Transplantation of autologous bone marrow mononuclear cells with VEGF gene improves diabetic critical limb ischaemia

Wstęp: Celem pracy była ocena bezpieczeństwa i skuteczności skojarzonej terapii autogennymi jednojądrzastymi komórkami szpiku kostnego oraz terapii genowej plazmidem VEGF165 u chorych z krytycznym niedokrwieniem kończyn dolnych spowodowanym cukrzycą. Materiał i metody: U 16 chorych z bólami spoczynkowymi oraz niedokrwiennym owrzodzeniem kończyny dolnej w przebiegu cukrzycy zdecydowano o podaniu komórek jednojądrzastych i plazmidu VEGF165. Komórki jednojądrzaste szpiku oraz plazmid były podawane drogą iniekcji domięśniowych do mięśni niedokrwionej kończyny. Do oceny wyniku zastosowanej terapii określano poziom VEGF w surowicy oraz wskaźnik kostka-ramię. Do określenia stopnia odczuwania bólu została użyta skala wzrokowo-analogowa (VAS). Do wykazania w naczyniach wykonywano CT angiografię przed i po 3 miesiącach terapii. Wyniki: Średnie (&#177; SD) stężenie VEGF w osoczu wzrastało nieistotnie statystycznie z 257 &#177; 80 pg/l przed terapią do 391 &#177; 82 pg/l (P > 0,05) po 2 tygodniach od zakończenia leczenia. Wskaźnik kostka&#8211;ramię wzrósł istotnie statystycznie z poziomu 0,26 &#177; 0,22 przed terapią do 0,49 &#177; 0,30 (p < 0,001) po 3 miesiącach terapii. Zmniejszenie bólu spoczynkowego obserwowano u wszystkich pacjentów, średnia wartość VAS zmniejszyła się z 6,3 &#177; 1,4 przed terapią do 1,2 &#177; 1,1 po 3 miesiącach (p < 0,002). Angiogramy wykazały rozwój naczyń krążenia obocznego w 12 kończynach. Niedokrwienne owrzodzenie zostało całkowicie wyleczone w przepadku 12 chorych. Amputacje przeprowadzono tylko u 4 pacjentów z powodu zaawansowanego zakażenia rany, jakkolwiek poziom amputacji obniżono w tych przypadkach poniżej kolana. Powikłania były ograniczone do przemijających obrzęków podudzi u 2 chorych i gorączki u 2 pacjentów. Wniosek: Domięśniowa autotransplantacja komórek jednojądrzastych szpiku kostnego w połączeniu z podaniem phVEGF165 genu jest bezpieczną oraz skuteczną metodą leczenia pacjentów z cukrzycą i krytycznym niedokrwieniem kończyn dolnych. (Endokrynol Pol 2013; 64 (2): 129&#8211;138)Introduction: The aim of this study was to assess the safety and efficacy of combined autologous bone marrow mononuclear cell and VEGF165 gene therapy in patients with diabetes mellitus suffering from critical limb ischaemia (CLI). Material and methods: The administration of mononuclear cells (MNCs) and naked VEGF165 plasmid was performed in 16 limbs of 16 patients with rest pain and ischaemic ulcers due to diabetes. MNCs and plasmid were injected into the muscles of the ischaemic limbs. The levels of VEGF in serum and the ankle-brachial index (ABI) were measured before and after treatment. The Visual Analogue Scale (VAS) was used to evaluate pain sensation. CT angiography was performed before and after three months of therapy. Results: Mean (&#177; SD) plasma levels of VEGF increased non-significantly from 257 &#177; 80 pg/L to 391 &#177; 82 pg/L (p > 0.05) two weeks after therapy. The ABI improved significantly from 0.26 &#177; 0.22 to 0.49 &#177; 0.30 (p < 0.001) three months after therapy. A decrease in rest pain was observed in all patients; mean VAS decreased from 6.3 &#177; 1.4 to 1.2 &#177; 1.1 after three months (p < 0.002). Angiograms showed the development of collateral vessels in 12 limbs. Ischaemic ulcers healed in 12 limbs. Amputation was performed in four patients only, because of advanced wound infection. However, the level of amputations was lowered below knee level in these cases. Complications were limited to transient leg oedema in two patients and fever in two patients. Conclusions: Intramuscular bone marrow MNCs autotransplantation combined with the administration of phVEGF165 gene is safe, feasible and effective for patients with diabetes and CLI. (Endokrynol Pol 2013; 64 (2): 129&#8211;138

Intimal hyperplasia in vascular anastomosis

Wstęp. Celem pracy była próba lepszego poznania procesów wgajania się protezy naczyniowej w układ tętniczy i powstawania neointimy. Materiał i metody. Badania prowadzono na 16 psach, którym wszczepiono protezę z PTFE w odcinku aortalno-udowym o średnicy 6 mm. Po upływie 6 miesięcy zwierzęta sekcjonowano, a protezę wraz z aortą i tętnicą udową pobierano i wykonywano badania okolicy zespolenia naczyniowego proksymalnego i dystalnego w mikroskopie świetlnym i transmisyjnym mikroskopie elektronowym. Wyniki. Stwierdzano, że błona wewnętrzna zespolenia dystalnego była grubsza. Wyniki były wysoce znamienne statystycznie (p = 0,0000000002824). W obrębie zespolenia dystalnego w porównaniu z proksymalnym stwierdzano makrofagi i granulocyty - wykazano cechy czynnej reakcji zapalnej. Liczniejsze były miofibroblasty i większe złogi kolagenu. Wnioski. Różnice w zespoleniu proksymalnym i dystalnym są wynikiem oddziaływań hemodynamicznych, bardziej nasilonych w zespoleniu dystalnym naprężeń stycznych (tangential stress), działających równolegle do prądu krwi wobec słabszych naprężeń stycznych (shear stress) działających prostopadle do naczynia. Przerost neointimy w obrębie zespolenia dystalnego jest więc spowodowany przez czynniki hemodynamiczne, biologiczną niezgodność protezy i tętnicy (compliance mismatch). Wydłużenie pobudzenia procesów zapalnych wywołuje powstanie neointimy, ale także wzmożoną produkcję kolagenu, przerost, pogrubienie błony wewnętrznej. Wywołuje to zaburzenia hemodynamiczne, wzrost oporu naczyniowego, turbulentny przepływ, co może powodować powikłania kliniczne - zakrzepicę protezy, infekcję protezy późną, tętniak zespolenia.Background. The study was performed to examine the healing process of a vascular prosthesis implanted into the arterial system and the formation of neointima. Materials and methods. The study was conducted on 16 dogs in which PTFE prostheses of 6 mm diameter had been implanted in the aortofemoral region. The animals underwent autopsy after 6 months. The prosthesis together with the aorta and the femoral artery was explanted in order to perform tests of the proximal and distal anastomosis area under the light and the transmission electron microscopes. Results. It was confirmed that the inner membrane (intima) of the distal anastomosis was thicker. The results were highly statistically significant (p = 0.0000000002824). Contrary to the proximal anastomosis, the area of the distal anastomosis revealed the presence of macrophages and granulocytes, being the markers of the active inflammatory reaction. Additionally, there were more myofibroblasts in this area and collagen deposits were bigger. Conclusions. The differences between the proximal and distal anastomosis are the result of haemodynamic processes. Tangential stress, appearing parallel to the blood flow, is much more prominent in the distal anastomosis area as opposed to the shear stress, appearing perpendicular to the vessel. Therefore, neointimal thickening in the area of distal anastomosis is caused by haemodynamic factors and the biological incompatibility of the prosthesis and the artery (compliance mismatch). Prolonged stimulation of the inflammatory process causes the formation of neointima as well as the increased collagen production, hypertrophy and thickening of the inner membrane. All of these result in haemodynamic disturbances, the increase of the vascular resistance and the turbulent blood flow. They, in turn, may become the cause of clinical complications such as thrombosis of the prosthesis, late prosthesis infection and formation of the aneurysm in the area of anastomosis

Investigating harbor porpoise (Phocoena phocoena) population differentiation using RAD-tag genotyping by sequencing

The population status of the harbor porpoise ( Phocoena phocoena ) in the Baltic Sea and adjacent regions is still not fully resolved. Here, we present a pilot study using the double digest restriction-site associated DNA sequencing (ddRAD-seq) genotyping- by -sequencing method on specimens from the Baltic Sea, eastern North Sea, Spain and the Black Sea. From a single Illumina lane and a set of 49 individuals, w e obtained around 6000 SNPs. We used these markers to estimate population structure and differentiation, and identified splits between porpoises from the North Sea and the Baltic, and within regions in the Baltic Sea (between the Belt Sea and the Inner Baltic Sea). The SNP analysis confirms population structure elucidated by previous mtDNA/microsatellite studies. We demonstrate the feasibility of SNP analysis on opportunistically sampled cetacean samples, with varying DNA quality, for population diversity and divergence analysis

Mitochondrial Control Region and microsatellite analyses on harbour porpoise (Phocoena phocoena) unravel population differentiation in the Baltic Sea and adjacent waters

The population status of the harbour porpoise (Phocoena phocoena) in the Baltic area has been a continuous matter of debate. Here we present the by far most comprehensive genetic population structure assessment to date for this region, both with regard to geographic coverage and sample size: 497 porpoise samples from North Sea, Skagerrak, Kattegat, Belt Sea, and Inner Baltic Sea were sequenced at the mitochondrial Control Region and 305 of these specimens were typed at 15 polymorphic microsatellite loci. Samples were stratified according to sample type (stranding vs. by-caught), sex, and season (breeding vs. non-breeding season). Our data provide ample evidence for a population split between the Skagerrak and the Belt Sea, with a transition zone in the Kattegat area. Among other measures, this was particularly visible in significant frequency shifts of the most abundant mitochondrial haplotypes. A particular haplotype almost absent in the North Sea was the most abundant in Belt Sea and Inner Baltic Sea. Microsatellites yielded a similar pattern (i.e., turnover in occurrence of clusters identified by STRUCTURE). Moreover, a highly significant association between microsatellite assignment and unlinked mitochondrial haplotypes further indicates a split between North Sea and Baltic porpoises. For the Inner Baltic Sea, we consistently recovered a small, but significant separation from the Belt Sea population. Despite recent arguments that separation should exceed a predefined threshold before populations shall be managed separately, we argue in favour of precautionary acknowledging the Inner Baltic porpoises as a separate management unit, which should receive particular attention, as it is threatened by various factors, in particular local fishery measures. © Springer Science+Business Media B.V. 2009

ProTheRaMon : a GATE simulation framework for proton therapy range monitoring using PET imaging

Objective. This paper reports on the implementation and shows examples of the use of the ProTheRaMon framework for simulating the delivery of proton therapy treatment plans and range monitoring using positron emission tomography (PET). ProTheRaMon offers complete processing of proton therapy treatment plans, patient CT geometries, and intra-treatment PET imaging, taking into account therapy and imaging coordinate systems and activity decay during the PET imaging protocol specific to a given proton therapy facility. We present the ProTheRaMon framework and illustrate its potential use case and data processing steps for a patient treated at the Cyclotron Centre Bronowice (CCB) proton therapy center in Krakow, Poland. Approach. The ProTheRaMon framework is based on GATE Monte Carlo software, the CASToR reconstruction package and in-house developed Python and bash scripts. The framework consists of five separated simulation and data processing steps, that can be further optimized according to the user’s needs and specific settings of a given proton therapy facility and PET scanner design. Main results. ProTheRaMon is presented using example data from a patient treated at CCB and the J-PET scanner to demonstrate the application of the framework for proton therapy range monitoring. The output of each simulation and data processing stage is described and visualized. Significance. We demonstrate that the ProTheRaMon simulation platform is a high-performance tool, capable of running on a computational cluster and suitable for multi-parameter studies, with databases consisting of large number of patients, as well as different PET scanner geometries and settings for range monitoring in a clinical environment. Due to its modular structure, the ProTheRaMon framework can be adjusted for different proton therapy centers and/or different PET detector geometries. It is available to the community via github (Borys et al 2022)