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Multiple sclerosis (MS) is a heterogeneous disease. With several disease modifying treatments of different mechanisms of action in use now and in development, it is important to identify reliable biomarkers to identify those higher risk MS patients in whom stronger but riskier treatments might be used, as well as to identify those for whom safer treatments of lower efficacy would be sufficient. Here we review cerebrospinal fluid (CSF) and blood biomarkers that show promise for differentiating people with MS who are at risk for severe disease and disability from those with more benign disease. We reviewed published literature for studies reporting biomarkers with predictive value in MS. Most studies of MS CSF found the presence of oligoclonal bands (both IgG and IgM), high IgG index and high levels of kappa light chains to each be associated with worse prognosis. Neurofilament light chain (NfL) and two markers of glial activation, glial fibrillary acidic protein (GFAP) and YKL-40, were higher in CSF of people with subsequent clinical progression or imaging evidence for neurodegeneration. Few reports have been made yet on the prognostic significance of blood NfL, but in one early report baseline, serum NfL (sNfL) predicted subsequent brain volume loss.

Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. The International Mouse Phenotyping Consortium (IMPC) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (ERG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the PLC-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.

The use of immunomodulatory and immunosuppressive therapies in multiple sclerosis (MS) has allowed practitioners to regulate MS disease activity, with the caveat that these potent medications may render patients susceptible to opportunistic infections. The approval of fingolimod presented the first oral option for relapsing MS. Since 2015, postmarketing safety data have documented several published cases of cryptococcal meningitis and disseminated cryptococcosis associated with fingolimod use. However, surveillance mechanisms for opportunistic infections and management of active demyelinating disease with ongoing infection have not been adequately addressed. We present a case of isolated pulmonary cryptococcosis with the use of fingolimod to highlight the hurdles in balancing efficacious disease-modifying therapies for MS while treating an opportunistic infection associated with that therapy.

Variants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+/J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D-induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.

Background: Recent genome-wide association and cross-sectional studies have identified novel human genetic polymorphisms associated with alterations in severe malaria risk. Some of the strongest associations have been identified in the ATP2B4 gene. ATP2B4 codes for PMCA4b, the primary regulator of erythrocyte calcium concentration. Methods: To investigate the cellular mechanisms by which polymorphisms in ATP2B4 alter malaria pathogenesis, we performed in vitro assays that model malaria pathogenesis on red blood cells (RBCs) collected from individuals (n=96) with and without copies of ATP2B4 variant haplotype. Findings: We found reduced rates of PMCA4b expression (p

Background: Human neonates elicit a profound hypoferremia to protect against bacterial and fungal sepsis on their first day of life. We examined the transience of this effect by measuring iron and its chaperone proteins, inflammatory and hematological parameters over the first post-partum week. Methods: We prospectively studied term (>37 completed gestational weeks), normal weight (>2500g) newborns at Kanifing General Hospital, The Gambia. Blood was sampled from the umbilical cord vein (CDV) and artery (CDA). Neonatal venous blood was sampled at 6-24h (V1) in all babies who were then randomized to a second blood draw at 25-80h (V2), 81-136h (V3) or 137-192h (V4). Hepcidin, serum iron, transferrin saturation, transferrin, haptoglobin, CRP, AGP, sTfR, ferritin, TIBC, UIBC and full blood count were assayed. Findings: 278 neonates (54.3% males, gestational age 39.4±1.3wk, birth weight 3299±368g) were enrolled. We confirmed the profound early post-natal decrease in serum iron (CDV=22.7±7.0µmol/L to V1=7.3±4.3µmol/L, P

Multiple sclerosis (MS) is clearly linked to the immune system. This has been demonstrated by the beneficial responses to treatments that dampen the immune system. This chapter summarizes the numerous changes in the immune system that are associated with the development of MS. The human immune system has two major divisions, the innate and the adaptive arms, and each is implicated in the pathogenesis of MS. Herein, we discuss mechanisms by which both arms contribute to lesion formation in MS. The adaptive immune system, which responds to specific antigenic targets, provides immune “memory” for subsequent exposures and thereby protecting against future exposures to the same or similar targets. This arm of the immune system is implicated in MS because its major cells, lymphocytes, are found in active central nervous system (CNS) MS lesions and also in the cerebrospinal fluid during MS activity. T lymphocytes can be divided into CD4+ and CD8+ T cells, and each is present in active MS lesions. In addition, the main animal model of MS, experimental autoimmune encephalomyelitis (EAE), can be transferred by myelin-specific T cells. B lymphocytes are also critical to MS, as deleting them in the blood can greatly reduce MS clinical and radiological activity. Innate immune cells process and present antigens to T cells in order to initiate and propagate tissue-specific damage. In addition, effector mechanisms employed by a variety of different innate cells, including macrophages and microglia, directly lead to myelin and neuronal injury during the genesis of MS lesions. Divisions between various innate cells impart features that contribute to inflammatory destruction as well as recovery from injury in MS. Overall, a complex interplay between immune cells serves to coordinate a complex cascade of inflammatory events that contribute to development of disease in MS.

OBJECTIVES: Full-term newborns elicit an immediate postnatal hypoferremia at birth. We examined whether preterm and low birthweight babies are capable of a similar response. The transience of this innate defence in full-term babies was assessed by measuring iron, inflammatory and haematological parameters over the first postpartum week. METHODS: We conducted an observational study of 152 babies who were either preterm (>32 to

Background: Neonates, particularly those born preterm (PTB) and with low birthweight (LBW), are especially susceptible to bacterial and fungal infections that cause an estimated 225,000 deaths annually. Iron is a vital nutrient for the most common organisms causing septicaemia. Full-term babies elicit an immediate postnatal hypoferremia assumed to have evolved as an innate defence. We tested whether PTB and LBW babies are capable of the same response. Methods: We conducted an observational study of 152 babies who were either PTB (born ≥32 to

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye., Author summary Having the correct eye size is important, too large and you will be short-sighted and too small and you will be far-sighted. Nanophthalmos, literally very small eye from the Greek, is a condition where the eye is very small but structurally normal. In addition to being farsighted such eyes are prone to glaucoma which can lead to loss of vision. Here we studied a protein called TMEM98 which is found in the membranes of the cells which form a layer at the back of eye called the retinal pigment epithelium (RPE). Mutations in TMEM98 have been found in nanophthalmos patients. Patients have one normal copy of the gene and one carrying a mutation. We removed Tmem98 from the RPE of mice in order to ascertain its function. We found, surprisingly, that rather than having small eyes this led to the development of very large eyes that were structurally fragile. We went on to identify protein partners of TMEM98 and found that it interacts with a protein called MYRF, mutations in which also cause nanophthalmos. This work demonstrates the importance of TMEM98 in eye size specification.

Background and ObjectiveTo describe the impact of coronavirus disease 2019 (COVID-19) on people with neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody disease (MOGAD).MethodsThe COVID-19 Infections in Multiple Sclerosis (MS) and Related Diseases (COViMS) Registry collected data on North American patients with MS and related diseases with laboratory-positive or highly suspected SARS-CoV-2 infection. Deidentified data were entered into a web-based registry by health care providers. Data were analyzed using t-tests, Pearson χ2 tests, or Fisher exact tests for categorical variables. Univariate logistic regression models examined effects of risk factors and COVID-19 clinical severity.ResultsAs of June 7, 2021, 77 patients with NMOSD and 20 patients with MOGAD were reported in the COViMS Registry. Most patients with NMOSD were laboratory positive for SARS-CoV-2 and taking rituximab at the time of COVID-19 diagnosis. Most patients with NMOSD were not hospitalized (64.9% [95% CI: 53.2%–75.5%]), whereas 15.6% (95% CI: 8.3%–25.6%) were hospitalized only, 9.1% (95% CI: 3.7%–17.8%) were admitted to the ICU and/or ventilated, and 10.4% (95% CI: 4.6%–19.5%) died. In patients with NMOSD, having a comorbidity was the sole factor identified for poorer COVID-19 outcome (OR = 6.0, 95% CI: 1.79–19.98). Most patients with MOGAD were laboratory positive for SARS-CoV-2, and almost half were taking rituximab. Among patients with MOGAD, 75.0% were not hospitalized, and no deaths were recorded; no factors were different between those not hospitalized and those hospitalized, admitted to the ICU, or ventilated.DiscussionAmong the reported patients with NMOSD, a high mortality rate was observed, and the presence of comorbid conditions was associated with worse COVID-19 outcome. There were no deaths reported in the patients with MOGAD, although these observations are limited due to small sample size.

PURPOSEWe previously found a dominant mutation,Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to beTmem98.In humans, mutations in the orthologous gene cause nanophthalmos. We modelled these mutations in mice and characterised the mutant eye phenotypes of these andRwhs.METHODSTheRwhsmutation was identified to be a missense mutation inTmem98by genetic mapping and sequencing. The humanTMEM98nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry and electron microscopy techniques were used to study adult eyes.RESULTSAn I135T mutation ofTmem98causes the dominantRwhsphenotype and is perinatally lethal when homozygous. Two dominant missense mutations ofTMEM98, A193P and H196P are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to theRwhsphenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganised outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions.CONCLUSIONSMutations in the mouse orthologue of the human nanophthalmos geneTMEM98do not result in small eyes. Rather, there is localised disruption of the laminar structure of the photoreceptors.

Background: Iron deficiency is the most prevalent nutritional deficiency worldwide. WHO recommends daily iron supplementation for all pregnant women where anaemia exceeds 40%, but adherence is limited by side-effects, effectiveness is low, and there are concerns around possible harm. The iron-regulatory hormone, hepcidin, signals 'ready-and-safe' to receive iron. We tested a hepcidin-guided screen-and-treat (S&T) approach to combat iron deficiency anaemia (IDA) aimed at achieving equivalent efficacy to universal administration but at lower levels of supplemental iron exposure. Methods: We conducted a 3-arm randomised-controlled double-blind trial in rural Gambia to assess non-inferiority of two ST b) daily UNIMMAP containing 60mg iron for 7d if weekly hepcidin was

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss., Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.

Summary Dietary interventions have not been effective in the treatment of multiple sclerosis (MS). Here, we show that periodic 3-day cycles of a fasting mimicking diet (FMD) are effective in ameliorating demyelination and symptoms in a murine experimental autoimmune encephalomyelitis (EAE) model. The FMD reduced clinical severity in all mice and completely reversed symptoms in 20% of animals. These improvements were associated with increased corticosterone levels and regulatory T (Treg) cell numbers and reduced levels of pro- inflammatory cytokines, TH1 and TH17 cells, and antigen-presenting cells (APCs). Moreover, the FMD promoted oligodendrocyte precursor cell regeneration and remyelination in axons in both EAE and cuprizone MS models, supporting its effects on both suppression of autoimmunity and remyelination. We also report preliminary data suggesting that an FMD or a chronic ketogenic diet are safe, feasible, and potentially effective in the treatment of relapsing-remitting multiple sclerosis (RRMS) patients (NCT01538355).

Background Antibodies to myelin oligodendrocyte glycoprotein (MOG) are associated with a CNS inflammatory disorder distinct from multiple sclerosis (MS) and aquaporin-4 antibody-positive neuromyelitis optica (NMO). Knowledge of the clinical spectrum of MOG antibody disease (MOGAD) remains incomplete, particularly in comparison to two related inflammatory demyelinating diseases, MS and NMO. Objective Compare demographics, clinical characteristics, estimated disability, laboratory results, and treatment responses of a U.S. MOGAD cohort with age- and sex-matched MS and NMO patients. Design, setting, and participants This observational, case-control, single-center study identified each group via ICD-10 diagnosis code searches through the electronic medical records of adult patients seen at the John L. Trotter MS Center between January 1, 2019 and January 1, 2020. MOGAD and NMO patients were confirmed to have at least one positive antibody test; those in the MS group had a confirmed diagnosis by a physician with MS subspecialty training. Data were collected after IRB approval. Results Twenty-six patients were included in each group. MOGAD patients were predominantly Caucasian (88.5%) with mean onset age of 43.9 years. MOGAD patients had no comorbid other autoimmune diseases and comparatively lower rates of family members with autoimmune disease (20.0%) than either MS (40.0%) or NMO (34.6%) matched cohorts. 91% of MOGAD attacks were monofocal, and over 70% presented with optic neuritis. Severity of MOGAD attacks was similar to that of seropositive NMO, but the robust degree of recovery was more similar to MS. Four MOGAD patients converted to negative antibody status, with no attacks occurring after conversion. Serum ANA and ENA were less frequently elevated in MOGAD (21.7%, 5.0%) than in seropositive NMO patients (66.7%, 42.9%). Elevated IgG synthesis rate and positive CSF-restricted oligoclonal bands were not seen in our MOGAD cohort, and only one MOGAD patient had an elevated IgG index. Despite anti-CD20 therapy, 28.6% of MOGAD patients continued to suffer relapses. Conclusions MOGAD was characterized by a predominantly monofocal presentation (typically optic neuritis) and severe attacks with better recovery than seen with seropositive NMO attacks. Lack of CSF-restricted oligoclonal bands distinguished MOGAD from MS.

Neonates regulate iron at birth and in early postnatal life. We reviewed literature from PubMed and Ovid Medline containing data on umbilical cord and venous blood concentrations of hepcidin and iron, and transferrin saturation (TSAT), in human neonates from 0 to 1 mo of age. Data from 59 studies were used to create reference ranges for hepcidin, iron, and TSAT for full-term-birth (FTB) neonates over the first month of life. In FTB neonates, venous hepcidin increases 100% over the first month of life (to reach 61.1 ng/mL; 95% CI: 20.1, 102.0 ng/mL) compared with umbilical cord blood (29.7 ng/mL; 95% CI: 21.1, 38.3 ng/mL). Cord blood has a high concentration of serum iron (28.4 μmol/L; 95% CI: 26.0, 31.1 μmol/L) and levels of TSAT (51.7%; 95% CI: 46.5%, 56.9%). After a short-lived immediate postnatal hypoferremia, iron and TSAT rebounded to approximately half the levels in the cord by the end of the first month. There were insufficient data to formulate reference ranges for preterm neonates.

Multiple sclerosis is a common, complex neurological disease, where almost 20% of risk heritability can be attributed to common genetic variants, including >230 identified by genome-wide association studies (Patsopoulos et al., 2017). Multiple strands of evidence suggest that the majority of the remaining heritability is also due to the additive effects of individual variants, rather than epistatic interactions between these variants, or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that as much as 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common variant signals, which highlight a key role for regulatory T cell homeostasis and regulation, IFNγ biology and NFκB signaling in MS pathogenesis. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

Background: Dimethyl fumarate (DMF) alters the phenotype of circulating immune cells and causes lymphopenia in a subpopulation of treated multiple sclerosis (MS) patients. Objective: To phenotypically characterize circulating leukocytes in DMF-treated MS patients. Methods: Cross-sectional observational comparisons of peripheral blood from DMF-treated MS patients ( n = 17 lymphopenic and n = 24 non-lymphopenic), untreated MS patients ( n = 17) and healthy controls ( n = 23); immunophenotyped using flow cytometry. Longitudinal samples were analyzed for 13 DMF-treated patients. Results: Lymphopenic DMF-treated patients had significantly fewer circulating CD8+ and CD4+ T cells, CD56dim natural killer (NK) cells, CD19+ B cells and plasmacytoid dendritic cells when compared to controls. CXCR3+ and CCR6+ expression was disproportionately reduced among CD4+ T cells, while the proportion of T-regulatory (T-reg) cells was unchanged. DMF did not affect circulating CD56hi NKcells, monocytes or myeloid dendritic cells. Whether lymphopenic or not, DMF-treated patients had a lower proportion of circulating central and effector memory T cells and concomitant expansion of naïve T cells compared to the controls. Conclusions: DMF shifts the immunophenotypes of circulating T cells, causing a reduction of memory cells and a relative expansion of naïve cells, regardless of the absolute lymphocyte count. This may represent one mechanism of action of the drug. Lymphopenic patients had a disproportionate loss of CD8+ T-cells, which may affect their immunocompetence.

We assembled and analyzed genetic data of 47,351 multiple sclerosis (MS) subjects and 68,284 control subjects and establish a reference map of the genetic architecture of MS that includes 200 autosomal susceptibility variants outside the major histocompatibility complex (MHC), one chromosome X variant, and 32 independent associations within the extended MHC. We used an ensemble of methods to prioritize up to 551 potentially associated MS susceptibility genes, that implicate multiple innate and adaptive pathways distributed across the cellular components of the immune system. Using expression profiles from purified human microglia, we do find enrichment for MS genes in these brain - resident immune cells. Thus, while MS is most likely initially triggered by perturbation of peripheral immune responses the functional responses of microglia and other brain cells are also altered and may have a role in targeting an autoimmune process to the central nervous system.One Sentence Summary:We report a detailed genetic and genomic map of multiple sclerosis, and describe the role of putatively affected genes in the peripheral immune system and brain resident microglia.

Apolipoprotein E (ApoE) functions as a ligand in receptor-mediated endocytosis of lipoprotein particles and has been demonstrated to play a role in antigen presentation. To explore the contribution of ApoE during autoimmune central nervous system (CNS) demyelination, we examined the clinical, cellular immune function, and pathologic consequences of experimental autoimmune encephalomyelitis (EAE) induction in ApoE knockout (ApoE(-/-)) mice. We observed reduced clinical severity of EAE in ApoE(-/-) mice in comparison to WT mice that was concomitant with an early reduction of dendritic cells (DCs) followed by a reduction of additional innate cells in the spinal cord at the peak of disease without any differences in axonal damage. While T cell priming was enhanced in ApoE(-/-) mice, reduced severity of EAE was also observed in ApoE(-/-) recipients of encephalitogenic wild type T cells. Expression of ApoE during EAE was elevated within the CNS of wild type mice, particularly by innate cells such as DCs. Overall, ApoE promotes clinical EAE, likely by mediation of inflammation localized within the CNS.

Purpose: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs. // Methods: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes. // Results: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions. // Conclusions: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.

Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-β expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment.

MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel-Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin-filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from Flna(Dilp2) null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin-filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

Objective: To determine the prevalence of serum antibodies to the ionotropic glutamate receptor 3 (GluR3) in patients with Rasmussen encephalitis (RE), a severe epileptic disorder, and to compare with serum from control subjects and patients with intractable epilepsy (IE). Methods: The authors looked for serum immunoglobulin (Ig) G antibodies to GluR3 in 30 patients with RE, including two patients who had plasma exchange and 12 who had been treated with IV Igs with varying results, and 49 patients with IE and 23 healthy individuals, using ELISA with GluR3B peptide, Western blot analysis of recombinant full-length GluR3, immunoprecipitation of [ 35 S]- and [ 125 I]-labeled GluR3 extracellular domains, immunohistochemistry on rat brain sections, and electrophysiology of GluR3 expressed in Xenopus oocytes. Results: Low levels of antibodies to the GluR3B peptide were detected using ELISA in only 4 of the 79 patients with epilepsy (2 with RE and 2 with IE); binding to GluR3B in other sera was shown to be nonspecific. One other patient with IE had antibodies to recombinant GluR3 on Western blot analysis. However, none of the sera tested precipitated either the [ 35 S]- or the [ 125 I]-labeled GluR3 domains; none bound to rat brain sections in a manner similar to rabbit antibodies to GluR3; and none of the nine sera tested affected the electrophysiologic function of GluR3. Conclusions: GluR3 antibodies were only infrequently found in Rasmussen encephalitis or intractable epilepsy.

Phase images obtained with gradient echo MRI provide image contrast distinct from T1- and T2-weighted images. It is commonly assumed that the local contribution to MRI signal phase directly relates to local bulk tissue magnetic susceptibility. Here, we use Maxwell’s equations and Monte Carlo simulations to provide theoretical background to the hypothesis that the local contribution to MRI signal phase does not depend on tissue bulk magnetic susceptibility but tissue magnetic architecture—distribution of magnetic susceptibility inclusions (lipids, proteins, iron, etc.) at the cellular and subcellular levels. Specifically, we show that the regular longitudinal structures forming cylindrical axons (myelin sheaths and neurofilaments) can be locally invisible in phase images. Contrary to an expectation that the phase contrast in multiple sclerosis lesions should always increase in degree along with worsening of lesion severity (which happens for all known MR magnitude-based contrast mechanisms), we show that phase contrast can actually disappear with extreme tissue destruction. We also show that the phase contrast in multiple sclerosis lesions could be altered without loss of nervous system tissue, which happens in mild injury to the myelin sheaths or axonal neurofilaments. Moreover, we predict that the sign of phase contrast in multiple sclerosis lesions indicates the predominant type of tissue injury—myelin damage (positive sign) vs. axonal neurofilament damage (negative sign). Therefore, our theoretical and experimental results shed light on understanding the relationship between gradient echo MRI signal phase and multiple sclerosis pathology.

The contribution of B cells and their products to the pathogenesis of MS has long been debated. The presence of B cells, plasma cells and excess immunoglobulins in MS lesions and in the cerebrospinal fluid of MS patients implicate the humoral immune system in the disease. Correlations of higher levels of CSF B cells and immunoglobulins found in some studies with a more aggressive clinical course of MS have bolstered the notion that the humoral immune system is involved in MS pathogenesis. However, until the advent of rituximab, a monoclonal antibody therapy that specifically lyses B cells, confirmation of the key role of B cells and their products in MS had been lacking. Development of this therapeutic monoclonal antibody to CD20, a cell surface molecule confined to B cells, allowed determination of the effects of B cell depletion. Perhaps somewhat unexpectedly, depletion of circulating B cells led to rapid and profound reduction in gadolinium enhancing lesions on brain MRI in three separate studies in relapsing MS subjects. When examined, depletion of B cells in the blood was accompanied by depletion of B cells in CSF. Notably, reduction of enhancing brain lesions was not accompanied by reduction in CSF immunoglobulins. Whether the critical role of B cells occurs in the periphery, in the CNS, or in both locations has not yet been determined.

Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS- 1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.

Multiple sclerosis (MS) is a central nervous system (CNS) inflammatory demyelinating disease impacted by the interplay of genetic and environmental factors. It is presumed to be autoimmune. MS is more common in western countries and differences in diet could contribute to its particular geographical distribution. Studies relating diet to MS have been inconclusive. A mechanism through which diet can influence immune responses is the gut microbiome, which is emerging as a critical contributor in numerous human diseases. Here we show that intermittent fasting (IF) ameliorated clinical course and pathology of the MS animal model, experimental autoimmune encephalomyelitis (EAE), leading to less inflammation, demyelination and axonal damage. IF changed the gut microbiome resulting in increased bacteria richness and enrichment of the Lactobacillaceae, Bacterioidaceae and Prevotellaceae families. Gut microbiome richness was inversely correlated with leptin levels. Microbial metabolic pathway analysis revealed that IF-induced changes to the gut microbiome increased ketone formation and glutathione metabolism, enhancing anti-oxidative pathways. Furthermore, IF had direct effects on the composition of T cells in the gut lamina propria with a reduction of IL-17 producing T cells and an increase in the number of regulatory T cells. These effects might modulate systemic immune responses. Importantly, fecal microbiome transplantation from mice on IF ameliorated EAE in immunized recipient mice on a normal diet, suggesting that IF immunomodulatory effects are at least partially mediated by the gut flora. We translated our findings to MS patients in a pilot clinical trial in MS patients undergoing relapse to test the safety, feasibility and effects of IF on clinical and laboratory measures. Potentially beneficial effects on levels of several immune inflammatory parameters as well as gut flora that resembled the protective changes observed in mice in EAE were observed. In conclusion, IF has potent immunomodulatory effects that are at least partially mediated by the gut microbiome.

Glutamate excitotoxicity plays a role in white matter injury in many neurological diseases. Oligodendrocytes in particular are highly vulnerable to excitotoxicity, mediated through activation of AMPA/Kainate receptors. Myelin may also be injured independently via NMDA (N-methyl-D-aspartic acid) receptors located on peripheral oligodendroglial processes. Central axons are susceptible to glutamate receptor activation in vivo, but it is unclear whether this is mediated directly by activation of receptors expressed on axons, or indirectly through glutamate toxicity of myelin or neighboring glial cells. We examined axonal vulnerability in mice deficient in myelin basic protein (shiverer), also expressing yellow fluorescent protein (YFP) in a subset of axons. YFP fluorescence, EM, and mouse behavior were assessed 24 hours after microstereotactical injections of S-AMPA or NMDA into lumbar dorsal columns. S-AMPA injection led to impaired rotarod performance and widespread axonal degeneration and was more pronounced in shiverer mice than controls. In contrast, NMDA injection did not cause axonal injury or behavioral changes in either group. These results indicate that spinal cord axons in vivo are vulnerable to toxicity mediated by AMPA but not NMDA receptors. The presence of compact myelin is not required for excitotoxic axon damage, and its absence may increase vulnerability. Further understanding of AMPA receptor-mediated axonal toxicity may provide new targets for neuroprotective therapy in WM diseases.

Genome-wide association studies (GWASs) have proven highly effective, identifying hundreds of associations across numerous complex diseases. These studies typically test hundreds of thousands of variations and identify hundreds of potential associations. However, to date, follow-up attempts have generally only concentrated on just the few most significant initial associations, leaving the majority of true associations in any GWAS study without replication. Here, we present a substantially more comprehensive follow-up of the first genome-wide association screen performed in multiple sclerosis (MS), a complex genetic disease with central nervous system inflammation. We genotyped approximately 30 000 single-nucleotide polymorphisms (SNPs) that demonstrated mild-to-moderate levels of significance (Por = 0.10) in the initial GWAS in an independent set of 1343 MS cases and 1379 controls. We further replicated several of the most significant findings in another independent data set of 2164 MS cases and 2016 controls. We find considerable evidence for a number of novel susceptibility loci including KIF21B [rs12122721, combined P = 6.56 x 10(-10), odds ratio (OR) = 1.22] and TMEM39A (rs1132200, P = 3.09 x 10(-8), OR = 1.24), both of which meet genome-wide significance. Both of these loci were overlooked in the initial replication, despite being among the top 3000 ( approximately 1%) SNP hits in the original screen.

Specialized glia, termed reactive astrocytes, accompany numerous pathologic conditions affecting the central nervous system, including stroke, multiple sclerosis, and neoplasia. To better define this important cell type, we employed high-density microarray gene expression profiling using two in vitro models of reactive gliosis (stimulation with dbcAMP or IL-1beta/IFNgamma). We identified 44 differentially expressed transcripts common to both in vitro models and demonstrated that a subset of these genes are also differentially expressed in response to experimental autoimmune encephalomyelitis and focal cerebral ischemia in vivo. Moreover, this pattern of differential gene expression is not observed in hyperproliferating or neoplastic glia.

Dysregulation of blood-brain barrier (BBB) function and transendothelial migration of leukocytes are essential components of the development and propagation of active lesions in multiple sclerosis (MS). Animal studies indicate that polarized expression of the chemokine CXCL12 at the BBB prevents leukocyte extravasation into the central nervous system (CNS) and that disruption of CXCL12 polarity promotes entry of autoreactive leukocytes and inflammation. In the present study, we examined expression of CXCL12 and its receptor, CXCR4, within CNS tissues from MS and non-MS patients. Immunohistochemical analysis of CXCL12 expression at the BBB revealed basolateral localization in tissues derived from non-MS patients and at uninvolved sites in tissues from MS patients. In contrast, within active MS lesions, CXCL12 expression was redistributed toward vessel lumena and was associated with CXCR4 activation in infiltrating leukocytes, as revealed by phospho-CXCR4-specific antibodies. Quantitative assessment of CXCL12 expression by the CNS microvasculature established a positive correlation between CXCL12 redistribution, leukocyte infiltration, and severity of histological disease. These results suggest that CXCL12 normally functions to localize infiltrating leukocytes to perivascular spaces, preventing CNS parenchymal infiltration. In the patient cohort studied, altered patterns of CXCL12 expression at the BBB were specifically associated with MS, possibly facilitating trafficking of CXCR4-expressing mononuclear cells into and out of the perivascular space and leading to progression of disease.

In this study, axial (lambda(parallel)) and radial (lambda(perpendicular)) diffusivities derived from diffusion tensor imaging (DTI) were used to evaluate white matter injury in brains of mice affected by experimental autoimmune encephalomyelitis (EAE). Sixteen female C57BL/6 mice were immunized with amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG(35-55)). Three months after immunization, optic nerve and tract were severely affected with 19% and 18% decrease in lambda(parallel) respectively, suggesting the presence of axonal injury. In addition, a 156% and 86% increase in lambda( perpendicular) was observed in optic nerve and tract respectively, suggestive of myelin injury. After in vivo DTI, mice were perfusion fixed and immunohistochemistry for the identification of myelin basic protein (MBP) and phosphorylated neurofilament (pNF) was performed to verify the presence of axonal and myelin injury. The present study demonstrated that the visual pathway is selectively affected in MOG(35-55) induced murine EAE and these injuries are non-invasively detectable using lambda(parallel) and lambda( perpendicular).

Background: African Americans (AAs) experience greater disability from multiple sclerosis (MS) compared with Caucasian Americans (CAs). Interethnic immunologic differences in MS and their relationship to disease-related disability have not been described. Objective: To compare measures of CSF humoral immunity between AAs and CAs with MS. Methods: Using a case–control design, all AA MS patients with CSF immune studies at the Washington University MS center were compared with randomly selected CAs with MS. Two CA controls were selected for every AA case. Immunoglobulin G (IgG) index and synthesis rates, oligoclonal band positivity, white blood cell count, and CSF protein were compared between groups. Survival analysis was conducted to compare times to ambulatory assistance. Results: Sixty-six AA cases and 132 CA controls were identified. Measures of CSF humoral activity were all higher in the AA group. The mean IgG index of AAs was 1.35 (SD 0.62), and that of CAs was 1.05 (SD 0.55), for a mean difference of 0.30 ( p = 0.001). The median IgG synthesis rate was also higher among AAs (13.55 vs 8.20 mg/day), for a median difference of 5.35 mg/day ( p = 0.010). Survival analysis confirmed previous reports of earlier ambulatory assistance requirement among AAs. Despite differences in both humoral immune response and times to ambulatory assistance, Cox proportional hazards modeling did not show IgG index as predictive of earlier ambulatory assistance. Conclusions: The CSF humoral immune response is more active among African Americans (AAs) than among Caucasian Americans (CAs) with multiple sclerosis. AAs also progress to ambulatory assistance earlier than CAs, but high immunoglobulin G index does not predict earlier progression to the disability endpoint.

The role B cells and humoral immunity play in multiple sclerosis (MS) is continually evolving. Recent discoveries include advances in lesion classification, identification of B cell clonal expansion in the central nervous system with evidence of antigen targeting, identification of chemokines in MS lesions, and expansion of information on the roles of autoantibodies and complement. Strong indications are accumulating that a greater degree of humoral dysfunction may lead to worsened long-term prognosis in MS. Effects of established MS treatments on B cells and their products have been further defined. Treatments that specifically target B cells are being implemented and hold promise.

Triggering receptor expressed on myeloid cells (TREM-2) is a membrane receptor associated with DAP12 that is expressed primarily in myeloid cells, including dendritic cells and microglia, and promotes fusion of osteoclast precursors into multinucleated cells. A rare autosomal recessive condition, Nasu-Hakola disease (NHD) is associated with loss-of-function mutations in DAP12 and TREM-2. The brain pathology observed in NHD patients suggests that disruption of the TREM-2/DAP12 pathway leads to neurodegeneration with demyelination and axonal loss. In this study, we have characterized TREM-2 protein expression on microglia using a newly produced monoclonal antibody directed against the mouse TREM-2 receptor. We report that TREM-2 expression is up-regulated in the spinal cord during both the early inflammatory and chronic phases of myelin oligodendrocyte glycoprotein (MOG)(35-55)peptide-induced experimental autoimmune encaphalomyelitis (EAE). We also demonstrate that TREM-2 is highly expressed on microglial cells in the central nervous system (CNS) during EAE and that blockade of TREM-2 during the effector phase of EAE results in disease exacerbation with more diffuse CNS inflammatory infiltrates and demyelination in the brain parenchyma. These results demonstrate a critical role for TREM-2 during inflammatory responses in the CNS.

Iron deficiency is the most common nutrient deficiency worldwide and routine supplementation is standard policy for pregnant mothers and children in most low-income countries. However, iron lies at the center of host-pathogen competition for nutritional resources and recent trials of iron administration in African and Asian children have resulted in significant excesses of serious adverse events including hospitalizations and deaths. Increased rates of malaria, respiratory infections, severe diarrhea and febrile illnesses of unknown origin have all been reported, but the mechanisms are unclear. We here investigated the ex vivo growth characteristics of exemplar sentinel bacteria in adult sera collected before and 4 h after oral supplementation with 2 mg/kg iron as ferrous sulfate. Escherichia coli, Yersinia enterocolitica and Salmonella enterica serovar Typhimurium (all gram-negative bacteria) and Staphylococcus epidermidis (gram-positive) showed markedly elevated growth in serum collected after iron supplementation. Growth rates were very strongly correlated with transferrin saturation (p Staphylococcus aureus, which preferentially scavenges heme iron, was unaffected. These data suggest that even modest oral supplements with highly soluble (non-physiological) iron, as typically used in low-income settings, could promote bacteremia by accelerating early phase bacterial growth prior to the induction of immune defenses.

Previous work demonstrated that EAE induced by recombinant human MOG was B cell-dependent. Data presented here reveal a T cell response to MOG61–85 in human rMOG-immunized B cell −/− mice not observed in WT mice. Further study revealed this peptide to be a cryptic epitope in WT mice. Co-immunization of B cell −/− mice with MOG35–55 and MOG61–85 peptides led to less severe disease compared to mice immunized with MOG35–55 alone. Disease amelioration was associated with decreased production of Interferon-γ by lymph node cells. Thus, MOG61–85 represents a protective epitope to human rMOG induced EAE in B cell −/− mice.

In an international effort to assign function to all genes, and to identify new models of human disease, cohorts of mice produced from mutant embryonic stem cells are being screened in large-scale phenotyping pipelines. Hundreds of mouse lines, each mutant for a different gene, have been analyzed for developmental, hematological, immunological, metabolic, neurological, reproductive, and sensory parameters, which, thus, provide a comprehensive assessment of the consequences of gene ablation.1 Included in the sensory analysis is slit-lamp examination of the anterior chamber of the eye, and indirect ophthalmoscopy to view the retina and retinal vessels. The phenotype data can be viewed online and the mice are freely available. We describe here the detailed pathological and functional analysis of a mouse line mutant in the sodium/hydrogen ion exchange protein gene, Slc9a8, which was identified as having a retinal defect and no other abnormality other than male sterility. Sodium/hydrogen ion exchange proteins (NHEs) or cotransporter solute channels are encoded by the Slc9 gene family, comprising nine members: Slc9a1-9, encoding NHE1-9. Proteins NHE1 to 5 are located in the plasma membrane while NHE6 to 9 are found in membranes of cell organelles (reviewed previously2). These proteins contain 12 membrane spanning domains and catalyze the electroneutral transport of cations down a concentration gradient. Typically sodium ions are transported in and hydrogen ions out of the cell or organelle. Simultaneous transport of anions creates osmotic pressure and results in influx of water, regulating the pH and volume of cells and organelles. Sodium/hydrogen ion exchange is involved in aqueous humor dynamics and maintenance of intraocular pressure (IOP). Inhibition of sodium/hydrogen ion exchange in mice3 reduces IOP and at least one cotransporter, NHE1, is found expressed in the ciliary body, where aqueous dynamics are regulated. The single yeast NHE is necessary for correct sorting of proteins to the vacuole4 and, indeed, depletion of NHE8 in mammalian cells disrupts endosome trafficking.5 Six of the Slc9 family members have been mutated in mice and give rise to a range of phenotypes, from hyperactivity or ataxia and seizures (Slc9a1 and 6)6–8 to relatively mild gastric secretion and absorption defects (Slc9a2, 3, and 4).9–11 The gene Slc9a8 is widely expressed in adult mouse tissues, but is enriched in kidney, intestine, muscle, liver, and testes.12,13 The gene is expressed in the kidney proximal tubules, where reabsorption of secreted bicarbonate requires NHE function, although this is mostly via Slc9a3 (NHE3). However, Slc9a8 is expressed at higher levels in the neonatal kidney, and may partially compensate for NHE3 in Slc9a3 knockout animals, which have only a mild absorption defect.14 The Slc9a8 gene previously has been knocked out and the authors report that the mice have normal levels of serum sodium ions. They also report an increase in the length of the intestine, but a reduction in the number of goblet cells in the mutant mice with a concomitant reduction in mucin secretion and pH.13,15 Furthermore, it has been reported recently that the gene is expressed in the epithelial cells of the conjunctiva, cornea, and lacrimal glands and may have a role in the protection of these ocular epithelia.16 The male mutants also are sterile. Here, we identified and characterized a previously undescribed retinal defect of Slc9a8 knockout mice and located the origin of the phenotype to be abnormalities in the retinal pigmented epithelium.

The Mitogen-Activated Protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signaling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9-weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a novel signaling molecule involved with hair cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes MAP3K1 is known to act upon, further investigation of this model may help elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

Effects of B cell depletion by rituximab, a monoclonal antibody to CD20, were studied in patients with relapsing MS that had not responded optimally to standard immunomodulatory therapies. Flow cytometry demonstrated reduced cerebrospinal fluid (CSF) B cells and T cells in most patients at 6 months post-treatment. ELISAs demonstrated modest reductions in serum antibodies to myelin oligodendrocyte glycoprotein and myelin basic protein in some subjects. Beta-interferon neutralizing antibodies were reduced in three subjects, but developed anew after treatment in three others, suggesting caution in considering rituximab as a means to eliminate NABs. In summary, rituximab depleted B cells from CSF at 24 weeks after initial treatment, and this B cell depletion was associated with a reduction in CSF T cells as well.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.

Transcriptional activity of a 573-bp fragment of HSP101 (At1g74310) incorporated into a Mutator-like element (MULE) transposon was investigated in Arabidopsis thaliana Columbia. Sequence identity between the HSP101-MULE arrangement and a continuous segment of the original HSP101 promoter, 5' UTR exon, and open reading frame (ORF) was high (87%) but lower in the 5' UTR intron (69%). Collectively, the HSP101 ORF, the MULE 5' terminal inverted repeat (TIR), and the 1.3 kb immediately upstream of the TIR is located on chromosome IV, and we refer to it as HSP101B. Located within the HSP101B promoter, upstream of 2 heat shock elements (HSEs), are 4 COR15a-like low-temperature response elements (LTREs). The HSP101B ORF was transcribed in the leaves and inflorescences of high-temperature stress (HTS) treated Arabidopsis thaliana but not in low-temperature stress (LTS) and control plants. Transiently transformed Arabidopsis seedlings, as well as stable transformed lines of Linum usitatissimum (flax) and Brassica napus (canola) containing a HSP101B promoter:GUS construct, showed either LTS-, or LTS- and HTS-, induced β-glucuronidase expression. Results from PCR amplifications of HpaII- and MspI-digested Arabidopsis genomic DNA suggest that endogenous expression of HSP101B may be downregulated by partial methylation of the HSP101B sequence between the TIRs of the associated MULE.Key words: promoter function, low temperature stress, high temperature stress; Arabidopsis HSP101, Mutator-like element, transposon.

Bacteria-containing commercial products, sold to facilitate biodegradation of human and animal wastes, consist of complex mixtures of bacteria. These mixtures are often of undetermined composition and grown in batch cultures from diverse bacteria-rich inocula of proprietary origins. In order to provide a means of testing for the presence of small numbers of microorganisms, pathogenic to terrestrial or avian wildlife, in bacteria-containing biodegradation products, five DNA extraction protocols were tested for their ability to purify total genomic DNA from nine biodegradation products of different formulations. A diatomaceous earth and guanidine thiocyanate-based DNA extraction method was found to be the most reliable. Fourteen microorganism-specific polymerase chain reaction (PCR)-based assays were developed. Each PCR assay was demonstrated to be specific using DNA from 61 other species of microorganisms (105 different isolates). The mean detection limit for 10 assays using cultured organisms spiked into biodegradation products was assessed. The mean was 1 × 102.62 ± 1 × 101.58 c.f.u.g−1 (bacteria) and 1 × 103.88 ± 1 × 101.14 cells g−1 (protozoa). There were no target wildlife pathogens detected by the 14 PCR assays in unspiked biodegradation products. This study has demonstrated that molecular diagnostic means can be used to detect small numbers of selected wildlife pathogens in complex biodegradation products.

Cyclospora cayetanensis is an emerging protozoan parasite capable of causing a protracted diarrheal illness in both immunocompromised and immunocompetent individuals. Ingestion of fresh produce and water sources contaminated with mature sporulated oocysts results in acquisition of cyclosporiasis. Currently, no animal model exists for the study of this pathogenic parasite and the only confirmed reservoir host for C. cayetanensis in nature is humans. Previously, Cyclospora-like oocysts had been detected by microscopy in several animals including non-human primates. However, their phylogenetic relationship to C. cayetanensis remained uncertain due to the limited availability of molecular techniques to differentiate and speciate these isolates. In the present study, we examined a series of fecal isolates obtained from dogs, chickens, and monkeys collected between May and September 2002 from several geographic regions of Nepal. All samples were examined by microscopy and a polymerase chain reaction (PCR) for the presence of C. cayetanensis. Both microscopic and conventional PCR/restriction fragment length polymorphism (RFLP) analysis demonstrated the presence of Cyclospora sp. in the fecal samples of two dogs, one chicken, and one monkey. Application of a species-specific multiplex PCR assay confirmed the presence of both Eimeria sp. and C. cayetanensis in the positive chicken sample and only C. cayetanensis in the dog and monkey samples. However, in the absence of tissue analysis, the assignment of these animals as a natural reservoir host for C. cayetanensis remains to be determined.

Dilp1 is a semi-dominant mouse mutation that causes dilated pupils when heterozygous and is lethal when homozygous. We report here that it is caused by a point mutation that introduces a stop codon close to the start of the coding sequence of the paired-like homeobox transcription factor Phox2b. Mice carrying a targeted allele of Phox2b also have dilated pupils and the two alleles do not complement. Phox2b is necessary for the development of the autonomic nervous system and when absent one of the consequences is that all parasympathetic ganglia fail to form. Constriction of the pupil is a parasympathetic response mediated by the ciliary ganglion and we find that in Phox2b heterozygous mutants it is highly atrophic. The development of other parasympathetic and sympathetic ganglia appears to be largely unaffected indicating that the ciliary ganglion is exquisitely sensitive to a reduction in dose of this transcription factor. PHOX2B has been implicated in human disease. Mutations, principally leading to polyalanine expansions within the protein, have been found in patients with congenital central hypoventilation syndrome (CCHS), the cardinal feature of which is an inability to breathe unassisted when asleep. Additionally, some CCHS patients have ocular abnormalities, including pupillary defects, although they principally have constricted rather than dilated pupils. The apparent phenotypic differences observed between mice carrying a loss-of-function mutation of Phox2b and CCHS patients indicate that PHOX2B mutations found in CCHS patients, all of which can produce proteins with intact DNA-binding domains, are gain-of-function mutations that alter rather than abolish protein function.