Highly efficient CRISPR/Cas9-mediated exon skipping for recessive dystrophic epidermolysis bullosa.

Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient-specific skin equivalents. Precise gene editing using homology-directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non-homologous end joining (NHEJ) repair aimed at excising dispensable, mutation-harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal-epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long-read Nanopore sequencing detected large on-target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short-read sequencing may underestimate the full range of unexpected Cas9-mediated editing events.

Proliferating macrophages in human tumours show characteristics of monocytes responding to myelopoietic growth factors.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-ß. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Elicitation of potent neutralizing antibodies in obese mice by ISA 51-adjuvanted SARS-CoV-2 spike RBD-Fc vaccine.

Vaccination is considered to be the most effective countermeasure to prevent and combat the global health threats of COVID-19. People with obesity are at a greater risk of hospitalization, life-threatening illness, and adverse outcomes after having COVID-19. Therefore, a safe and effective COVID-19 vaccine for obese individuals is urgently needed. In the study, the vaccine composed of the ISA 51 adjuvant and the SARS-CoV-2 spike (S) receptor-binding domain (RBD) in conjugation with the human IgG1 Fc fragment (named as ISA 51-adjuvanted RBD-Fc vaccine) was developed and inoculated in the regular chow diet (RCD) lean mice and the high-fat diet (HFD)-induced obese mice. The S protein-specific IgG titers were largely induced in an increasing manner along with three doses of ISA 51-adjuvanted RBD-Fc vaccine without causing any harmful side effect. In the HFD mice, the S protein-specific IgG titers can be quickly observed 2 weeks post the first inoculation. The antisera elicited by the ISA 51-adjuvanted RBD-Fc vaccine in the RCD and HFD mice exhibited potent SARS-CoV-2 neutralizing activities in the plaque reduction neutralization test (PRNT) assays and showed similar specificity for recognizing the key residues in the RBD which were involved in interacting with angiotensin-converting enzyme 2 (ACE2) receptor. The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine in the HFD mice can be sustainably maintained with the PRNT50 values of 1.80-1.91×10-3 for at least 8 weeks post the third inoculation. Collectively, the RBD-Fc-based immunogen and the ISA 51-adjuvanted formulation can be developed as an effective COVID-19 vaccine for obese individuals. KEY POINTS: • The ISA 51-adjuvanted RBD-Fc vaccine can induce potent SARS-CoV-2 neutralizing antibodies in the obese mouse • The antibodies elicited by the ISA 51-adjuvanted RBD-Fc vaccine can bind to the key RBD residues involved in interacting with ACE2 • The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine can be sustainably maintained for at least 8 weeks post the third inoculation.

Aspergillus oryzae Fermentation Extract Alleviates Inflammation in Mycoplasma pneumoniae Pneumonia.

The filamentous fungus Aspergillus oryzae, also known as koji mold, has been used for centuries in the production of fermented foods in East Asia. A. oryzae fermentation can produce enzymes and metabolites with various bioactivities. In this study, we investigated whether A. oryzae fermentation extract (AOFE) has any effect on Mycoplasma pneumoniae (Mp) pneumonia. We performed solid-state fermentation of A. oryzae and obtained the ethanol extract. AOFE was analyzed by HPLC, and the major component was identified to be kojic acid. In vitro, AOFE suppressed Mp growth and invasion into A549 lung epithelial cells as determined by the gentamicin protection assay. AOFE treatment also suppressed Mp-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 at mRNA and protein levels in murine MH-S alveolar macrophages. In a mouse model of Mp pneumonia, Mp infection induced a marked pulmonary infiltration of neutrophils, which was significantly reduced in mice pre-treated orally with AOFE. AOFE administration also suppressed the production of proinflammatory cytokines and chemokines in the lungs. Collectively, our results show that AOFE has the potential to be developed into a preventive/therapeutic agent for Mp pneumonia.

Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium.

BACKGROUND AIMS: The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing. RESULTS: We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. "Kelch's medium" supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media. CONCLUSIONS: This new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

Detection of Anticipatory Dynamics between a Pair of Zebrafish.

Anticipatory dynamics (AD) is unusual in that responses from an information receiver can appear ahead of triggers from the source, and direction of information flow (DIF) is needed to establish causality. Although it is believed that anticipatory dynamics is important for animals' survival, natural examples are rare. Time series (trajectories) from a pair of interacting zebrafish are used to look for the existence of AD in natural systems. In order to obtain the DIF between the two trajectories, we have made use of a special experimental design to designate information source. However, we have also used common statistical tools such as Granger causality and transfer entropy to detect DIF. In our experiments, we found that a majority of the fish pairs do not show any anticipatory behaviors and only a few pairs displayed possible AD. Interestingly, for fish in this latter group, they do not display AD all the time. Our findings suggest that the formation of schooling of fish might not need the help of AD, and new tools are needed in the detection of causality in AD system.

Photoinduced arylation of chloroarenes in flow: synthesis of unsymmetrical biaryls.

A photoflow method is presented for a radical-based coupling of unactivated arenes and aryl chlorides. The process proceeded smoothly at ambient temperature under metal-free conditions. Of note is that the reaction conditions are fine-tuned for chloroarenes with different electronic properties. While the reactivity profile of aryl chlorides is generally known to be inferior to those of the corresponding iodides and bromides, we demonstrate that the title protocol is efficient in converting readily available and inexpensive chloroarenes into unsymmetrical biaryl products.

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

An objective index for spinal cord compression on computed tomography in Thoroughbred horses.

BACKGROUND: Computed tomographic myelography can be a useful tool for evaluating vertebral canal stenosis. However, an index of spinal cord compression is yet to be established. OBJECTIVES: This observational descriptive study aimed to establish an index for spinal cord compression using computed tomography (CT). METHODS: Twenty-three Thoroughbred horses (age, 155-717 days; weight, 205-523 kg) with suspected cervical vertebral malformation were subjected to computed tomographic myelography in dorsal recumbency using large-bore gantry CT to define the entire cervical vertebrae from C1 to C7. Subsequently, the height of the spinal cord was measured in the sagittal plane reformatted using curved multi-planar reformation (MPR), thereby comparing it with stenotic ratio (i.e. dividing the area of spinal cord by that of the subarachnoid space) measured in the transverse plane. The measurement was performed at the level of each of six intervertebral spaces, for a total of 138 sites. Accordingly, the appropriate cut-off value for spinal cord height was determined using the receiver-operating characteristic curve, from which the area under the curve with 95% confidence interval was estimated. RESULTS: The spinal cord compression cut-off value was 7.06 mm, with an area under curve of 0.84. A weak relationship was observed between spinal cord height and stenotic ratio (R2  = 0.08, p < 0.05). CONCLUSIONS: Following curved MPR, a cut-off value of 7.06 mm may serve as an index for spinal cord compression.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Photo-Fries rearrangement in flow under aqueous micellar conditions.

A flow edition of photo-Fries rearrangement for the synthesis of 2-acylphenols in an aqueous micellar medium has been described. We take advantage of a narrow channel reactor and micelle-induced confinement effect to refine both the efficiency and selectivity of the parent photoreaction.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

Immunogenicity and safety of an inactivated enterovirus A71 vaccine in children 3-6 years and 2-35 months of age- an open-label, randomized phase IIb clinical trial.

BACKGROUND: Enterovirus A71 (EV-A71) infection can cause severe debilitating complications and even death in young children. The immunogenicity and safety of an inactivated whole EV-A71 virus vaccine were assessed in children 2 months to 6 years of age. METHODS: This was an open-label, multi-center and randomized phase IIb study, which divided into part A and B. In part A, children 36 months to 6 years of age were enrolled and randomized into 3 groups, receiving 0.5 µg total viral protein (TP) with adjuvant Al(OH)3, 1.0 µg TP with Al(OH)3 or 1.0 µg TP only. Two doses of vaccines were administered at a 28-day interval and blood was taken before immunization, at week 4, 8, 28 and 52 (optional) for virus neutralization assay. Safety profiles were also monitored. After safety profiles had shown no concerns, children 2 months to 35 months of age (part B) were subsequently enrolled following the same protocol. RESULTS: A total of 135 children completed two doses of immunization, including 58 in part A and 77 in part B. Both adjuvanted 0.5 µg and 1.0 µg TP elicited significant raise of neutralizing antibody titers and seroconversion rate was up to 93.75-100.0% after 2 doses of immunization. Adjuvanted 1.0 µg TP induced higher titers of neutralizing antibodies than adjuvanted 0.5 µg TP. By contrast, non-adjuvanted 1.0 µg TP was not immunogenic. No major adverse events were reported. CONCLUSIONS: This EV-A71 vaccine containing adjuvant is immunogenic and safe in children 2 months to 6 years of age. CLINICAL TRIALS REGISTRATION: NCT03268083.

Risk of cancer in patients with genital warts: A nationwide, population-based cohort study in Taiwan.

BACKGROUND: Condyloma acuminata currently affects around 1% of sexually active adults, and its incidence is increasing. The coexistence of genital warts (GW) and certain cancers and an association between human papillomavirus (HPV) and various malignancies have been reported. Therefore, we conducted this large national study to analyze the risk of malignancies among men and women with GW in Taiwan. METHODS AND FINDINGS: Between January 2000 and December 2013, approximately 3 million patients were reported to the National Health Insurance Research Database of Taiwan. Of these patients, 21,763 were diagnosed with GW. In the same time period, a total of 213,541 cancer cases were reported to the registry, of which 1002 were recorded among patients with GW. The age-specific incidence rates of GW and standardized incidence ratios (SIRs) of malignancies compared to the general population were calculated. Women acquired GW earlier than men, with a mean age at diagnosis of 32.63±12.78 years. The highest incidence rate for both genders peaked at 20-29 years. Of the 1002 patients with GW and malignancies, the SIR was 1.95 (95%CI 1.83-2.07). The most markedly increased risk was found for HPV-related cancers, with a SIR of 9.74 (95%CI 3.70-15.77). Significantly elevated risks were also noted for smoking-related cancers, anogenital cancers, cervix in situ, colon, rectum, lung, kidney, and prostate cancers. Most cancers developed within 10 years after the diagnosis of GW. CONCLUSIONS: Patients with GW have an increased risk of HPV-related cancers, especially anogenital malignancies in Taiwan. The elevated risk of other cancers highlights differences in exposure and risk factors among patients with GW compared to the general population. Cancer screening and HPV vaccination programs should be emphasized for at-risk patients.

Recent trends in prescribing antibiotics for acute tonsillitis in pediatric ambulatory care in Taiwan, 2000-2009: A nationwide population-based study.

BACKGROUND/PURPOSE: Acute tonsillitis is the leading diagnosis in pediatric ambulatory care, and group A beta-hemolytic streptococcus is the main reason for antibiotic prescriptions in patients with acute tonsillitis. The aim of this study was to analyze trends in prescribing antibiotics and to investigate the prescription patterns for acute tonsillitis in pediatric ambulatory care in Taiwan from 2000 to 2009. METHODS: Data on children younger than 18 years with a primary diagnosis of acute tonsillitis were retrieved from the National Health Insurance Research Database of Taiwan from 2000 to 2009. Concomitant bacterial infections were excluded. Sex, age, seasonality, location, level of medical institution, and physician specialty were analyzed. Annual and monthly changes in antibiotic prescriptions and classification were also evaluated. RESULTS: A total of 40,775 cases were enrolled, with an overall antibiotic prescription rate of 16.8%. There was a remarkable decline in the antibiotic prescription rates for tonsillitis from 28.4% in 2000 to 10.9% in 2009. Factors associated with a higher prescription rate included older age, visits from eastern Taiwan, medical centers, and nonpediatrician physicians. Otolaryngologists had higher antibiotic prescription rate, whereas pediatricians had the lowest (21.9% vs. 11.6%). The rates of obtaining throat cultures were low although the culture performing rate in the medical centers was significantly higher (12.3%, p < 0.001). CONCLUSION: From 2000 to 2009, there was a remarkable decline in the antibiotic prescription rates for tonsillitis. Further studies to evaluate diagnostic tools such as rapid antigen detection tests or throat cultures to decrease antibiotic prescriptions are warranted.

Clinical analysis of fever of unknown origin in children: A 10-year experience in a northern Taiwan medical center.

BACKGROUND: Fever of unknown origin (FUO) was first described in 1961 as fever >38.3°C for at least 3 weeks with no apparent source after 1 week of investigations in the hospital. Infectious disease comprises the majority of cases (40-60%). There is no related research on FUO in children in Taiwan. The aim of this study is to determine the etiologies of FUO in children in Taiwan and to evaluate the relationship between the diagnosis and patient's demography and laboratory data. METHODS: Children under 18 years old with fever >38.3°C for >2 weeks without apparent source after preliminary investigations at Taipei Veterans General Hospital during 2002-2012 were included. Fever duration, symptoms and signs, laboratory examinations, and final diagnosis were recorded. The distribution of etiologies and age, fever duration, laboratory examinations, and associated symptoms and signs were analyzed. RESULTS: A total of 126 children were enrolled; 60 were girls and 66 were boys. The mean age was 6.7 years old. Infection accounted for 27.0% of cases, followed by undiagnosed cases (23.8%), miscellaneous etiologies (19.8%), malignancies (16.6%), and autoimmune disorders (12.7%). Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were the most commonly found pathogens for infectious disease, and Kawasaki disease (KD) was the top cause of miscellaneous diagnosis. CONCLUSIONS: Infectious disease remains the most common etiology. Careful history taking and physical examination are most crucial for making the diagnosis. Conservative treatment may be enough for most children with FUO, except for those suffering from malignancies.

Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

BACKGROUND: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. RESULTS: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. CONCLUSIONS: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.