Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

The liver is important in the biotransformation of various drugs, where hepatic transporters facilitate uptake and excretion. Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery, and the developing oxidative stress can lead to graft failure. We used intravital multiphoton tomography, with fluorescence lifetime imaging, to characterize metabolic damage associated with hepatic I/R injury and to model the distribution of fluorescein as a measure of liver function. In addition to measuring a significant increase in serum alanine transaminase levels, characteristic of hepatic I/R injury, a decrease in the averaged weighted lifetime of reduced nicotinamide adenine dinucleotide phosphate was observed, which can be attributed to a changed metabolic redox state of the hepatocytes. I/R injury was associated with delayed uptake and excretion of fluorescein and elevated area-under-the-curve within the hepatocytes compared to sham (i.e., untreated control) as visualized and modeled using images recorded by intravital multiphoton tomography. High-performance liquid chromatography analysis showed no differences in plasma or bile concentrations of fluorescein. Finally, altered fluorescein distribution was associated with acute changes in the expression of liver transport proteins. In summary, multiphoton intravital imaging is an effective approach to measure liver function and is more sensitive in contrasting the impact of I/R injury than measuring plasma and bile concentrations of fluorescein

Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery. In orthotopic transplantation, the donor liver is exposed to periods of ischemia and when oxygenated blood is reintroduced to the liver, oxidative stress may develop and lead to graft failure. The aim of this project was to investigate whether noninvasive multiphoton and fluorescence lifetime imaging microscopy, without external markers, were useful in detecting early liver damage caused by I/R injury. Localized hepatic ischemia was induced in rats for 1 h followed by 4 h reperfusion. Multiphoton and fluorescence lifetime imaging microscopy was conducted prior to ischemia and up to 4 h of reperfusion and compared to morphological and biochemical assessment of liver damage. Liver function was significantly impaired at 2 and 4 h of reperfusion. Multiphoton microscopy detected liver damage at 1 h of reperfusion, manifested by vacuolated cells and heterogeneous spread of damage over the liver. The damage was mainly localized in the midzonal region of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity. Multiphoton and fluorescence lifetime imaging microscopy detected evidence of early I/R injury both structurally and functionally. This provides a simple noninvasive technique useful for following progressive liver injury without external markers. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3647597

Computational Mapping of Antibody Sequence and Structure Space

Vores verden er fuld af mikroorganismer som bakterier og virus. Men selvom vi konstant kommer i kontakt med dem, bliver vi sjældent syge. Selv ved mødet med en ny influenzavariant, vil immunforsvaret i de fleste tilfælde have slå sygdommen ned inden for en uge eller to. I dette tidsrum kan immunforsvarets B celler finde den nye virusvariant og ved hjælp af en række mutationer i deres DNA skabe sygdomsbekæmpende antistoffer. Mutationsprocessen ændrer i antistoffernes gener og medfører at det menneskelige antistof repertoire er nærmest uendeligt. Dette gør kroppen i stand til næsten altid kan finde en måde at bekæmpe nye sygdomme. Ved at studere menneskers og dyrs antistoffer er forskere lykkedes med at designe kunstige antistoffer, der både kan produceres i store mængder og bruges til at bekæmpe sygdomme, som kroppens immunforsvar selv er ude af stand til at bekæmpe. En af de væsentligste hæmsko i jagten på nye antistoffer, er at fremmede antistoffer i de fleste tilfælde opdages af kroppens immunforsvar ved behandlingsforløbets start. Den grundlæggende udfordring her er, at antistoffer der har andre egenskaber end de menneskelige, også har en væsentligt anderledes opbygning. Dette tvinger os til at afveje to modsatrettede hensyn: På den ene side er det nødvendigt at ændre væsentligt i antistoffet for at gøre det kompatibelt med immunforsvaret. På den anden side er der risiko for at store ændringer, også ødelægger immunforsvarets evne til at bekæmpe den sygdom, hvilket de ellers er designet til.Denne afhandling forsøger at afdække, hvad der gør et antistof kompatibelt med det menneskelige immunforsvar. Ved at beskrive så stor en andel af diversiteten i den menneskelige antistof population som muligt, bliver det nemmere at tilpasse kunstige antistoffer til den menneskelige population uden at ødelægge deres evne til sygdomsbekæmpelse. Vi laver derfor en statistisk oversigt over den største tilgængelig samling af offentligt tilgængelige antistoffer.På samme måde som det kan være gavnligt at have overblik over så stor en del af det menneskelige antistof repertoire som muligt, er det også gavnligt at have overblik over antistof population fra de dyr, hvis antistoffer inspirerer designs af nye lægemidler. Kameler, dromedarer og lamaers antistoffer; nanobodies, adskiller sig fra de menneskelige antistoffer ved at være meget kompakte og anvende en lidt anderledes mekanisme til at spore virus og bakterier. Desværre har vi endnu ikke overblik over deres gener og det gør det vanskeligt at beskrive dem så grundigt, som det er muligt med menneskelige antistoffer. Vi præsenterer derfor en model, der anvender kunstig intelligens til at hjælpe med at designe funktionelle nanobodies. Endelig beskriver vi hvordan måden hvorpå vi observerer det menneskelige antistof repertoire bærer præg af, at mange antistoffer minder om de gener, de er muteret væk fra. Dette kan medføre at både vi og de statistiske modeller, vi bygger, kommer til at fokuserer ensidigt på de typer antistoffer, som kroppen i forvejen producerer flest af. Den type antistoffer er ganske vist funktionelle og effektive, men det kan være at kommende lægemidler bedre understøtter sygdomsbekæmpelse, hvis de designes og virker anderledes end de antistoffer, som kroppen selv har lettest ved at udvikle. Derfor præsenterer vi et alternativt værktøj til at designe antistoffer, som ikke fokuserer på hele antistoffet på en gang, men i stedet anvender de byggeklodser, antistoffer er lavet af, til at foreslå nye designs.Antibodies are small proteins produced by immune cells to target and neutralize alien agents in the body. Utilizing a combination of semi random genetic recombination and antigen target mutation, human antibodies are estimated to have a diversity of at least 1015 unique sequences. The high diversity enables the human adaptive immune system to generate antibodies that specifically target a wide range of antigens, and have made antibodies an attractive platform for computational drug design. The thesis consists of 3 main parts. In the first part the largest volume of publicly available antibody sequences are characterized in terms of gene distribution, positional entropy, composition of the CDRH3 region, conditions by length, VJ gene context and subregions. Finally we chart co mutations between all non CDRH3 positions of the V gene to identify independent residues.In the second part we present a language model for predicting residue content in nanobodies. Language models offer a means to predict the positional likelihood of amino acid residues without the guidance of a reliable germline reference. We demonstrated that our model outperforms both general antibody models and protein models.In the third part, we propose a novel paradigm for computational antibody design. Language models are trained using natural populations and may exhibit bias towards the densely populated regions of the antibody natural space observed around the germlines. Template based methods may be affected by the same bias when combining multiple sequences to create a reference sequence for mutation. In contrast, merMap aims at estimating a larger proportion of the functional space by charting germline independent building blocks within natural antibody populations.Finally we discuss the potential advance in antibody engineering that may arise from sampling strategies that estimate and mitigate bias towards densely populated regions of the natural antibody space.<br/

Report from the working group on diet and cancer.

No abstract available

Enhjørningen er på plads

Den 23. marts 2000 blev Enhjørningen afsløret ved en festlighed på Steno Museets repos