmental intrauterine disorders.

Artrogryposis multiplex congenita -- a rare fetal condition caused by maternal myasthenia gravis.

OBJECTIVES: To look at the occurrence of arthrogryposis multiplex congenita in newborn of mothers with myasthenia gravis (MG) and factors connected to this. MATERIAL AND METHODS: We retrospectively studied 176 births by 79 MG mothers, recorded in the Medical Birth Registry of Norway (MBRN). Four (2.2%) newborns (including one pair of twins) born with severe skeletal anomalies were identified. RESULTS: All four children died. Three had findings consistent with arthrogryposis multiplexa congenita (AMC), one had a fetal akinesia deformation sequence (FADS). The mother of the child with FADS had previously given birth to a child with neonatal MG. She was now in complete MG remission. The mother of the twins with AMC later gave birth to a child with neonatal MG. CONCLUSION: Siblings of an affected child -- either with neonatal MG or AMC -- have an increased risk to develop either neonatal MG or AMC. As this appears to be independent of the MG mother's clinical state, it is important to discuss previous pregnancy outcomes with all female MG patients.

Antibodies in myasthenia gravis and related disorders.

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

Antibodies to neuronal targets in neurological and psychiatric diseases.

The role of antibodies to specific neuronal and muscle ion channels in the etiology of neuromuscular transmission disorders is now well accepted. In addition, maternal antibodies can cross the placenta and cause neonatal disease or even alter the development of the infant, raising the possibility that some neurodevelopmental conditions could be caused by maternal antibodies. Voltage-gated ion channels are expressed in the brain as well as at the neuromuscular junction, and in recent years it has become clear that antibodies to some central nervous system (CNS) channels can be associated with CNS disease. This review highlights features of these conditions, preliminary investigations into neurodevelopmental disorders, and areas for further study.

Antibodies affecting ion channel function in acquired neuromyotonia, in seropositive and seronegative myasthenia gravis, and in antibody-mediated arthrogryposis multiplex congenita.

A new autoimmune disease affecting the neuromuscular junction has been defined. Acquired neuromyotonia is associated with antibodies to voltage-gated potassium channels that act, at least in part, by reducing potassium channel function with resulting neuronal hyperactivity. This condition is quite frequently associated with thymoma and, in many cases, antibodies to acetylcholine receptors are present as well as antibodies to VGKC. Improvements in techniques and the availability of cloned DNA and recombinant forms of the AChR subunits have led to new observations concerning the specificity and roles of antibodies in myasthenia gravis. The transfection of a cell line with the epsilon subunit means that we can now accurately compare antibodies reactive with adult and fetal human AChR. This may help to determine the relationship between AChR subunit expression in different tissues and the induction of antibodies that bind specifically to the two forms, as well as to clarify the role of antibodies to fetal or adult AChR in causing ocular muscle symptoms. Serum antibodies from a few mothers with obstetric histories of recurrent arthrogryposis multiplex congenita in their babies specifically inhibit the function of fetal AChR. These observations not only explain the cause of some cases of arthrogryposis multiplex congenita, but also suggest that other fetal-specific antibodies might be responsible for other fetal or neonatal conditions. An animal model has been established to enable us to investigate the role of maternal serum factors in causing such disorders. Seronegative MG has been the subject of many studies from our laboratory over the last ten years. The transience of the effects of SNMG plasmas on AChR function strongly suggests that the plasma antibodies do not bind directly to the AChR, but inhibit function by some indirect mechanism. They do not appear to act via the cAMP-dependent protein kinase pathway, and studies are in progress to investigate the involvement of other second messenger systems.

Association of arthrogryposis multiplex congenita with maternal antibodies inhibiting fetal acetylcholine receptor function.

Arthrogryposis multiplex congenita (AMC), characterized by multiple joint contractures developing in utero, results from lack of fetal movement. Some cases are genetically determined, but AMC occasionally complicates pregnancy in patients with myasthenia gravis (MG) suggesting involvement of circulating maternal antibodies. We previously demonstrated antibodies that inhibited the function of fetal acetylcholine receptor (AChR) in one healthy woman with an obstetric history of recurrent AMC. Here we study sera from this woman, from one other with a similar history, and from three (one asymptomatic) whose babies had neonatal MG and AMC. All five maternal sera had high titers of antibodies that inhibited alpha-Bungarotoxin (alpha-BuTx) binding to fetal AChR, and their sera markedly inhibited fetal AChR function with little effect on adult AChR function. Moreover, in a further survey, 3 of 20 sera from anti-AChR negative AMC mothers inhibited fetal AChR function significantly at 1:100 dilution. These results demonstrate the role of antibodies to fetal AChR and perhaps other muscle antigens in some cases of AMC. More generally, they suggest that placental transfer of antibodies directed at fetal antigens should be considered as a cause of other recurrent fetal or perinatal disorders.

Arthrogryposis multiplex congenita with maternal autoantibodies specific for a fetal antigen.

Fetal arthrogryposis multiplex congenita (AMC) is characterised by non-progressive multiple joint contractures, which may result in fetal death, and is heterogeneous in origin. It can associate with maternal myasthenia gravis and autoantibodies to muscle acetylcholine receptor (AChR). We found maternal antibodies that selectively inhibit the fetal form of the AChR in a mother who herself had no features of myasthenia gravis. Maternal autoantibodies specific for fetal antigens could be an unrecognised cause of other congenital disorders.