Pentosan polysulfate induces low-level persistent prion infection keeping measurable seeding activity without PrP-res detection in Fukuoka-1 infected cell cultures.

Each prion strain has its own characteristics and the efficacy of anti-prion drugs varies. Screening of prion disease therapeutics is typically evaluated by measuring amounts of protease-resistant prion protein (PrP-res). However, it remains unclear whether such measurements correlate with seeding activity, which is evaluated by real-time quaking-induced conversion (RT-QuIC). In this study, the effects of anti-prion compounds pentosan polysulfate (PPS), Congo red, and alprenolol were measured in N2a58 cells infected with Fukuoka-1 (FK1) or 22L strain. The compounds abolished PrP-res and seeding activity, except for N2a58/FK1 treated with PPS. Interestingly, the seeding activity of N2a58/FK1, which was reduced in the presence of PPS, was not lost and remained at low levels. However, upon removal of PPS, both were gradually restored to their original levels. These results indicate that low-level persistent prion infection keeping measurable seeding activity is induced by PPS in a strain-dependent manner. Furthermore, for protein misfolding cyclic amplification (PMCA), the anti-prion effect of PPS decreased in FK1 compared to 22L, suggesting that the differences occur at the level of the direct conversion. Our findings demonstrate that the advantages of RT-QuIC and PMCA can be exploited for more accurate assessment of therapeutic drug screening, reflecting strain differences.

Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system.

Prion diseases are transmissible and progressive neurodegenerative disorders characterized by abnormal prion protein (PrPSc) accumulation in the central nervous system. Generation of synthetic PrPSc in a cell-free conversion system and examination of its transmissibility to animals would facilitate testing of the protein-only hypothesis and the understanding of the molecular basis of sporadic prion diseases. In this study, we used recombinant prion protein from a baculovirus-insect cell expression system (Bac-rPrP) and insect cell-derived cofactors to determine whether Bac-rPrPSc is spontaneously produced in intermittent ultrasonic reactions. No spontaneous generation of Bac-rPrPSc was observed at 37 °C, but when the reaction temperature was increased to 45 °C, Bac-rPrPSc was generated in all trials. Some Bac-rPrPSc variants were transmissible to mice, but when the reaction was repeated for 40 rounds, the transmissibility was lost. Notably, a variety of Bac-rPrPSc variants, including non-transmissible ones, differing in resistance to proteinase K and cofactor dependence during amplification, was generated under the same experimental conditions, including the same sonication settings and cofactors. However, their characteristics also disappeared after 40 reaction rounds and the variety converged onto a single variant. These results indicate that various Bac-rPrPSc variants with different transmissibility to mice and structural properties are generated, which compete with each other and gradually converge onto a variant with a slightly faster amplification rate.

The complete mitochondrial genome of a bagrid catfish, Tachysurus nudiceps, and its phylogenetic implications for the classification of the bagrid genera.

The complete sequence of the mitochondrial genome (mitogenome) of Tachysurus nudiceps (family Bagridae; order Siluriformes) was determined using next-generation sequencing. The composition of its mitogenome is the same as that observed in most other vertebrates and consists of 37 genes, an L-strand replication origin and a control region. As in previous studies, our phylogenetic analyses revealed that many of the bagrid genera are not monophyletic, emphasizing the necessity for reviewing and revising the taxonomy of this family at the genus level.

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in Arabidopsis thaliana.

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks.

Suppression of MYC transcription activators by the immune cofactor NPR1 fine-tunes plant immune responses.

Plants tailor immune responses to defend against pathogens with different lifestyles. In this process, antagonism between the immune hormones salicylic acid (SA) and jasmonic acid (JA) optimizes transcriptional signatures specifically to the attacker encountered. Antagonism is controlled by the transcription cofactor NPR1. The indispensable role of NPR1 in activating SA-responsive genes is well understood, but how it functions as a repressor of JA-responsive genes remains unclear. Here, we demonstrate that SA-induced NPR1 is recruited to JA-responsive promoter regions that are co-occupied by a JA-induced transcription complex consisting of the MYC2 activator and MED25 Mediator subunit. In the presence of SA, NPR1 physically associates with JA-induced MYC2 and inhibits transcriptional activation by disrupting its interaction with MED25. Importantly, NPR1-mediated inhibition of MYC2 is a major immune mechanism for suppressing pathogen virulence. Thus, NPR1 orchestrates the immune transcriptome not only by activating SA-responsive genes but also by acting as a corepressor of JA-responsive MYC2.

Ethanolamine Is a New Anti-Prion Compound.

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.

[Primary Pulmonary Amyloidosis with a Localized Consolidation:Report of Two Cases].

We experienced two cases of primary pulmonary amyloidosis with a localized consolidation. Case 1 is a 80-year-old man, who was found to have an abnormal chest nodular shadow with blurred margin at a medical examination. Chest computed tomography( CT) showed a localized consolidation on the periphery of the upper lobe of the right lung. A CT-guided biopsy was performed. Case 2 is a 66-year-old woman, who was found to have an abnormal chest opacity at a medical examination. Chest CT showed a localized gathering of small nodules in the right lower lobe. Gradual enlargement was noted by follow up CT and the accumulation of fluorodeoxyglucose (FDG) was shown by PET/CT. In consideration of primary lung cancer or malignant lymphoma, right lower lobectomy was performed. Both cases were pathologically diagnosed as pulmonary amyloidosis. Since no findings of amyloid deposits in other organs or of existence of any blood disorders, a diagnosis of primary pulmonary amyloidosis was made.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice.

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

[Elucidation of the Molecular Basis of Abnormal Prion Protein (PrP) Formation in a Cell-Free System Using Baculovirus and Insect Cell-derived Recombinant PrP].

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.

Infiltration of CD4, CD8, CD56, and Fox-P3-positive lymphocytes in breast carcinoma tissue after neoadjuvant chemotherapy with or without trastuzumab.

BACKGROUND: Trastuzumab (Tz) is assumed to prime antibody-dependent cellular cytotoxicity (ADCC); however, it remains unclear whether Tz therapy can clinically induce adaptive cellular immunity. OBJECTIVE: Adaptive Cellular Immune Effect of Tz Therapy. METHODS: This study included 29 surgical invasive breast carcinomas administered neoadjuvant chemotherapy with Tz (15 cases) or without Tz (14 cases). The numbers of immunoreactive cells (CD4, CD8, CD56, and Fox-P3) in three different compartments (intratumoral, adjacent stromal, and distant stromal) were determined. RESULTS: The average number of adjacent stromal CD4-positive, CD8-positive, and Fox-P3-positive cells in the Tz+ group was significantly greater than that in the Tz- group (p = 0.036, 0.0049, and 0.043, respectively). However, the number of Fox-P3-positive cells was much less than that of CD4-positive cells. Moreover, distant stromal CD4-positive and CD8-positive cells in the Tz+ group was also significantly greater than that of the Tz- group (p = 0.029 and 0.032, respectively). Only a small number of CD56-positive natural killer cells, playing a main role in ADCC, accumulated at the tumor site after Tz therapy. CONCLUSIONS: The results suggest that Tz therapy induces adaptive cellular immunity, including infiltration of both CD4-positive helper T cells and CD8-positive cytotoxic T cells into the breast carcinoma lesion.

Type I interferon protects neurons from prions in in vivo models.

Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

Progression Potential of Ductal Carcinoma in situ Assessed by Genomic Copy Number Profiling.

BACKGROUND: Ductal carcinoma in situ (DCIS) of the breast is heterogeneous in terms of the risk of progression to invasive ductal carcinoma (IDC). To treat DCIS appropriately for its progression risk, we classified individual DCIS by its profile of genomic changes into 2 groups and correlated them with clinicopathological progression factors. METHODS: We used surgically resected, formalin-fixed, paraffin-embedded tissues of 22 DCIS and 30 IDC lesions. We performed immunohistochemical intrinsic subtyping, array-based comparative genomic hybridization, and unsupervised clustering. RESULTS: The samples were divided into 2 major clusters, A and B. Cluster A showed a greater number of gene and chromosome copy number alterations, a larger IDC/DCIS ratio, a higher frequency of nonluminal subtype, a lower frequency of luminal subtype, and a higher nuclear grade, when compared with cluster B. However, there was no difference in the frequencies of lymph node metastasis between clusters A and B. We identified 9 breast-cancer-related genes, including TP53 and GATA3, that highly contributed to the discrimination of A and B clusters. CONCLUSION: Classification of breast tumors into rapidly progressive cluster A and the other (cluster B) may contribute to select the treatment appropriate for their progression risk.

[Two Cases of Unresectable/Advanced Breast Cancer with Pathological Complete Response after Combination Chemotherapy with Pertuzumab. Trastuzumab, and Docetaxel].

Combination chemotherapy with pertuzumab, trastuzumab, and docetaxel is recommended as the first-line treatment for patients with HER2-positive unresectable or metastatic breast cancer. We report 2 cases of unresectable breast cancer for which pertuzumab, trastuzumab, and docetaxel therapy was effective. Case 1: A woman in her 40s was diagnosed with TxN3aM0, Stage ⅢC, HER2-positive, hormone receptor-positive advanced breast cancer. After administration of 6 courses of pertuzumab, trastuzumab, and docetaxel therapy, she underwent surgery(Bt+Ax[Ⅱ]). Histopathological examination revealed that chemotherapy effect was Grade 3. Case 2: A woman in her 60s was diagnosed with de novo Stage Ⅳ, HER2- positive, hormone receptor-negative breast cancer. She was administered 8 courses of pertuzumab, trastuzumab, and docetaxel therapy as the third-line treatment, because she initially refused treatment. Thereafter, she underwent surgery(Bt+Ax [Ⅰ]). In both cases, histopathological examination revealed complete response after chemotherapy. Thus, combination therapy of pertuzumab and trastuzumab may improve the prognosis in patients with HER2-positive breast cancer.

Age of donor of human mesenchymal stem cells affects structural and functional recovery after cell therapy following ischaemic stroke.

Cell transplantation therapy offers great potential to improve impairments after stroke. However, the importance of donor age on therapeutic efficacy is unclear. We investigated the regenerative capacity of transplanted cells focusing on donor age (young vs. old) for ischaemic stroke. The quantities of human mesenchymal stem cell (hMSC) secreted brain-derived neurotrophic factor in vitro and of monocyte chemotactic protein-1 at day 7 in vivo were both significantly higher for young hMSC compared with old hMSC. Male Sprague-Dawley rats subjected to transient middle cerebral artery occlusion that received young hMSC (trans-arterially at 24 h after stroke) showed better behavioural recovery with prevention of brain atrophy compared with rats that received old hMSC. Histological analysis of the peri-infarct cortex showed that rats treated with young hMSC had significantly fewer microglia and more vessels covered with pericytes. Interestingly, migration of neural stem/progenitor cells expressing Musashi-1 positively correlated with astrocyte process alignment, which was more pronounced for young hMSC. Aging of hMSC may be a critical factor that affects cell therapy outcomes, and transplantation of young hMSC appears to provide better functional recovery through anti-inflammatory effects, vessel maturation, and neurogenesis potentially by the dominance of trophic factor secretion.

[A Case of Small Intestinal GIST with Long-Term Survival after Tumor Resection for Repeated Peritoneal Recurrence].

A 70-year-old woman presenting with abdominal pain was admitted to our hospital. Abdominal contrast CT revealed a small intestine tumor of 10 cm with active bleeding and performed partial resection of the small intestine including tumor. Pathological findings were high risk GIST of the small intestine because of spindle cells and c-kit positive. Imatinib 400mg/day as adjuvant chemotherapy was administered. However administration was stopped for 15 days because of the Grade 4 erythema multiforme. Recurrence of peritoneal dissemination was observed in 2 years after surgery and tumor resection was performed, but complete resection was difficult. Within 5 years after surgery, tumor resection was performed on a total of 5 times peritoneal disseminative recurrences, and it was possible to avoid the appearance of symptoms due to tumor augmentation.

Prion-Seeding Activity Is widely Distributed in Tissues of Sporadic Creutzfeldt-Jakob Disease Patients.

Human prion diseases are neurodegenerative disorders caused by abnormally folded prion proteins in the central nervous system. These proteins can be detected using the quaking-induced conversion assay. Compared with other bioassays, this assay is extremely sensitive and was used in the present study to determine prion distribution in sporadic Creutzfeldt-Jakob disease patients at autopsy. Although infectivity of the sporadic form is thought to be restricted within the central nervous system, results showed that prion-seeding activities reach 106/g from a 50% seeding dose in non-neuronal tissues, suggesting that prion-seeding activity exists in non-neural organs, and we suggested that non-neural tissues of 106/g SD50 did not exist the infectivity.

Sequential Washing with Electrolyzed Alkaline and Acidic Water Effectively Removes Pathogens from Metal Surfaces.

Removal of pathogenic organisms from reprocessed surgical instruments is essential to prevent iatrogenic infections. Some bacteria can make persistent biofilms on medical devices. Contamination of non-disposable equipment with prions also represents a serious risk to surgical patients. Efficient disinfection of prions from endoscopes and other instruments such as high-resolution cameras remains problematic because these instruments do not tolerate aggressive chemical or heat treatments. Herein, we develop a new washing system that uses both the alkaline and acidic water produced by electrolysis. Electrolyzed acidic water, containing HCl and HOCl as active substances, has been reported to be an effective disinfectant. A 0.15% NaCl solution was electrolyzed and used immediately to wash bio-contaminated stainless steel model systems with alkaline water (pH 11.9) with sonication, and then with acidic water (pH 2.7) without sonication. Two bacterial species (Staphylococcus aureus and Pseudomonas aeruginosa) and a fungus (Candida albicans) were effectively removed or inactivated by the washing process. In addition, this process effectively removed or inactivated prions from the stainless steel surfaces. This washing system will be potentially useful for the disinfection of clinical devices such as neuroendoscopes because electrolyzed water is gentle to both patients and equipment and is environmentally sound.

A direct assessment of human prion adhered to steel wire using real-time quaking-induced conversion.

Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt-Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 10(2.8) to 10(5.8) SD50. Using this method, we also confirmed that the seeding activities on the wire were removed following treatment with NaOH. As seeding activity closely correlated with the infectivity of prions using the bioassay, this wire-QuIC assay will be useful for the direct evaluation of decontamination methods for human prions.