Anti-neurofascin antibodies

Neurofascin (NF) is a cell-adhesion molecule that is found at the nodes of Ranvier. The 186 kDa isoform of neurofascin (NF186) is expressed on the axon in the exposed node, and the 155 kDa isoform (NF155) is expressed on myelinating glia at the paranode. NF186 is essential for clustering of sodium channels to the nodes while NF155 is needed for close paranodal interactions between myelinating glia and axons. The neurofascins are found in both the peripheral and central nervous system (PNS and CNS). NF-specific autoantibodies were identified in serum of multiple sclerosis (MS) patients using a proteomics approach with two-dimensional Western blotting of human myelin glycoproteins. A monoclonal antibody (mAb) specific for NF was shown to induce axonal injury in an animal model of MS, experimental autoimmune encephalomyelitis. This indicated that NF is a relevant autoantibody target in patients with inflammatory diseases of the nervous system (central and peripheral), but actual abundance of anti-NF autoantibodies is unknown. The objectives of the thesis were the following: 1) Develop assays to detect autoantibodies against human NF. 2) Determine the prevalence in patients with MS and with inflammatory diseases of the PNS. 3) Characterize the reactivity by immunoglobulin isotyping, serial dilution, epitope mapping, and staining of nodal structures in tissue sections. 4) Affinity purify anti-NF antibodies from plasma exchange material. 5) Determine possible in vivo effects of anti-NF antibodies in the PNS using a neuritis animal model. First, we expressed the complete human NF155 and NF186 on the surface of stable human cell lines, produced the complete extracellular portion of the NFs in HEK293 cells, and expressed truncated variants of the NFs in E. coli. With these reagents, we set up three antibody detection assays: cell-based assay by flow cytometry, ELISA, and Western blot. These assays were validated using NF-specific monoclonal and polyclonal antibodies, and optimized with a test cohort of serum samples. We screened 687 serum and 48 plasma exchange samples from patients with MS (n = 233), inflammatory diseases in the PNS (n = 294), and controls (n = 208). From serum analysis, we observed low prevalence of anti-NF reactivity (3%) by flow cytometry and/or ELISA despite broad reactivity in almost half of the serum samples analyzed by Western blot. Reactivity observed by flow cytometry and by ELISA were congruent only in the patients with the highest reactivities. The anti-NF antibodies were NF-isoform specific, mainly IgG subclasses, and at high titres in some cases. Using truncated variants of NF fused to super green fluorescence protein (sGFP), we showed that reactivity of anti-NF Abs was largely directed towards the membrane proximal extracellular domains that are unique to each isoform, while the membrane distal immunoglobulin-like domains and fibronectin domains were not recognized. A small proportion (3%; 8/254) of patients with GBS and CIDP showed reactivity to human NF by ELISA. A few showed a particularly high reactivity (up to 1:10 000 dilution) to NF. Two CIDP patients showed a particularly high (up to 1:10 000 dilution) anti-NF155 reactivity by FACS and ELISA, recognized paranodes in tissue sections, and exhibited dominant IgG4 subclass usage. Another CIDP patient who benefited from plasma exchange had a persistent anti-NF155 reactivity by ELISA in serum, and after affinity purification, anti-NF186 and -NF155 reactivity by FACS and ELISA were detected in addition. These antibodies were mainly IgG3, with minor contribution of IgM and IgA. To investigate possible functions of anti-NF antibodies in inflammatory PNS diseases, we injected two different monoclonal antibodies (mAbs) into a P2-peptide induced experimental autoimmune neuritis (EAN) animal model at disease onset. We found that while the anti-NF mAbs prolonged and exacerbated clinical disease in these animals, they could not induce disease on their own. We detected NF-reactivity in a small proportion of MS samples (3%; 7/225) by ELISA and flow cytometry. We obtained follow-up material from two NF-reactive patients and saw a persistent NF reactivity in one of them. To increase detection sensitivity, we affinity purified anti-NF antibodies from plasma exchange material of patients with MS (n = 8). IgG, IgM, and IgA were isolated from most of the samples; they were found to recognize NF155 and to a lower extent NF186 by ELISA and in a few also by flow cytometry. This indicates that low levels of anti-NF antibodies exist in a proportion of MS patients. In conclusion, 3% of serum samples from patients with PNS inflammatory neuropathies (GBS and CIDP) showed reactivity by ELISA and none of the controls. In an animal model of autoimmune peripheral nerve inflammation, we showed, using two anti-NF mAbs, that antibody targeting of NF can enhance and prolong disease course. This suggests that antibodies to NF may be relevant for a small group of patients with peripheral inflammatory neuropathies. In MS patients, 3% showed anti-NF reactivity by flow cytometry and ELISA. Furthermore, low levels of anti-NF antibodies that could be detected by ELISA and flow cytometry after affinity purification were additionally found in some MS patient samples that were unreactive by serum screening. This raises the possibility that low levels of antibodies to NF are present in some MS patients and may contribute to the pathogenesis of this chronic disease

Neurofascin-155 IgM autoantibodies in patients with inflammatory neuropathies

Objectives Recently, IgG autoantibodies against different paranodal proteins have been detected and this has led to important advances in the management of inflammatory neuropathies. In contrast, not much is known on IgM autoantibodies against paranodal proteins. Methods In the present study, we screened a large cohort of patients (n=140) with inflammatory neuropathies for IgM autoantibodies against neurofascin-155, neurofascin-186 or contactin-1. Results IgM autoantibodies against neurofascin-155 were detected by ELISA in five patients, four with inflammatory demyelinating polyradiculoneuropathy (CIDP) and one with Guillain-Barre syndrome (GBS), and were confirmed by ELISA-based preabsorption experiments and Western blot. Titres ranged from 1:100 to 1:400. We did not detect IgM anti-neurofascin-186 or anti-contactin-1 antibodies in this cohort. All patients presented with distally accentuated tetraparesis and hypesthesia. Remarkably, tremor was present in three of the patients with CIDP and occurred in the patients with GBS after the acute phase of disease. Nerve conduction studies revealed prolonged distal motor latencies and F wave latencies. Nerve biopsies showed signs of secondary axonal damage in three of the patients, demyelinating features in one patient. Teased fibre preparations did not demonstrate paranodal damage. Conclusion In summary, IgM neurofascin-155 autoantibodies may be worth testing in patients with inflammatory neuropathies. Their pathogenic role needs to be determined in future experiments

The liver-enriched transcription factor CREB-H is a growth suppressor protein underexpressed in hepatocellular carcinoma

We have previously characterized transcription factor LZIP to be a growth suppressor targeted by hepatitis C virus oncoprotein. In search of proteins closely related to LZIP, we have identified a liver-enriched transcription factor CREB-H. LZIP and CREB-H represent a new subfamily of bZIP factors. CREB-H activates transcription by binding to cAMP responsive element, box B, and ATF6-binding element. Interestingly, CREB-H has a putative transmembrane (TM) domain and it localizes ambiently to the endoplasmic reticulum. Proteolytic cleavage that removes the TM domain leads to nuclear translocation and activation of CREB-H. CREB-H activates the promoter of hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase. This activation can be further stimulated by cAMP and protein kinase A. CREB-H transcript is exclusively abundant in adult liver. In contrast, the expression of CREB-H mRNA is aberrantly reduced in hepatoma tissues and cells. The enforced expression of CREB-H suppresses the proliferation of cultured hepatoma cells. Taken together, our findings suggest that the liver-enriched bZIP transcription factor CREB-H is a growth suppressor that plays a role in hepatic physiology and pathology

Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues

Current available tissue clearing techniques are mostly used for rodent tissues. Here, the authors develop OPTIClear solution for fresh and archival human brain tissue clearing and establish associated protocols for three-dimensional histological investigations

Rationalisation and Validation of an Acrylamide-Free Procedure in Three-Dimensional Histological Imaging

201810_a bcmapublished_fina

Who reports insufficient and disturbed sleep? Results from a representative population-based health survey in Hong Kong.

OBJECTIVES: To highlight the prevalence of sleep problems and identify associated risk factors among a representative sample recruited from the general population of Hong Kong. DESIGN, SETTING AND PARTICIPANTS: Participants included 12 022 individuals (aged 15 or above) who took part in the Population Health Survey 2014/15, a territory-wide survey conducted by the Department of Health of the Government of the Hong Kong Special Administrative Region. PRIMARY AND SECONDARY OUTCOME MEASURES: Outcomes were the prevalence of (1) insufficient sleep (<6 hours sleep per day) and (2) any sleep disturbance (difficulty initiating sleep, intermittent awakenings, early awakening) ≥3 times per week in the past 30 days. Multivariable logistic regression identified associations between sleep problems and sociodemographic, clinical and lifestyle factors. RESULTS: 9.7% of respondents reported insufficient sleep and 10.5% reported sleep disturbances ≥3 times a week. Female gender, monthly household income <$12 250 (Hong Kong dollar), lower education level, mental health condition and physical health condition were significantly associated with both insufficient and disturbed sleep (all p<0.05). Unemployment, homemaker, insufficient physical activity, current/former smoking status and harmful alcohol consumption were associated with sleep disturbances only (all p<0.01). CONCLUSIONS: Sleep problems are highly prevalent in Hong Kong. As such problems are associated with a range of health conditions, it is important to facilitate improvements in sleep. Our results show that harmful alcohol consumption, insufficient physical activity and current smoking are modifiable risk factors for sleep disturbances. Public health campaigns should focus on these risk factors in order to promote a healthy lifestyle and ultimately reduce sleep disturbances. Targeted interventions for high-risk groups may also be warranted, particularly for those with doctor-diagnosed physical and mental health conditions

Herpes zoster related hospitalization after inactivated (CoronaVac) and mRNA (BNT162b2) SARS-CoV-2 vaccination: A self-controlled case series and nested case-control study

BACKGROUND: Stimulation of immunity by vaccination may elicit adverse events. There is currently inconclusive evidence on the relationship between herpes zoster related hospitalization and COVID-19 vaccination. This study aimed to evaluate the effect of inactivated virus (CoronaVac, Sinovac) and mRNA (BNT162b2, BioNTech/Fosun Pharma) COVID-19 vaccine on the risk of herpes zoster related hospitalization. METHODS: Self-controlled case series (SCCS) analysis was conducted using the data from the electronic health records in Hospital Authority and COVID-19 vaccination records in the Department of Health in Hong Kong. We conducted the SCCS analysis including patients with a first primary diagnosis of herpes zoster in the hospital inpatient setting between February 23 and July 31, 2021. A confirmatory analysis by nested case-control method was also conducted. Each herpes zoster case was randomly matched with ten controls according to sex, age, Charlson comorbidity index, and date of hospital admission. Conditional Poisson regression and logistic regression models were used to assess the potential excess rates of herpes zoster after vaccination. FINDINGS: From February 23 to July 31, 2021, a total of 16 and 27 patients were identified with a first primary hospital diagnosis of herpes zoster within 28 days after CoronaVac and BNT162b2 vaccinations. The incidence of herpes zoster was 7.9 (95% Confidence interval [CI]: 5.2–11.5) for CoronaVac and 7.1 (95% CI: 4.1–11.5) for BNT162b2 per 1,000,000 doses administered. In SCCS analysis, CoronaVac vaccination was associated with significantly higher risk of herpes zoster within 14 days after first dose (adjusted incidence rate ratio [aIRR]=2.67, 95% CI: 1.08–6.59) but not in other periods afterwards compared to the baseline period. Regarding BNT162b2 vaccination, a significantly increased risk of herpes zoster was observed after first dose up to 14 days after second dose (0-13 days after first dose: aIRR=5.23, 95% CI: 1.61–17.03; 14–27 days after first dose: aIRR=5.82, 95% CI: 1.62–20.91; 0-13 days after second dose: aIRR=5.14, 95% CI: 1.29–20.47). Using these relative rates, we estimated that there has been an excess of approximately 5 and 7 cases of hospitalization as a result of herpes zoster after every 1,000,000 doses of CoronaVac and BNT162b2 vaccination, respectively. The findings in the nested case control analysis showed similar results. INTERPRETATION: We identified an increased risk of herpes zoster related hospitalization after CoronaVac and BNT162b2 vaccinations. However, the absolute risks of such adverse event after CoronaVac and BNT162b2 vaccinations were very low. In locations where COVID-19 is prevalent, the protective effects on COVID-19 from vaccinations will greatly outweigh the potential side effects of vaccination. FUNDING: The project was funded by Research Grant from the Food and Health Bureau, The Government of the Hong Kong Special Administrative Region (Ref. No.COVID19F01). FTTL (Francisco Tsz Tsun Lai) and ICKW (Ian Chi Kei Wong)’s posts were partly funded by D(2)4H; hence this work was partly supported by AIR@InnoHK administered by Innovation and Technology Commission

Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy

Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients’ sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

First narrow-band search for continuous gravitational waves from known pulsars in advanced detector data

Spinning neutron stars asymmetric with respect to their rotation axis are potential sources of continuous gravitational waves for ground-based interferometric detectors. In the case of known pulsars a fully coherent search, based on matched filtering, which uses the position and rotational parameters obtained from electromagnetic observations, can be carried out. Matched filtering maximizes the signalto- noise (SNR) ratio, but a large sensitivity loss is expected in case of even a very small mismatch between the assumed and the true signal parameters. For this reason, narrow-band analysis methods have been developed, allowing a fully coherent search for gravitational waves from known pulsars over a fraction of a hertz and several spin-down values. In this paper we describe a narrow-band search of 11 pulsars using data from Advanced LIGO’s first observing run. Although we have found several initial outliers, further studies show no significant evidence for the presence of a gravitational wave signal. Finally, we have placed upper limits on the signal strain amplitude lower than the spin-down limit for 5 of the 11 targets over the bands searched; in the case of J1813-1749 the spin-down limit has been beaten for the first time. For an additional 3 targets, the median upper limit across the search bands is below the spin-down limit. This is the most sensitive narrow-band search for continuous gravitational waves carried out so far