We previously found thatMertkand its ligandGas6, astrocytic genes involved in phagocytosis, are upregulated after acute sleep deprivation. These results suggested that astrocytes may engage in phagocytic activity during extended wake, but direct evidence was lacking. Studies in humans and rodents also found that sleep loss increases peripheral markers of inflammation, but whether these changes are associated with neuroinflammation and/or activation of microglia, the brain's resident innate immune cells, was unknown. Here we used serial block-face scanning electron microscopy to obtain 3D volume measurements of synapses and surrounding astrocytic processes in mouse frontal cortex after 6–8 h of sleep, spontaneous wake, or sleep deprivation (SD) and after chronic (∼5 d) sleep restriction (CSR). Astrocytic phagocytosis, mainly of presynaptic components of large synapses, increased after both acute and chronic sleep loss relative to sleep and wake. MERTK expression and lipid peroxidation in synaptoneurosomes also increased to a similar extent after short and long sleep loss, suggesting that astrocytic phagocytosis may represent the brain's response to the increase in synaptic activity associated with prolonged wake, clearing worn components of heavily used synapses. Using confocal microscopy, we then found that CSR but not SD mice show morphological signs of microglial activation and enhanced microglial phagocytosis of synaptic elements, without obvious signs of neuroinflammation in the CSF. Because low-level sustained microglia activation can lead to abnormal responses to a secondary insult, these results suggest that chronic sleep loss, through microglia priming, may predispose the brain to further damage.SIGNIFICANCE STATEMENTWe find that astrocytic phagocytosis of synaptic elements, mostly of presynaptic origin and in large synapses, is upregulated already after a few hours of sleep deprivation and shows a further significant increase after prolonged and severe sleep loss, suggesting that it may promote the housekeeping of heavily used and strong synapses in response to the increased neuronal activity of extended wake. By contrast, chronic sleep restriction but not acute sleep loss activates microglia, promotes their phagocytic activity, and does so in the absence of overt signs of neuroinflammation, suggesting that like many other stressors, extended sleep disruption may lead to a state of sustained microglia activation, perhaps increasing the brain's susceptibility to other forms of damage.

Background: MxA is an antiviral protein exclusively induced by type I interferons (IFN) and some viruses, and MxA gene expression is one of the most appropriate markers for measuring the biologic activity of exogenous IFNβ. Methods: A new quantitative-competitive PCR method was used to quantify MxA mRNA in peripheral blood mononuclear cells of 99 treatment-naïve and 92 IFNβ-treated patients with MS (22 Avonex, 17 Betaferon, and 53 Rebif-22). Every 3 months, IFNβ-induced neutralizing antibodies (NAb) were evaluated in sera using a cytopathic effect assay. Three categories of patients were identified: NAb negative (NAb−), persistent NAb positive (NAb+, ≥2 consecutive positive samples), and isolated NAb+ (one positive sample). Results: Treatment-naïve patients expressed detectable MxA mRNA levels (mean = 36 ± 32 fg MxA/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH); range 1 to 160) and an upper normal threshold was established (mean + 3 SD = 132 fg MxA/pg GAPDH). IFNβ-treated patients exhibited more than 11-fold higher levels (mean = 412 ± 282 fg MxA/pg GAPDH; range 16 to 1,172). However, 17 patients did not exhibit an increase in MxA mRNA level; 15 of these 17 patients showed a concurrent Nab+ titer. Moreover, 13 were persistent NAb+. Isolated NAb+ patients did not show a decrease in bioavailability of IFNβ (n = 9; mean = 567 ± 366 fg MxA/pg GAPDH; range 83 to 1,120). In NAb− patients, bioavailability was comparable among the three different IFNβ preparations 12 hours after injection. Conclusion: During IFNβ therapy, the presence of NAb reduced or abolished bioavailability in a relevant percentage of patients. These data could be important for the early detection of patients with MS who are not responsive to IFNβ therapy.

Objective: To analyze the impact of neutralizing antibodies (NAbs) on the clinical efficacy of IFNβ. Methods: This was an open-label study involving 78 patients with multiple sclerosis treated with Betaferon 8 million IU (MIU) subcutaneously (SC) every other day (n = 20), Rebif 22 μg SC 3 times weekly (n = 25), or Avonex 30 μg IM once weekly (n = 33). Every 3 months, blood samples were collected and sera were analyzed for NAbs using an antiviral cytopathic effect assay. Patients were categorized according to their NAb status: NAb negative (NAb−); isolated NAb positive (NAb+), patients with ≥1 positive sample (titer ≥ 20); and persistent NAb+, patients with ≥2 consecutive positive samples (titer ≥ 20). Patients who were NAb− and persistent NAb+ were compared for relapse rate, time between first and second relapse, percentage of relapse-free patients, and percentage of patients who had a sustained progression of ≥1 point on the Expanded Disability Status Scale (EDSS). Results: The incidence of persistent NAb+ patients was 35% for Betaferon, 20% for Rebif, and 3% for Avonex. During IFNβ treatment, both NAb+ and NAb− patients showed a reduction in relapse rate; this reduction (25%) was not significant in NAb+ patients but was significant (67%; p < 0.0001) in NAb− patients. In addition, the mean relapse rate was higher (p = 0.039), mean time between first and second relapse was shorter (13 vs 21 months; p = 0.0064), and there was a trend suggesting that NAbs affected the probability of remaining relapse free (p = 0.08). A higher percentage of NAb+ patients versus NAb− patients had worsening of EDSS scores during follow-up (p = 0.013). Conclusion: Persistent NAbs significantly reduce the clinical efficacy of IFNβ.

Purpose To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice. Methods Untreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H₂O₂-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured. Results All rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H₂O₂-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H₂O₂-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a. Conclusions Oxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.

Pregnancy is associated with reduced activity of multiple sclerosis (MS). However, the biological mechanisms underlying this pregnancy-related decrease in disease activity are poorly understood. We conducted a genome-wide transcription analysis in peripheral blood mononuclear cells (PBMCs) from 12 women (7 MS patients and 5 healthy controls) followed during their pregnancy. Samples were obtained before, during (i.e. at the third, sixth, and ninth month of gestation) and after pregnancy. A validation of the expression profiles has been conducted by using the same samples and an independent group of 25 MS patients and 11 healthy controls. Finally, considering the total group of 32 MS patients, we compared expression profiles of patients relapsing during pregnancy (n = 6) with those of relapse-free patients (n = 26). Results showed an altered expression of 347 transcripts in non-pregnant MS patients with respect to non-pregnant healthy controls. Complementary changes in expression, occurring during pregnancy, reverted the previous imbalance particularly for seven inflammation-related transcripts, i.e. SOCS2, TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and STAG3L1. Longitudinal analysis showed that the overall deregulation of gene expression reverted to “normal” already within the third month of gestation, while in the post-partum gene expressions rebounded to pre-pregnancy levels. Six (18.7%) of the 32 MS patients had a relapse during pregnancy, mostly in the first trimester. The latter showed delayed expression profiles when compared to relapse-free patients: in these patients expression imbalance was reverted later in the pregnancy, i.e. at sixth month. Specific changes in expression during pregnancy were associated with a decrease in disease activity assessed by occurrence of relapses during pregnancy. Findings might help in understanding the pathogenesis of MS and may provide basis for the development of novel therapeutic strategies.

Purpose To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNβ) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory. Methods RhIFNβ-1b and three rhIFNβ-1a preparations with different aggregate levels were injected intraperitoneally in mice 15× during 3 weeks, and the mice were rechallenged with rhIFNβ-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored. Results Bulk rhIFNβ-1a contained large, mainly non-covalent aggregates and stressed rhIFNβ-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNβ-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNβ-1b showed the highest and reformulated rhIFNβ-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. Conclusions The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNβ in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.