Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53+/+ cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53

”VI VILL INTE HA SJUKVÅRD, ISTÄLLET VILL VI HA FRISKVÅRD”

Allt fler familjer lever med neuropsykiatriska funktionsnedsättningar. Tidigare forskning visar på att många föräldrar till barn med neuropsykiatrisk funktionsnedsättning upplever en hög stressnivå i vardagen på grund av diagnosen / diagnoserna. Detta kan leda till att föräldrar utvecklar tillstånd som depression, utmattningssyndrom och liknande problematik. Författarna till denna uppsats har haft ett intresse av att se vidare på den viktiga roll familjen har för att skapa en hög livskvalitet för personer med funktionsnedsättning. Syftet med denna studie var att undersöka ideella föreningars företrädares erfarenheter av det stöd som finns tillgängligt från den offentliga sektorn till medlemmarna i deras förening. Eftersom författarna har valt att intervjua ideella föreningars företrädare har det varit möjligt att uppnå en bred kunskap om ämnet då föreningarna når ut till personer med funktionsnedsättningar och deras släktingar. Syftet med denna uppsats var också att ta upp frågor om huruvida ett holistiskt tillvägagångssätt tillämpas i insatser riktade mot personer med funktionsnedsättning och deras familjer. Den teoretiska ramen för denna studie är baserad på Goffmans stigmatiska teori samt Hochschilds teori om emotionellt arbete.An exceeding amount of families today are affected by neuropsychiatric disabilities. Previous research shows that many parents of children with neuropsychiatric disabilities experience a high stress level in everyday life due to the diagnosis / diagnoses. This can result in that parents develop conditions such as depression, chronic fatigue syndrome and similar problems. The authors of this study had an interest to look further into the important role the family has in creating a high quality of life for individuals with disability. The aim of this study was to investigate non- profit associate representatives experience of the support available from the public sector to the members of their organization. Since the authors have chosen to interview non-profit associate representatives, it has been possible to achieve a broad knowledge of the subject as the associations target people with disabilities and their relatives. The purpose of this study was also to raise questions concerning if a holistic approach is applied in interventions aimed at individuals with disability and their families. The theoretical framework for this study is based on Goffmans stigma theory as well as Hochschild's theory on emotional work

”VI VILL INTE HA SJUKVÅRD, ISTÄLLET VILL VI HA FRISKVÅRD”

Allt fler familjer lever med neuropsykiatriska funktionsnedsättningar. Tidigare forskning visar på att många föräldrar till barn med neuropsykiatrisk funktionsnedsättning upplever en hög stressnivå i vardagen på grund av diagnosen / diagnoserna. Detta kan leda till att föräldrar utvecklar tillstånd som depression, utmattningssyndrom och liknande problematik. Författarna till denna uppsats har haft ett intresse av att se vidare på den viktiga roll familjen har för att skapa en hög livskvalitet för personer med funktionsnedsättning. Syftet med denna studie var att undersöka ideella föreningars företrädares erfarenheter av det stöd som finns tillgängligt från den offentliga sektorn till medlemmarna i deras förening. Eftersom författarna har valt att intervjua ideella föreningars företrädare har det varit möjligt att uppnå en bred kunskap om ämnet då föreningarna når ut till personer med funktionsnedsättningar och deras släktingar. Syftet med denna uppsats var också att ta upp frågor om huruvida ett holistiskt tillvägagångssätt tillämpas i insatser riktade mot personer med funktionsnedsättning och deras familjer. Den teoretiska ramen för denna studie är baserad på Goffmans stigmatiska teori samt Hochschilds teori om emotionellt arbete.An exceeding amount of families today are affected by neuropsychiatric disabilities. Previous research shows that many parents of children with neuropsychiatric disabilities experience a high stress level in everyday life due to the diagnosis / diagnoses. This can result in that parents develop conditions such as depression, chronic fatigue syndrome and similar problems. The authors of this study had an interest to look further into the important role the family has in creating a high quality of life for individuals with disability. The aim of this study was to investigate non- profit associate representatives experience of the support available from the public sector to the members of their organization. Since the authors have chosen to interview non-profit associate representatives, it has been possible to achieve a broad knowledge of the subject as the associations target people with disabilities and their relatives. The purpose of this study was also to raise questions concerning if a holistic approach is applied in interventions aimed at individuals with disability and their families. The theoretical framework for this study is based on Goffmans stigma theory as well as Hochschild's theory on emotional work

”VI VILL INTE HA SJUKVÅRD, ISTÄLLET VILL VI HA FRISKVÅRD”

Allt fler familjer lever med neuropsykiatriska funktionsnedsättningar. Tidigare forskning visar på att många föräldrar till barn med neuropsykiatrisk funktionsnedsättning upplever en hög stressnivå i vardagen på grund av diagnosen / diagnoserna. Detta kan leda till att föräldrar utvecklar tillstånd som depression, utmattningssyndrom och liknande problematik. Författarna till denna uppsats har haft ett intresse av att se vidare på den viktiga roll familjen har för att skapa en hög livskvalitet för personer med funktionsnedsättning. Syftet med denna studie var att undersöka ideella föreningars företrädares erfarenheter av det stöd som finns tillgängligt från den offentliga sektorn till medlemmarna i deras förening. Eftersom författarna har valt att intervjua ideella föreningars företrädare har det varit möjligt att uppnå en bred kunskap om ämnet då föreningarna når ut till personer med funktionsnedsättningar och deras släktingar. Syftet med denna uppsats var också att ta upp frågor om huruvida ett holistiskt tillvägagångssätt tillämpas i insatser riktade mot personer med funktionsnedsättning och deras familjer. Den teoretiska ramen för denna studie är baserad på Goffmans stigmatiska teori samt Hochschilds teori om emotionellt arbete.An exceeding amount of families today are affected by neuropsychiatric disabilities. Previous research shows that many parents of children with neuropsychiatric disabilities experience a high stress level in everyday life due to the diagnosis / diagnoses. This can result in that parents develop conditions such as depression, chronic fatigue syndrome and similar problems. The authors of this study had an interest to look further into the important role the family has in creating a high quality of life for individuals with disability. The aim of this study was to investigate non- profit associate representatives experience of the support available from the public sector to the members of their organization. Since the authors have chosen to interview non-profit associate representatives, it has been possible to achieve a broad knowledge of the subject as the associations target people with disabilities and their relatives. The purpose of this study was also to raise questions concerning if a holistic approach is applied in interventions aimed at individuals with disability and their families. The theoretical framework for this study is based on Goffmans stigma theory as well as Hochschild's theory on emotional work

Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs

Monoclonal antibodies (MAbs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed

A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism

High Cell Density Cultivation Process for the Expression of Botulinum Neurotoxin a Receptor Binding Domain

The receptor-binding domain of botulinum neurotoxin (HC fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale HC fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in HC fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the HC fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified HC fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the HC fragment in E. coli for use in anti-botulism vaccines

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins