Type I diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreas. Destruction of the body's own proteins, cells, and tissues is precipitated by the dysfunction of cytokine production, protein modification, and signaling pathways in immune cell subtypes. Tumor Necrosis Factor α (TNFα) and its receptors Tumor Necrosis Factor 1 (TNFR1) also known as p55 and TNFRSF1A, and Tumor Necrosis Factor 2 (TNFR2) also known as P75 and TNFRSF1B play a crucial role in this autoimmune process. TNFα has been shown to stimulate cell death through TNFR1 signaling by the caspase system, while promoting cell survival through TNFR2 signaling using the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-𝜅B) pathway. Recent findings show a defect in immuno-proteasomes found in autoreactive T cells in people with T1D. This defect causes improper signaling transduction when TNFα binds to TNFR2. The inability to save the cell by activating the NF-𝜅B pathway eventually leads instead to apoptosis using the caspase system. A decrease in TNFα or increase in soluble TNFα receptors might be an explanation for these autoreactive T cells to evade the host immune system, and allow them to cause destruction of the pancreas. We hypothesize that patients with T1D will show abnormal distribution of TNFα and its receptors at basal levels, as well as when stimulated with interleukins, cytokines, and bacteria such as interleukin-2 (IL-2), lipotechoic acid (LTA), granulocyte macrophage-colony stimulating factor (GM-CSF), and Bacillus Calmette-Guérin (BCG). To test this hypothesis, we obtained peripheral blood from T1D patients (n=102) and controls (n=89) and performed in vitro stimulation assays. After a 48-hour incubation, tissue culture supernatants were collected and analyzed for TNF and its receptors production by ELISA, as well as densities of cell membrane receptors by flow cytometry. The data from this study showed significant differences in basal levels of TNFα, TNFR1, and TNFR2 on both the membrane and in the serum between patients and controls. Patients contained a greater percentage of CD4, 8, and 14 - TNFR2 and not TNFR1 double positive cells than their healthy control counterparts. Patient's sera also contained higher levels of all three markers, sTNFα, sTNFR1, and sTNFR2 than the controls. However, no significant differences were found between patient and controls when stimulated with the various compounds listed above.

Introdução: A artrite reumatoide é uma doença inflamatória autoimune associada à complicações sistêmicas como fadiga e perda muscular. Perda muscular pode estar relacionada com a ativação do sistema ubiquitina-proteossomo. O objetivo deste trabalho foi avaliar a perda muscular e o evolvimento do proteossomo no modelo de artrite induzida por colágeno (CIA), com ou sem o tratamento de metotrexato ou inibidor de TNF (etanercepte). Métodos: Camundongos DBA1/J machos foram divididos em 4 grupos (n=8 cada): CIA (salina); ETN (etanercepte, 5.5 /) e MTX (metotrexato, 35 /), tratados duas vezes por semana por 6 semanas, e um grupo controle saudável (CO). Tratamentos iniciaram uma semana após a injeção do booster. Escore clínico, edema da pata traseira e peso corporal foram analisados durante o período experimental. Músculo gastrocnêmio (GA) foi pesado após a morte e usado para quantificar a atividade, níveis proteicos e expressão de mRNA das diferentes subunidades do proteossomo através de ensaio fluorogênico, Western blot e rtPCR, respectivamente. Resultados: Tratamentos reduziram o desenvolvimento da doença, observado através do menor escore clínico e edema da pata traseira nos grupos ETN e MTX. ETN apresentou maior peso corporal do que MTX nas semanas 5 e 7. Músculo GA estava aumentado em ETN do que CIA e MTX, um resultado também observado no peso muscular normalizado. As propriedades catalíticas do proteossomo 26S muscular mostraram um aumento na atividade do tipo caspase nos grupos CIA e MTX. Tecidos musculares de animais MTX demonstraram maiores níveis proteicos das subunidades do proteossomo PSMB8 e PSMB9 e maior expressão gênica de Psmb5, Psmb8 e Psmb9. Por outro lado, a expressão de Psmb6 estava diminuída e de Psmb9 estava aumentada em CIA. Conclusões: Apesar de ambos os medicamentos melhorarem o escore da doença, ETN apresentou um afeito anti-artrítico mais forte e foi o único tratamento capaz de prevenir parcialmente a perda muscular. Ao contrário de ETN, CIA e o tratamento com MTX apresentaram perda muscular e atividade e expressão do proteossomo aumentadas. / Background: Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. The aim of this study was to evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods: Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 /) and MTX (methotrexate, 35 /), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR, respectively. Results: Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX groups. ETN presented higher body weight compared to MTX group at weeks 5 and 7. GA weight was heavier in ETN than CIA and MTX, a result also observed in the normalized muscle weight. The catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups. Muscles tissues of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and higher gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions: Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, CIA and MTX treatment did not prevent muscles loss due to increased proteasome expression and activity.

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine that is predominantly produced by activated macrophages. The effects of TNFα are as diverse as the cells with which it interacts, e.g., stimulating fibroblast growth, exerting cytotoxic/cytostatic; activity against various human and murine cell lines, promoting inflammation through upregulation of endothelial adhesion molecules and IL-8 production. Yet TNFα is best known, and in fact was originally described, for its role in the bacterial-induced hemorrhagic necrosis of tumors and exacerbation of septic shock in which aberrant TNFα production leads to vascular collapse, cachexia, multiple organ failure, and ultimately death in as many as 100,000 people each year in the United States alone. LPS, a component of the outer cell wall of gram-negative bacteria, is the principal inducer of macrophage TNFα production. TNFα production can be enhanced by IFNγ which also induces upregulation of TNFα receptors allowing for the establishment of a TNFα autocrine loop. It has been hypothesized that autocrine TNFα stimulation plays a critical role in the induction of macrophage effector function, e.g., nitric oxide production. This dissertation represents efforts to evaluate the respective roles of the TNFα receptors in the induction of macrophage effector function, in addition to examining the mechanism by which autocrine TNFα exerts its effects on macrophages. Exploiting the species specificity of the murine TNFα receptor type 2 (TNF-R2), splenic macrophages were stimulated with human TNFα (which binds to TNF-R1 but not TNF-R2), in the presence of IFNγ. Human TNFα was effective in the induction of nitric oxide production, albeit at concentrations 12.5-fold greater than those required by murine TNFα to achieve the same result. Addition of anti-TNF-R1 completely inhibited the murine TNFα mediated induction of macrophage effector function. However, treatment with anti-TNF-R2 resulted in partial inhibition of macrophage activation. Taken together this data suggests that the primary TNFα mediated signals involved in macrophage activation are transduced through TNF-R1, although TNF-R2 appears to contribute to the intensity of the macrophage response. To evaluate the role of autocrine TNFα signaling in the induction of macrophage effector function, immortalized macrophages from normal C57Bl/6J mice (B6/J2) and C57Bl/6J mice containing gene targeted disruptions of the TNF-R1 and TNF-R2 genes (TRN) were stimulated under CD14-dependent and CD14-independent conditions. Although the B6/J2 and TRN clones mounted similar NO responses to LPS in the presence of serum, the TRN macrophages generated a weak nitric oxide response as compared to B6/J2 when stimulated with LPS under serum-free conditions. The involvement of TNFα autocrine stimulation in the CD14-independent activation was corroborated by the ability of soluble TNF-R1 to inhibit the response of B6/J2 macrophages to LPS in serum-free medium. CD14-independent LPS stimulation of TRN and B6/J2 resulted in equivalent levels of IL-1β, TNFα, and NOS gene expression, as determined by RT-PCR, and in release of equivalent amounts of biologically active TNFα. However, western blot analysis revealed that NOS protein production by TRN was as much as 50% less than that produced by B6/J2. These results indicate that autocrine TNFα stimulation contributes to the signaling pathways initiated by ligation of CD14-independent LPS receptors and may be involved in NOS post-transcriptional regulation.

As the largest endocrine organ, adipose tissue secretes many bioactive molecules that circulate in blood, collectively termed adipokines. Efforts to identify such metabolic regulators have led to the discovery of a family of secreted proteins, designated as C1q tumor necrosis factor (TNF)-related proteins (CTRPs). The CTRP proteins, adiponectin, TNF-alpha, as well as other proteins with the distinct C1q domain are collectively grouped together as the C1q/TNF superfamily. Reflecting profound biological potency, the initial characterization of these adipose tissue-derived CTRP factors finds wide-ranging effects upon metabolism, inflammation, and survival-signaling in multiple tissue types. CTRP3 (also known as CORS26, cartducin, or cartonectin) is a unique member of this adipokine family. In this review we provide a comprehensive overview of the research concerning the expression, regulation, and physiological function of CTRP3. © 2017 American Physiological Society. Compr Physiol 7:863-878, 2017.

abstract: Nicotine self-administration is associated with decreased expression of the glial glutamate transporter 1 (GLT-1) and the cystine-glutamate exchange protein xCT in the nucleus accumbens core (NAcore). N-acetylcysteine (NAC), which is an antioxidant, anti-inflammatory, and glutamatergic agent, restores these proteins associated with increased relapse vulnerability. However, the specific molecular mechanisms driving NAC inhibitory effects on cue-induced nicotine reinstatement are unknown. Thus, the present study assessed NAC’s effects on cue-induced nicotine reinstatement are dependent on NAcore GLT-1 expression. Here, rats were treated with NAC in combination with intra-NAcore vivo-morpholinos to examine the role of GLT-1 in NAC-mediated inhibition of cue-induced nicotine seeking. Subchronic NAC treatment attenuated cue-induced nicotine seeking in male rats and an antisense vivo-morpholino (AS) designed to selectively suppress GLT-1 expression in the NAcore blocked this effect. NAC treatment was also associated with an inhibition of pro-inflammatory tumor necrosis factor alpha (TNFα) expression in the NAcore. As well, GLT-1 AS markedly increased expression of CD40, a known marker of pro-inflammatory M1 activation of microglia and macrophages. To further examine whether NAC-induced decreases in nicotine seeking involve suppression of TNFα, we manipulated a downstream mediator of this pathway, nuclear factor kappa B (NF-kB). Considering the putative role of NF-κB in learning, memory, and synaptic plasticity, separate experiments were performed where rats were treated with herpes simplex virus (HSV) vectors designed to increase (HSV-IKKca) or decrease (HSV-IKKdn) NF-κB signaling through interactions with IκB Kinase (IKK). The goal was to examine the role of NF-κB signaling in mediating nicotine seeking behavior and if NF-κB signaling regulates GLT-1 expression. HSV-IKKdn alone and in combination with NAC inhibited cue-induced nicotine reinstatement, while HSV-IKKca blocked the attenuating effect of NAC on reinstatement. Interestingly, both HSV-IKKdn and HSV-IKKca, regardless of NAC treatment, inhibited GLT-1 expression. Taken together, these results suggest that while GLT-1 may be a conserved neurobiological substrate underlying relapse vulnerability across drugs of abuse, immunomodulatory mechanisms may regulate drug-induced alterations in glutamatergic plasticity that mediate cue-induced drug-seeking behavior through GLT-1-independent mechanisms. / Dissertation/Thesis / Masters Thesis Psychology 2019

Anti-TNF alpha antibodies were among the first approved antibody drugs and now belongs to the best-selling drugs. Today, several companies are developing biosimilars to those drugs which will increase the access of medications and potentially reduce health care costs. There is a great demand for pharmacokinetic assays for anti-TNF-alpha drugs and the bridging assay format is a potential tool, mostly due to its high serum tolerance. This project at Gyros Protein Technologies AB aimed to investigate the properties of the solid phase on the Gyrolab and to utilize this to optimize the bridging assay to be used as a pharmacokinetic assay for a human antibody in the presence of serum. The solid phase was optimized by incorporating three reagents with increasing molecular weight and examining the column profiles generated. Furthermore, the capture reagent was titrated with b-BSA to avoid cross-binding of both arms of the antibody to the capture reagent. Since the background was relatively high, further optimization was done to reduce background and increase the signal to noise ratio. The performance of the optimized bridging assay was compared to alternative PK assay formats. The estimated sensitivity of the bridging assay was 5 ng/ml compared to 250 ng/ml for the indirect antibody assay and 2.5 ng/ml for the bridging assay using an anti-idiotypic antibody as detect. The optimized bridging assay performed well without dilution in buffer and was therefore used for affinity determination of Humira in neat serum. Variable concentrations of TNF-alpha were added to a fix concentration of Humira to compete with the interaction. Calculated KD-values were similar regardless of whether the measurements were performed in neat serum or after dilution in Rexxip buffer.

Glioblastoma multiforme (GBM) is the most lethal primary central nervous system tumor, with median survival after diagnosis of less than 12 months because dissemination into the brain parenchyma limits the long-term effectiveness of surgical resection, and because GBM cells are resistant to radiation and chemotherapy. This sad dismal prognosis for patients with GBM emphasizes the need for greater understand of the fundamental biology of the disease. Invasion is one of the major causes of treatment failure and death from glioma, because disseminated tumor cells provide the seeds for tumor recurrence. Inflammation is increasingly recognized as an important component of invasion. In the brain, inflammation can occur by activation of microglia, the resident macrophages of the brain, or by tumor-associated blood macrophages. Therefore, we hypothesize that activity of the innate immune system in the brain can influence tumor progression by secreting cytokines such as Tumor Necrosis Factor alpha (TNF-α). In this study, we show that patient-derived glioma spheres undergo morphological changes in response to TNF‑α that are associated with changes in migration behavior in vitro. These morphological changes include appearance of tumor islands in site different from where the primary tumor cells were seeded. We further showed that TNF‑α treated cells significantly increased expression of cell adhesion molecules such as CD44 and VCAM-1. Furthermore, we demonstrate increased cell density also caused increased in expression of cell adhesion molecules. The extent to which these are recapitulated in vivo will be investigated.

Antipsychotic medications remain the first line of treatment for individuals diagnosed with schizophrenia (SCZ). However, antipsychotic treatment is often not compliant due to dysregulation of both the central (CNS) and autonomic (ANS) nervous systems, resulting in debilitating dose-dependent side effects. Recent work suggests a new approach for treatment of SCZ that could potentially lower treatment doses and reduce side effects. Increased neuroinflammation has been shown in patients diagnosed with SCZ, particularly within the prefrontal cortex (PFC) and hippocampal (HPC) regions of the brain. Tumor necrosis factor-alpha (TNFa) is one of the key pro-inflammatory cytokines observed to be secreted during the inflammatory response. When TNFa is chronically secreted, resident CNS microglia become pro-inflammatory and toxic to the local environment. Microglial activation alongside of dopamine dysregulation thereby results in both the behavioral and neuroinflammatory aspects of SCZ. In this study, we hypothesized dietary administration of two different novel TNFamodulators (PD2024 – Experiment 1 and PD340 – Experiment 2) developed by our collaborators from P2D Bioscience, Inc. (Cincinnati, OH) would alleviate auditory sensorimotor gating deficits and reduce microglial cell activation caused by neonatal polyinosinic:polycytidylic acid (Poly I:C) treatment in rats, which is a validated rodent model of SCZ. Four groups (Experiment 1: Poly IC/PD2024, Poly IC/Control, Saline/PD2024, Saline/Control and Experiment 2: Poly IC/PD340, Poly IC/Control, Saline/PD340, Saline/Control) were intraperitoneally administered either Poly I:C (2 mg/kg) or saline (0.9% NaCl) from postnatal days 5-7. From P30-67, animals were placed on the experimental diet containing either low (10 mg/kg) or high (30 mg/kg) doses of either PD2024 or PD340, whereas the control animals remained on a normal diet. Prepulse inhibition (PPI) was used to test for auditory sensorimotor gating (behavioral abnormalities) in both adolescence (P44-46) and in adulthood (P60-66). At P67, immunohistochemistry (IHC) and confocal microscopy were used to evaluate and examine microglial cell activation using the Iba1-GFP antibody (neuroinflammatory abnormalities) in the PFC and HPC. Results revealed auditory sensorimotor gating deficits in Poly IC/Controls were alleviated in both adolescence and adulthood with either PD2024 or PD340. It was also found that both TNFa modulators significantly reduced microglial activation in the HPC, but not the PFC. The data supports our hypothesis that dietary administration of PD2024 or PD340 alleviates behavioral deficits and decreases neuroinflammation generated from the Poly I:C rodent model of SCZ. Therefore, an approach with a TNFa modulator alongside of current antipsychotic medications could treat both the behavioral and neuroinflammatory aspects of SCZ.

Ce travail suggère que l'apotose régule différentiellement la demi-vie des populations lymphocytaires T productrices de cytokines. Processus anti-inflammatoire physiologique, l'apoptose exerce principalement son contrôle négatif sur les cellules T productrices de TNFa. Lors des traitements anti-rétroviraux combinés, la réduction de l'apoptose lymphocytaire est associée à une polarisation pro-inflammatoire qui pourrait favoriser la constitution des anomalies métaboliques mais aussi la survenue des pathologies inflammatoires associées à la restauration immunitaire.

Le TNF-α est une cytokine pro-inflammatoire jouant un rôle délétère dans de nombreuses pathologies. Nous nous sommes intéressés à l'inhibition du TNF-α à l'aide de variants du récepteur soluble de type I du TNF-α (hTNFR-Is) administrés par thérapie génique non virale. Trois variants ont été étudiés: un monomère hTNFR-Is, correspondant au récepteur soluble physiologique, une protéine chimérique hTNFR-Is/mIgG1, dont l'efficacité par administration protéique est reconnue, et une forme dimérique obtenue par association de deux fragments hTNFR-Is à l'aide d'un espaceur polyglycine. La vectorisation des plasmides codant ces variants a été étudiée par différentes voies afin d'obtenir un effet systémique (électrotransfert intramusculaire, greffe de cellules autologues), ou un effet local (électrotransfert intra-articulaire (genou de souris), électrotransfert intra-oculaire). L'électrotransfert intramusculaire permettait d'obtenir une expression de protéine à long terme (supérieure à 6 mois) et dose-dépendante. La protéine chimérique était très stable dans la circulation, et était sécrétée à un taux élevé. Cette procédure n'induisait pas de réponse immune contre la protéine transgénique. Nous avons démontré l'obtention, par électrotransfert intra-articulaire, d'une expression dosedépendante du transgène durant deux semaines. Le passage de la protéine dans la circulation était faible, ce qui était l'objectif de cette approche locale. Aux fortes doses, une réponse immune contre la protéine chimérique était observée. Nous avons montré la faisabilité de l'électrotransfert dans un muscle lisse, le muscle ciliaire de l'œil. L'expression obtenue était uniquement localisée dans le muscle ciblé, et la procédure était sûre (pas d'inflammation ni de dommages observables). L'expression du transgène était supérieure à un mois, sans passage de la protéine dans la circulation. L'électrotransfert intramusculaire de variants du hTNFR-Is était efficace (forme chimérique principalement) dans un modèle de polyarthrite rhumatoïde (arthrite expérimentale au collagène) sur les signes cliniques et histologiques de la maladie, par un unique électrotransfert à l'apparition des signes cliniques de la maladie. La comparaison de ce traitement à l'injection répétée de protéine recombinante illustrait la potentialité d'une approche par thérapie génique, de part son efficacité à long terme. Les approches locale et cellulaire restent à tester dans ce modèle. L'électrotransfert intramusculaire de plasmide codant les hTNFR-Is ne permettait pas d'améliorer la récupération des fonctions motrices après un traumatisme crânien, dans un modèle murin, même si la protéine était détectée dans le cerveau, et capable d'inhiber le TNF-α. Ces résultats sont cependant très préliminaires. L'électrotransfert intra-oculaire du plasmide codant la forme chimérique hTNFR-Is/mIgG1 permettait d'inhiber efficacement les signes cliniques et histologiques dans un modèle expérimental d'uvéite (uvéite expérimentale aux endotoxines). Nos résultats mettent en évidence l'efficacité de l'électrotransfert pour délivrer un gène thérapeutique et obtenir une production locale ou systémique (selon la stratégie utilisée) de protéine thérapeutique. Nos résultats illustrent le potentiel de nouvelles cibles (articulation, muscle lisse de l'œil) pour cette technologie, ce qui est encourageant pour l'application future de l'électrotransfert à d'autres tissus/organes cibles et à d'autres pathologies.