T cell delivery of immune-stimulatory cytokines to enhance cancer immunotherapy

Adoptive cell therapy using TCR-engineered T cells is an exciting area of research and has emerged as a promising strategy for treating cancer patients. However, the effector function of TCR-engineered T cells can be tuned down by local mechanisms of tumour-associated immunosuppression. The potential of cytokines to reverse local immune suppression and enhance tumour immunity has been described in the past. The main aim of this project was to engineer T cell specificity as well as effector cytokine production as a strategy to enhance cancer immunotherapy. This was achieved by combining TCR gene transfer with genetic engineering to achieve IL-12 and IL-27 production in therapeutic T cells. In vitro validation data demonstrated not only an enhanced production of IL-12 and IL-27 by the engineered T cells but also an enhanced effector function upon antigen-specific stimulation. In order to circumvent previously described toxic side effects observed with systemic IL-12 delivery, a tet-regulated gene expression system was utilised to regulate cytokine production by engineered T cells in vivo. Adoptive transfer of TCR-redirected T cells expressing regulated IL-12 in B16F10 melanoma-bearing mice resulted in an enhanced accumulation of transferred CD8+ T cells in the tumour and in a change of the innate immune cell composition in the tumour microenvironment. Importantly, regulated IL-12 delivery resulted in enhanced therapeutic efficacy of the transferred T cells without causing systemic toxicity. IL-27 delivery in engineered T cells also showed some effectiveness when combined with TCR gene therapy, although the therapeutic benefit of IL-27 was inferior to IL-12. The data in this study demonstrate the potency of additional genetic manipulation to tailor the TCR-redirected T cell effector function which can result in a substantial enhancement in their therapeutic efficacy, and thus, enhanced antitumor immune response

Contribution of new thiol antioxidant in the treatment of acetaminophen toxicity

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. It is safe at therapeutic doses, but an overdose can result in severe hepato-nephrotoxicity, a leading cause of drug-induced acute liver failure in the U.S. Although a few different mechanisms have been proposed for APAP-induced toxicity, a significant amount of evidence has pointed to the potential involvement of oxidative stress in acetaminophen toxicity. Depletion of glutathione (GSH) is one of the initiating steps in APAP-induced toxicity; therefore, one strategy for restricting organ damage is to restore GSH levels by using GSH prodrugs like N-acetylcysteine (NAC). Although NAC is the treatment of choice for APAP-induced toxicity, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and IV administration of NAC in a hospital setting are laborious and costly. With limited therapeutic options, other than NAC, it is important to develop therapeutic alternatives to effectively protect against APAP-induced toxicity and to improve treatment outcomes and prevent death. Therefore, we studied the protective effects of N-acetylcysteine amide (NACA), a novel antioxidant with higher bioavailability, and compared it with NAC in APAP-induced toxicity in C57BL/6 mice. Our results showed that a lower dose of NACA is better than NAC in combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA, in protecting against APAP-induced toxicity, suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose. --Abstract, page iii

Comparative evaluation of N-acetylcysteine and N-acetylcysteine amide in acetaminophen-induced oxidative stress

Acetaminophen (APAP) is the most widely used pharmaceutical analgesic-antipyretic agent in the world, but its toxicity is a common cause of drug-induced hepatotoxicity. With APAP toxicity, cellular glutathione (GSH) is depleted. This results in the availability of N-acetyl-p-benzoquinone imine (NAPQI), is a toxic metabolite of APAP that binds to cellular macromolecules, which leads to cell necrosis. N-acetyl cysteine (NAC), a GSH precursor, is the only approved antidote for an acetaminophen overdose. It is a negatively charged molecule that diminishes its penetration into the cells, thereby requiring fairly high doses that increase the severity of side effects. In addition, oral and I.V. administration of NAC in a hospital setting is laborious and costly. Recently, NACA, an amide form of NAC, which is neutral at physiological pH has been developed to improve NAC\u27s bioavailability. Therefore, in this study, we conducted an investigation to determine the mechanism of APAP-induced hepatotoxicity. We also evaluated the hepatoprotective effectiveness of NACA and compared it with NAC in the hepatic cell line, HepaRG. This comparison was based on several oxidative stress parameters, including the levels of intracellular reactive oxygen species, GSH, various antioxidant enzyme activities, and lactate dehydrogenase levels. In conclusion, NACA protected HepaRG cells against damage induced by acetaminophen toxicity and may, therefore, be a more useful antidote than NAC (the only approved antidote) --Abstract, page iii

Comparison of curette and paper point sampling of subgingival plaque bacteria as analyzed by real-time PCR

Because of their important etiological role in periodontitis the microbiological identification of subgingival plaque bacteria is essential for clinical diagnostics. However, only few studies have systematically evaluated microbiological sampling methods of periodontal bacteria. The present study has been conducted to compare for the first time two widely used sampling techniques (paper point and curette) using the novel method of quantitative real-time PCR. Twenty adult patients with chronic periodontitis participated in a prospective study using a cross-over design. In each patient one periodontal pocket with a probing depth of more than 6 mm was selected for microbial sampling. In patients of group A the first sample was obtained with a paper point and the second with a Gracey-curette. In group B the sampling sequence was reversed. Samples were analysed by a blinded examiner in a specialised microbiological laboratory using real-time PCR technology. The analysis enabled the quantitative evaluation of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum ssp., Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia as well as total bacterial counts. Eight weeks after anti-infective periodontal therapy the sites were sampled again, using the same sampling sequence as before. Statistical analysis included t-test, Kappa and Spearman correlation. In this study higher total bacterial counts could be harvested by use of curettes than by paper points. The ratios between paper point and curette samples in group A were 1:4 before and 1:1 after therapy, in group B the respective values were 1:4 before and 1:3 after therapy. In contrast, the relative proportions of target bacteria in the total sample were similar. Following therapy, both sampling techniques showed a reduction of total bacterial counts as well as of the relative proportion of periodontopathogens. Overall, there was a relatively good agreement for the results of the investigated sampling techniques for the analysis of subgingival plaque bacteria. Thus, both techniques appear to well suited for microbiological diagnostics of patients with periodontitis.Evaluation mikrobiologischer Entnahmetechniken subgingivaler Plaque-Bakterien mittels Real-Time PCR (Curette - Paper point) Aufgrund ihrer wichtigen ätiologischen Bedeutung bei der Parodontitis hat der mikrobiologische Nachweis subgingival lokalisierter parodontalpathogener Bakterien eine wichtige Bedeutung in der klinischen Diagnostik. Nur wenige Studien haben bisher allerdings Entnahmetechniken subgingivaler Plaquebakterien systematisch untersucht. Diese Studie diente dem erstmaligen Vergleich von zwei häufig verwendeten Probeentnahmetechniken (Papierspitze bzw. Kürette) mit Hilfe der Methode der quantitativen Real-time PCR. Zwanzig Patienten mit chronischer Parodontitis nahmen an einer prospektiven Studie im cross-over Design teil. Bei jedem Patienten wurde eine Tasche tiefer als 6 mm an einem Frontzahn zur Probeentnahme ausgewählt. Bei der Gruppe A wurde die erste Probe mit einer Papierspitze und die zweite mit einer Gracey-Kürette entnommen. Bei der Gruppe B wurde die Probeentnahme in der umgekehrten Reihenfolge durchgeführt. Die Proben wurden in einem mikrobiologischen Fachlabor durch einen geblindeten Untersucher mit einem auf Real-Time PCR basierten Test analysiert. Dies erlaubte die quantitative Bestimmung von Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum ssp., Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia sowie der Gesamtbakterienzahl. Acht Wochen nach antiinfektöser Therapie der Patienten wurden in jeweils derselben Reihenfolge nochmals Proben entnommen. Die statistische Auswertung erfolgte mit t-Test, Kappa und Spearman Korrelation. In dieser Studie konnten mit Küretten mehr Gesamtbakterien als mit Papierspitzen entnommen werden. Die Verhältnisse zwischen Papierspitze und Kürette lagen in Gruppe A vor Therapie bei 1:4 und nach Therapie bei 1:1, in Gruppe B waren sie 1:4 vor und 1:3 nach Therapie. Hingegen waren die relativen Anteile der Zielbakterien an der Gesamtprobe vergleichbar. Beide Entnahmetechniken zeigten übereinstimmend sowohl eine Reduktion der Gesamtbakterienzahl als auch des relativen Anteils der Parodontalpathogene an der Gesamtprobe nach Therapie. Insgesamt bestand eine relativ gute Übereinstimmung der Ergebnisse nach den untersuchten Entnahmetechniken zur Analyse subgingivaler Plaque-Bakterien, so dass beide für die klinisch-mikrobiologische Diagnostik bei Patienten mit Parodontitis geeignet erscheinen

Congenital agenesis of internal carotid artery with ipsilateral Horner presenting as focal neurological symptoms

Internal carotid artery (ICA) agenesis is a rare developmental anomaly and is most frequently asymptomatic, but it may also present as cerebrovascular accidents. The association with Horner’s syndrome is exceptional. We present three cases of agenesis of ICA associated with Horner’s syndrome and hypochromia iridum presenting as focal neurological symptoms. A system of collaterals develops as a consequence of agenesis of the ICA, making the majority of cases asymptomatic. Three types of collateral circulations have been described. These collaterals increase the risk of aneurysm formation and the occurrence of life-threatening subarachnoid hemorrhages. The association of congenital Horner’s syndrome and hypochromia iridum without anhidrosis is highly suggestive of sympathetic pathway injury early in life. Such signs should prompt further diagnostic evaluation to demonstrate the presence of the agenesis of the carotid canal. Early diagnosis is essential to rule out potentially life-threatening associated vascular anomalies

Glucose-Insulin Dynamical Model for Type 2 Diabetic Patients

In this paper, a literature review is made for the current models of glucose-insulin dynamics of type 2 diabetes patients. Afterwards, a model is proposed by combining and modifying some of the available models in literature to take into account the effect of multiple glucose meals, multiple metformin doses, insulin injections, physical exercise, and stress on the glucose-insulin dynamics of T2D patients. The model is proposed as a candidate to be validated with real patients data in the future

An Online Stochastic Optimization Approach for Insulin Intensification in Type 2 Diabetes with Attention to Pseudo-Hypoglycemia

In this paper, we present a model free approach to calculate long-acting insulin doses for Type 2 Diabetic (T2D) subjects in order to bring their blood glucose (BG) concentration to be within a safe range. The proposed strategy tunes the parameters of a proposed control law by using a zeroth-order online stochastic optimization approach for a defined cost function. The strategy uses gradient estimates obtained by a Recursive Least Square (RLS) scheme in an adaptive moment estimation based approach named AdaBelief. Additionally, we show how the proposed strategy with a feedback rating measurement can accommodate for a phenomena known as relative hypoglycemia or pseudo-hypoglycemia (PHG) in which subjects experience hypoglycemia symptoms depending on how quick their BG concentration is lowered. The performance of the insulin calculation strategy is demonstrated and compared with current insulin calculation strategies using simulations with three different models.Comment: Preprint for a paper accepted and presented at CD