Group A Streptococcus Subcutaneous Infection-Induced Central Nervous System Inflammation Is Attenuated by Blocking Peripheral TNF

Group A streptococcus (GAS) infection causes a strong inflammatory response associated with cytokine storms, leading to multiorgan failure, which is characterized as streptococcal toxic shock syndrome. However, little is known about GAS subcutaneous infection-mediated brain inflammation. Therefore, we used a bioluminescent GAS strain and reporter mice carrying firefly luciferase under transcriptional control of the nuclear factor-kappa B (NF-κB) promoter to concurrently monitor the host immune response and bacterial burden in a single mouse. Notably, in addition to the subcutaneous inoculation locus at the back of mice, we detected strong luminescence signals from NF-κB activation and increased inflammatory cytokine production in the brain, implying the existence of central nervous system inflammation after GAS subcutaneous infection. The inflamed brain exhibited an increased expression of glial fibrillary acidic protein and nicotinamide adenine dinucleotide phosphate oxidase components and greater microglial activation and blood–brain barrier (BBB) disruption. Furthermore, Fluoro-Jade C positive cells increased in the brain, indicating that neurons underwent degeneration. Peripheral tumor necrosis factor (TNF), which contributes to pathology in brain injury, was elevated in the circulation, and the expression of its receptor was also increased in the inflamed brain. Blockage of peripheral TNF effectively reduced brain inflammation and injury, thereby preventing BBB disruption and improving survival. Our study provides new insights into GAS-induced central nervous system inflammation, such as encephalopathy, which can be attenuated by circulating TNF blockage

Optimization of the in situ detection of viral protein and nucleic acid in tissue: mouse hepatitis virus and canine distemper virus

鼠肝炎病毒（mouse hepatitis virus；MHV）是小鼠族群高度傳染性首要病原。 本研究在缺乏特異性冠狀病毒初級抗體情況下，利用血清酵素免疫吸附法（ELISA）鼠肝炎病毒陽性檢體血清，結合卵白素(avidin)與生物素化 (biotinylated)的二級抗體，在發病免疫缺陷鼠之福馬林固定組織，如肝、胃、脾臟、 盲腸、及結腸等偵測鼠肝炎病毒抗原存在(Chapter II)。犬瘟熱病毒（canine distemper virus, CDV）為一年代久遠的疾病，本病之發生，己遍及全世界。犬瘟熱病毒為一高傳染性疾病，且存在臺灣許多年。然而；犬瘟熱病毒分離，基因型卻不曾有報告。組織病變，免疫化學染色診斷，病理學分析及併發症等均缺乏全面性調查資料。本研究目的為建立快速及正確的犬瘟熱病毒感染診斷方法，分離犬瘟熱病毒株及分析其H基因之遺傳差異，及經免疫化學染色(IHC)或反轉錄聚合酶反應(RT-PCR)確診下之自發病例，分析其中樞神經脫髓鞘發生率及其它病理變化。研究發現經非生物素性聚合山葵過氧化酶 (non-biotin HRP)免疫化學染色方法，對福馬林固定、石蠟包埋組織之犬瘟熱感染病例，其診斷率可達87.5%。犬瘟熱病毒特異性抗原可在下列組織發現：大腦皮層外錐狀細胞及樹狀突細胞質、小腦白質層星狀細胞及細胞質、軟腦膜、神經膠質細胞、神經元、血管內皮細胞、腦室周圍、室管膜細胞、脈胳叢；脊髓灰、白質及中央導水管；腎盂上皮及腎小管上皮細胞；膀胱黏膜上皮細胞；脾臟白髓及淋巴結巨噬細胞及淋巴球；皮膚海綿層細胞及細胞質；肺臟小支氣管黏膜上皮及肺泡巨噬細胞；肝臟巨噬細胞；胃腸黏膜上皮細胞；扁桃腺及食道複層扁平上皮細胞等。組織經高溫高壓滅菌鍋前處理的免疫化學染色方法，其抗原復舊程度遠遠超過傳統微波爐法，大大提高犬瘟熱病毒診斷率(Chapter III,VI)。為了要確認免疫化學染色訊號，本研究建立一個改良的原位雜交染色步驟，應用於上述病例。此方法除了傳統的蛋白水解酶K的雜交前處理外；另外比較了將切片置入於不同(Trilogy, TBS S3006, H3301, S1700)溶液中進行高溫高壓滅菌鍋前處理。同時原位雜交訊號使用平均光密度(IOD)及組織形態完整性進行半定量性效果分析。結果發現進行原位雜交前，切片如果配合蛋白質水解酶K先處理，再以Trilogy溶液高溫高壓處理後進行原位雜交，其訊號及組織完整性均較其它組強，此一方法建立，未來將廣泛應用於犬瘟熱病例或其它病毒性疾病之原位雜交診斷(Chapter IV)。為了解台灣地區犬瘟熱病毒基因型分析，2003-2005年間進行病毒分離，自17隻未經疫苗注射之發病幼犬，應用患犬血液單核球與 B95a 細胞株共同培養之技術，分離出兩株具誘發融合細胞病變之病毒，經免疫螢光染色及抗原測試確認為犬瘟熱病毒。將此兩株病毒之血球凝集素基因（H），與同期間自台灣大學動物醫院臨床送檢病例之另外4株犬瘟熱病毒之H基因進行核酸定序。經比對序列與樹狀圖分析發現，本土病毒株皆有9個N連結配醣位，其中第7個配醣位為日本或中國大陸流行之亞洲1型犬瘟熱病毒所特有。本研究顯示臺灣地區所流行之犬瘟熱病毒，其H基因經分析屬亞洲1型 (Chapter V)。為了解台灣地區是否有犬瘟熱病毒感染之病理變化差異，2000-2009年分析來自收容所或臨床動物醫院之犬瘟熱患犬計52隻(經IHC或RT-PCR確診)。診斷方法包括肉眼檢查、組織病理、脫髓鞘特殊染色(LFB-CEV)、IHC、RT-PCR等。結果發現32隻來自臨床動物醫院，20隻來自收容所。來自臨床動物醫院之犬，75%為6月齡或以下，但來自收容所之犬，85%為大於6月齡。共計11種品種，但67% 為混種犬。在病理學分析，淋巴減少(79%)、間質性肺炎(71%)、中樞神經脫髓鞘(65%)、卡他性腸炎(32%)為主要病變。臨床動物醫院組相對地在淋巴減少(96%)、病毒包涵體(87%)、間質性肺炎(81%)及中樞神經脫髓鞘(81%)發生率很高，且統計差異顯著。腸炎占二組別之30-34%，無統計差異顯著。犬瘟熱病毒病毒包涵体在膀胱、淋巴組織、肺臟及消化系統，二組別有顯著差異。在29種犬瘟熱病毒感染之併發症或協同病變分析中，除了間質性腎炎外，二組間皆無顯著性統計差異。總結而言，淋巴減少、肺炎、中樞神經脫髓鞘是主要病變，然而病毒包涵體出現却以淋巴組織及黏膜上皮如腎臟、膀胱、肺臟及消化系統較高(ChapterIII,VI)。綜合以上結果，本研究證明台灣犬瘟熱病毒至少存在一種以上H基因型(亞洲1型)，其彼此間的胺基酸差異小於5%。同時本研究所建立的免疫化學染色及原位雜交染色方法使回溯性犬瘟熱病毒及鼠肝炎病毒之研究及診斷更簡單及正確。犬瘟熱病毒分離之方法可應用於未來臺灣不同地區之病毒分離。犬瘟熱病毒感染之伴隨病變視動物來源、治療病史、年齡及組織分布而定。Mouse hepatitis virus (MHV) is the leading viral pathogen of laboratory mice in Taiwan. This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method for detection of MHV in tissues. Mouse hepatitis virus antigen was clearly detected in samples of liver, stomach, caecal and colonic mucosa, and spleen. This method may prove useful in diagnosis when commercial antisera are unavailable or when immunosuppression prevents serological diagnosis. (Chapter II). Canine distemper virus (CDV) causes a highly contagious disease, which has been reported in Taiwan for many years; however the causative and its genes have never been identified and pathogenesis are poorly understood. The objectives of the dissertation were to set up a fast and easy diagnostic method of CDV infection, to isolate the field virus and do phylogenetic analysis of the viral H gene, to characterize the pathology of CD in Taiwan, and to assess the frequency of CNS demyelination and other pathological lesions in cases of CDV infection confirmed by immunohistochemistry (IHC) and/or reverse transcription polymerase chain reaction (RT-PCR). This study describes a modified non-biotin polymerized horseradish peroxidase (HRP) immunohistochemical method for the diagnosis of CDV infection from formalin-fixed, paraffin-embedded tissues. This method confirmed seven out of eight (87.5%) suspected cases. Labeled CDV antigen was observed in cerebrum, cerebellum, meninges, glial cells, neurons, vascular endothelium, periventricular areas and pericytes, and choroid plexus; grey and white matter and central canal of the spinal cord; renal pelvis and tubular epithelium, and urinary bladder epithelium; macrophages and lymphocytes in splenic white pulp and lymph nodes; skin epidermis; bronchiolar epithelium and alveolar macrophages; hepatic Kupffer cells, and gastric and intestinal mucosal epithelium; stratified squamous epithelium of the tongue and oesophagus.With the non-biotin HRP detection system, pretreatment by autoclaving followed by microwave heating gave better labeling results than did microwave pretreatment alone. No obvious difference was noted between the labeling results produced by the non-biotin HRP detection system and the Super Sensitive TM Link-Label IHC detection system (Chapter III, VI). For the purpose of confirming the IHC labeling and improving the detection of CDV nucleoprotein RNA, in situ hybridization (ISH) was applied in paraffin-embedded tissues from selected dogs with spontaneous CDV infections. In addition to proteinase K, autoclaving in various solutions (Trilogy, TBS S3006, H-3301, and S1700) for pre-treatments were compared. The intensity was assessed by using the integrated optical density (IOD) and the integrity of the tissue morphology. The combination of proteinase K digestion and autoclaving in a Trilogy solution resulted in a 5- to 100-fold ISH signal enhancement of CDV RNA. This modified technique can be useful in the retrospective viral studies across a broad range in the future (Chapter IV). For the purpose of CDV genotyping, during the period from 2003 to 2005, two CDV strains were isolated from 17 non-vaccinated puppies with suspected canine distemper by co-culture with peripheral blood mononuclear leucocytes and B95a cells. In addition, four cloned hemagglutinin (H) genes were obtained from 166 dogs infected with CDV. Indirect immunofluorescence assays and antigen tests confirmed that they were CDV. Analysis of the H genes of the six identified strains revealed that the deduced amino acid sequences contained nine potential sites for N-linked glycosylation, as had been found for the H proteins of Japanese isolates. The seventh site is characteristic to the Taiwan strains described in this report and to the recently reported Japanese strains. Furthermore, phylogenetic analysis of the H gene showed that the six isolates belong to the Asia-1 group and are closely related to the recently reported Japanese and Chinese strains (Chapter V). To realize the histopathological lesions of CDV in Taiwan, fifty two (IHC or RT-PCR positive) affected dogs were obtained from either animal clinics or dog shelters from 2000 to 2009, within which 32 were from clinics and 20 were from shelters. Postmortem and laboratory examination included, gross findings, histopathology, Luxol-fast blue cresyl echt violet (LFB-CEV) histochemistry, non-biotin HRP anti-CDV immunohistochemistry, and phosphoprotein gene RT-PCR. Clinic cases had histories of treatment and or vaccination. Twenty four clinic cases (75%) were puppies less than 6 months old. Seventeen shelter cases (85%) were identified as ‘adults’ greater than 6 months old. There were 27 males and 25 females. Eleven dog breeds were represented, but most dogs (35/52, 67%) were crossbred. Totally, 79% (41/52) showed lymphoid depletion, 71% (37/52) had interstitial pneumonia, 65% (34/52) had CNS demyelination, 32% (17/52) had catarrhal enteritis. Younger dogs from clinic group frequently had lymphoid depletion (31/32, 96%), inclusion bodies (28/32, 87%), pneumonia 81% (26/32, 81%), and CNS demyelination (26/32, 81%), which were all statistically significantly different from those from shelter. Enteritis was identified in about one third of the animals in both groups. The distribution of inclusion bodies also showed significant difference in urinary bladder, lymphoid tissues, lung, and alimentary tract between the two groups. Twenty nine co-infections and other associated lesions were identified. However, no significant difference was seen in the frequency of occurrence between two groups with the exception of interstitial nephritis. In conclusion, lymphoid depletion, pneumonia, and CNS demyelination were the most common CDV-infected principal lesions, and the inclusion bodies had a high occurrence in lymph nodes, spleen as well as mucosa epithelium of lung, stomach, urinary bladder and kidney (Chapter III, VI). In the present study, it has been demonstrated that CDV in Taiwan has at least one clade of the phylogenetic tree showing more than 95% amino acid similarity (< 5% amino acid variation) in the H gene. The modified non-biotin polymerlized horseradish peroxidase (HRP) IHC labeling and ISH method are easy, optimal and accurate for retrospective diagnosis of CDV and MHV. The occurrence of CDV-associated pathological lesions depend on the source of the animals, treatment history, age and tissue distribution

Performance Study of Multipath Effect in 5G Millimeter-Wave Channel

5G has been essentially a buzzword for several years, but according to the experts, from 2022 onward, there will be an inflection point between network maturity and the availability of 5G. To make 5G a reality, we must minimize all propagation losses. One of the possible factors that reduces the performance of 5G transmission is the multipath effect. In this paper, we investigate the severity of the multipath effect in the 5G millimeter-wave (mmWave) channel and mitigate the multipath effect using adaptive equalization based on the least mean square (LMS) algorithm to improve the performance of 5G wireless signal transmission. A mmWave channel simulator, NYUSIM, provides complete data for all resolvable multipaths in a specific channel configuration. An analysis of bit-error-rate (BER) based on the minimum BER (MBER) and minimum mean square error (MMSE) optimization criterion is performed to measure the improved performance of a 5G data channel simulated under line-of-sight (LOS) and non-LOS (NLOS) paths. A good overall performance of BER based on the MBER and MMSE criteria is attained using the LMS equalization method in a micro-urban area at a maximum data rate of 50 Mbps. For both LOS and NLOS conditions, the increase in data rate to 55.56 Mbps and 62.5 Mbps causes a significant decrease in BER performance. In conclusion, the primary factor affecting the BER performance is the data rate, not the frequency or transmitter-to-receiver distance

Performance Study of Multipath Effect in 5G Millimeter-Wave Channel

5G has been essentially a buzzword for several years, but according to the experts, from 2022 onward, there will be an inflection point between network maturity and the availability of 5G. To make 5G a reality, we must minimize all propagation losses. One of the possible factors that reduces the performance of 5G transmission is the multipath effect. In this paper, we investigate the severity of the multipath effect in the 5G millimeter-wave (mmWave) channel and mitigate the multipath effect using adaptive equalization based on the least mean square (LMS) algorithm to improve the performance of 5G wireless signal transmission. A mmWave channel simulator, NYUSIM, provides complete data for all resolvable multipaths in a specific channel configuration. An analysis of bit-error-rate (BER) based on the minimum BER (MBER) and minimum mean square error (MMSE) optimization criterion is performed to measure the improved performance of a 5G data channel simulated under line-of-sight (LOS) and non-LOS (NLOS) paths. A good overall performance of BER based on the MBER and MMSE criteria is attained using the LMS equalization method in a micro-urban area at a maximum data rate of 50 Mbps. For both LOS and NLOS conditions, the increase in data rate to 55.56 Mbps and 62.5 Mbps causes a significant decrease in BER performance. In conclusion, the primary factor affecting the BER performance is the data rate, not the frequency or transmitter-to-receiver distance

The FYVE Domain of Smad Anchor for Receptor Activation (SARA) Is Required to Prevent Skin Carcinogenesis, but Not in Mouse Development

<div><p>Smad Anchor for Receptor Activation (SARA) has been reported as a critical role in TGF-β signal transduction by recruiting non-activated Smad2/3 to the TGF-β receptor and ensuring appropriate subcellular localization of the activated receptor-bound complex. However, controversies still exist in previous reports. In this study, we describe the expression of two SARA isoforms, SARA₁ and SARA₂, in mice and report the generation and characterization of SARA mutant mice with FYVE domain deletion. SARA mutant mice developed normally and showed no gross abnormalities. Further examination showed that the TGF-β signaling pathway was indeed altered in SARA mutant mice, with the downregulation of Smad2 protein expression. The decreasing expression of Smad2 was caused by enhancing Smurf2-mediated proteasome degradation pathway. However, the internalization of TGF-β receptors into the early endosome was not affected in SARA mutant mouse embryonic fibroblasts (MEFs). Moreover, the downregulation of Smad2 in SARA mutant MEFs was not sufficient to disrupt the diverse cellular biological functions of TGF-β signaling, including growth inhibition, apoptosis, senescence, and the epithelial-to-mesenchymal transition. Our results indicate that SARA is not involved in the activation process of TGF-β signal transduction. Using a two-stage skin chemical carcinogenesis assay, we found that the loss of SARA promoted skin tumor formation and malignant progression. Our data suggest a protective role of SARA in skin carcinogenesis.</p></div

Dietary chemoprevention of PhIP induced carcinogenesis in male Fischer 344 rats with tomato and broccoli.

The heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-B]pyridine (PhIP), found in meats cooked at high temperatures, has been implicated in epidemiological and rodent studies for causing breast, prostate, and colorectal cancers. A previous animal study using a xenograft model has shown that whole tomato and broccoli, when eaten in combination, exhibit a marked effect on tumor reduction compared to when eaten alone. Our aim was to determine if PhIP-induced carcinogenesis can be prevented by dietary consumption of whole tomato + broccoli powders. Male Fischer 344 rats (n = 45) were randomized into the following treatment groups: control (AIN93G diet), PhIP (200 ppm in AIN93G diet for the first 20 weeks of the study), or tomato + broccoli + PhIP (mixed in AIN93G diet at 10% each and fed with PhIP for 20 weeks, and then without PhIP for 32 weeks). Study animals were monitored for 52 weeks and were euthanized as necessary based on a set of criteria for health status and tumor burden. Although there appeared to be some hepatic and intestinal toxicity due to the combination of PhIP and tomato + broccoli, these rodents had improved survival and reduced incidence and/or severity of PhIP-induced neoplastic lesions compared to the PhIP-alone treated group. Rats eating tomato + broccoli exhibited a marked decrease in the number and size of cribiform prostatic intraepitheilial neoplasia/carcinoma in situ (cribiform PIN/CIS) lesions and in the incidence of invasive intestinal adenocarcinomas and skin carcinomas. Although the apparent toxic effects of combined PhIP and tomato + broccoli need additional study, the results of this study support the hypothesis that a diet rich in tomato and broccoli can reduce or prevent dietary carcinogen-induced cancers