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.

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.

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.

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.

[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.

[Stage IV Gastric Cancer with Positive Peritoneal Washing Cytology or Peritoneal Dissemination Was Successfully Treated with Gastrectomy and Hyperthermic Intraperitoneal Chemotherapy(HIPEC)Followed by Systemic Chemotherapy - A Report of Two Cases].

Survival of Stage IV gastric cancer is poor. We report 2 cases of Stage IV gastric cancer with positive peritoneal washing cytology or peritoneal dissemination that were successfully treated with gastrectomy and hyperthermic intraperitoneal chemotherapy( HIPEC)followed by systemic chemotherapy. Case 1: A 59-year-old woman. She was diagnosed with advanced gastric cancer and underwent gastrectomy with HIPEC. Her peritoneal washing cytology was positive during the gastrectomy. After the surgery, she underwent chemotherapy consisting of 8 courses of combination S-1 plus CPT-11 and 19 courses of PTX. It has been 5 years and 7 months since she had the surgery and she survives without recurrence of the cancer. Case 2: A 60-year-old woman. She was diagnosed with advanced gastric cancer and peritoneal dissemination(peritoneal cancer index: 3 points). She underwent gastrectomy, hemi-colectomy, and HIPEC. After the surgery, she underwent chemotherapy, 35 courses of combination S-1 plus PSK/DOC, and 13 courses of S-1 plus PSK. It has been 5 years since her surgery and she survives without exacerbation of the cancer. These cases suggest a gastrectomy and HIPEC followed by systemic chemotherapy may represent an effective treatment for advanced gastric cancer with a small amount of peritoneal metastasis.

[Salvage Surgery after CRT for Advanced Esophageal Cancer Resulting in a Pathological Complete Response and More Than Five Years' Survival].

A 62-year-old woman visited our hospital because of dysphagia. She was diagnosed with upper-middle esophageal type 4 cancer, which was 9 cm in length, according to the results of endoscopy. Squamous cell carcinoma was demonstrated using endoscopic biopsy. A CT scan revealed that the tumor had directly invaded into the trachea(cT4). Chemoradiotherapy(CRT) (5-FU and CDDP with 50 Gy of radiation)was administered. Although CT after CRT resulted in shrinkage of the tumor and no further tracheal invasion, esophageal stenosis remained. Therefore, salvage surgery(subtotal esophagectomy with 3-field lymph node dissection)was performed. Pathologically, no carcinoma cells were found in the resected specimen and a com- plete response(grade 3)was diagnosed. The patient received adjuvant chemotherapy(tegafur/uracil at 300mg/day per os) for 1 year. The patient is alive with no relapse of carcinoma more than 5 years after the first treatment.

[A Case of Breast Metastasis of Eccrine Porocarcinoma].

An 80's woman was diagnosed with eccrine porocarcinoma of the head in 2010.T he tumor was removed surgically but relapsed in the cervical and axillary lymph nodes 2 years later.The patient underwent surgery, and received systemic chemotherapy and radiation.Chest CT after treatment revealed an irregular mass and thickened skin in the left breast.Core needle biopsy specimens were used to diagnose metastasis of eccrine porocarcinoma.A wide excision with a 1 cm margin was performed under local anesthesia.After surgery, supraclavicular lymph node recurrence was detected.The patient received palliative care because there was no effective treatment available.Eccrine porocarcinoma is a rare malignant tumor of the intraepidermal sweat duct.Breast metastasis from malignant disease is also rare.To our knowledge, breast metastasis of eccrine porocarcinoma has not been reported.

[Surgery for Multiple Gastrointestinal Stromal Tumors of the Small Intestine in Patients with Neurofibromatosis Type 1 - A Report of Three Cases].

We report 3 cases of multiple GIST of the small intestine in 3 patients with NF1 who have been followed for over 5 years. All patients presented with melena, and tumors of the small intestine suspected to be GIST were found on endoscopy. We performed partial resections of the small intestine for all 3 patients. After surgery, 1 patient had residual tumors that gradually enlarged during 8 year 2 months and another had residual tumors that have been stable for 8 years. In the third patient, we resected all the tumors, and there has been no sign of recurrence in 6 year 1 month.

Umbilical metastasis derived from breast cancer: report of a case.

Umbilical metastases mainly arise from malignancies of the digestive and gynecological systems, but rarely from breast cancer. A 64-year-old woman with a history of breast cancer was referred to us for investigation of a painful lesion in the umbilicus. Immunohistochemical staining of a specimen obtained by biopsy from the nodule showed umbilical metastasis of breast cancer. After a work up, she was successfully treated with a combination of surgery and endocrine therapy. We report this case to reinforce that not all periumbilical tumoral deposits are consistent.

[Evaluation of breath alcohol concentration after the administration of alcohol-based docetaxel].

In the present study, we measured breath alcohol concentration(BAC)after the administration of alcohol-containing docetaxel(OTX)in breast cancer patients, and examined the safety of OTX outpatient administration. Twenty breast cancer patients who received OTX chemotherapy at our outpatient facility were included. The administered doses were 100mg/m2 in 5 cases, 75mg/m / 2 in 13 cases, and 60 mg/m2 in 2 cases. BAC was measured 3 times: immediately after infusion, 30 minutes after infusion, and 60 minutes after infusion. No symptoms of hot flash or drunkenness due to alcohol were observed. BAC was detected in 10 cases(50%)immediately after infusion, in 7 cases(35%)at 30 minutes after infusion, and in 1 case(5%) at 60 minutes after infusion. BAC was more than 0.15mg/L in only 1 case(5%)and reduced to less than 0.15mg/L in all cases after 30 minutes. Our results suggest that the effects of alcohol are alleviated 60 minutes after infusion and that patients receiving OTX could return home safely.

[A case of breast cancer with repeated cardiac dysfunction due to trastuzumab].

Here we present a case of breast cancer in which cardiac dysfunction had previously been observed on trastuzumab(TRS) administration; the condition then improved but reoccurred on readministration of TRS. A 52-year-old woman received preoperative chemotherapy for StageIIIC left breast cancer(fluorouracil, epirubicin and cyclophosphamide followed by docetaxel and TRS), and then underwent partial mastectomy and axillary lymph node dissection. For adjuvant therapy, she received endocrine therapy and TRS. Radiation therapy was administered to the left residual breast. The patient complained about palpitation in the 5th cycle of TRS, and left ventricle ejection fraction(LVEF)decreased to 45.3% from 64%. Therefore, we stopped TRS administration. Palpitation improved, and LVEF increased to 53% after 2 months. TRS was administered again; however, palpitation reoccurred and LVEF decreased to 44%. TRS administration was once again discontinued. However, according to the HERA trial report regarding patients with a history of anthracycline and radiation therapy, TRS administration could be resumed when LVEF is greater than 50%, but we should be more careful during readministration of TRS.

The complete mitochondrial genome of a Biwa goby, Gymnogobius isaza (Tanaka, 1916).

We determined the complete mitochondrial DNA sequence of a Biwa goby, Gymnogobius isaza (Tanaka, 1916) using next-generation sequencing methods. The composition of its mitogenome is the same as that observed in most other vertebrates, comprising of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and two control regions. Our molecular phylogenetic analysis confirmed the close phylogenetic relationship between G. isaza and G. petschiliensis. This mitogenome information will be useful for distribution surveys using environmental DNA and the development of conservation strategies for this species.

Feasibility and toxicity of docetaxel before or after fluorouracil, epirubicin and cyclophosphamide as adjuvant chemotherapy for early breast cancer.

BACKGROUND: The tolerance and safety associated with the administration order of the anthracycline and taxane drugs have not been evaluated. PATIENTS AND METHODS: Breast cancer patients with node-positive or high-risk patients with node-negative were eligible. The feasibility and toxicity were evaluated in the following regimens--arm A, 3 courses of fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2) and cyclophosphamide 500 mg/m(2) (FEC) followed by 3 courses of docetaxel 100 mg/m(2) (DOC); arm B, 3 courses of DOC followed by 3 courses of FEC. RESULTS: Forty-two patients were registered. The relative dose intensity was 94.2 % for FEC and 97.8 % for DOC in arm A, and 98.9 % for DOC and 95.2 % for FEC in arm B. In arm A, grade 3 or higher hematological toxicity was observed in nine patients, and febrile neutropenia developed in three patients with FEC. In arm B, grade 3 or higher hematological toxicity was observed in seven patients, but febrile neutropenia was not noted in any patient. CONCLUSION: The regimens in both arms A and B were safe regarding adjuvant chemotherapy for early breast cancer. However, DOC followed by FEC might be more tolerable. Further studies will maximize the results obtained with DOC followed by FEC.

Intraductally administered pegylated liposomal doxorubicin reduces mammary stem cell function in the mammary gland but in the long term, induces malignant tumors.

Previously, we have shown that the intraductal (i.duc) administration of pegylated liposomal doxorubicin (PLD) to Her2/neu transgenic mice is associated with mammary tumor regression and prevention. Exploring the mechanism underlying the protection afforded by PLD, we studied: the effects of i.duc PLD-treatment with a subsequent pregnancy on outgrowth of tumors in Her2/neu mice; whether the i.duc PLD antitumor effect was mediated partially through changes in normal mammary stem cells (MaSCs); and the long-term safety of i.duc PLD into the normal mouse mammary gland. Her2/neu mice were treated with two i.duc injections of PLD given four weeks apart; pregnancy was induced and mice were followed up for changes in physiology, and tumor formation. We found that all pups born to i.duc PLD-treated Her2/neu mice died without weight gain within 7 days after birth. Despite an additional pregnancy, compared to vehicle control PLD-treated Her2/neu mice had a significantly longer latency and lower frequency of tumor development. Mammary epithelial cells isolated from untreated and i.duc PLD-treated 6-8 months-old multiparous FVB/N mice were analyzed for their repopulating ability in mammary fat pads of naïve recipients. Mice were also monitored for abnormalities in mammary gland morphology and function, including tumor formation. PLD-treated FVB/N mice displayed histomorphologic changes and a significant reduction in the outgrowth potential of cells from the mammary glands. Thus, i.duc PLD administration altered the mammary gland structurally and functionally by reducing the MaSC population, which could compromise milk production. Followed long term, i.duc PLD-treated FVB/N mice developed malignant mammary tumors, confirming similar published findings on doxorubicin injected into the mammary gland of rats. Unless there are fundamental species differences in PLD metabolism in rodents and humans, this finding seriously limits the consideration of i.duc PLD use in the clinic for treatment or prevention of breast cancer.

Orally administered prion protein is incorporated by m cells and spreads into lymphoid tissues with macrophages in prion protein knockout mice.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer's patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer's patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer's patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer's patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer's patches to other lymphoid tissues and the peripheral nervous system.

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.