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Wstęp: Przewlekłe krytyczne niedokrwienie tętnicze kończyn dolnych w razie braku możliwości wykonania zabiegów odtwórczych na układzie tętniczym, mimo intensywnego leczenia zachowawczego, w 30-50% przypadków prowadzi do amputacji kończyny. Zdolność komórek macierzystych szpiku do różnicowania w kierunku różnych komórek, w tym komórek śródbłonka i mięśni gładkich, stwarza podstawy do ich wykorzystania w celu indukowania angiogenezy w niedokrwionych obszarach kończyn. Materiał i metody: W okresie od grudnia 2003 roku do sierpnia 2005 roku wykonano 19 autotransplantacji mezenchymalnych komórek macierzystych szpiku u 16 chorych na przewlekłe krytyczne niedokrwienie kończyn dolnych. Poza dwiema kobietami pozostali chorzy płci męskiej, w wieku: 41--61lat (śr. 50,33 + 10,07) zgłaszali bóle spoczynkowe, wymagające stosowania leków przeciwbólowych, u wszystkich obserwowano obecność owrzodzeń niedokrwiennych, wskaźnik ABI wynosił 0-0,5. U wszystkich chorych wykonano arteriografię, która była podstawą do dyskwalifikacji z leczenia chirurgicznego. U 3 chorych wykonano 2-krotne transplantacje, w tym u 2 do mięśni tej samej kończyny. Okres obserwacji wynosił 6-26 miesięcy. Szpik kostny w ilości 500 ml pobierano metodą biopsji aspiracyjnej przez mnogie punkcje kości biodrowych w znieczuleniu ogólnym. Szpik zabezpieczano płynem konserwującym ACD-A w stosunku 10:1. Mezenchymalne komórki macierzyste izolowano w Pracowni Aferezy Dolnośląskiego Centrum Transplantacji Komórkowych przy użyciu separatora komórkowego COBE Spectra do uzyskania 90-procentowej czystości ocenianej metodą cytometrii przepływowej. Uzyskany preparat komórek macierzystych w końcowej objętości 40-50 ml podawano metodą wielokrotnych (50-60) wstrzyknięć objętości około 1 ml/Wstrzyknięcie do mięśni brzuchatych łydki kończyny niedokrwionej na głębokość około 1,5 cm przy użyciu igieł iniekcyjnych 0,7 x 25 mm. Wyniki: Obserwowano szybkie -- w ciągu 3-7 dni -- ustępowanie lub złagodzenie dolegliwości bólowych pozwalające odstawić bądź istotnie zredukować dawki analgetyków. Ustępowaniu bólów towarzyszyło gojenie owrzodzeń z redukcją o ponad około 75% powierzchni ocenianej planimetrycznie po miesiącu i o około 80% po 2 miesiącach obserwacji. Obserwowano zahamowanie postępów gojenia po około 3 miesiącach od implantacji. Po roku od autoprzeszczepu redukcję/ustąpienie bólów zgłaszało 15/16 leczonych, pełne wygojenie owrzodzeń uzyskano u 8/16 pacjentów, częściowe wygojenie u 4/10, u 2 pacjentów w okresie obserwacji doszło do progresji zmian martwiczych i amputacji kończyny. Cechy neorewaskularyzacji w badaniach arteriograficznych wykazano u 8/16 leczonych. Nie obserwowano powikłań procedury. Na podstawie obserwacji klinicznej i zauważalnego zaniku korzystnego efektu klinicznego autotransplantacji po około 3 miesiącach zmodyfikowano program terapeutyczny, wdrażając powtarzane implantacje komórek macierzystych. … [ABSTRACT FROM AUTHOR]

Wstęp: Pierwsze obserwacje korzystnego działania larw much Lucilla sericata w leczeniu zainfekowanych ran pochodzą z czasów amerykańskiej wojny secesyjnej. Stosowali je także brytyjscy chirurdzy wojskowi podczas I wojny światowej. Wprowadzenie do lecznictwa sulfonamidów, a następnie antybiotyków spowodowało zarzucenie tej metody leczenia ran. Obecnie w związku z rosnącym problemem antybiotykooporności bakterii w Europie i w Stanach Zjednoczonych coraz częściej larwy Lucilla sericata znajdują zastosowanie kliniczne. W Polsce dotychczas larwy medyczne nie były komercyjnie dostępne. Celem pracy było zbadanie przydatności i bezpieczeństwa stosowania larw Lucilla sericata pochodzących z własnej hodowli w aseptycznych i antyseptycznych warunkach laboratoryjnych. Materiał i metody: Larwy zastosowano u 22 pacjentów, w tym u 2 pacjentów z niegojącymi się kikutami po amputacjach kończyn, 7 pacjentów z zainfekowanymi owrzodzeniami żylnymi, 13 z martwicą i owrzodzeniami niedokrwiennymi. W tej grupie było 6 pacjentów z cukrzycą powikłaną zespołem stopy cukrzycowej. Larwy w wieku 12-24 godzin aplikowano na rany przemyte 0,9-procentowym NaCl w ilości około 50-200 sztuk, arbitralnie w zależności od powierzchni rany. Do opatrunku używano jałowej gazy zwilżonej solą fizjologiczną, dodatkowo na gazę zakładano odpowiedniej wielkości warstwę gęstego szyfonu, bandaż i standardową, krótką, stylonową pończochę. Opatrunek utrzymywano 3-6 dni. Objawem skłaniającym do zdjęcia opatrunku była migracja larw na zewnątrz. Stosowano 2-6 aplikacji larw do istotnego oczyszczenia ran z tkanek martwiczych i zropiałych. Wyniki: 2 pacjentów odmówiło kontynuacji leczenia po pierwszej aplikacji z przyczyn psychologiczno-estetycznych i organoleptycznych. U 2 zaprzestano leczenia po 2 aplikacji z powodu postępu martwicy. Poprawę wyrażoną oczyszczaniem się owrzodzeń i oddzielaniem tkanek martwiczych obserwowano u 18 pacjentów. U obu chorych z niegojącymi się kikutami amputacyjnymi obserwowano szybki postęp gojenia. U chorych z zespołem stopy cukrzycowej gojenie przez naskórkowanie brzeżne towarzyszyło oczyszczaniu się ran. Mimo oczyszczenia z tkanek marwiczo-ropnych nie obserwowano znamiennej redukcji powierzchni owrzodzeń żylnych. U 2 z tych chorych wystąpiły przejściowe objawy przyrannej alergizacji skórnej. Wszyscy chorzy z owrzodzeniami niedokrwiennymi bez cukrzycy zgłaszali nasilenie bólów niedokrwiennych od 3. dnia aplikacji larw. Wnioski: Miejscowe stosowanie larw Lucilla sericata jest korzystne w leczeniu przewlekłych ran u chorych naczyniowych, zwłaszcza trudno gojących się kikutów amputacyjnych i u chorych z zespołem stopy cukrzycowej. Metoda jest dobrze tolerowana przez większość pacjentów. Pozwala na ograniczenie stosowania antybiotyków. Nadaje się do stosowania ambulatoryjnego. [ABSTRACT FROM AUTHOR]

Copyright of Acta Angiologica is the property of VM Medica-VM Group (Via Medica) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Angiologica is the property of VM Medica-VM Group (Via Medica) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Angiologica is the property of VM Medica-VM Group (Via Medica) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.

Adipose tissue is a reliable source of mesenchymal stromal cells (MSC) for use in regenerative medicine. The aim of this pilot study was to describe the method, and assess the safety and the potential efficacy of transplantation of autologous adipose tissue-derived MSC for the treatment of chronic venous stasis ulcers. Study group consisted of 11 patients (mean age: 66.6 ± 9.5 years) with chronic venous stasis ulcers. Adipose tissue was harvested by tumescent-aspiration method. Stromal cells were separated using a dedicated closed system in a real-time bedside manner. The phenotype of cells was determined immediately after separation. Cell concentrate was implanted subcutaneously around the wound and the wound bed. All ulcers were assessed planimetrically before autotransplantation and every two weeks during the six-month follow-up. During the study all patients received standard local and general treatment. The preparation contained an average of 5.6 × 10 6  ± 4 × 10 6 cells per milliliter. The phenotype of 65-82% of transplanted cells expressed MSC markers: CD73 + CD90 + and CD34 + . An improvement was observed in 75% of ulcers. The data showed highly significant negative correlation (p < 0.0001) between wound size and wound closure degree. There was no correlation of ulcer healing with other parameters evaluated, including age of the patients. No serious side effects were observed. Autotransplantation of adipose tissue stromal cells may be a safe and promising treatment method for chronic venous ulcers.

Background: Non-healing wounds are becoming a growing concern for public health as a result of their increasing prevalence in progressively aging societies., Objectives: The aim of this article is to evaluate the effects of wound etiology on a panel of circulating cytokines in patients with non-healing wounds of the lower extremities., Material and Methods: This prospective case-control study involved 104 individuals: healthy elderly people (n = 46) and patients with diabetes and/or cardiovascular disease (n = 58; among them 38 with chronic wounds of venous, ischemic or neurotrophic etiology). Selected serum cytokines - i.e. IL-1β, IL-4, IL-6, IL-8, FGF-2, G-CSF, GM-CSF, MCP-1, MIP-1α, TNF-α, VEGF-A, and PDGF-BB - were measured using the Luminex platform., Results: Compared to healthy elderly people, presence of diabetes and/or cardiovascular disease was associated with elevated IL-6, IL-8, MCP-1 and G-CSF while non-healing wounds coexisted with the increase in the levels of all examined cytokines/growth factors except for G-CSF and GM-CSF. Among diseased elderly people, having wounds was associated with increased levels of IL-1β, IL-4, IL-6, IL-8, FGF-2, MIP-1α, PDGF-BB, and VEGF-A. Interleukin 1β elevation was a sole independent predictor of chronic wounds with an odds ratio (OR) of 6.3. Cytokines in healthy seniors were loosely interrelated, while the levels of cytokines in diseased patients with wounds displayed a tight pattern of association. When stratified by their etiology, the association pattern for IL-6, IL-8, MCP-1, and VEGF-A was disrupted in neurotrophic wounds., Conclusions: The results presented herein may improve our understanding of the pathomechanisms which lead to chronic wounds and of the effects they exert on a systemic level, as well as providing potential targets for more effective therapies.

Objective: The status of metabolites of the nitric oxide (NO) pathway in patients with chronic wounds in the course of cardiometabolic diseases is largely unknown. Yet arginine supplementation and citrulline supplementation as novel therapeutic modalities aimed at increasing NO are tested., Material and Methods: Targeted metabolomics approach (LC-MS/MS) was applied to determine the concentrations of L-arginine, L-citrulline, asymmetric and symmetric dimethylarginines (ADMA and SDMA), and arginine/ADMA and arginine/SDMA ratios as surrogate markers of NO and arginine availability in ulnar and femoral veins, representing systemic and local levels of metabolites, in patients with chronic wounds in the course of cardiometabolic diseases ( n = 59) as compared to patients without chronic wounds but with similar cardiometabolic burden ( n = 55) and healthy individuals ( n = 88)., Results: Patients with chronic wounds had significantly lower systemic L-citrulline and higher ADMA and SDMA concentrations and lower L-arginine/ADMA and L-arginine/SDMA as compared to healthy controls. The presence of chronic wounds in patients with cardiometabolic diseases was associated with decreased L-arginine but with increased L-citrulline, ADMA, and SDMA concentrations and decreased L-arginine/ADMA and L-arginine/SDMA. Serum obtained from the ulnar and femoral veins of patients with chronic wounds differed by L-arginine concentrations and L-arginine/SDMA ratio, both lower in the femoral vein. Wound etiology affected L-citrulline and SDMA concentrations, lower and higher, respectively, in patients with venous stasis, and the L-arginine/SDMA ratio-lower in venous stasis. The wound type affected L-arginine/ADMA and citrulline-lower in patients with ulcerations or gangrene. IL-6 was an independent predictor of L-arginine/ADMA, VEGF-A of ADMA, G-CSF of L-arginine/SDMA, and GM-CSF of L-citrulline and SDMA., Conclusion: Chronic wounds in the course of cardiometabolic diseases are associated with reduced NO and arginine availability due to ADMA and SDMA accumulation rather than arginine deficiency, not supporting its supplementation. Wound character seems to affect NO bioavailability and wound etiology-arginine bioavailability. Arginine concentration and its availability are more markedly reduced at the local level than the systemic level., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper.

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 μL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R 2  = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline., (Copyright © 2017 John Wiley & Sons, Ltd.)

Surgical and endovascular revascularization of ischemic legs in patients with peripheral arterial disease (PAD) can damage the arterial wall (endothelial and smooth muscle cells). Hemostatic factors released during endothelial dysfunction can lead to restenosis. 1. Determination of selected endothelial hemostatic factors in PAD patients and a reference group. 2. Prospective observation of new restenosis appearance in PAD patients after endovascular revascularization. 3. Comparison of selected endothelial hemostatic factors between non-restenotic and restenotic PAD patients. 150 PAD patients after endovascular revascularization - 90 men and 60 women, aged 44-88 (mean 65.5) years - were examined. During one-year observation after the revascularization procedures in 38 PAD patients restenosis occurred, when blood samples were also collected. The reference group consisted of 53 healthy persons - 44 men and 9 women, aged 20-56 years. Blood was drawn in the morning into 3.2% sodium citrate at a ratio of 9:1. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) were measured in plasma with commercial tests using the enzyme immunoassay. In the plasma of PAD patients after revascularization, the concentrations of TF and vWF were significantly higher, TM lower, TFPI and t-PA similar compared to the reference group. Six months after revascularization the level of TF had increased and vWF had significantly decreased. The endothelial hemostatic factors before and after restenosis did not significantly differ except TF, which after restenosis was higher. Increased TF and vWF levels in PAD patients indicate arterial endothelial cell damage, by atherosclerotic and revascularization processes. In PAD patients with restenosis compared to these patients before restenosis the determined endothelial hemostatic factors, except TF level, did not significantly differ. Perhaps TF participates in restenosis formation.

Background: In patients with peripheral artery disease (PAD) a hypercoagulable state and thromboembolic complications occur. Revascularization procedures increase this state, sometimes leading to restenosis. Restenosis following balloon angioplasty (PTA)and stent implantation is ≥ 50% of artery stenosis., Objectives: To determine the concentration of tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombin-antithrombin (TAT) complexes, fibrinogen and D-dimers in the blood of patients with PAD after peripheral endovascular revascularization of the lower legs and in PAD patients with restenosis., Material and Methods: The study included 150 patients with PAD, 90 men and 60 women, aged 44-88 (mean 65.5) years, after successful peripheral angioplasty (PTA) and/or with stenting. During the 6 months after the revascularization procedures, restenosis occurred in 27 patients. The reference group consisted of 53 healthy persons (44 men and 9 women, aged 20-56 years). Blood was drawn in the morning into 3.2% natrium citrate at a ratio of 9 : 1. The concentration of TF, TFPI, TAT complexes and D-dimers were measured in plasma with commercial tests using an enzyme immunoassay. Fibrinogen was determined with coagulometer., Results: In the plasma of patients with PAD after endovascular revascularization, the concentrations of TF, TAT complexes, fibrinogen and D-dimers were significantly higher compared to the reference group. During the six months of observation, 27 patients developed restenosis. The results of hemostatic factors in patients with restenosis were compared with the same patients before restenosis and the group of 123 PAD patients after endovascular revascularization. TF and fibrinogen levels in the 27 patients with restenosis were significantly higher than in the group of PAD patients before restenosis., Conclusions: Statistically significantly higher levels of tissue factor (TF) and fibrinogen in PAD patients with new restenosis, compared to those without restenosis after endovascular revascularization, indicate they can participate in the formation of restenosis.

Enolase, is a glycolytic enzyme ubiquitous in higher organisms, where it forms tissue specific dimers of isoforms, also found in the cytoplasm of fermentative bacteria. The aim of this work was to identify enolase-like proteins in the cell wall of some Gram-negative bacteria using antibodies against human beta-enolase, an isoenzyme specific to skeletal and heart muscles. Cell wall outer membrane protein (OMP) preparations were obtained from 9 strains of Enterobacteriaceae and one of Pseudomonas aeruginosa. Specific enzymatic enolase activity was detected in the supernatant fractions of cytosolic and inner membrane material, but not in purified OMP preparations. Rabbit polyclonal antibodies specific against human beta-enolase were prepared and purified using immobilized human beta-enolase in affinity chromatography. In SDS-polyacrylamide gel electrophoresis and immunoblotting assay of purified OMP preparations, rabbit anti-enolase antibody interacted specifically with a few OMPs, of which a 45-kDa band also interacted with human sera of patients presenting Buerger disease and atherosclerosis. The most distinct interaction of human sera was observed with a 45-kDa OMP of Klebsiella pneumoniae. This protein was further isolated from K. pneumoniae cell mass in two ways, namely preparative SDS-polyacrylamide gel electrophoresis and specific affinity chromatography using immobilized affinity-purified rabbit antibody raised against human beta-enolase. The data obtained from tandem mass spectrometry tryptic peptide analysis and sequence comparison of human and bacterial enolases using protein databases, could reveal the similarity in the epitopes between membrane enolase-like protein from Klebsiella and human beta-enolase. The results show that the protein present in all studied strains has a common epitope on human beta-enolase. These data raise the question whether such a bacterial protein might be a marker for detecting and monitoring damage to skeletal and heart muscles.

Unlabelled: High serum level of lipoprotein(a) [Lp(a)] is one of the risk factors of atherosclerosis and so do high total cholesterol (TC) or LDL-cholesterol (LDL-ch) serum levels or diabetes mellitus. Structure of the protein part of Lp(a) is similar to plasminogen (PL). The aim of the study was estimation serum concentrations of: Lp(a), TC, LDL-ch, HDL-cholesterol (HDL-ch), triglycerides (TG) and PL in diabetic patients macroangiopathy hypertension. 63 patients (35 females and 28 males) with diabetes mellitus were included into the study. 36 of them suffered from chronic limb ischaemia caused by diabetic macroangiopathy 44 persons of the group were hypertensive. Control group consisted of 15 healthy people in the similar age. PL was estimated by turbidimetric method, Lp(a)--by ELISA, other lipids--by routine methods., Results: In diabetic patients in comparison to healthy persons were significantly higher: Lp(a) serum (p<0.003), PL (p<0.001), TC, LDL-ch, and significantly lower: HDL-ch. There were no statistically significant differences in Lp(a), other lipids and PL levels in patients with chronic leg ischaemia vs without that complication and in hypertensive vs normotensive diabetics.

Lipoprotein(a) [Lp(a)] is the specific lipoprotein which physiological role has not been explained. Similarity between apolipoprotein(a) and plasminogen structure suggests, that Lp(a) can be the bridge connecting the lipid metabolism and the coagulation system. The aim of the study was to evaluate if there is any correlation between Lp(a) and plasminogen serum concentrations in patients with diabetes t.2. 20 males and females with diabetes t.2 in the age 19-75 years (mean: 54.9 years). The control group consisted of 15 healthy men and woman in the age of 23 years (medical students). Lp(a) was estimated by ELISA, plasminogen by turbidimetric methods. Lp(a) serum level in the examined group was: 40.06 + 59.45 mg%. In 7 patients it was over 30 mg%. In the control group: Lp(a) concentration was: 13.23 + 10.5 mg%, no one was above 30 mg%. The different was significant. Plasminogen concentration was: in patients: 94.08 + 53.08%, in the control group: 89.08 + 40.7%. There was no significant difference. Correlation index between Lp(a) and plasminogen concentrations was in the patient's group: 0.3, in the control group: 0.2. In patients with diabetes we can find increased Lp(a) commentation in serum and normal plasminogen concentration. Concentration of Lp(a) didn't demonstrate significant correlation with plasminogen serum level.

In order to find an objective marker of disease intensity, neutrophil elastase activity was determined in patients with ulcerative colitis. The study included 41 patients with ulcerative colitis who were divided into two groups depending on tge stage of the disease on the basis of the activity index according to Maier et al. The obtained results of studies were compared a group of 18 healthy persons. The values of neutrophil elastase activity in the patients and in the control group were subjected to statistical analysis. It was found that neutrophil elastase activity in the patients with high degree of disease intensity was lower than that in the patients with low degree of disease intensity and in healthy subjects. The obtained differences were statistically significant. From the results of the study the conclusion was drawn that the neutrophil elastase activity may be an indicator of ulcerative colitis activity.

Rheographic investigations and segmental measurements of pressure by Doppler method were done in 49 patients with type II diabetes or with impaired glucose tolerance who had chronic ischaemia of lower extremities in all stages, after Fontaine. The diagnostic usefulness of both methods was demonstrated, similarly as the correlation of their results with clinically determined progression of arterial ischaemia of the lower extremities. Parallel application of both methods was particularly useful in diabetic since it made possible detection of Mönckeberg's arteriosclerosis and evaluation of collateral circulation development.