Type 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy.were isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N- amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.hTenoImmunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.At  Type 2 diabetes mellitus (T2DM) is one of the most common worldwide endocrine lifelong diseases, especially among Malaysian . T2DM isThe pathogenesis of diabetic tendinopathy involves both inflammation and degeneration process. Weaker tendon healing ability is more prominent in T2DM patients . To dateBoth in vitro and in vivo models have been developed to study the interference of impaired insulin action and hyperglycemia on the major target tissues (or cells) for insulin, i.e., adipose tissues (adipocytes), liver (hepatocytes) and muscle (myoblasts), little has been done on other tissues eg. tendon. The effects of insulin on glucose metabolism vary depending on the target tissues. No study has yet explained the impacts of IR in the tendon (or tenocytes). An in vivo study on the T2DM murine model (fed with high-fat diet) had reported the tendon as one of the insulin target tissues, where the blunted phosphorylation of Akt was observed in the cells isolated from the T2DM tendons, indicating loss of insulin sensitivity . Howeverα  on hTeno were evaluated namely glucose uptake, total collagen expression, type I Collagen (COL-I) expression, mRNA gene expression levels of candidate tenogenic markers and extracellular matrix (ECM) metabolism-related markers, as well as apoptosis assay.Elevated levels of inflammatory cytokines in serum, particularly the tumour necrosis factor-alpha (TNF-α), have been reported in T2DM patients  and in tThis study was conducted in accordance with the recommendations and approval by the University of Malaya Medical Center (UMMC) Medical Research Ethics Committee (MREC reference number: 20157-1486 and 20164-2398). Tendon specimens were collected from donors with informed consent. Remnants of hamstring tendon grafts were obtained from patients undergoing anterior cruciate ligament reconstruction  for humaHamstring tendon specimens were collected into sterile phosphate-buffered saline , supplemented with penicillin-streptomycin , stored at 4° C for same day processing. Tendon explant cultures were prepared as previously described , the priFor immunocyto-staining, hTeno were seeded at 2000 cells per chamber in the 8-well chamber slide , and cultured for 24 h. Then, the cells were fixed with ice-cold 100% methanol for 5 min at room temperature, and proceed to immunofluorescence staining . Antibod® 488 donkey anti-mouse IgG H&L , Alexa Fluor® 647 goat anti-rabbit  and Alexa Fluor® 555 donkey anti-rabbit IgG H&L . All images were captured using the Leica TCS SPII confocal laser scanning microscope  with LAS AF Lite software. Images were captured with sequential scanning to avoid signal cross-talk and enhance image quality.For both tendon tissue or hTeno cells, the secondary antibodies used were from Abcam, USA, namely Alexa Fluor2 and cultured for 24 h with low glucose DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1% GlutaMAX. Then, the cells were synchronized with 0.5% FBS supplemented DMEM for 24 h. Next, different concentrations of recombinant human TNF-α  were added to the hTeno culture: 0.008, 0.08, 0.8 and 8 µM, and cultured for another 24 h  at 37 °C. For hTeno cultured with insulin supplement, human recombinant insulin  was added into the culture at 30 min before the end point. In all the assays, the hTeno without TNF-α and insulin was used as the untreated control , and the positive control  was also included in each experiment. For glucose uptake assay, total collagen assay and type I collagen assay, the end points were at 24 h after adding the TNF-α. For the apoptosis assay and gene expression analysis, the end points were at 24, 48 and 72 hr.To determine the effects of the different concentrations of recombinant human TNF-α, hTeno were plate at a cell density of 210 cells per mmFor glucose uptake assay, the hTeno cells were seeded in a 24-well plate and analysed using the 2-[N- amino]-2-deoxy-d-glucose (2-NBDG) glucose uptake assay kit , as described in Gene expression analysis was performed for candidate tenogenic markers [Scleraxis (SCX) and Mohawk (MKX)] and extracellular matrix (ECM) metabolism-related markers . The qPCR analysis was completed using the Integrated Fluidic Circuit (IFC) plate with Biomark HD real-time PCR system  .Apoptosis assay was performed using the FITC Annexin V/Dead cell apoptosis kit  or non-parametric tests  were used based on the normality of the data sets  was considered statistically significant. Different significance symbols were applied, where:At least 3 independent experiments were performed for each assays. Data were analyzed using SPSS software (version 22). All raw data were normalized with their basal group. Either parametric test  versus the basal group , * indicates  2.p < 0.05 and § indicates p < 0.01, versus paired-treated groups without insulin stimulation.For the pairwise comparison between the insulin-stimulated groups versus their respective paired-treated groups without insulin stimulation, ‡ indicates  3.p < 0.05 and ¶ indicates p < 0.01, versus non-TNF-α treated basal group.For the comparison between fold change of different concentrations of TNF-α versus the non-TNF-α treated basal group, ∥ indicates ). The fibroblastic cells started to attach to the plastic surface of the cell culture flask on day-14 of the explant culture. The hTeno cells grow in colonies (1C) and proliferate to reach confluence within about 30 days after the cells attached to the culture flasks  were cultured in monolayer culture. At day-0 when the tendon explants were placed in the cell culture flask, the cells were floating in suspension and some were within the digested tendon collagen fibers . The fibcolonies  and 1C ae flasks .–The single transmembrane domain of the INSR-β was positively stained in the tendon cells in the human Achilles tendons  as well Figs. 3DThe hTeno treated with TNF-α showed a significant increase in the relative 2-NBDG uptake compared to the basal group regardless of the TNF-α concentrations, with and without insulin supplement . In the The hTeno treated with TNF-α showed a significant increase in relative total collagen expression compared to basal group (1.000 ± 0.000), regardless of the TNF-α concentrations with and without insulin . The hTeThe hTeno treated with TNF-α showed a significant reduced in relative COL-I expression compared to basal group (1.000 ± 0.000) when treated with lesser than 8 µM TNF-α without insulin supplement, or at 0.008 µM and 0.8 µM TNF-α with insulin supplement . The hTeThe hTeno treated with TNF-α showed a significant downregulation in their insulin-mediated normalized SCX mRNA gene expression levels compared to non-TNF-α treated basal group (1.000 ± 0.000), regardless of the TNF-α concentrations . A signiThe hTeno treated with TNF-α showed a significant downregulation in their insulin-mediated normalized COL1A1 mRNA gene expression levels compared to non-TNF-α treated basal group (1.000 ± 0.000), regardless of the TNF-α concentrations .Fig. 9B and Fig. 9C and Fig. 9D and In contrast, insulin-mediated normalized COL3A1 mRNA gene expression levels were significantly upregulated at all the different TNF-α concentrations . In this study, 0.008 µM TNF-α is selected as the optimum concentration to induced the IR condition in hTeno.The findings of this study were summarized in the schematic diagram , with the constant apposition of collagenous and non-collagenous matrix production by tenocytes . In thisTenocytes are crucial in maintaining the tendon homeostasis by synthesizing essential collagen . Reducedα induced IR in hTeno is summarized in In summary, the hypothetical pathomechanism involved in TNF-In summary, our study demonstrated that 0.008 µM of TNF-α induces IR condition in hTeno. Following the 0.008 µM of TNF-α treatment, the cellular changes include: (i) a significant reduce in insulin-mediated glucose uptake; (ii) a significantly reduce in the fold change of insulin-mediated total collagen and COL-I expression levels; (iii) a significant down-regulation in the candidate tenogenic marker genes (SCX and MKX) and ECM anabolic–related genes (COL1A1), and up–regulation in ECM catabolic–related genes ; (iv) a significant increase in apoptotic events. Future studies utilizing this hTeno IR model would allow us to understand the cellular mechanisms of IR on tenocytes functionality, which would lead to the discovery of better preventive measures and future therapeutic interventions in the tendon related pathological conditions in T2D.10.7717/peerj.8740/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8740/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8740/supp-3Supplemental Information 3INSR-β is indicated by green colour; GLUT 4 is indicated by red colour; while the nucleus is indicated by blue colour in the image.Click here for additional data file.10.7717/peerj.8740/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.8740/supp-5Supplemental Information 5Click here for additional data file.


Фактор некроза опухолей (TNF) – один из основных цитокинов, медиаторов иммунной системы,обеспечивающих защиту организма человека от вирусных инфекций. В процессе эволюции антропогенныйвирус натуральной оспы  освоил эффективные механизмы преодоления иммунологическихбарьеров человека, кодируя в своем геноме белки, способные взаимодействовать с рецепторами цитокиноворганизма-хозяина, блокируя таким образом их активность. В частности, ген G2R этого вируса кодирует белокCrmB, который эффективно связывает TNF человека и мышей. При этом данный белок является двухдоменными наряду с TNF-связывающим N-концевым доменом содержит С-концевой хемокин-связывающий домен. Прииспользовании методологии молекулярного клонирования нами ранее получен рекомбинантный бакуловирус, продуцирующий в клетках насекомых рекомбинантный белок CrmB вируса натуральной оспы (VARV-CrmB),и показан его TNF-нейтрализующий потенциал в различных тестах in vitro и in vivo. С целью снижения иммуногенности этого вирусного белка при его многократном введении для терапии хронических воспалительных заболеваний получена рекомбинантная плазмида, направляющая в клетках Escherichia coli биосинтез укороченногооднодоменного TNF-связывающего (TNF-BD) белка VARV. Методом металл-хелатной аффинной хроматографиииз клеток выделен рекомбинантный белок TNF-BD. Терапевтический потенциал белка TNF-BD изучен в экспериментальной модели септического шока, индуцированного в организме мышей введением бактериальноголипополисахарида (ЛПС). После индукции септического шока животным вводили разные дозы рекомбинантного белка TNF-BD и регистрировали их гибель в течение 7 сут. Все мыши, не получавшие препарат белка TNF-BDпослеинъекции ЛПС, погибли через 3 сут, а в группах животных, которым вводили TNF-BD, в зависимости отдозы этого белка выжили 30, 40 или 60 % мышей. Результаты исследования демонстрируют наличие специфической фармакологической активности у рекомбинантного белка TNF-BD, синтезированного в бактериальныхклетках, в мышиной модели ЛПС-индуцированного септического шока. В процессе коэволюции у вируса натуральной оспы (ВНО)появились многочисленные эффективные механизмыпреодоления иммунологических барьеров человека.В частности, вирусный геном детерминирует синтезсекретируемых белков, имеющих структурное сходствос клеточными рецепторами цитокинов. Вирусные белкифункционируют как связывающие цитокины рецепторы,блокируя таким образом их активность . Способность вирусных белков эффективно взаимодействовать с иммунной системой человекаоткрывает перспективу их использования для разработкииммуномодулирующих терапевтических средств новогопоколения.Некоторые TNF-блокаторы – Remicade® , Enbrel® , Humira™ , действующим началом которых являются моно-клональные антитела или химерные белки, состоящиеиз TNF-рецепторных и иммуноглобулиновых доменов,прошли клинические испытания и разрешены для применения в медицинской практике, но не один из перечисленных препаратов не оказался эффективным противсептического шока . Продемонстрированная in vivo и in vitro высокая TNF-нейтрализующаяактивность белка VARV-CrmB вируса натуральной оспыпозволяет надеяться, что основой терапевтических пре-паратов нового поколения может стать этот белок.Эндотоксический шок, индуцированный липополисахаридом (ЛПС) грамотрицательных бактерий (реакцияШварцмана), – широко используемая экспериментальнаямодель септического шока . Введение рекомбинантногобелка VARV-CrmB мышам с ЛПС-индуцированным септическим шоком приводило к достоверному увеличениювыживаемости мышей и отчетливому снижению гистопатологических изменений внутренних органов по срав-нению с контрольными животными .Полноразмерный рекомбинантный белок VARV-CrmBимеет молекулярную массу 47 кДа, и в его структуре выделяютN-концевой TNF- (TNF-BD) и С-концевой хемокин-связывающий (Сh-BD) домены .С целью уменьшения иммуногенности рекомбинантногобелка VARV-CrmB ранее нами получен его укороченныйоднодоменный вариант TNF-BD. Показаны биологическаяактивность белка TNF-BD in vitro , а такжепониженная иммуногенность относительно полнораз-мерного белка .Цель настоящего исследования – изучение специфи-ческой фармакологической активности укороченногооднодоменного рекомбинантного белка TNF-BD вирусанатуральной оспы in vivo, а именно – в экспериментальноймодели ЛПС-индуцированного септического шока.Реагенты. Для приготовления жидких и твердых пита-тельных средств использовали бактопептон, дрожжевойэкстракт и бактоагар , антибиотики ампициллин,тетрациклин и канамицин . Ре-комбинантную плазмидную ДНК выделяли с помощьюнабора Qiagen Plasmid Midi Kit .В работе были применены также реактивы фирмы Sigma(США): имидазол, ЛПС Escherichia coli 055:B5, мочевина,фенилметилсульфонилфторид (PMSF), IPTG.Рекомбинантные плазмиды и бактериальные штаммы.Рекомбинантная плазмида pQE60-TNF-BD любезнопредоставлена Т.В. Трегубчак .Для амплификации этой плазмиды использовали штаммE. coli XL10-Gold , а для нара-ботки рекомбинантного белка TNF-BD – штамм E. coliSG13009[pRep4] . Компетентныеклетки E. coli XL10-Gold и E. coli SG13009 получали пометоду .Бактериальные среды. Для культивирования штаммаE. coli SG13009[pRep4], содержащего pQE60-TNF-BD, ис-пользовали стандартную LB-среду .Электрофорез, хроматография. Фракционированиебелков проводили в SDS-ПААГ по стандартной методике, с помощью маркеров молекулярных массбелков PageRuler™ Plus Prestained Protein Ladder  .Металл-хелатную афинную хроматографию проводили,используя Ni-NTA агарозу , на ко-лонке объемом 1 мл. Концентрацию белка определяли пометоду .Животные. В эксперименты брали самок мышейлинии BALB/c с массой тела 19–21 г (возраст 8–9 нед),полученных из вивария Государственного научногоцентра вирусологии и биотехнологии «Вектор» Роспо-требнадзора .Мышей содержали при естественном световом режиме и постоянном доступе к воде и пище. Животных выводилииз эксперимента в соответствии с правилами, принятымиЕвропейской конвенцией по защите животных, используемыхдля экспериментальных целей.r-, Kmr-, TcMr-трансформантов E. coli SG13009[pRep4].Инокулят разводили свежим L-бульоном (1:50), содержа-щим 100 мкг/мл ампициллина и по 30 мкг/мл канамицинаи тетрациклина. Бактериальную культуру инкубировалипри 37 °С и интенсивной аэрации до достижения оптической плотности культуры А550 = 0.3–0.5, затем добавлялииндуктор IPTG до концентрации 1 мМ (продолжительность индукции 4 ч). Клетки собирали низкоскоростнымцентрифугированием и ресуспендировали в 5 мл буфераследующего состава: 10 % сахароза, 100 мМ Трис-HCl,pH 7.3, 5 мМ ЕДТА, 20 мкг/мл PMSF. Затем бактериальныеклетки разрушали ультразвуком на установке Soniprep. Как было показано ранее , рекомбинантный белок TNF-BD синтезируетсяв клетках E. coli в виде «телец включения». Для выделенияцелевого продукта тельца включения солюбилизовали вбуфере А  и суперна-тант наносили на колонку с Ni-NTA агарозой (V = 1 мл),уравновешенную 5 мл этого же буфера. Колонку про-мывали (2 мл × 3) буфером В  и элюировали целевой продукт буфером С (0.3 мл × 5). Фракции выделения анализировалив 10 % ПААГ. Результаты выделенияпредставлены на рис. 1. Фракции, содержащие целевойпродукт, объединяли. Таким образом, был выделен пре-парат рекомбинантного белка TNF-BD, чистота которого,по данным электрофореза, составляла около 90 %.Наработка рекомбинантного белка TNF-BD. Для полу-чения инокулятов были взяты индивидуальные колонииApИсследование токсичности рекомбинантного белкаTNF-BD. Для того чтобы определить способность пре-парата укороченного однодоменного белка TNF-BD предотвращатьвзаимодействие мышиного TNF со специфи-ческими рецепторамиэукариотических клеток, изучалиего активность в тесте нейтрализации цитотоксическогодействия mTNF на культуре клеток мышиных фибробластов L929, как описано в работе .Для определения токсичности рекомбинантного белкавзяли две группы мышей линии BALB/c (каждая группасодержала пять животных). Одной группе вводили внутрибрюшинно физиологическийраствор, другой – ре-комбинантный белок в дозе 50 мкг/мышь. Наблюдениевели в течение 7 сут, за этот период не было обнаруженопроявлений токсичности рекомбинантного белка.Исследование специфической фармакологическойактивности белка TNF-BD в экспериментальной моделисептического шока. Предварительно для мышейлинии BALB/c была определена доза ЛПС E. coli 055:B5,вызывающая гибель 100 % животных (ЛД100 ), равная20 мг/ кг веса. Животные были объединены в пять групп (по 10 особей в группе). Мышам в группах 1–3 внутрибрюшинновводили рекомбинантный белок TNF-BD вдозах 0.2, 2 или 20 мкг/мышь. Через 30 мин этим живот-ным внутрибрюшинно инъецировали раствор ЛПС E. coli(O55:B5) в дозе 1 ЛД100. Группе сравнения (группа 4)вводили только ЛПС, а группе отрицательного контроля(группа 5) – натрий-фосфатный буфер. После введенияпрепаратов мышам регистрировали их гибель в течение7 сут. Результаты эксперимента показаны на рис. 2: наконец эксперимента выживаемость животных в группе 5составила 100 %, в группе 4 – 0 %, а в группах 1–3 – 30,40 и 60 % соответственно.Cигнальная система, активируемая TNF, – один из основных факторов, обусловливающих развитие воспалительного процесса. Гиперпродукция TNF может приводить кгибели организма в результате развития системной воспалительнойреакции или септического шока. Сепсис исептический шок до настоящего времени представляютодну из основных проблем здравоохранения. Ежегодноони являются причиной смерти более миллиона человек. TNF-антагонисты, действующимначалом которых были моноклональные антитела или аналог клеточных рецепторов (Etanercept), прошли клинические испытания и разрешены для применения в медицинской практике, но неодин из этих препаратов не проявил терапевтической активностипротив септического шока .Поэтому поиск новых классов соединений, обладающихTNF-нейтрализующей активностью, остается актуальнойпроблемой.Существование вирусных белков, способных связыватьцитокины и, в частности TNF, блокируя таким образом егоактивность , позволяетпредположить возможность разработки нового поколенияTNF-антагонистов на основе TNF-связывающего белкавируса натуральной оспы (VARV-CrmB). Ранее нами по-лучен рекомбинантный бакуловирус BVi67, несущий генбелка VARV-CrmB, а из инфицированных им клеток насекомых линии Sf21 выделен рекомбинантный вирусныйбелок. Рекомбинантный белок VARV-CrmB эффективнонейтрализует эффекты TNF в экспериментальных системах in vitro и in vivo . Особенно следуетподчеркнуть его выраженный терапевтический эффект напроявление ЛПС-индуцированного септического шока умышей, что отчетливо снижает гистопатологические из-менения внутренних органов и достоверно увеличиваетвыживаемость животных . НовыйTNF-антагонист, кроме TNF-нейтрализующего потенциала,должен обладать низкой иммуногенностью, так какэффективностьприменения препарата может быть сниженаиз-за развития иммунного ответа на терапевтическийбелок .Полноразмерный рекомбинантный белок VARV-CrmB(47 кДа) обладает способностью к олигомеризации, этоможет снизить его терапевтический потенциал при по-вторном или многократном применении. Белок VARVCrmBсостоит из TNF- и хемокин-связывающего доменов. Нами сделано предположение, что однодоменный укороченный TNF-связывающий белоксохранит свою терапевтическую активность и будет обладать существенно меньшей иммуногенностью. Былполучен бактериальный продуцент укороченного белкаTNF-BD, соответствующего TNF-связывающему доменубелка VARV-CrmB, и экспериментально доказана его способность ингибировать цитотоксичность TNF на клеткахмышиных фибробластов L929 и TNF-индуцированнуюокислительно-метаболическую активность лейкоцитовкрови мышей . При этом рекомбинантный TNF-BD проявлялсниженную иммуногенность относительно белка VARVCrmBпри многократном введении экспериментальнымживотным .В настоящем исследовании изучен терапевтический потенциал короткого рекомбинантного однодоменного белкаTNF-BD в экспериментальной модели септического шока.С этой целью белок TNF-BD выделили из клеток E. coliметодом металл-хелатной аффинной хроматографии, какэто показано на рис. 1, а и б. При его внутрибрюшинномвведении мышам линии BALB/c установлено отсутствиетоксичности. Для мышей этой линии экспериментальноопределена доза ЛПС E. coli (O55:B5), вызывающая 100 %гибель животных (ЛД100 ). При внутрибрюшинном введении ЛПС и белка TNF-BD в нашей работе впервыепродемонстрирована дозозависимая специфическая фармакологическаяактивность низкоиммуногенного укороченногорекомбинантного белка TNF-BD в модели ЛПС-индуцированногосептического шока (см. рис. 2).Следует отметить, что вопрос правомерности использованиямышиной модели эндотоксимии (реакции Щварцмана)для выявления новых препаратов для терапиисептического шока у людей остается дискуссионным.Основанием для скептического отношения к мышиноймодели служат отличия результатов транскриптомныханализов, механизмов развития врожденного и приобретенного иммунных ответов, гетерогенность человеческойпопуляции versus гомогенности инбредных линий мышей. Не исключено, чтопрепараты, проявившие высокую эффективность в мыши-ной модели сепсиса, не будут эффективными для человека,как это случилось с препаратом Centocor .Однако простота проведения эксперимента и воспроизводимость результатов делают эту модель незаменимойдля первичного скрининга TNF-связывающих терапевтических препаратов.На основании проведенного исследования можно сделатьвывод, что рекомбинантный белок TNF-BD вируса нату-ральной оспы является перспективным для разработкинового поколения TNF-антагонистов. Этот белок можетслужить модельным объектом для изучения взаимо-действия вирусных белков с компонентами иммуннойсистемыорганизма человека. Методами делеционногоанализаи мутагенеза, рентгеноструктурного анализа,компьютерного моделирования возможно идентифициро-вать в структуре белка TNF-BD аминокислотные остатки,осуществляющие взаимодействие с TNF и составляющиеконкуренцию клеточным рецепторам.The authors declare no conflict of interest.Непомнящих Т.С., Трегубчак Т.В., Якубицкий С.Н., Таранов О.С.,Максютов Р.А., Щелкунов С.Н. Кандидатные антиревматиче-ские плазмидные конструкции обладают низкой иммуноген-ностью. Вавиловский журнал генетики и селекции. 2017;21(3):317-322. DOI 10.18699/VJ17.249.Трегубчак Т.В., Шеховцов С.В., Непомнящих Т.С., Пельтек С.Е.,Колчанов Н.А., Щелкунов С.Н. TNF-связывающий домен белкаVARV-CrmB, синтезированный в клетках Escherichia coli, эф-фективно взаимодействует с TNF человека. Докл. Акад. наук.2015;462(4):488-492. 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N-hydroxysuccinimide (NHS). The evaluation of the efficiency of the coupling reaction was carried out by UV-vis spectrophotometry. The developed antibodies coupled to MNPs were tested for the pre-concentration of two biomarkers tumor necrosis factor alpha (TNF-α) and Interleukin-10 (IL-10). Both biomarkers were assessed in the matrix based on phosphate-buffered saline solution (PBS) and artificial saliva (AS) to carry out the demonstration of the format assay. Supernatants were used to determine the number of free biomarkers for both studies. Reduction of the nonspecific saliva protein adsorption on the surface of the complex antibodies-MNPs to levels low enough to allow the detection of biomarkers in complex media has been achieved.Magnetic nanoparticles (MNPs) were synthesized using the colloidal co-precipitation method and further coated with silica using the Stöber process. These were functionalized with carboxylic and amine functionalities for further covalent immobilization of antibodies on these MNPs. The procedure for covalent immobilization of antibodies on MNPs was developed using 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and  Superparamagnetic iron oxide nanoparticles (SPIONs) are of great importance when grafted with biomarkers for applications in modern biological and biotechnology areas. The surface-modified magnetic nanoparticles (MNPs) can be used both in in-vitro and in-vivo systems effectively. The size of the MNPs needs to be controlled and innovative functionalization techniques need to be utilized for the effective implementation of these modified MNPs in medical applications ,3,4,5,6.The small size of MNPs (a few nanometers) is essential for them to be able to interact, bind, or penetrate biological entities. This is because in the nano range, their dimensions are comparable to those of proteins, cells, or viruses, and this facilitates their movement through biological structures . These M3O4 core in a substantial way. Among the different types of MNPs, iron oxides (magnetite (Fe3O4), maghemite (γ-Fe2O3), and hematite (α-Fe2O3)) are by far the most commonly employed ones for in-vivo applications since iron is physiologically well tolerated. Silica is inorganic but bio-friendly and it is known for its chemical stability and ease of formation. The biggest advantage of having a surface enriched in silica is the presence of silanol groups, which can easily react with coupling agents, providing strong attachment of surface ligands on MNPs [The successful design of MNPs for biological applications needs a careful selection of magnetic core and surface coating material, where the former mainly determines the MNPs’ heating and sensing capabilities with regards to application efficiency and the latter specifies the interaction of these MNPs with a physiological environment. To enable the direct use of MNPs in biomedical applications, the MNPs should be further functionalized by conjugating them with functional groups. The surface coating provides a suitable base for the attachment of these functional groups on MNPs. These groups such as antibodies, peptides, polysaccharides, etc., permit specific recognition of cell types and direct the MNPs to a specific tissue or cell type by binding to a cell surface receptor. The silica coating facilitates the functionalization of the surface of the MNPs with either amine groups or carboxylic groups, which help in bonding with biological entities for medical applications. Also, the silica coating does not affect the magnetic property of the Fe on MNPs . There a on MNPs . In thisThe coupling strategy used for linking the functionalizing agent to the particle is a pivotal component for success, and will largely depend both on the coating of the MNPs and the available functional groups on the target moiety ,24,25,26To learn if salivary diagnosis is applicable in the conditions defined earlier on pre-concentration in saliva we tested tumor necrosis factor alpha , responsible for a diverse range of signaling events within cells, and Interleukin 10 (IL-10) that plays a central role in limiting host immune response to pathogens, thereby preventing damage to the host. To study the feasibility of saliva and, in these preliminary tests, avoid the variability in concentration of the saliva, we considered adequate to test the immunoreagents in artificial saliva and ensure that there is no cross-reactivity between the immunoreagents used. In this research, we have immobilized anti-TNF-α and anti-IL-10 antibodies, which are present in saliva on MNPs as a proof of concept. The 3D nano-collectors generated were evaluated using UV-vis spectrophotometry (JENWAY 7205) for their ability to pre-concentrate the antigens TNF-α and IL-10, respectively, by employing an external magnetic field. Tests were performed both in phosphate buffered saline (PBS) and artificial saliva (AS) as the matrix. The obtained results have proven that our MNPs can represent a promising tool for rapid pre-concentration of heart failure biomarkers (TNF-α/IL-10) in saliva. Further investigation will be carried out to integrate our MNPs with sensors to increase the sensitivity, selectivity, and decrease matrix effect.On the other hand, the colloidal stability is a big issue with the MNPs or magnetic nanocomposites, since their advent there remains a big issue surrounding the stability of the MNPs in colloids. The more stable they are in a colloidal state, the less easy it is to use them for magnetic separation, while on the other hand, if they are less stable then they are easier to isolate by external magnetic field application. So, for this a compromise has to be made regarding them and based on their application they are synthesized as needed. In order to improve magnetization, larger particles can be synthesized; these show higher magnetization but much less stability in colloidal state as, due to their large sizes, they have a greater tendency to aggregate. MNPs are more prone to aggregation compared to other nanoparticles and agglomeration is even more significant for MNPs as there exists magnetic dipole–dipole attraction among themselves . In partIn order to improve stability and also preserve the magnetic properties, the following few things can be done: Sonication, which is a mechanical way and very effective in disaggregating the particles and dispersing them in colloidal state. One of the other methods is using inorganic shell coating like Silica or carbon coating to improve the stability. Also, surface coating with macromolecules like polymers or surfactants can be used for improving stability. Increasing viscosity of the medium can also prevent aggregation as the particles cannot get dispersed easily and travel through the viscous medium, thus preventing aggregation. This can be done by gum gelation or by emulsion formation.2 layer on the iron oxide core. This strategy coupled with the concave nature of the reaction flask used results in more homogenized coating of the magnetic core with the Silica layer. The traditional method of using a magnetic bead in this step results in inefficient coating as a major part of the magnetic core becomes attracted to the bead and does not fully participate in the reaction. Using our strategy of a specifically designed mechanical stirrer in the concave flask with Teflon coating on top leads to an increase in the chemical kinetics of the reaction by creating a vortex that leads to homogenized coating with almost negligible reactant losses. Usually, when magnetic nanoparticles tend to aggregate, this causes difficulty in magnetic separation as it is well established that the smaller the size of the particles, the faster is the separation time, while phenomenon like aggregation and flocculation slow down the process [In this paper, one of the first studies done using saliva as a diagnostic fluid for heart failure research is being reported. There lies a novelty in the synthesis procedure in the use of a non-magnetic mechanical stirrer for the coating of SiO process .Furthermore, besides a modification in the synthesis procedure of the MNPs and artificial saliva as the fluid for heart failure detection, colloidal stability of MNPs has been studied here with different surfactants and by increasing the viscosity of the medium by using a surfactant. Relevant issues like colloidal stability of MNPs in long-term use for varied applications and how the medium affects their stability are avoided in most studies dealing with MNPs. In this paper, preliminary results of how the stability varies with the change in viscosity of the medium and how difficult it is to extract with an external magnet is reported. Certain common issues with MNPs, which are not reported when its synthesis and application is stated like its long-term usage, stability, and extraction time using external magnets and stirrer systems, are dealt with here.3O4@SiO2-NH2 and Fe3O4@SiO2-NH2-COOH were done using co-precipitation method, followed by Stober process and functionalization [2 and COOH groups. The Fe3O4@SiO2 MNPs are shown in TEM images . 2+ ions of the MNPs. Also, further chemical composition was analyzed using the IR spectrum. Fe3O4@SiO2 and Fe3O4@SiO2-COOH was measured with IR and the characteristic peaks of Si-O and C=O was observed in the respective samples  nanoparticles is dependent on the distribution of Fe ions in octahedral and tetrahedral sites of the spinel structure [3+ ions in the octahedral sites and the tetrahedral sites are the same, their magnetic spins cancel out each other. Consequently, the magnetic spins of only Fe2+ ions in the octahedral sites contribute to the net magnetic moment in a spinel structure. R) nor coercivity  was observed. The saturation magnetization value of MNPs at 300K was 38.66 emu/g for Fe3O4@SiO2-NH2 and 40.76 emu/g for Fe3O4@SiO2-NH2-COOH. Also, the magnetization curve for Fe3O4@SiO2 is shown where the saturated magnetization is 41.2 emu/g. (Magnetic characterization of the MNPs was carried out in a superconductive quantum interference device (SQUID) magnetometer (Quantum Design MPMS5XL). Magnetization vs. magnetic field measurements were performed at 300K in a 6T field. The samples were prepared using a polycarbonate capsule each filled with 1 mg of Fetructure . The mag2 emu/g.  The colloidal stability as mentioned at the start is an issue with the MNPs. The MNPs we synthesized were extremely small in size and hence had a tendency to aggregate due to the magnetic dipole–dipole interaction and precipitate over the course of time. This also aided in easily extracting them with the help of an external magnet. To be able to use these MNPs in biosensors, it was important to test their stability and, if possible, increase it without affecting their facile extraction using an external magnet. Five different surfactants were used for this purpose and they are as follows: Citric Acid, Tricaprylylmethyl ammonium chloride, Cetyl trimethyl ammonium chloride (CTAC), Tetrabutyl ammonium chloride, and Dimethyl di-octadecyl ammonium chloride. Out of these five, the last one was successful in providing long-term stability. This was so because it increased the viscosity of the medium and, hence, stopped the MNPs from aggregating. The surfactant prevented the diffusion of the MNPs through the solvent by increasing the viscosity and hence hindering their aggregation. However, this came with a major drawback, as these MNPs were harder to extract using an external magnet. Different concentrations of the surfactant were studied but the trend observed was the same; the more stable they became, the harder it was to extract them. The zeta-potential values obtained for each surfactant is given in Up until 15 mg of the surfactant, the dispersion was stable, and with time, a small number of MNPs could be extracted by the magnet. However, to obtain them completely dry and free of the surfactant is difficult as it makes the suspension extremely viscous. The edges of toroidal external magnets have been used in all the cases to extract the magnets as they comprise of the maximum magnetic fields passing through them. The magnets were used on the sidewalls of the vials or reaction flasks to extract the MNPs. 2-NH2-COOH activated with EDC/NHS was incubated for a fixed period of time with a known concentration of anti-TNF-α antibody. Following a magnetic separation, the supernatant liquid was analyzed by UV-vis spectrophotometry. The bio-functionalization efficacy was evaluated by comparing the absorbance reading to that obtained for the supernatants of the same concentration of anti-TNF-α antibody after incubation with non-activated MNPs. Consequently, the physisorption of antibodies onto MNPs was taken into account and differences in absorbance can only be attributed to the concentration of unreacted antibody remaining in the supernatants. An illustration of the steps involved in the bio-functionalization of MNPs is presented in 2-NH2-CO-anti-TNF-α was incubated with the antigen at different concentrations.To learn about the bio-functionalization efficacy of MNPs with anti-TNF-α antibody, the following experiments were performed: A known amount of MNP@SiO2-NH2-COOH”. Therefore, based on these results, the concentration of anti-TNF-α antibody to run the incubation was fixed at 10 ng/mL to ensure the successful bio-functionalization of MNPs.This was confirmed by the reproducibility of our three replication measurements. In addition, our bio-functionalized MNPs are vortexed before the removal of supernatant so that the weakly bonded by physical interaction are removed and only the strongly bonded by covalent interaction remain, enabling us to evaluate the effectiveness of our chemistry. These results confirm that the bio-functionalization of MNPs with the antibodies was successfully achieved. Moreover, the concentration of anti-TNF-α in the supernatant decreased by 7%, 16%, and 8% for an initial concentration of 2, 10, and 20 ng/mL, respectively, using the "MNP@SiOThe bio-functionalization efficacy was calculated using Beer–Lambert law. Beer–Lambert Law is difficult for quantitative measurement for wavelengths near 200 nm though it has been used here and has provided satisfactory results.Concentrations (ng/mL)21020Absorbance Sample0.0660.1350.315Absorbance Reference0.0710.1600.341A is the absorbance, ε is the molar attenuation coefficient, l is the optical path length, and c is the concentration of the attenuation species (Equation (1)).The methodology that has been used is the following:sample/Areference = Csample/CreferenceEquation (2)/Equation (3): Asample = Asample × Creference/AreferenceC2 ng/mLsample = Asample × Creference/Areference = 0.066 × 2/0.071 = 1.85 ng/mLCThis is means that the concentration of Antibodies fixed on MNPs is 0.15 ng/mL and the percentage of antibodies fixed in the MNPs is 7%.10 ng/mLsample = Asample × Creference/Areference = 0.135 × 10/0.160 = 8.43 ng/mLCThis is means that the concentration of Antibodies fixed on MNPs is 1.57 ng/mL and the percentage of antibodies fixed in the MNPs is 16%.20 ng/mLsample = Asample × Creference/Areference = 0.315 × 20/0.341 = 18.47 ng/mLCThis means that the concentration of Antibodies fixed on MNPs is 1.53 ng/mL and the percentage of antibodies fixed in the MNPs is 8%.2-NH2-CO-anti-TNF-α described in the previous section was used to pre-concentrate TNF-α in PBS. This section details the experiments performed to incubate TNF-α at different concentrations with the complex MNP@SiO2-NH2-CO-anti-TNF-α and afterwards to measure the supernatants containing the unreacted TNF-α by UV-vis spectrophotometry. Thus, a comparison with the initial concentration of the antigen could be carried out and the percentage of TNF-α, which has been bonded to the complex MNP@SiO2-NH2-CO-anti-TNF-α, could be assessed.The complex MNP@SiO2-NH2-CO-anti-TNF-α at three different concentrations of TNF-α: 2, 5 and 10 ng/L. The curves, corresponding to reference (reference 1 and 2) to which the former plot was compared, are also shown. Reference (1) depicts supernatants containing unreacted TNF-α after incubation with MNPs that were non-activated with EDC/NHS during the functionalization with anti-TNF-α. Since -COOH groups present on the MNPs were not activated, we did not expect any covalent bonding between anti-TNF-α-MNPs. Therefore, we only expected interaction of TNF-α with those antibodies physisorbed on the MNPs. Reference (2) depicts Supernatants containing unreacted TNF-α after incubation with the initial MNPs that have only been washed four times with 1mL of 10 mM PBS. In this case, the only physisorption of TNF-α on the MNPs was expected.2-NH2-CO-anti-TNF-α is lower than the concentration of TNF-α after incubation with the other two references.Based on the results, it can be asserted that the concentration of unreacted TNF-α in the supernatants after incubation with the complex MNP@SiO2-NH2-CO-anti-TNF-α binds the antigen TNF-α more effectively as antibodies are both covalently bonded and physisorbed to the MNPs. Therefore, the pre-concentration effect when incubating TNF-α with “Sample” is much more important. As shown in (1)There is an effective coupling between MNPs and anti-TNF-α antibody when the MNPs were activated using EDC/NHS.(2)2-NH2-CO-Anti-TNF-α can bind the antigen TNF-α and it can be followed by UV-vis spectrophotometry.The complex MNP@SiOThus, we can confirm these two hypotheses: 2HPO4, 0.6 g/L anhydrous CaCl2, 0.4 g/L KCl, 0.4 g/L NaCl, 4 g/L mucin, and 4 g/L urea  in deionized water. The pH was adjusted to 7.2 by adding NaOH and it was stored at 4 °C until use.The previous experiment as described above was tested using artificial saliva as a matrix. First, a stock solution of artificial saliva (AS) was prepared by dissolving 0.6 g/L NaA dilution profile was performed by UV-vis spectrophotometry to find out the optimal dilution range of artificial saliva at which the matrix effect is removed. For this purpose, AS was diluted using 10 mM PBS to obtain saliva/PBS ratios of 1/1, 1/10, 1/100, 1/500, and 1/1000. The results are shown in 2-NH2-CO-anti-TNF-α was obtained as described above. Subsequently, TNF-α was prepared in AS diluted in PBS 1/500 at three different concentrations: 2, 5, and 10 ng/mL. The solutions prepared were incubated with the complex MNP@SiO2-NH2-CO-Anti-TNF-α as described above and the supernatants containing the unreacted TNF-α were measured by UV-vis spectrophotometry.In the first stage, the complex MNP@SiOOnce again, the solution used for the reference was based on the supernatants containing unreacted TNF-α after incubation with MNPs that were non-activated with EDC/NHS during the functionalization with anti-TNF-α antibody.2-NH2-CO-anti-TNF-α was lower than the supernatant of TNF-α after incubation with non-activated MNP (without activation with EDC/NHS) used as a reference. Therefore, it can be concluded that the pre-concentration effect when incubating TNF-α with the complex MNP@SiO2-NH2-CO-anti-TNF-α is much more important. The concentration of TNF-α decreased by 9%, 12%, and 9% when compared to the initial concentration of 2, 5, and 10 ng/mL, respectively, using the MNP@SiO2-NH2-COOH.Based on these results shown in The same experiment described with TNF-α was repeated for the cytokine IL-10 and its corresponding anti-IL-10 antibody. First, the concentration range at which IL-10 can be measurable by UV-vis spectrophotometry in a fixed background of AS diluted 1/500 with PBS was obtained. In the UV region, IL-10 was shown to absorb 10 times less than TNF-α for the same concentration. Consequently, the results showed a working range of IL-10 at a concentration between 10 and 100 ng/mL .2-NH2-COOH. Once again, the results demonstrated the ability of the complex antibody-MNPs to pre-concentrate the antigen.The concentration of IL-10 decreased by 7%, 6%, and 4% when compared to the initial concentration of 10, 40, and 100 ng/mL, respectively, using MNP@SiO2) 0.1% in 10 mM PBS, and ethanolamine cyanoborohydride (NaBH3CN) 0.1% in 10 mM PBS.A comparative study of different deactivating compounds was carried out. The aim was to assess the adsorption effect of the TNF-α antigen when using different chemicals/proteins for the deactivation step performed after bio-functionalization of MNPs with an antibody. For this purpose, four different deactivation mixtures were used: 0.1% bovine serum albumin (BSA) in 10 mM PBS, ethanolamine 0.1% in 10mM PBS, poly (ethylene glycol) methyl ether amine  used as a reference. Therefore, it can be concluded that the pre-concentration effect when incubating TNF-α with the complex anti-TNF-α-MNPs is much more important. The concentration of TNF-α decreased by 5%, 16%, and 17% when compared to the initial concentration of 2, 5, and 10 ng/mL, respectively.Based on the results shown in A magnetic rack purchased from Sigma Aldrich  and a tube revolver/rotator purchased from Thermo Fisher Scientific  were used during the bio-functionalization process of MNPs. All experiments for indirect measurements of the biomarkers were carried out at room temperature (22 ± 2 °C). The indirect measurements were carried out using UV-vis spectrophotometry (JENWAY 7205) purchased from Jeulin . Transmission electron microscopy (TEM) studies were carried out using JEOL JEM 1210 at 120 kV. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis was done using the QUANTA FEI 200 FEG-ESEM device. The solid sample was analyzed for this. Magnetization hysteresis cycle was measured using Quantum Design MPMS-XL system at 300K with a maximum of 60 kOe.2HPO4, PharmaGrade), magnesium nitrate (Mg(NO3)2, purity 98%), calcium chloride , mucin , sodium hydroxide , Urea, phosphate buffer solution (PBS) tablets, hydrochloric acid , 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), ethanolamine, poly (ethylene glycol) methyl ether amine (PEG-NH2), and sodium cyanoborohydride (NaBH3CN) were purchased from Sigma-Aldrich (France). Anti-TNF-α antibody , Anti-IL-10 antibody , TNF-α , and IL-10  were purchased from R&D Systems (France). Millipore Milli-Q nanopure water (resistivity > 18 MΩ cm) was produced bya Millipore Reagent Water System (France). The PBS buffer used in this study was prepared by dissolving PBS tablets in the nanopure water as indicated by the supplier by yielding 0.01 M phosphate buffer (pH 7.4) containing 0.0027 M potassium chloride and 0.137 M sodium chloride. All reagents used in the present work for the synthesis of magnetic nanoparticles and its functionalization were obtained from Aldrich Chemical Co and were used without further purification. Aqueous ammonia (30% as NH3) was purchased from Panreac AppliChem and used as received.Sodium chloride , potassium chloride , sodium phosphate dibasic  (2 g) and Iron (III) chloride hexahydrate (FeCl3.6H2O) (5.2 g) was added to 25 mL of distilled water. The NaOH solution previously made was added slowly to the mixture of the iron salts with vigorous stirring. Then, 1 mL of concentrated hydrochloric acid (HCl) was added dropwise along with vigorous stirring to make a black solid product. The resultant mixture was heated using an oil bath for 6 h at 80 °C. The black magnetite solid MNPs were isolated using an external magnet and washed three times with distilled water and then dried at 80 °C for overnight. The Iron oxide (Fe3O4) nanoparticles were formed. In the step for the addition of the NaOH, the base is added slowly to the iron salts to obtain smaller sized MNPs while if the iron salts are added directly into the base the sizes of the particle obtained are larger, so it was avoided.First, 25 mL of a 15M solution of sodium hydroxide (NaOH) in distilled water was made. Then, a mixture of Iron (II) chloride tetrahydrate (FeCl3O4 as-synthesized was dispersed in a mixture of ethanol (100 mL) and distilled water (25 mL) for 30 min. Then, 3 mL of NH3 was added followed by the dropwise addition of tetraethoxysilane (TEOS) (1.7 mL). This solution was stirred mechanically for 8 h at room temperature (r.t). Then, the product Fe3O4@SiO2 was separated using an external magnet, was washed three times with distilled water and three times with ethanol, and then dried at 80 °C overnight. First, 5 mmol of Fe3O4@SiO2 was suspended using an ultra-sound bath in 45 mL of ethanol. Then, 5 mL of 3-(2-aminoethylamino) propyl methyl dimethoxy silane (APMS) was dissolved in 20 mL ethanol (20 wt%), and was added to the suspension of Fe3O4@SiO2 in ethanol and stirred vigorously. The pH of the solution was then maintained at 11 using tetramethyl ammonium hydroxide (TMAH). After that, the solution was stirred for 5 h at 50 °C. Then, the Fe3O4@SiO2-NH2 was obtained by magnetic separation, and was washed with distilled water thrice. Some of the particles were not isolated at this stage, and were kept in dispersed form, so sodium chloride (NaCl) (20 mg in 50 mL of deionized water) was added to the dispersion and with this, extra precipitation was achieved. After redispersion, these particles were magnetically decanted and washed thrice. Both crops were mixed together and dried at the oven at 80 °C. Then, 100 mg of Fe3O4@SiO2-NH2 was dispersed in 45 mL dry dimethylformamide (DMF) and 60 mg of succinic anhydride was added to this suspension. The mixture was then stirred with a magnetic stirrer for 22 h at room temperature r.t. The MNPs were then washed thrice with acetone, separated with a magnet, and dried under vacuum. The Fe3O4@SiO2-NH2-COOH MNPs were ready to be used.First, 125 mg of Fe2-NH2-COOH in water were washed following magnetic separation with 1 mL of 10 mM PBS. The washing was repeated three times to ensure all the residues were washed out. The activation of carboxylic acid groups present on the MNPs was achieved by incubation of MNPs in a 500 µL mixture of EDC/NHS (250 µL of 100 mM EDC + 250 µL of 100 mM NHS both prepared in 10 mM PBS) at r.t, and under soft stirring using tube revolver/rotator for 90 min. Then, the remaining EDC/NHS was removed and the MNPs were washed for three consecutive times using 1 mL of cold HCl 1 mM. Subsequently, 500 µL of purified anti-TNF-α antibody was added. Accordingly, three different concentrations of antibody  were used to assess the impact of the concentration on the bio-functionalization efficacy. The activated mixture of MNPs + anti-TNF-α antibody was slowly stirred at r.t for 2 h 30 min. Finally, the complex MNPs@SiO2-NH2-CO-Antibody was obtained. It was further immobilized using the magnetic rack and the supernatants were collected and measured by UV-vis spectrophotometry.(I)- 100 µL, 0.5% solid content of the mother solution containing MNPs@SiO2-NH2-CO-anti-TNF-α) to the absorbance corresponding to the number of antibodies covalently bonded to the MNPs. (II)- For reference, the same procedure was repeated. However, the activation step with EDC/NHS was skipped. Consequently, the supernatants collected after incubation of anti-TNF-α antibody with MNPs correspond to the initial concentration of antibody minus the number of antibodies that were physisorbed to the MNPs. This phenomenon also occurs in (I)-. Therefore, the final concentration measured in the supernatants corresponding to (I) represents the initial concentration of anti-TNF-α antibody minus the amount of anti-TNF-α antibody covalently bonded to the MNPs minus the amount of anti-TNF-α antibody physisorbed. In summary, the absorbance read for (I) and (II) are as follows (Equation (4) and Equation (5)):2-NH2-COOH in water were washed following magnetic separation with 1 mL of 10 mM PBS. The washing was done thrice to ensure all the residues were washed out. The activation of carboxylic acid groups present on the MNPs was achieved by incubation of MNPs in a 500 µL mixture of EDC/NHS (250 µL of 100 mM EDC + 250 µL of 100 mM NHS both prepared in 10 mM PBS) at r.t, and under soft stirring using tube revolver/rotator for 90 min. Then, the remaining EDC/NHS was removed and the MNPs were washed for three consecutive times using 1 mL of cold HCl 1 mM. Subsequently, 500 µL of purified Anti-IL-10antibody (10 ng/mL) was added. The activated mixture of MNPs + Anti-IL-10 was slowly stirred at r.t for 2 h 30 min. Finally, the complex MNPs@SiO2-NH2-CO-Anti-IL-10 was obtained.First, 100 µL, 0.5% solid content of the mother solution containing MNPs@SiO2-NH2-CO-anti-TNF-α obtained was washed with 1 mL of 10 mM PBS. A magnetic rack was placed to immobilize the complex and allow the discharge of supernatants and the added PBS. The cleaning was repeated three times to ensure all the residues were washed out. Afterwards, the unreacted sites were deactivated using a solution of 0.1% BSA in PBS (500 µL in each Eppendorf). The mixture was stirred (33 rpm) using tube revolver/rotator at r.t for 45 min. Then, the unreacted BSA was removed and the complex was washed three times with 1 mL of 10 mM PBS. Again, magnetic separation was done. At this point, the complex was incubated with TNF-α at three different concentrations: 2, 5, and 10 ng/mL. Incubation was performed at r.t and stirred at 33 rpm for 45 min. Finally, after 45 min, the supernatants containing the unreacted TNF-α were collected and the absorbance was measured for the respective concentrations. 2-NH2-CO-anti-TNF-α.(I) The complex MNP@SiO(II) For the reference, two approaches were compared:Reference (1) Supernatants containing unreacted TNF-α after incubation with MNPs that were NOT ACTIVATED with EDC/NHS during the functionalization with anti-TNF-α. Since -COOH groups present on the MNPs were not activated, we do not expect any covalent bonding anti-TNF-α-MNPs. Therefore, we only expect interaction of TNF-α with those antibodies physisorbed on the MNPs. Reference (2) Supernatants containing unreacted TNF-α after incubation with the initial MNPs that have only been washed four times with 1mL of 10 mM PBS. In this case, the only physisorption of TNF-α on the MNPs is expected.2-NH2-CO-Anti-IL-10 obtained was washed with 1 mL of 10 mM PBS. A magnetic rack was placed to immobilize the complex and allow the discharge of supernatants and the added PBS. The cleaning was done thrice to ensure all the residues were washed out. Afterwards, the unreacted sites were deactivated using a solution of 0.1% BSA in PBS (500 µL in each Eppendorf). The mixture was stirred (33 rpm) using tube revolver/rotator at room temperature for 45 min. Then, the unreacted BSA was removed and the complex was washed three times with 1 mL of 10 mM PBS. Again, magnetic separation was done. At this point, the complex was incubated with IL-10 at three different concentrations: 10, 40, and 100 ng/mL. Incubation was performed at r.t and stirred at 33 rpm for 45 min. Finally, after 45 min, the supernatants containing the unreacted IL-10 were collected and the absorbance was measured for the respective concentrations. The complex MNP@SiO2-NH2  at r.t, and under soft stirring, for 2 h 30 min to create the complex MNPs@SiO2-NH-Antibody. Afterwards, MNPs were washed for three consecutive times using 1 mL of cold HCl 1 mM, followed by washing for three times using 1 mL of PBS 10 Mm. Finally, the complex was used for the pre-concentration with different concentrations of TNF-α  and the supernatants were collected and measured by UV-vis spectrophotometry.First, 100 µL of the anti-TNF-α antibody at 10 ng/mL were activated by the incubation in a 500 µL mixture of EDC/NHS (250 µL of 100 mM EDC + 250 µL of 100 mM NHS both prepared in 10 mM PBS) at r.t, and under soft stirring for 90 min. Then, the activated anti-TNF-α antibody was added to the MNP@SiOIn the first stage, a calibration curve was obtained in PBS in order to determine the optimal range of concentration at which both TNF-α and Anti-TNF-α can be analyzed by UV-vis spectrophotometry.Firstly, Anti-TNF-α antibody was prepared in PBS 10 mM at three different concentrations: 20, 100, and 200 ng/mL; 10 mM solution of PBS was used as blank. All the measurements were carried out in duplicates.As shown in Here, the absorbance increased linearly with the concentration of antibody in conformity with the Beer–Lambert’s law (A = εcl). The saturation effect disappeared and therefore the concentration range was fixed between 2 and 20 ng/mL. Measurements were repeated three times and the calibration curve obtained is shown in Likewise, a calibration study was carried out to assess the concentration range at which the antigen TNF-α is measurable by UV-vis spectrophotometry. For this purpose, TNF-α was prepared in PBS 10 mM at different concentrations: 2, 5, and 10 ng/mL. A 10mM solution of PBS was used as blank. All the measurements were carried out in duplicates. A peak for TNF-α was found at λ = 205 nm. In this case, no saturation was observed and the concentration range of antigen was fixed from 2 to 10 ng/mL. The calibration curve obtained is shown in 3O4 core has been synthesized and coated with a layer of SiO2 using the Stöber process. A good SiO2 layer homogeneity has been achieved using an overhead mechanical stirrer with a glass bar containing two blades in a beaker with a Teflon lid to prevent alcohol evaporation while other processes have been performed using typical magnetic stirrers with a magnetic bead. The SiO2 coating has been decorated with –NH2 and –COOH free end groups. The MNPs thus produced were Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Fe3O4@SiO2-NH2-COOH that show a gradual increase in size with the additional functionalized layers. Colloidal stability of the MNPs have also been studied using different surfactants and observing the change in the zeta-potential values of the colloids. The –COOH and –NH2 MNPs have been further functionalized with anti-TNF-α and anti-IL-10 antibodies using (EDC) and (NHS) reagents. The magnetic conjugates produced can be easily extracted using an external magnet. These MNPs have been prepared for testing the tumor necrosis factor alpha (TNF-α), while the others have been prepared for Interleukin-10 (IL-10). To have the highest selectivity towards TNF-α, and IL-10, the non-functionalized MNPs surface sites have been neutralized with the BSA protein. The anti-TNF-α and anti-IL-10 antibodies MNPs have been incubated with TNF-α and IL-10, respectively, in PBS, and after magnetic separation the supernatant solution has been tested for TNF-α and IL-10, respectively, in PBS. They have been measured by UV-vis spectrophotometry where a lower absorbance reading indicates better MNPs capture. The supernatant containing unreacted TNF-α that had been incubated with MNPs whose –COOH reagents has been used as the main reference. It had not been activated with EDC/NHS, although anti-TNF-α or anti-IL-10 antibodies had been added (named Reference 1). Although these did not presumably have any bonded anti-TNF-α antibody or anti-IL-10 antibody, they may indeed have some anti-TNF-α antibody or anti-IL-10 antibody physisorbed, thus interacting with TNF-α or IL-10. Further, there could have been the physisorption on the MNPs by the very same TNF-α and IL-10. To test this, TNF-α and IL-10 were incubated with MNPs before EDC/NHS activation (named Reference 2). Without any exception, the MNPs incorporating the antibodies gave the lowest value of TNF-α and IL-10 in solution, which implies their highest uptake by the MNPs, whereas the highest value of TNF-α and IL-10 was for Reference 2. All this implies that antibodies can be adsorbed, in part, on the surface of silica, a factor that has to be taken in consideration. All these studies were done in PBS, but it was sought to use saliva as the body fluid. To apply the UV-vis spectrophotometry technique it was observed that the saliva had to be diluted with PBS to record the amount of TNF-α and IL-10. Dilution was necessary to 1:500 with PBS. By using Fe3O4@SiO2-NH2-CO-anti-TNF-α antibody or anti-IL-10 antibody the pre-concentration process for TNF-α and IL-10 was clear.We would like to conclude that MNPs with FeWhen the coupling with the anti-TNF-α antibody or anti-IL-10 antibody occurs, not all activated COOH generate the desired CO-anti-TNF-α or anti-IL-10; some remain “unreacted” and to avoid later reaction with TNF-α or IL-10 that would obscure the reaction, it is necessary to use one deactivating agent. Additionally, these deactivating agents also fill the cavities in the MNPs preventing their occupation by physisorbed antibodies or antigens. Among the several compounds/mixtures utilized like BSA, ethanolamine, ethanolamine+ sodium cyanoborohydride (NaBH3CN), and poly(ethylene glycol) methyl ether amine (PEG-NH2), the BSA and ethanolamine + NaBH3CN were found to be the most efficient. To conclude, in this work, we demonstrate the successful synthesis of two different kind of MNPs  as well as the characterization of these MNPs using different techniques. Then, the high selectivity of these MNPs to pre-concentrate two kind of Heart failure biomarkers: TNF-α (2–20 ng/mL) and IL-10 (10–100 ng/mL) in a complex medium  after five hours of biofunctionalization process was studied. In this paper, other than the use of the new set-up using mechanical stirrer with glass blades having a Teflon cover for homogeneous coating of the Silica layer, it reports one of the first studies done using saliva as a diagnostic fluid for heart failure research. Furthermore, colloidal stability of MNPs has been studied here with different surfactants and by increasing the viscosity of the medium by using a surfactant. Preliminary results of how the stability varies with the change in viscosity of the medium and how difficult it is to extract with an external magnet is reported here. This work tries to point out the little concern that exists on the numerous studies of MNPs about taking into consideration the time required for magnetic extraction, or the medium and how it affects the magnetic extraction and the stability of the MNPs for long time shell storage in case they are intended to be used in automatic machines without trained personnel. Further, it deals with the kind of stirrers needed for synthesizing MNPs and that simple UV-vis spectroscopy could be used to determine the loading of MNPs. This paper aims towards showing certain basic fundamental things needed for using MNPs, rather than just reporting a synthesis of MNPs and the detection of antigens. Some basic fundamentals of magnetic nanoparticles, which are mostly excluded from the papers dealing with such topics have been addressed here.


Anti-tumor necrosis factor alpha (TNF-α) antibodies are effective in patients with inflammatory bowel disease (IBD). However, the effect is not optimal because a sufficient concentration of antibodies cannot be maintained at the site of inflammation. Thus, a macromolecular complex was developed with schizophyllan (SPG) and antisense oligonucleotides. In the present study, an SPG-antisense TNF-α complex was prepared, and its therapeutic efficacy was examined using a dextran sodium sulfate (DSS)-induced colitis model. The TNF-α production in CD11b+ macrophages significantly increased in the colon of DSS-treated mice. Dectin-1, a receptor of SPG, binds with SPG and is subsequently taken into the cells via phagocytosis. The expression of dectin-1 by CD11b+ macrophages significantly increased in DSS-treated mice. Flow cytometry revealed that the uptake of SPG-antisense TNF-α in the macrophages was efficient. TNF-α production was suppressed significantly by SPG-antisense TNF-α in vitro, which was administered via enema to evaluate its efficacy. The intrarectal administration of SPG-antisense TNF-α ameliorated the intestinal inflammation. In this study, we showed that the delivery system that conjugates SPG and antisense can have higher therapeutic efficacy. Thus, the new therapeutic approach presented in this study may be used in the management of IBD. Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. The two major IBD phenotypes are ulcerative colitis (UC) and Crohn’s disease (CD) . The patAlthough anti-TNF-α Abs were found to be effective in the treatment of IBD , high doSchizophyllan (SPG), which belongs to the β-(1-3) glucan family A, forms In our study, the topical administration of SPG-antisense TNF-α suppressed TNF-α production by macrophages and significantly improved dextran sodium sulfate (DSS)-induced colitis.The expressions of TNF-α and other proinflammatory cytokines, such as interleukin (IL)-1β and IL-6, in the mucosa of DSS-treated mice were examined via real-time polymerase chain reaction (PCR). The expression of these proinflammatory cytokines in the DSS-treated mice increased significantly A. Since Dectin-1 is a pathogen pattern-recognition receptor (PRR) in macrophages and DCs, and it binds with β-glucans, including SPG . The expWe examined the uptake rate of SPG-antisense TNF-α at different time points A. The CDThe results showed that SPG-antisense TNF-α inhibited the production of TNF-α in vitro. To investigate the therapeutic effect of SPG-antisense TNF-α in vivo, 0.2 mg/kg SPG-antisense TNF-α was administered via enema twice per week in DSS-treated mice A; 0.2 mgTNF-α is produced by macrophages and T cells, and is a key cytokine in the development of inflammation. Moreover, it plays a role in the pathogenesis of IBD since the level of TNF-α increases in the serum of patients with UC and CD . FurtherMacrophages and DCs recognize pathogens such as fungi and mycobacteria using PRRs, activate innate immunity, and eliminate the pathogens. Dectin-1 is a type II C-type lectin receptor that belongs to the PRRs. It is highly expressed in macrophages and DCs, and binds with the carbohydrate structures of pathogens ,28. PrevASOs have been used in patients with IBD to inhibit specific targets, including Smad7, intercellular adhesion molecule (ICAM)-1, and toll-like receptor (TLR) 9. Mongersen, a Smad7 ASO targeting RNA, encoded in the 107–128 DNA region, was developed for CD therapy. In a phase II clinical trial of patients with CD, Mongersen significantly improved Crohn’s Disease Activity Index scores . AlthougBased on these results, the use of ASO and siRNA has several disadvantages. Hydrolysis mediated by deoxyribonuclease causes the instability of ASO and siRNA. Thus, additional strategies are required for them to reach the intestine and stay in the intestinal mucosa, despite inflammation causing a loss of mucous gel layers and the disruption of the epithelial cell barrier in the intestine. The administration of ASOs with SPG directly to an inflammatory site forming an ulcer effectively acts on the inflammatory site and does not affect the whole body. The SPG-based antisense delivery system had several advantages. The SPG complex was stable in vivo and did not dissolve in the presence of deoxyribonuclease, and it was effectively taken up by macrophages via dectin-1 and phagocytosis. Therefore, the topical administration of the SPG-antisense TNF-α significantly inhibited the production of TNF-α by the macrophages and improved intestinal inflammation. These complexes are considered effective against human IBD when administered orally in the form of PH-dependent capsules or via enema. Thus, the efficacy and safety of SPG-based antisense therapy in individuals with IBD must be further investigated.Mw = 1.5 × 105, via gel-permeation chromatography) was provided by Mitsui Sugar Co., Ltd. . ASO for TNF-α (AACCCATCGGCTGGCACCAC-(dA)60) was synthesized at FASMAC Co., Ltd.  and purified using high-performance liquid chromatography. SPG can dissociate from a triple helix to a single chain in 0.25 N NaOH for 3–5 days. The single chains of SPG were mixed with antisense TNF-α in a phosphate buffer solution (pH 7.4) and were stored overnight at 4 °C. The final DNA concentrations of the complex were measured using ultraviolet absorbance. The formation of SPG-based antisense complex was confirmed via polyacrylamide gel electrophoresis.SPG  and the Kyudo Laboratory . All experiments were performed under specific pathogen-free conditions at Kurume University and Fukuoka University. The mice were used according to the approved protocols of the Animal Care and Use Committees of Kurume University Animal  and Fukuoka University .w/v) DSS   drinking water. Eight-week-old female mice were provided with 3% DSS water for 5 days. The body weights of the mice were assessed to evaluate the development of colitis. They were sacrificed on day 14 and the colon was removed and used in the experiments. A part of the colon was fixed in 4% formaldehyde and was stained with hematoxylin and eosin. The histology was scored, as previously described [Colitis was induced with 3% . The production of TNF-α in the culture supernatants was measured using an enzyme-linked immunosorbent assay kit  for TNF-α ). The absorbance was measured using an ELISA reader at a wavelength of 450 nm. The relative TNF-α productions, with several concentrations of SPG, antisense TNF-α, and SPG-antisense TNF-α normalized to TNF-α production without them, were measured as the inhibition rate.For the cell culture, mononuclear cells were isolated from the LP in the colon. The intestinal epithelial cells were removed from the colon with 1 mmol/L EDTA. Then, colonic tissues were shredded and digested by 100 U/mL of collagenase type 2. The LP cells were passed through a 100 µm nylon membrane and were purified using a 45%/72% Percoll gradient . The CD11b+ cells were isolated via magnetic activated cell sorting using anti-CD11b MicroBeads . Moreover, 1 × 10−ΔCt.RNA was extracted from the colon and CD11b+ cells using Trizol . The RNA was reverse transcribed into cDNA with ReverTra Ace qPCR RT Master Mix . The cDNA was quantified using real-time PCR (RT-PCR) with an ABI PRISM 7000 . The RT-PCR for TNF-α, IL-1β, IL-6, dectin-1, and GAPDH was performed using the TaqMan probe. For the relative quantification of gene expression, the comparative Ct method was used. The final amount of the target gene, normalized to an endogenous reference gene as GAPDH (ΔCt = Ct target gene − Ct reference gene) was determined using the formula 2LPMCs were prepared in a phosphate-buffered saline buffer with 1% bovine serum albumin and 0.5% sodium azide, and were incubated with FITC labeled rat anti-mouse CD11b mAb (clone M1/70) and PE-labeled rat anti-mouse dectin-1 mAb (clone 2A11). The expression of CD11b and dectin-1 was examined via flow cytometry analysis . CD11b+ cells isolated from LPMCs were incubated with Alexa546-labeled SPG-antisense TNF-α at different time points . The uptake of the complex was determined via flow cytometry. For immunofluorescence, CD11b+ cells were incubated with Alexa546-labeled antisense TNF-α or FITC labeled-SPG-Alexa546-antisense TNF-α for 4 h. Fluorescence microscope analyses were performed to detect antisense TNF-α or SPG-antisense TNF-α in CD11b+ cells using a BZ-9000 digital fluorescence microscope .For therapeutic experiments in DSS-treated mice, 0.2 mg/kg of SPG, antisense TNF-α, or SPG-antisense TNF-α was administered to the colon via enema twice a week . To evaluate the activity of colitis, a video endoscopic system for mice was used  . The endt-test or the Mann–Whitney U test was used for the analysis of quantitative variables, and the chi-square test for qualitative variables. Two-sided p values of <0.05 were considered statistically significant.The student TNF-α production both in vitro and in vivo was significantly inhibited by SPG-antisense TNF-α. The topical therapy by SPG-antisense TNF-α ameliorated intestinal inflammation. A more effective therapeutic effect can be expected using β-glucan-based drug delivery system that conjugates SPG and antisense. Our result demonstrated the possibility of new topical therapeutic approach against the inflammatory bowel disease.


Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum.The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis.We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices.Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction.Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy. Patients with liver cirrhosis may develop minimal hepatic encephalopathy (MHE) with mild cognitive impairment, attention deficits, motor incoordination, and psychomotor slowing, which impair quality of life, reduce the lifespan, and increase accidents, falls, and hospitalizations. MHE affects several million people and is a serious health, social, and economic problem .Hyperammonemia and peripheral inflammation play synergistic roles in inducing cognitive and motor alterations in MHE –5.Studies in animal models of hyperammonemia and MHE have shown that the emergence of neurological alterations is mediated by the induction of neuroinflammation, which alters glutamatergic and GABAergic neurotransmission, leading to cognitive and motor impairment –15. BothIn agreement with the early emergence of neuroinflammation in the cerebellum, one of the earliest alterations in hyperammonemia and MHE is the impairment of motor coordination, which is modulated mainly by GABAergic neurotransmission in the cerebellum. We have recently shown that in hyperammonemic rats, motor incoordination is a consequence of increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum, which increases GABAergic neurotransmission. The administration of extracellular cGMP normalizes the activation of TNFR1, GABAergic neurotransmission, and motor coordination in hyperammonemic rats .However, it remains unclear how hyperammonemia induces an increase in TNF-a in the cerebellum and which cell types show increased TNF-a content.We recently showed that hyperammonemia per se induces peripheral inflammation, which is the main contributor to neuroinflammation, in the hippocampus and to cognitive impairment . SustainIt is therefore likely that hyperammonemia-induced peripheral inflammation is the trigger of TNF-a induction in the cerebellum of rats. The first aim of this study was to assess whether peripheral inflammation is responsible for the induction of neuroinflammation and TNF-a in the cerebella of hyperammonemic rats. To answer this question, we assessed whether preventing peripheral inflammation by treating rats with an anti-TNF-a antibody (infliximab) prevents neuroinflammation and TNF-a induction in hyperammonemic rats.In the central nervous system, microglia and astrocytes are usually the main sources of TNF-a. However, a few studies have shown that TNF-a synthesis is induced in neurons in some pathological situations, including stroke , intraceOver the course of the study, we found that sustained hyperammonemia induced the expression of TNF-a in Purkinje neurons.NF-κB is a major transcription factor that controls the expression of many genes, is involved in multiple aspects of innate and adaptive immune responses, inducing the expression of many proinflammatory genes, including TNF-a , 32, andIn resting cells, NF-κB dimers (predominantly p50/p65 complexes) are retained in the cytoplasm by binding to IkB. The activation of the IkB kinase (IKK) complex leads to the phosphorylation of IkB, dissociation of NF-κB, and translocation of NF-κB to the nucleus; NF-κB binds to specific DNA sequences and promotes the transcription of target genes, including TNF-a . We asseThe activation of NF-κB may be induced by the activation of different receptors, including the TNF-a receptor TNFR1. As we have previously shown that the activation of TNFR1 is increased in the cerebella of hyperammonemic rats , we asseFollowing TNFR1 stimulation, the intracellular death domain (DD) recruits RIP1 and TNFR-associated death domain (TRADD). TRADD engages TNFR-associated factor 2 (TRAF2), inhibitor of apoptosis protein 1 (cIAP1), and inhibitor of apoptosis protein 2 (cIAP2), leading to the formation of complex I , which aTo assess whether the activation of NF-κB and induction of TNF-a expression in Purkinje neurons is mediated by the activation of TNFR1, we assessed whether these processes are prevented by R7050. R7050 does not affect the binding of TNF-a to TNFR1 but selectively inhibits the association of TNFR1 with intracellular adaptor molecules  and thus inhibits subsequent cellular responses after TNF-a binding .We also assessed whether TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis and have suffered sustained hyperammonemia.To assess whether peripheral inflammation is responsible for the induction of neuroinflammation and TNF-a in the cerebella of hyperammonemic rats.To assess the cell type(s) in which TNF-a is increased in the cerebella of hyperammonemic rats.To provide some insights into the mechanisms by which sustained hyperammonemia induces TNF-a in Purkinje neurons to assess whether (a) the induction of TNF-a by hyperammonemia is associated with increased nuclear expression of NF-κB or (b) the activation of NF-κB and induction of TNF-a in Purkinje neurons are mediated by the activation of TNFR1.To analyze the time course of TNF-a induction in Purkinje neurons and glial cells following the induction of hyperammonemia.To assess whether TNF-a is also induced in the Purkinje neurons of patients who die with liver cirrhosis.Therefore, the aims of this work were as follows:Formalin-fixed paraffin-embedded sections (5-μm thick) from the cerebella of 4 patients who died with liver cirrhosis and 3 matched controls (subjects with no liver or neurodegenerative diseases) were obtained from the Instituto de Medicina Legal y Ciencias Forenses , Hospital Universitario Fundación Alcorcon and A Coruña Biobank. For each case, appropriate authorization to collect the tissue for research was obtained. The characteristics of the subjects, cause of death, and postmortem delay are detailed in Table Male Wistar rats were made hyperammonemic by feeding them an ammonium-containing diet as previously described . The expThe rats were randomly distributed into four groups: the control, control+infliximab, hyperammonemic, and hyperammonemic+infliximab group. Infliximab is an anti-TNF-a antibody that does not cross the blood-brain barrier , binds tRats were fed an ammonium-containing diet as described above and sacrificed at different times . In vivo analysis of microglial and astrocyte activation was performed after 1 week of hyperammonemia (blue box), and in vivo analysis of nuclear NF-kB and TNF-a protein expression in glia and Purkinje neurons was performed after 1 and 2 weeks of hyperammonemia (green box) . The brains were removed and postfixed in the same fixative solution for 24 h at 4 °C. Paraffin-embedded sections (5-μm thick) from the rats or humans were cut and mounted on coated glass slides, processed with the Envision Flex+ Kit (Dako) to block endogenous peroxidase activity for 5 min, and incubated with an anti-Iba-1 , anti-CD-68 , anti-GFAP , or anti-TNF-a  antibody. The reaction was visualized by incubation with Envision Flex+ horseradish peroxidase for 20 min and then with diaminobenzidine for 10 min. The sections were counterstained with Mayer’s hematoxylin for 5 min.+ cells was counted using the cell counter function of ImageJ, and the results are expressed as cells/mm2.Analysis of Iba-1, CD68, and GFAP staining in the white matter of the cerebellum was performed using ImageJ software. Brain sections from six animals per group were used. Microglial activation was assessed by measuring the perimeter of Iba-1-stained cells in 10 randomly selected 56x fields per section. The results are expressed in micrometers. To further assess microglial activation, staining for CD68, a marker of activated microglia , was anaFor GFAP quantification, the area of interest was selected. Using the auto local threshold and analyze particle functions, intensity thresholds and size filters were applied. To measure the total amount of GFAP, no size filter was applied. For each rat, at least 10 56x fields were quantified. The results are expressed as the percentage of the area stained with GFAP.2. For the analysis of Purkinje neurons, this region was manually selected using the freehand selection tool of the ROI manager function, and the mean intensity (M.I.) of TNF-a staining was recorded. Analysis of each region was performed in at least 10 56x fields (for the white matter) and 10 40x fields (for the Purkinje layer) from each rat or subject.Analysis of TNF-a staining in the white matter and Purkinje layer of the cerebellum was performed using ImageJ software. The number of cells expressing TNF-a in the white matter of the cerebellum was manually counted using the cell counter plugin of ImageJ, and the results are expressed as cells/mmFormalin-fixed paraffin-embedded sections from the cerebella of 4 cirrhotic patients and 3 control subjects were used for the immunohistochemical analysis of TNF-a expression in human Purkinje neurons as described above Fig. .Fluorescence in situ hybridization was performed to detect TNF-a mRNA expression in 5-μm cerebellar sections as previously described [2.escribed . In brieDouble immunofluorescence staining was performed to confirm the localization of TNF-a in microglia , astrocytes , and Purkinje neurons .Analysis of the p50 and p65 subunits of NF-κB was performed by immunofluorescence. Sections from six different animals from each group were selected, washed in 0.1 M phosphate buffer, and blocked with normal serum from the same species as the secondary antibody before being incubated overnight with primary antibodies (NF-κB p50 (1:200), NF-κB p65 (1:100), and Iba-1 (1:300); Abcam; GFAP (1:400); Sigma-Aldrich) diluted in blocking buffer and fluorescent secondary antibodies . The nuclei were counterstained with DAPI (Sigma-Aldrich), and the sections were coverslipped. The sections were observed under a confocal microscope (Leica TCS-SP2-AOBS) and imaged.The p50 and p65 subunits may be located in the nucleus, nucleolus, or cytosol. The nuclear, nucleolar, and cytoplasmic intensities of the p50 and p65 subunits were analyzed using ImageJ (1.48v). Nuclei and nucleoli were outlined on the blue (DAPI) channel using the ROI manager function, and the selection was applied on the green channel (p50 or p65) to measure fluorescence. The mean intensity (M.I.) for each nucleus or nucleolus was measured. For analysis of cytoplasmic NF-κB p50 and p65 subunits, the green channel was used; the cytosol of each cell was manually outlined using the freehand selection tool of ImageJ, and the mean intensity (M.I.) was recorded. The results are expressed as the nuclear/cytoplasmic ratios of the p50 and p65 subunits of NF-κB. In addition, the percentage of cells expressing the p50 subunit of NF-κB in the nucleolus was analyzed.Double immunofluorescence of the microglial marker Iba-1  or the astroglial marker GFAP  and the p50 subunit of NF-κB were to confirm the expression of NF-κB p50 in these glial cell types.2PO4, 26.2 NaHCO3, 2.5 CaCl2, and 11 glucose) aerated with 95% O2 and 5% CO2 at pH 7.4. Cerebellar slices  were cut and incubated for 20 min at 35.5 °C in Krebs buffer for stabilization. To assess whether the nuclear translocation of NF-κB and the induction of TNF-a synthesis in neurons are mediated by the activation of the TNFR1-TRADD/RIP1/TRAF2 complex, we blocked the formation of this complex by incubation with 20 μM R7050 [Control and hyperammonemic rats were sacrificed after 1, 2, or 4 weeks of hyperammonemia, and the cerebella were immediately immersed in ice-cold Krebs buffer  followed by Tukey’s post hoc test when there were more than two groups, while Student’s p < 0.001) in perimeter (164 ± 1 μm) compared with that observed in control rats (196 ± 6 μm)  Fig. d. Treatmμm) Fig. d.Fig. 2p < 0.0001) the number of cells stained with CD68 in the white matter to 11 ± 2 cells/mm2 compared to 4 ± 1 cells/mm2 in control rats  in hyperammonemic rats (28 ± 1%) compared to control rats (24 ± 1%)  to 233 ± 12 mm2 in hyperammonemic rats compared to 171 ± 4 mm2 in control rats   and astrocytes  to a similar extent  compared to control rats   compared to that in control subjects who died without liver or neurodegenerative diseases.Patients with liver cirrhosis show sustained hyperammonemia. We assessed whether this also leads to an increased level of TNF-a in Purkinje neurons. As shown in Fig. This study shows that sustained (4 weeks), but not short-term (2 weeks), hyperammonemia induces the expression of TNF-a in Purkinje neurons in rats. Moreover, TNF-a expression is also increased in Purkinje neurons of patients who die with liver cirrhosis and are exposed to sustained hyperammonemia. As discussed below, this induction of TNF-a may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy by altering the function of Purkinje neurons and neurotransmission and by inducing neuronal death.The induction of TNF-a in Purkinje neurons is associated with increased nuclear localization of NF-κB, a transcription factor that promotes the transcription of TNF-a mRNA . We showBoth the nuclear translocation of NF-κB and the increased level of TNF-a in Purkinje neurons are reversed by blocking the TNFR1-TRADD/RIP1/TRAF2 signal transduction pathway through the activation of the TNF-a receptor TNFR1 Fig. b. This sIn fact, the data reported here show that hyperammonemia induces the synthesis of TNF-a in activated microglia and astrocytes after 2 weeks of hyperammonemia, when TNF-a was not increased in Purkinje cells. We also show that after 1 week of hyperammonemia, both microglia and astrocytes are activated, but TNF-a expression is not yet induced. This indicates that the process follows a time course in which hyperammonemia induces the activation of microglia and astrocytes at 1 week. This is followed by the induction of TNF-a in both types of glial cells (but not in Purkinje cells) at 2 weeks. TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to the induction of TNF-a expression. It is entirely plausible that activated microglia or astrocytes produce a plethora of signals that can cause an increase in TNFa expression in neurons and that treatment with R7050 may act on glia to prevent the release of a number of factors in a TNFR1-dependent manner that may then act on neurons. Further in vitro studies and/or the use of cell-specific TNFR1 knockout models will help to better elucidate the exact mechanisms involved.We have recently shown using the same animal model used here that hyperammonemia per se induces peripheral inflammation by increasing proinflammatory TNF-a, IL-6, and PGE2 and decreasing anti-inflammatory IL-10 . These fHere, we show that blocking peripheral TNF-a with an anti-TNF-a antibody (infliximab) prevents the activation of microglia and astrocytes and the induction of TNF-a, indicating that peripheral TNF-a and inflammation trigger glial activation and all the above process, leading to TNF-a expression in Purkinje neurons. However, the mechanisms by which peripheral inflammatory signals are able to induce glial cell activation and TNF-a expression in the cerebellum are unclear. There are several mechanisms by which systemic inflammation may induce neuroinflammation, including (a) the active transport of certain cytokines into the brain ; (b) thePhoneutria nigriventer spider venom causes blood-brain barrier permeability and induces the expression of TNF-a in Purkinje neurons in rats [TNF-a synthesis in neurons has already been reported in some pathological situations, including spinal cord injury , stroke  in rats .These studies suggest that the induction of TNF-a expression in neurons, including Purkinje neurons, may occur in different pathological situations and is preceded by TNF-a induction in glial cells. However, the underlying mechanisms remain unclear.We show here that sustained hyperammonemia and peripheral inflammation induce TNF-a expression in Purkinje neurons in rats and in patients who die with chronic liver cirrhosis and hyperammonemia. Moreover, we provide some insights into the time course of and mechanisms involved in this process Fig. . HyperamThe induction of TNF-a by sustained hyperammonemia may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy by altering the function of Purkinje neurons and neurotransmission and by inducing neuronal death.The trafficking and surface membrane expression of AMPA and NMDA receptors is altered by high levels of TNF-a –54. IncrMoreover, TNF-a has been shown to induce excitotoxicity-mediated neuronal loss in some neurodegenerative diseases . Under nIn summary, this study shows that sustained (but not short-term) hyperammonemia induces TNF-a expression in Purkinje neurons; this may be due to the activation of TNFR1 by TNF-a produced in glia , which induces the nuclear translocation of NF-κB and the induction of TNF-a mRNA transcription. The induction of TNF-a in Purkinje neurons alters the function of these cells and contributes to altered neurotransmission and neurological impairment. TNF-a is also induced in the Purkinje neurons of patients who die with liver cirrhosis and contributes to the loss of Purkinje cells in these patients. The induction of TNF-a in the Purkinje neurons of hyperammonemic rats is prevented in vivo by peripheral treatment with an anti-TNF-a antibody and ex vivo by treatment with R7050, which prevents the transduction of TNFR1 signals into Purkinje neurons, the nuclear translocation of NF-κB and the induction of TNF-a. This suggests that treatments that reduce peripheral inflammation or the activation of NF-κB may reduce the deleterious effects of sustained hyperammonemia and hepatic encephalopathy. The use of molecules to inhibit elements of the NF-κB signaling pathway has also been suggested as a therapeutic option for neurological disorders such as  multiple