Tomohiro Kawano

Intracerebral hemorrhage and deep microbleeds associated with cnm -positive Streptococcus mutans ; a hospital cohort study

Oral infectious diseases are epidemiologically associated with stroke. We previously showed that oral Streptococcus mutans with the cnm gene encoding a collagen-binding Cnm protein induced intracerebral hemorrhage (ICH) experimentally and was also associated with cerebral microbleeds (CMBs) in our population-based cohort study. We therefore investigated the roles of cnm-positive Streptococcus mutans in this single hospital-based, observational study that enrolled 100 acute stroke subjects. The cnm gene in Streptococcus mutans isolated from saliva was screened using PCR techniques and its collagen-binding activities examined. CMBs were evaluated on T2* gradient-recalled echo MRI. One subject withdrew informed consent and 99 subjects (63 males) were analyzed, consisting of 67 subjects with ischemic stroke, 5 with transient ischemic attack, and 27 with ICH. Eleven cases showed Streptococcus mutans strains positive for cnm. The presence of cnm-positive Streptococcus mutans was significantly associated with ICH [OR vs. ischemic stroke, 4.5; 95% CI, 1.17–19.1] and increased number of deep CMBs [median (IQR), 3 (2–9) vs. 0 (0–1), p = 0.0002]. In subjects positive for Streptococcus mutans, collagen binding activity was positively correlated with the number of deep CMBs (R2 = 0.405; p < 0.0001). These results provide further evidence for the key role of oral health in stroke.

A Novel Therapeutic Peptide as a Partial Agonist of RANKL in Ischemic Stroke

The enhanced receptor activator of nuclear factor-κB (NFκB) ligand (RANKL) and its receptor (RANK) signal have been reported to attenuate ischemic brain injury through inhibition of Toll-like receptor (TLR) 4-mediated inflammation. However, augmentation of the RANKL/RANK signal also accelerates osteoporosis, which is a potential problem in clinical use of RANKL. Therefore, we developed novel peptides, microglial healing peptides (MHPs), which were based on the DE and/or EF loop of RANKL. Among them, MHP1 was the most effective inhibitor of TLR4-induced inflammations in microglia/macrophages. The effects depended on RANK, as confirmed by knockdown experiments. In contrast to RANKL, MHP1 did not stimulate osteoclast differentiation. Unexpectedly, MHP1 inhibited RANKL-induced osteoclast differentiation. These findings suggested that MHP1 was a partial agonist of RANKL, and administration of MHP1 attenuated ischemic injury by decreasing inflammation. MHP1 could be a novel therapeutic agent for treating ischemic stroke.

Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes

Glial fibrillary acidic protein (GFAP) is expressed in peri‐islet Schwann cells, as well as in glia cells, and has been reported to be an autoantigen candidate for type 1 diabetes mellitus (T1DM). We confirmed that the production of the autoantibodies GFAP and glutamic acid decarboxylase 65 (GAD65) was increased and inversely correlated with the concentration of secreted C peptide in female nonobese diabetic mice (T1DM model). Importantly, the development of T1DM in female nonobese diabetic mice at 30 wk of age was predicted by the positive GFAP autoantibody titer at 17 wk. The production of GFAP and GAD65 autoantibodies was also increased in KK‐Ay mice [type 2 diabetes mellitus (T2DM) model]. In patients with diabetes mellitus, GFAP autoantibody levels were increased in patients with either T1DM or T2DM, and were significantly associated with GAD65 autoantibodies but not zinc transporter 8 autoantibodies. Furthermore, we identified a B‐cell epitope of GFAP corresponding to the GFAP autoantibody in both mice and patients with diabetes. Thus, these results indicate that autoantibodies against GFAP could serve as a predictive marker for the development of overt autoimmune diabetes.—Pang, Z., Kushiyama, A., Sun, J., Kikuchi, T., Yamazaki, H., Iwamoto, Y., Koriyama, H., Yoshida, S., Shimamura, M., Higuchi, M., Kawano, T., Takami, Y., Rakugi, H., Morishita, R., Nakagumi, H. Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes. FASEB J. 31, 4053–4063 (2017). www.fasebj.org—Pang, Zhengda, Kushiyama, Akifumi, Sun, Jiao, Kikuchi, Takako, Yamazaki, Hiroki, Iwamoto, Yasuhiko, Koriyama, Hiroshi, Yoshida, Shota, Shimamura, Munehisa, Higuchi, Masayoshi, Kawano, Tomohiro, Takami, Yoichi, Rakugi, Hiromi, Morishita, Ryuichi, Nakagami, Hironori Glial fibrillary acidic protein (GFAP) is a novel biomarker for the prediction of autoimmune diabetes. FASEB J. 31, 4053–4063 (2017)

Development of vaccine for dyslipidemia targeted to a proprotein convertase subtilisin/kexin type 9 (PCSK9) epitope in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates expression of low-density lipoprotein (LDL) receptors via receptor internalization and subsequent lysosomal degradation. Thus, an anti-PCSK9 antibody is well known as an anti-hyperlipidemia drug. Here, we aimed to develop vaccine for a long-term treatment of dyslipidemia targeted to PCSK9. In This study, we designed a peptide vaccine for mouse PCSK-9, which consisted of short peptides conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Vaccines were administered to male apolipoprotein E (ApoE) deficient mice with adjuvants and significantly elicited an antibody response against PCSK9. The PCSK9 vaccines were administered to mice three times in 2-week intervals, and antibody titers and lipoprotein levels were evaluated up to 24 weeks after the first immunization to determine the therapeutic effect. Anti-PCSK9 antibody titers reached peak levels 6 weeks after the first immunization, and theses titers were maintained for up to 24 weeks. Decreased plasma levels of total cholesterol, very low-density lipoprotein (VLDL), and chylomicron (CM) were maintained for up to 24 weeks. Immunized mice exhibited a significant increase in cell-surface LDL receptor expression. Stimulation with KLH, but not PCSK9, induced the production of INF-gamma and interleukin-4 (IL-4), as determined with ELISPOT assays, thus indicating that PCSK9 vaccine did not elicit T-cell activation in our vaccine system. The present anti-PCSK9 vaccine induced long-lasting anti-PCSK9 antibody production and improved lipoprotein profiles. Thus, anti-PCSK9 vaccine could become a new option for the treatment of dyslipidemia as a long-acting therapy in future.

Direct aspiration first pass technique for a middle cerebral artery occlusion with a hidden aneurysm.

Hidden aneurysms within occluded vessels present a challenge for interventionists because vessel perforation can lead to life-threatening complications. We present a case of middle cerebral artery ischemic stroke, refractory to thrombolysis. A direct aspiration first pass technique (ADAPT) was employed for revascularization. Following thrombectomy, an aneurysm of the occluded vessel was revealed. Despite this, the patient recovered without hemorrhagic complication. ADAPT permits the minimal insertion of endovascular devices and might be a safe procedure when hidden aneurysms are suspected.

Aortic Transgraft Hemorrhage after Intravenous Tissue Plasminogen Activator Therapy in Patients with Acute Ischemic Stroke

BACKGROUND The safety of intravenous recombinant tissue plasminogen activator (IV tPA) therapy for patients with an aortic aneurysm or undergoing aortic graft replacement has not been established. We evaluated the incidence, bleeding site, coagulation factors, and clinical outcomes of patients treated with IV tPA for acute stroke. METHODS Between October 2005 and May 2013, 394 ischemic stroke patients were treated in our stroke center with IV tPA. Among these patients, we investigated those who had a history of aortic aneurysm with or without aortic graft replacement before IV tPA therapy and underwent computed tomography imaging. We compared the levels of D-dimer and hemoglobin (Hb) around IV tPA therapy between the patients with and without tPA-associated periaortic bleeding. RESULTS Seven patients with a history of aortic aneurysm (3 men; mean age: 80.4 years) were examined; 3 had undergone aortic graft replacement, and 2 had experienced tPA-associated bleeding around vascular grafts. The serum D-dimer levels in those with bleeding were only slightly higher before tPA than in those without (median: 10.5 vs. 1.5 μg/mL) but were elevated 1 day after tPA (107.4 vs. 8.6 μg/mL). The Hb levels 2 days after tPA were comparable with those before tPA (11.9 vs. 11.8 g/dL) but were lower in the patients with bleeding than in those without (8.5 vs. 11.7 g/dL). Surgical intervention was not required, although 1 patient required blood transfusion. CONCLUSIONS Our analysis provides reassurance regarding the risk of IV tPA therapy in patients undergoing aortic graft replacement.

Blood biomarkers associated with neurological deterioration in patients with acute penetrating artery territory infarction: A multicenter prospective observational study:

Background and purpose Neurological deterioration in acute penetrating artery territory infarction is unpredictable and associated with unfavorable clinical outcomes. The aim of this prospective study was to clarify the cause of neurological worsening and predict clinical outcomes using blood biomarkers. Methods Eight Japanese stroke centers participated. Blood samples were obtained within 24 h (the first sampling) and on day 7 in hospital (the second sampling) in patients with penetrating artery territory infarction, arriving within two days of stroke onset. Symptomatic worsening was defined as a minimum increase of one point on the National Institutes of Health Stroke Scale. Poor outcome was defined as a modified Rankin Scale score of ≥3 at 90 days after ictus. Results Of the 89 patients, 25 (28%) had symptomatic worsening, and 25 (28%) had a poor outcome. Although tumor necrosis factor-alpha, high-sensitivity C-reactive protein levels were significantly increased in both groups at the second sampling, soluble lectin-like oxidized low-density lipoprotein receptor-1, CD40 ligand, and pro-adrenomedullin levels were significantly increased and ADAMTS13 activity was decreased in symptomatic worsening patients (p < 0.05 for all). After multivariate adjustment, a low number of CD34+ cells at the first sampling was an independent predictor of poor outcome (odds ratio, 0.20; 95% confidence interval, 0.04–0.74, p = 0.011, per 1 cell/µl increase). Conclusions Blood biomarkers associated with atherosclerotic processes seem to be an indication for symptomatic worsening, and the number of CD34+ cells may help to predict three-month functional outcome in patients with penetrating artery territory infarction.

Therapeutic Effects of Systemic Administration of the Novel RANKL-Modified Peptide, MHP1, for Ischemic Stroke in Mice

Microglial healing peptide 1, “MHP1”, is a newly developed synthetic peptide composed of the DE and a part of the EF loop of the receptor activator of nuclear factor-кB (NFκB) ligand (RANKL). Our previous report demonstrated that MHP1 significantly inhibits Toll-like receptor (TLR) 2- and 4-induced inflammation in microglia/macrophages through RANK signaling without osteoclast activation. However, its inhibitory effects on ischemic stroke when administered intravenously have not been clarified. First, we examined whether MHP1 could penetrate the brain parenchyma. Intravenous injection of FITC-conjugated MHP1 demonstrated that MHP1 could cross the blood-brain-barrier in peri-infarct regions, but not in intact regions. Because MHP1 in the parenchyma was reduced at 60 minutes after injection, we speculated that continuous injection was necessary to achieve the therapeutic effects. To check the possible deactivation of MHP1 by continuous injection, the anti-inflammatory effects were checked in MG6 cells after incubation in 37°C for 24 hours. Although the inhibitory effects for IL6 and TNFα were reduced compared to nonincubated MHP1, its anti-inflammatory efficacy remained, indicating that continuous administration with pump was possible. The single and successive continuous administration of MHP1 starting from 4 or 6 hours after cerebral ischemia successfully reduced infarct volume and prevented the exacerbation of neurological deficits with reduced activation of microglia/macrophages and inflammatory cytokines. Different from recombinant RANKL, MHP1 did not activate osteoclasts in the paralytic arm. Although further modification of MHP1 is necessary for stabilization, the MHP1 could be a novel agent for the treatment ischemic stroke.