<p>Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets <i>in vitro</i>. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction.</p><p>In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed <i>in vivo</i> these mediators would probably contribute to β-cell destruction.</p><p>The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets.</p><p>We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells <i>in vitro</i> and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction.</p><p>To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.</p>

Les recherches menées au sein de notre laboratoire ont montré que la Nicotinamide Phosphoribosyltransférase (Nampt) est surexprimée lors de l’activation de plusieurs cellules immunes. Cette surexpression est surprenante si l’on considère que cette enzyme intervient dans le métabolisme de base de toute cellule (immune et non immune) en participant à la synthèse du cofacteur NAD. Au cours de ce travail, nous avons essayé de comprendre quelle est la contribution de cette enzyme aux fonctions immunes. Puisque les souris génétiquement invalidées pour le gène de la Nampt meurent à un stade embryonnaire précoce nous avons invalidé ce gène de façon conditionnelle uniquement dans les lymphocytes T et B. Nos résultats suggèrent que la délétion de la Nampt dans les cellules T et B compromet leur survie. De plus, nous avons testé l’effet d’un inhibiteur spécifique de la Nampt sur la sécrétion de TNF par des macrophages et des cellules dendritiques. Nos résultats montrent que l’inhibition de la Nampt s’accompagne d’une diminution du taux de NAD intracellulaire et, parallèlement, d’une réduction de la quantité de TNF synthétisé. De même, nous avons montré qu’en augmentant le taux de NAD cellulaire il est possible d’augmenter la quantité de TNF produit, ce qui laisse supposer que la capacité de la cellule à synthétiser du TNF serait directement liée à son contenu en NAD. Cette régulation semble impliquer une étape post-transcriptionnelle puisque l’ARNm du TNF ne paraît pas être affecté par ces traitements. Puisque le taux de NAD peut influencer directement l’activité d’enzymes NAD-dépendantes, et en particulier l’activité des enzymes de la famille des sirtuines, nous nous sommes demandés si une sirtuine était impliquée dans la synthèse du TNF. Une approche pharmacologique et une approche génétique nous ont permis de montrer que SIRT6 serait capable d’augmenter la production de TNF à une étape traductionnelle. Cette conclusion semble être confirmée par le fait que des cellules dendritiques dérivées de souris invalidées pour le gène SIRT6 produisent moins de TNF en réponse à un stimulus microbien par rapport à des cellules sauvages. En conclusion nos observations suggèrent la Nampt, en affectant le niveau intracellulaire de NAD, joue un rôle important dans le contrôle de la production du TNF par les cellules de système immunitaire

Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets in vitro. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction. In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed in vivo these mediators would probably contribute to β-cell destruction. The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets. We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells in vitro and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction. To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.

Thesis (doctoral)--Rijksuniversiteit te Groningen. / Description based on print version record.

Hypertonic saline (HS) is an accepted treatment for traumatic brain injury (TBI). However, the behavioral and cognitive consequences following HS administration have not thoroughly been examined. Recent preclinical evidence has suggested that nicotinamide (NAM) is beneficial for recovery of function following TBI. The first study compared the behavioral and cognitive consequences of HS and NAM as competitive therapeutic agents for the treatment of TBI. Following controlled cortical impact (CCI), bolus administrations of NAM (500 mg/kg), 7.5% HS, or 0.9% saline vehicle (1.0 mL/kg) were given at 2, 24, and 48 hrs post-CCI. Behavioral results revealed that animals treated with NAM and HS showed significant improvements in beam walk and locomotor placing compared to the Vehicle group. The Morris water maze (MWM) retrograde amnesia test was conducted on day 12 post-CCI and showed that all groups had significant retention of memory compared to injured, Vehicle-treated animals. Working memory was also assessed on days 18-20 using the MWM. The NAM and Vehicle groups quickly acquired the task; however, HS animals showed no acquisition of this task. Histological examinations revealed that the HS-treated animals lost significantly more cortical tissue than either the NAM or Vehicle-treated animals. HS-treated animals showed a greater loss of hippocampal tissue compared to the other groups. In general, NAM showed a faster rate of recovery than HS without this associated tissue loss. Study 1 suggested that future research into HS should include drug injection time course studies. Multiple injections may be responsible for the notable tissue damage. Therefore, it is possible that fewer injections will result in comparable behavioral recovery and less tissue damage that was observed. Due to the detrimental effects of 7.5% HS on cognition and hippocampal tissue following multiple administration in study 1, the proposed second study sought to study the behavioral and cognitive effects of HS using either single or multiple injection regime. The proposed study entailed a lengthier testing schedule than in study 1 and included the same histological examination to compare the different dosages. Additionally, edema formation was measured 24 hours following each drug endpoint in order to delineate the possible underlying mechanism of the observed deficits. In Study 2, HS tended to improve function on motor, sensorimotor and neurological tasks. Although this was a trend on all tests, animals treated with a single administration of HS overall performed better on all tasks compared to those receiving double or multiple injections. In the retrograde amnesia test, although not significant, the Sham, HS-2, and HS-24 animals showed improvement; whereas, the Vehicle and HS-48 animals showed no improvement in performance. This could possibly be linked to the additional hippocampal tissue loss that was noted in the HS-48 animals. In the working memory paradigm, the HS-2 and Vehicle groups had longer latencies to reach the platform than did the Sham group. However, after the first testing day, there were no significant differences between any of the groups. All animals treated with HS performed at the same rate and their performance either stayed the same over the three day testing period or became worse. It appears these animals were unable to learn and improve in the new memory acquisition task which is comparable to the results found in study 1. In study 1, there were again no observed hippocampal volume differences between the Sham and Vehicle-treated animals. However, there was extensive hippocampal tissue damage observed in all of the HS groups. Furthermore, animals treated with a single administration of HS had less hippocampal loss than those with double or multiple doses. Those animals receiving more than one dose of HS lost significantly more hippocampal tissue than the Vehicle group. The results of study 2 are comparable, and support, the results of study 1. Both studies support the strengths and weakness of HS therapy following TBI. Although there are potential benefits of HS therapy, there are also detrimental risks involved. Cognitive and structural damage could possible occur if the dosage amounts are not closely studied and monitored. Although the use of HS may be beneficial to reduce ICP following TBI, it appears that the use of HS may also lead to direct or indirect tissue loss possibly by chronic cellular dehydration. Stronger or more delineated effects may be noticed using higher doses or concentrations of HS in future studies. However, due to the nature of these results, caution should be advised with the use of all therapeutic usage of HS until further detailed studies are conducted.

Traumatic Brain Injury (TBI) is a leading cause of death and disability in the United States for which there are no federally approved pharmacological treatments. Preclinical trials with nicotinamide (NAM) and progesterone (Prog) treatment demonstrate beneficial neuroprotection and recovery of function following TBI. The primary goal of this study was to assess both neuroprotection and recovery of function in an animal model of TBI after combination treatment of both NAM and Prog. Animals received a cortical contusion injury over the sensorimotor cortex and were treated with either nicotinamide (75 mg/kg, i.p. NAM loading dose, 12 mg/kg/hr NAM, s.c. over 72 hrs), Prog (10 mg/kg Prog, i.p. over 72 hrs), NAM and Prog(75 mg/kg, i.p. NAM loading dose, followed by continuous infusion of 12 mg/kg/hr NAM, s.c. over 72 hrs; 10 mg/kg Prog, i.p. over 72 hrs) or Vehicle (75 mg/kg, i.p. sterile saline loading dose, followed by continuous infusion 12 mg/kg/hr sterile saline, s.c. over 72 hrs; 10 mg/kg peanut oil, i.p. over 72 hrs), and compared to a craniotomy only (Sham) group. Following this regimen they were assessed in a battery of behavioral (fine and gross motor, sensory, and cognitive) tasks or a histological assessment at 24 hrs post-injury assessing lesion cavity size, degenerating neurons, and reactive astrocytes. Our results replicate the beneficial effects of treatment with either NAM or Prog demonstrating significant improvements in recovery of function, and a reduction in lesion cavitation, degenerating neurons and reactive astrocytes 24 hours post-injury. The combination treatment of NAM and Prog led to a significant improvement in both neuroprotection at 24 hrs post-injury and recovery of function in sensorimotor related tasks when compared to each individual treatment (NAM or Prog). It is suggested here that further preclinical trials using NAM and Prog as a combination treatment should be done to identify any drug interactions, pharmacokinetics, and a new window of opportunity and proper dosing of this combination treatment.

Traumatic brain injury (TBI) is a serious problem facing the medical community. Every year, over 1.7 million TBIs occur in the United States alone (CDC, 2010). Over 25 years of research and 21 major clinical trials have failed to yield a pharmaceutical treatment for this complicated injury (Maas et al., 2007). One of the possible reasons for the many clinical failures is a lack of behavioral assessment at the level of animal models. In particular, there is typically only one type of cognitive measure employed in most studies, usually a measure of spatial learning (e.g. Morris water maze). In other fields, alternative measures have been utilized for many years, including non-spatial discriminations. The primary goal of this study was to evaluate the use of a visual discrimination in a model of frontal TBI and determine whether or not administering a neuroprotectant could alleviate deficits in discrimination. Long-Evans rats were trained on a simple visual discrimination task and a progressive ratio schedule of reinforcement (PR). After assessing their baseline discrimination performance, motor ability and PR performance, they were advanced to surgery and given either a bilateral frontal controlled cortical impact TBI or sham procedure. Following TBI, injured rats were given either doses of the neuroprotectant nicotinamide (NAM; 150 mg/kg, i.p.) or a saline solution (1 ml/kg, i.p.). They were then assessed for 35 days on the discrimination and progressive ratio. On days 7, 14 and 27, motor abilities were assessed in automated motor activity monitors. On days 15-18 and 21-23 post-surgery, rats were also assessed on the Morris water maze (MWM). On day 43 post-surgery, rats were transcardially perfused and brains extracted. Brains were briefly post-fixed and then sliced on a sliding microtome at 40 µm. These slices were then mounted to slides and stained with cresyl violet to examine the extent of the lesion. Brain injury impaired performance on the discrimination task and PR task. In the discrimination task, deficits were primarily driven by an inability to complete chains of responses. On the PR task, deficits were characterized by reduced break points and low rates of responding. Administration of NAM reduced deficits in discrimination and PR performance. There were no gross motor deficits as a result of the injury. On the MWM task and measures of lesion size, there were no improvements due to NAM administration. Based on the outcome of this study, operant measures such as discrimination or progressive ratio could be incorporated into the testing battery for experimental TBI. Despite some of the challenges of adapting tasks designed for single-subject analysis, it is well worth the time spent due to the robust ability to detect deficits. Additionally, this study showed that nicotinamide administration was neuroprotective across multiple tasks, suggesting that these tasks are indeed well suited for the assessment of pharmacological agents and that nicotinamide has treatment potential for clinical populations.

nÃo hà / A tuberculose à uma das principais causas de mortalidade no mundo, porÃm à uma doenÃa negligenciada por ser endÃmica de paÃses em desenvolvimento. Um dos principais pontos de tratamento da tuberculose à a morte do bacilo causador, o Mycobacterium tuberculosis, atravÃs da interrupÃÃo da produÃÃo de Ãcidos micÃlicos, componentes da parede celular do bacilo, usando como um dos alvos a enzima InhA, porÃm esta rota tambÃm à a principal causa de resistÃncia. O presente trabalho se propÃe a estudar a enzima InhA, realizando modelagens in silico de interaÃÃes entre a enzima e ligantes selecionados. Os ligantes estudados fazem parte de duas bibliotecas distintas, sendo uma de compostos orgÃnicos selecionados por sua similaridade com o substrato da enzima. A outra biblioteca à composta de complexos metÃlicos com o nÃcleo pentacianoferrato, variando-se o ligante auxiliar. A justificativa para esta classe de compostos ser utilizada se dà pelo fato de o complexo pentacianoisoniazidaferato (II) ter apresentado atividade anti-tuberculose tanto in vitro como por via oral em ratos. Os ensaios de docking foram realizados utilizando-se duas abordagens, uma completamente rÃgida e outra em que a proteÃna era rÃgida e o ligante era flexÃvel. Ambos os ensaios apresentaram boa correlaÃÃo entre os seus resultados, independentemente da funÃÃo de avaliaÃÃo utilizada. Observou-se que as melhores estruturas em termos de inibiÃÃo possuÃam uma quantidade razoÃvel de interaÃÃes hidrofÃbicas, de modo a manterem-se estÃveis no sÃtio de ligaÃÃo da enzima que possui baixa polaridade. / Tuberculosis is found among the main causes of mortality in the World, although is a neglected disease since it is endemic in developing countries. The main route of therapy of tuberculosis is the inhibition of InhA, enzyme that catalyses the production of mycolic acids, which is a component of bacillus cellular wall. This reaction also is the main point of resistance against TB drugs. In this work proposed the study of InhA enzyme, working specifically in silico modeling of enzyme-ligant interactions. These ligands distinguish themselves between two distinct libraries, one of them containing organic compounds selected by its structural similarity with the enzyme substrate, NADH. Due in vitro and orally activity in murine model against tuberculosis exhibited by the compound pentacianoisoniazideferrate (II), another library, containing the pentacianoferrate II moiety bind to an auxiliary ligand studied against que InhA target. The essays realized using ligand rigid and flexible docking both, although the protein always considered rigid. Both essays had acceptable correlation within its results, regardless the scoring function used. The leading inhibitors structures had in common a high stabilization of ligand-enzyme complex due hydrophobic interactions, something expected due polarity of the enzyme binding site

The vitamin B3 precursor nicotinamide riboside (NR) and the ketone DL-body beta-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. NR increases nicotinamide adenine dinucleotide (NAD) levels to activate sirtuin protein deacetylases and BHB is an anti-aging calorie restriction (CR) mimetic. Caenorhabditis elegans is a 1 mm long nematode worm used as a model system to study aging with a mean lifespan of roughly 2-3 weeks depending upon the exact temperature of culture. NR and BHB have previously been shown to increase lifespan when administered to C. elegans by roughly 20%. However, the effect on lifespan when both compounds are added together has not yet been studied. It is hypothesized that when added together the effect on lifespan will be slightly larger than when either compound is given alone, due to the activation of complementary signaling pathways. The results will help determine if humans could benefit from taking both compounds simultaneously as most signaling pathways that regulate lifespan are conserved from nematodes to humans. For these experiments cultures of mixed age C. elegans nematodes were first treated with alkaline-bleach to kill adult worms leaving only eggs that are protected by their thick eggshell. Using this protocol isolated eggs are age-synchronized to within 9 hours of each other. The eggs were then transferred into E. coli-permeable, but nematode impermeable 8 micron cell culture inserts placed in twelve well microplates with roughly 25-40 eggs per insert. 1.35 mL of liquid S-media containing 9 x109 E. coli cells/mL as food was added to each well. Microplates were shaken to provide culture aeration. After three days, the worms reached adulthood and 0.4 mM fluorodeoxyuridine (FUdR), a DNA synthesis inhibitor, was added to prevent C. elegans egg-laying to maintain age-synchrony. Every Monday, Wednesday, and Friday for the approximate 4 weeks of the lifespan experiments the worms were counted under a microscope and the culture media and bacteria replaced. Results were analyzed using Kaplan-Meier survival curves and Log-rank analysis. Results indicated that individual treatment with BHB or NR or both combined increased lifespan in the two trials performed thus far. Another trial is currently underway, and results will be analyzed after it is completed to determine if the combined treatment has a greater benefit on lifespan then either individual treatment. Future studies could also be performed to determine if either NR or BHB can further extend the lifespan of animals given rapamycin, a TOR kinase inhibitor, another promising anti-aging therapeutic.

no / Co-crystal screening is routinely undertaken using high-throughput solution growth. We report a low- to medium throughput approach, encompassing both a melt and solution crystallization step as a route to the identification of co-crystals. Prior to solution studies, a melt growth step was included utilizing the Kofler mixed fusion method. This method allowed elucidation of the thermodynamic landscape within the binary phase diagram and was found to increase overall screening efficiency. The pharmaceutically acceptable adduct nicotinamide was selected and screened against a small set of active pharmaceutical ingredients (APIs) (ibuprofen (both the racemic compound (R/S) and S-enantiomer), fenbufen, flurbiprofen (R/S), ketoprofen (R/S), paracetamol, piracetam, and salicylic acid) as part of a larger systematic study of synthon stability. From the screen, three new co-crystal systems have been identified (ibuprofen (R/S and S) and salicylic acid) and their crystal structures determined. Because of poor crystal growth synchrotron radiation was required for structure solution of the S-ibuprofen nicotinamide co-crystal. Two further potential systems have also been discovered (fenbufen and flurbiprofen), but crystals suitable for structure determination have yet to be obtained. A greater ability to control crystallization kinetics is required to yield phase-pure single-crystalline material for full verification of this crystal engineering strategy.