Albumin-binding recombinant human IL-18BP ameliorates macrophage activation syndrome and atopic dermatitis via direct IL-18 inactivation.

Given the clinical success of cytokine blockade in managing diverse inflammatory human conditions, this approach could be exploited for numerous refractory or uncontrolled inflammatory conditions by identifying novel targets for functional blockade. Interleukin (IL)-18, a pro-inflammatory cytokine, is relatively underestimated as a therapeutic target, despite accumulated evidence indicating the unique roles of IL-18 in acute and chronic inflammatory conditions, such as macrophage activation syndrome. Herein, we designed a new form of IL-18 blockade, i.e., APB-R3, a long-acting recombinant human IL-18BP linked to human albumin-binding Fab fragment, SL335, for extending half-life. We then explored the pharmacokinetics and pharmacodynamics of APB-R3. In addition to an extended serum half-life, APB-R3 alleviates liver inflammation and splenomegaly in a model of the macrophage activation syndrome induced in IL-18BP knockout mice. Moreover, APB-R3 substantially controlled skin inflammation in a model of atopic dermatitis. Thus, we report APB-R3 as a new potent IL-18 blocking agent that could be applied to treat IL-18-mediated inflammatory diseases.

Disruption of IL-18 signaling via engineered IL-18BP biologics alleviates experimental cholestatic liver disease.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis around intrahepatic and extrahepatic bile ducts leading to severe hepatic cirrhosis and high mortality. Although there is an urgent clinical unmet need for PSC, no effective medical therapy has been developed to delay the disease progression until today. IL-18 binding protein (IL-18BP) is well-known to be a natural negative feedback regulator for IL-18, and we have developed a recombinant long-acting IL-18BP referred to as APB-R3 as a therapeutic agent to treat IL-18-related inflammatory diseases. Here, we aimed to study whether disrupted IL-18 signaling by APB-R3 treatment can inhibit PSC injuries in the experimental DDC diet-induced PSC rodent model. First, we found that the amounts of free IL-18 are augmented under PSC condition with increased expression of biliary IL-18 receptors. Administration of APB-R3 effectively attenuated key diagnostic parameters of PSC such as plasma ALP and GGT levels as well as bile acids levels. We also observed that blockade of IL-18 suppressed ductular reactive and proliferative phenotypes of cholangiocytes. Additionally, APB-R3 significantly ameliorated DDC diet-induced periductal fibrosis and transcriptional expressions of pro-fibrotic marker genes. Enhanced senescence associated secretory phenotype (SASP) markers in cholestatic liver disease were diminished by APB-R3 treatment. Our findings clearly demonstrate that the administration of IL-18BP biologics, APB-R3, effectively alleviates DDC diet-induced biliary injuries in rodent PSC model, implying APB-R3 can be a promising therapeutic reagent which warrants clinical human trials as new therapeutic options.

Patient-derived glioblastoma cell lines with conserved genome profiles of the original tissue.

Glioblastoma (GBM) is the most lethal intracranial tumor. Sequencing technologies have supported personalized therapy for precise diagnosis and optimal treatment of GBM by revealing clinically actionable molecular characteristics. Although accumulating sequence data from brain tumors and matched normal tissues have facilitated a comprehensive understanding of genomic features of GBM, these in silico evaluations could gain more biological credibility when they are verified with in vitro and in vivo models. From this perspective, GBM cell lines with whole exome sequencing (WES) datasets of matched tumor tissues and normal blood are suitable biological platforms to not only investigate molecular markers of GBM but also validate the applicability of druggable targets. Here, we provide a complete WES dataset of 26 GBM patient-derived cell lines along with their matched tumor tissues and blood to demonstrate that cell lines can mostly recapitulate genomic profiles of original tumors such as mutational signatures and copy number alterations.

Long-acting recombinant human follicle-stimulating hormone (SAFA-FSH) enhances spermatogenesis.

Introduction: Administration of follicle-stimulating hormone (FSH) has been recommended to stimulate spermatogenesis in infertile men with hypogonadotropic hypogonadism, whose sperm counts do not respond to human chorionic gonadotropin alone. However, FSH has a short serum half-life requiring frequent administration to maintain its therapeutic efficacy. To improve its pharmacokinetic properties, we developed a unique albumin-binder technology, termed "anti-serum albumin Fab-associated" (SAFA) technology. We tested the feasibility of applying SAFA technology to create long-acting FSH as a therapeutic candidate for patients with hypogonadotropic hypogonadism. Methods: SAFA-FSH was produced using a Chinese hamster ovary expression system. To confirm the biological function, the production of cyclic AMP and phosphorylation of ERK and CREB were measured in TM4-FSHR cells. The effect of gonadotropin-releasing hormone agonists on spermatogenesis in a hypogonadal rat model was investigated. Results: In in vitro experiments, SAFA-FSH treatment increased the production of cyclic AMP and increased the phosphorylation of ERK and CREB in a dose-dependent manner. In animal experiments, sperm production was not restored by human chorionic gonadotropin treatment alone, but was restored after additional recombinant FSH treatment thrice per week or once every 5 days. Sperm production was restored even after additional SAFA-FSH treatment at intervals of once every 5 or 10 days. Discussion: Long-acting FSH with bioactivity was successfully created using SAFA technology. These data support further development of SAFA-FSH in a clinical setting, potentially representing an important advancement in the treatment of patients with hypogonadotropic hypogonadism.

First-Pass Recanalization with EmboTrap II in Acute Ischemic Stroke (FREE-AIS): A Multicenter Prospective Study.

OBJECTIVE: We aimed to evaluate the efficacy of EmboTrap II in terms of first-pass recanalization and to determine whether it could yield favorable outcomes. MATERIALS AND METHODS: In this multicenter, prospective study, we consecutively enrolled patients who underwent mechanical thrombectomy using EmboTrap II as a front-line device. The primary outcome was the first pass effect (FPE) rate defined by modified Thrombolysis In Cerebral Infarction (mTICI) grade 2c or 3 by the first pass of EmboTrap II. In addition, modified FPE (mFPE; mTICI grade 2b-3 by the first pass of EmboTrap II), successful recanalization (final mTICI grade 2b-3), and clinical outcomes were assessed. We also analyzed the effect of FPE on a modified Rankin Scale (mRS) score of 0-2 at 3 months. RESULTS: Two hundred-ten patients (mean age ± standard deviation, 73.3 ± 11.4 years; male, 55.7%) were included. Ninety-nine patients (47.1%) had FPE, and mFPE was achieved in 150 (71.4%) patients. Successful recanalization was achieved in 191 (91.0%) patients. Among them, 164 (85.9%) patients underwent successful recanalization by exclusively using EmboTrap II. The time from groin puncture to FPE was 25.0 minutes (interquartile range, 17.0-35.0 minutes). Procedure-related complications were observed in seven (3.3%) patients. Symptomatic intracranial hemorrhage developed in 14 (6.7%) patients. One hundred twenty-three (58.9% of 209 completely followed) patients had an mRS score of 0-2. Sixteen (7.7% of 209) patients died during the follow-up period. Patients who had successful recanalization with FPE were four times more likely to have an mRS score of 0-2 than those who had successful recanalization without FPE (adjusted odds ratio, 4.13; 95% confidence interval, 1.59-10.8; p = 0.004). CONCLUSION: Mechanical thrombectomy using the front-line EmboTrap II is effective and safe. In particular, FPE rates were high. Achieving FPE was important for an mRS score of 0-2, even in patients with successful recanalization.

APB-F1, a long-acting feline granulocyte colony-stimulating factor fusion protein, created by exploiting FL335, a chimeric Fab specific for feline serum albumin.

Off-label use of a human granulocyte colony stimulating factor (hG-CSF) has been allowed to treat dogs and cats with neutropenia. However, repeated administration of hG-CSF induces undesirable anti-drug antibody (ADA) responses, implying the necessity of animal-derived G-CSF as a therapeutic reagent, preferably with a long-acting capability. Herein, we generated a recombinant fusion protein by genetically combining FL335, a chimeric Fab specific for feline serum albumin (FSA), and feline G-CSF (fG-CSF), with the ultimate goal of developing a long-acting therapeutic fG-CSF for cats. The resulting FL335-fG-CSF fusion protein, referred to as APB-F1, was produced well as a functional form in a Chinese hamster ovary (CHO) expression system. In in vitro analyses, APB-F1 bound to FSA at high affinity (KD = 400 pM) and possessed 0.78 × 107 U/mg G-CSF biological activity, clearly proving its biological functionality. Pharmacokinetic (PK) and pharmacodynamic (PD) studies using healthy cats revealed that the serum half-life (t1/2) of APB-F1 was increased five times compared with that of fG-CSF (t1/2 = 13.3 h vs. 2.7 h) in subcutaneous (SC) injections. Additionally, APB-F1 induced a profound and sustained increase in white blood cell (WBC) and actual neutrophil count (ANC) up to 10 days, which was far superior to other G-CSF preparations, including filgrastim (Neupogen™) and even pegfilgrastim (Neulasta™). Conclusively, a long-acting fG-CSF with potent in vivo bioactivity was successfully created by using FL335; thus, we provided evidence that our "anti-serum albumin Fab-associated" (SAFA) technology can be applied reliably in developing valuable long-acting biologics in veterinary medicine.

Gelfoam embolization for distal, medium vessel injury during mechanical thrombectomy in acute stroke: A case report.

BACKGROUND: Arterial perforation has inevitably increased as endovascular treatments have become more common for intracranial large vessel occlusions, and even distal, medium vessel occlusions. A distal, medium vessel has a tortuous course and thinner wall compared to large arteries, making it more susceptible to damage. Here, we review the treatment strategies for arterial perforation during mechanical thrombectomy, and we report the case of a patient treated with gelfoam embolization. CASE SUMMARY: A 63-year-old woman presented to the emergency department with sudden neurologic symptoms of right hemiparesis and global aphasia. The initial National Institutes of Health Stroke Scale score was 15. Computed tomography (CT) and CT angiography revealed hyperacute infarction and emergent arterial occlusion of the left middle cerebral artery M2-3 portion. During endovascular mechanical thrombectomy, arterial rupture occurred. The patient's vital signs were stable, but delayed angiography showed persistent active bleeding. Therefore, selective embolization of the injured artery was performed using gelfoam. Subsequent left vertebral and internal carotid angiography was performed to confirm hemostasis. A localized subarachnoid hemorrhage (SAH) was confirmed on a follow-up CT scan. A repeated CT scan after 12 d showed resolution of the SAH, and rebleeding did not occur. CONCLUSION: Rescue embolization with gelfoam could be considered an additional option in distal, medium vessel perforation.

Sphenoid sinus pseudoaneurysm with carotid cavernous fistula presenting with delayed subarachnoid hemorrhage: A case report.

RATIONALE: Sphenoid sinus pseudoaneurysm arising from the cavernous segment of the internal carotid artery (ICA) caused by traumatic vessel injury is rare, and rarer is a concomitant carotid-cavernous fistula (CCF). In particular, delayed subarachnoid hemorrhage (SAH) due to pseudoaneurysm rupture has not been reported to-date in literature. Here, we report a case of sphenoid sinus pseudoaneurysm with CCF presenting with delayed SAH. PATIENT CONCERNS: A 73-year-old man presented with traumatic brain injury due to motorcycle accident. DIAGNOSES: Twenty-four days after admission, the patient's neurological status suddenly deteriorated. Brain computed tomography (CT) showed acute SAH along interhemispheric cisterns and suprasellar intracerebral hematoma. Brain CT angiography and digital subtraction angiography revealed giant sphenoid sinus pseudoaneurysm with CCF and the daughter sac of the pseudoaneurysm extended to the intracranial part via fracture in the superior wall of the sphenoid sinus. INTERVENTIONS: As the sphenoid sinus pseudoaneurysm and CCF shared one rupture point, endovascular treatment with intraarterial approach using coil and liquid embolic material by balloon assisted technique was performed simultaneously. OUTCOMES: The origin of the pseudoaneurysmal sac and CCF was sufficiently blocked after injection of liquid embolic material and the lesions completely resolved immediately after endovascular treatment. LESSONS: Sphenoid sinus pseudoaneurysm and CCF rarely occur following head trauma through a series of processes involving fracture of the lateral wall of the sphenoid sinus and ICA cavernous segment injury. Sphenoid sinus pseudoaneurysm may present as SAH through intracranial rupture with concomitant superior wall fracture of the sphenoid sinus. Therefore, early diagnosis using CT or magnetic resonance angiography and appropriate treatment through understanding the disease mechanism is necessary.

An engineered neurovascular unit for modeling neuroinflammation.

The neurovascular unit (NVU) comprises multiple types of brain cells, including brain endothelial cells, astrocytes, pericytes, neurons, microglia, and oligodendrocytes. Each cell type contributes to the maintenance of the molecular transport barrier and brain tissue homeostasis. Several disorders and diseases of the central nervous system, including neuroinflammation, Alzheimer's disease, stroke, and multiple sclerosis, have been associated with dysfunction of the NVU. As a result, there has been increased demand for the development of NVUin vitromodels. Here, we present a three-dimensional (3D) immortalized human cell-based NVU model generated by organizing the brain microvasculature in a collagen matrix embedded with six different types of cells that comprise the NVU. By surrounding a perfusable brain endothelium with six types of NVU-composing cells, we demonstrated a significant impact of the 3D co-culture on the maturation of barrier function, which is supported by cytokines secreted from NVU-composing cells. Furthermore, NVU-composing cells alleviated the inflammatory responses induced by lipopolysaccharides. Our human cell-based NVUin vitromodel could enable elucidation of both physiological and pathological mechanisms in the human brain and evaluation of safety and efficacy in the context of high-content analysis during the process of drug development.

Assessment of Hand Motor Function in a Non-human Primate Model of Ischemic Stroke.

Ischemic stroke results from arterial occlusion and can cause irreversible brain injury. A non-human primate (NHP) model of ischemic stroke was previously developed to investigate its pathophysiology and for efficacy testing of therapeutic candidates; however, fine motor impairment remains to be well-characterized. We evaluated hand motor function in a cynomolgus monkey model of ischemic stroke. Endovascular transient middle cerebral artery occlusion (MCAO) with an angiographic microcatheter induced cerebral infarction. In vivo magnetic resonance imaging mapped and measured the ischemia-induced infarct lesion. In vivo diffusion tensor imaging (DTI) of the stroke lesion to assess the neuroplastic changes and fiber tractography demonstrated three-dimensional patterns in the corticospinal tract 12 weeks after MCAO. The hand dexterity task (HDT) was used to evaluate fine motor movement of upper extremity digits. The HDT was modified for a home cage-based training system, instead of conventional chair restraint training. The lesion was localized in the middle cerebral artery territory, including the sensorimotor cortex. Maximum infarct volume was exhibited over the first week after MCAO, which progressively inhibited ischemic core expansion, manifested by enhanced functional recovery of the affected hand over 12 weeks after MCAO. The total performance time decreased with increasing success rate for both hands on the HDT. Compensatory strategies and retrieval failure improved in the chronic phase after stroke. Our findings demonstrate the recovery of fine motor skill after stroke, and outline the behavioral characteristics and features of functional disorder of NHP stroke model, providing a basis for assessing hand motor function after stroke.

Which pathologic staining method can visualize the hyperacute infarction lesion identified by diffusion MRI?: A comparative experimental study.

BACKGROUND: The study aimed to establish a staining method that could delineate the macroscopic lesion boundary of a hyperacute infarction depicted by diffusion-weighted MRI (DWI) and to validate the infarction boundary by comparing different staining methods. NEW METHOD: Thirteen rats with 1 -h middle cerebral artery (MCA) infarction were included. Five different staining methods (Hematoxylin and eosin (H&E), Nissl, 2,3,5-triphenyltetrazolium hydrochloride (TTC), microtubule associated protein 2 (MAP2), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stains) were used to identify whether the hyperacute infarction could be histopathologically identified. Dice indices were compared to evaluate similarities in the lesion area ascertained by DWI and the staining methods. Through macroscopic lesion delineation, each region was subdivided into abnormal regions in all three stains (ROIA), abnormal in two stains (ROIB), and abnormal in only one (ROIC). Microscopic cellular changes were evaluated and graded according to each region. RESULTS: Mean Dice indices of the H&E stain were significantly higher than those of the Nissl- and MAP2-stained specimens (0.83 ±â€¯0.052, 0.58 ±â€¯0.107, and 0.56 ±â€¯0.059, respectively; p = 0.000). Grading scores for ROIs in the DWI abnormal lesions varied by region: ROIA exhibited the most severe damage [median (IQR), 3 (1)], followed respectively by ROIB [median (IQR), 2 (0)] and ROIC [median (IQR), 1 (0)] COMPARISON WITH EXISTING METHODS: H&E stain best reflects 1 h hyperacute DWI abnormal lesions. CONCLUSIONS: H&E stain allowed for the macroscopic delineation of the 1 h DWI-abnormal lesions, while MAP2 and Nissl stains could only partially depict lesions.

Structural basis of serum albumin recognition by SL335, an antibody Fab extending the serum half-life of protein therapeutics.

Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.

Schwannoma of the thyroid bed: A case report and review of the literature.

RATIONALE: Schwannomas involving the thyroid gland are very rare and only a few cases have been reported in the literature. However, previous reports did not distinguish between thyroid bed schwannomas and intrathyroidal schwannomas. Here, we report a thyroid bed schwannoma mimicking a malignant thyroid nodule and review the literature on thyroid bed schwannomas. PATIENT CONCERNS: A 33-year-old woman presented at our hospital with mild neck swelling. DIAGNOSIS: Thyroid ultrasound revealed a well-defined, oval-shaped, markedly hypoechoic solid nodule with echogenic foci suggesting macro- and microcalcifications in the left thyroid gland. The lesion was considered a "highly suspicious" intrathyroidal nodule, based on the guidelines for the assessment of thyroid nodules. Fine needle aspiration was performed twice, but the cytological results were nondiagnostic. INTERVENTIONS: Left thyroidectomy was performed, and schwannoma of the thyroid bed was confirmed on histopathologic analysis. OUTCOMES: The patient was in a stable condition after surgery, and the thyroid function test results were within the normal range. LESSONS: Diagnosis of a schwannoma of the thyroid bed is challenging because its incidence is extremely low, and it is often misdiagnosed as an intrathyroidal nodule on ultrasonography. Therefore, it is advisable to adopt a diagnostic strategy to perform additional core needle biopsy in cases of thyroid nodules with nondiagnostic fine needle aspiration results and to consider the location of the lesion more carefully to determine the suitable therapy.

Increased CD68/TGFß Co-expressing Microglia/ Macrophages after Transient Middle Cerebral Artery Occlusion in Rhesus Monkeys.

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFß) was found co-localized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206+ cells, may exert anti-inflammatory effects by secreting TGFß after the subacute stage of ischemic stroke. CD68+ microglia/macrophages can therefore be used as a potential therapeutic target.

Dichotomizing Level of Pial Collaterals on Multiphase CT Angiography for Endovascular Treatment in Acute Ischemic Stroke: Should It Be Refined for 6-Hour Time Window?

PURPOSE: Although endovascular treatment is currently thought to only be suitable for patients who have pial arterial filling scores >3 as determined by multiphase computed tomography angiography (mpCTA), a cut-off score of 3 was determined by a study, including patients within 12 hours after symptom onset. We aimed to investigate whether a cut-off score of 3 for endovascular treatment within 6 hours of symptom onset is an appropriate predictor of good functional outcome at 3 months. MATERIALS AND METHODS: From April 2015 to January 2016, acute ischemic stroke patients treated with mechanical thrombectomy within 6 hours of symptom onset were enrolled into this study. Pial arterial filling scores were semi-quantitatively assessed using mpCTA, and clinical and radiological parameters were compared between patients with favorable and unfavorable outcomes. Multivariate logistic regression analysis was then performed to investigate the independent association between clinical outcome and pial collateral score, with the predictive power of the latter assessed using C-statistics. RESULTS: Of the 38 patients enrolled, 20 (52.6%) had a favorable outcome and 18 had an unfavorable outcome, with the latter group showing a lower mean pial arterial filling score (3.6±0.8 vs. 2.4±1.2, P=0.002). After adjusting for variables with a P-value of <0.1 in univariate analysis (i.e., age and National Institutes of Health Stroke Scale score at admission), pial arterial filling scores higher than a cut-off of 2 were found to be independently associated with favorable clinical outcomes (P=0.012). C-statistic analysis confirmed that our model had the highest prediction power when pial arterial filling scores were dichotomized at >2 vs. ≤2. CONCLUSION: A pial arterial filling cut-off score of 2 as determined by mpCTA appears to be more suitable for predicting clinical outcomes following endovascular treatment within 6 hours of symptom onset than the cut-off of 3 that had been previously suggested.

Intact bioactivities and improved pharmacokinetic of the SL335-IFN-ß-1a fusion protein that created by genetic fusion of SL335, a human anti-serum albumin fab, and human interferon-ß.

Recombinant human interferon beta (rIFN-ß) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-ß is desirable since only one pegylated version of long-acting rIFN-ß-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-®, we generated a fusion construct by linking human IFN-ß cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-ß-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/ß receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-ß-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-ß-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-ß-1a is worthy of further development as an alternative long-acting IFN-ß therapeutic.

A novel voxel-wise lesion segmentation technique on 3.0-T diffusion MRI of hyperacute focal cerebral ischemia at 1 h after permanent MCAO in rats.

To assess hyperacute focal cerebral ischemia in rats on 3.0-Tesla diffusion-weighted imaging (DWI), we developed a novel voxel-wise lesion segmentation technique that overcomes intra- and inter-subject variation in apparent diffusion coefficient (ADC) distribution. Our novel technique involves the following: (1) intensity normalization including determination of the optimal type of region of interest (ROI) and its intra- and inter-subject validation, (2) verification of focal cerebral ischemic lesions at 1 h with gross and high-magnification light microscopy of hematoxylin-eosin (H&E) pathology, (3) voxel-wise segmentation on ADC with various thresholds, and (4) calculation of dice indices (DIs) to compare focal cerebral ischemic lesions at 1 h defined by ADC and matching H&E pathology. The best coefficient of variation was the mode of the left hemisphere after normalization using whole left hemispheric ROI, which showed lower intra- (2.54 ± 0.72%) and inter-subject (2.67 ± 0.70%) values than the original. Focal ischemic lesion at 1 h after middle cerebral artery occlusion (MCAO) was confirmed on both gross and microscopic H&E pathology. The 83 relative threshold of normalized ADC showed the highest mean DI (DI = 0.820 ± 0.075). We could evaluate hyperacute ischemic lesions at 1 h more reliably on 3-Tesla DWI in rat brains.

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (VH+CH1) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli.

Sustained diffusion reversal with in-bore reperfusion in monkey stroke models: Confirmed by prospective magnetic resonance imaging.

Although early diffusion lesion reversal after recanalization treatment of acute ischaemic stroke has been observed in clinical settings, the reversibility of lesions observed by diffusion-weighted imaging remains controversial. Here, we present consistent observations of sustained diffusion lesion reversal after transient middle cerebral artery occlusion in a monkey stroke model. Seven rhesus macaques were subjected to endovascular transient middle cerebral artery occlusion with in-bore reperfusion confirmed by repeated prospective diffusion-weighted imaging. Early diffusion lesion reversal was defined as lesion reversal at 3 h after reperfusion. Sustained diffusion lesion reversal was defined as the difference between the ADC-derived pre-reperfusion maximal ischemic lesion volume (ADCD-P Match) and the lesion on 4-week follow-up FLAIR magnetic resonance imaging. Diffusion lesions were spatiotemporally assessed using a 3-D voxel-based quantitative technique. The ADCD-P Match was 9.7 ± 6.0% (mean ± SD) and the final infarct was 1.2-6.0% of the volume of the ipsilateral hemisphere. Early diffusion lesion reversal and sustained diffusion lesion reversal were observed in all seven animals, and the calculated percentages compared with their ADCD-P Match ranged from 8.3 to 51.9% (mean ± SD, 26.9 ± 15.3%) and 41.7-77.8% (mean ± SD, 65.4 ± 12.2%), respectively. Substantial sustained diffusion lesion reversal and early reversal were observed in all animals in this monkey model of transient focal cerebral ischaemia.