NKT-like cells are "non-classical", "CD1d-independent" NKT cells which represent highly differentiated, conventional T lymphocytes coexpressing several NK (natural killer) associated receptors. They are effector lymphocytes of both innate and adaptive immunity and simultaneously regulatory cells of the adaptive immune system. They reveal large granular lymphocyte morphology and can mediate both MHC-restricted and MHC-unrestricted cytotoxicity, secrete many cytokines and modulate Th1 immune responses. The aim of our study was to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in NKT-like cells compared to T lymphocytes during ageing.The study involved three groups of participants: the oldest seniors (n = 25; aged over 85; mean age 88 ± 0.5 ys), the old (n = 30; aged under 85; mean age 76 ± 0.9 ys) and the young (n = 32; mean age 21 ± 0.3 ys). Whole blood samples were analyzed by flow cytometry to assess the NKT-like (CD3+CD56+) and T (CD3+) cell populations.The group of the oldest seniors differed from the other age groups by much higher percentage of both NKT-like cells and T lymphocytes expressing SIRT1, HSP70 and SOD2. The expression of these proteins correlated positively with the age of the participants. Interestingly, the significantly higher expression of the studied protective proteins; i.e. HSP70 and SOD2 was found in CD3+CD56+ cells compared to CD3+ lymphocytes and this phenomenon concerned all the studied age groups. These differences were not significant regarding the expression of SIRT1; however, the same tendency was noticeable.The analysis of CD3+ and CD3+CD56+ lymphocytes showed the increase in the number of NKT-like cells and decreased number of T cells in the process of ageing. The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NKT-like and T-lymphocytes of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing cellular homeostasis specific for healthy ageing. Furthermore, the higher expression of the protective proteins in NKT-like cells compared to T lymphocytes may indicate their particular role in the interplay between innate and adaptive immunity responses during the process of ageing.
NKT-like cells are T lymphocytes coexpressing several NK cell-associated receptors. They are effector lymphocytes of innate and adaptive immunity, and their number increases with age. The study aimed to analyze the expression of cellular protective proteins, i.e. sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in NKT-like and T cells of the young ('young', 31 subjects, age range 19-24 years), seniors aged under 85 ('old'; 30 subjects, age range 65-84 years) and seniors aged over 85 ('oldest', 24 subjects, age range 85-94 years). Both NKT-like and T cells were cultured for 48 h and stimulated with IL-2, LPS and PMA with ionomycin and compared with unstimulated control cells.The oldest seniors varied from the other age groups by significantly increased expression of SIRT1 and HSP70 in both NKT-like and T cells observed in both stimulated and nonstimulated cells. The analyzed lymphocyte populations of the oldest revealed not only the highest expression of these proteins but also insensitivity to all types of applied stimulation. When NKT-like cells were compared to T cells, higher expression of the studied protective proteins was observed in both stimulated and unstimulated NKT-like cells. Neither CD3 + CD56+ nor CD3+ cells revealed elevated expression of SOD2, and these cells responded to stimulation until very advanced age. T cells revealed higher sensitivity to stimulation with IL-2 regarding SIRT1 and HSP70 expression. NKT-like cells were more sensitive to stimulation with PMA and ionomycin concerning the expression of these proteins. IL-2 did not induce a significant increase in SOD2 expression in the studied age groups.The oldest seniors developed an adaptive stress response in both T and NKT-like cells regarding the expression of SIRT1 and HSP70, which was increased and insensitive to further stimulation in contrast to SOD2, which showed a more inducible pattern of expression. CD3 + CD56+ cells exhibited higher expression of cellular protective proteins than CD3+ cells in both stimulated and control, nonstimulated cells. NKT-like and T cells showed a distinct sensitivity to the applied stimulatory factors in the respective age groups.
Chimeric antigen receptor T (CAR-T) cells are genetically modified autologous T cells that have revolutionized the treatment of relapsing and refractory haematological malignancies. In this review we present molecular pathways involved in the activation of CAR-T cells, describe in details the structures of receptors and the biological activity of CAR-T cells currently approved for clinical practice in the European Union, and explain the functional differences between them. Finally, we present the potential for the development of CAR-T cells in Poland, as well as indicate the possible directions of future research in this area, including novel modifications and applications of CAR-T cells and CAR-natural killer (NK) cells.
NK cells are a component of innate immunity which activity significantly correlates with health status. The aim of our study was to estimate a status of NK (natural killer) cells in the very old (mean age 92+/-2 ys) and old subjects (mean age 78+/-5 ys) as compared to a control group of young individuals (mean age 25+/-4 ys). NK cells were characterized by measurement of their cytotoxic activity, expression of intracellular interferon gamma, telomere length and telomerase activity in resting and activated cells. The results revealed that the oldest seniors did not differ from the other age groups in the number of NK cells and NK cytotoxic activity, however, they displayed the shortest telomeres and the lowest telomerase activity. Surprisingly, activated NK cells of the very old, similarly to the old subjects, were able to significantly increase intracellular level of IFNgamma. Moreover, activated with IL-2 NK cells of the old and oldest seniors showed increased telomerase activity. The results of our study suggest that the functional status of NK cells and their sensitivity to activation is well preserved until very advanced age and may contribute to longevity and successful ageing.
Pterostilbene, a polyphenolic compound present in grapes and other fruits, has been demonstrated to inhibit growth and induce apoptosis and autophagy in some cancer cell types. We found that pterostilbene at the IC90 concentration of 44 µM inhibited proliferation and induced apoptosis in MOLT4 human leukemia cells. Treatment with pterostilbene resulted in a transient accumulation of cells in the G 0 /G 1 -cell cycle phase followed by the S-phase arrest. Pterostilbene-induced apoptotic death of MOLT4 cells was mediated by caspase-3 activation and was accompanied by the disruption of mitochondrial membrane potential, phosphatidylserine externalization and internucleosomal DNA fragmentation. Our results suggest that pterostilbene could serve as a potential additional chemotherapeutic agent for the treatment of leukemia.
NK cells are cytotoxic lymphocytes of innate immunity composed of: cytotoxic CD56dim and immunoregulatory CD56bright cells. The study aimed to analyze the expression of cellular protective proteins: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in CD56dim and CD56bright NK cells of the young, seniors aged under 85 ('the old') and seniors aged over 85 ('the oldest'). We studied both non-stimulated NK cells and cells stimulated by IL-2, LPS or PMA with ionomycin. The expression level of proinflammatory cytokines TNF and IFN-γ was also assessed in NK cell subsets and some relationships between the studied parameters were analyzed.CD56bright cells showed sensitivity to most of the applied stimulatory agents until very advanced age in regards to the expression of SIRT1 and intracellular HSP70. On the contrary, CD56dim cells, sensitive to stimulation by most of the stimulatory agents in the young and the old, in the oldest lost this sensitivity and presented rather high, constant expression of SIRT1 and HSP70, resistant to further stimulation. With reference to SOD2 expression, CD56dim cells were insensitive to stimulation in the young, but their sensitivity increased with ageing. CD56bright cells were sensitive to most of the applied agents in the young and the old but in the oldest they responded to all of the stimulatory agents used in the study. Similarly, both NK cell subsets were sensitive to stimulation until very advanced age in regards to the expression of TNF and IFN-γ.CD56bright cells maintained sensitivity to stimulation until very advanced age presenting also an increased expression of SIRT1 and HSP70. CD56dim cells showed a constantly increased expression of these cellular protective proteins in the oldest, insensitive for further stimulation. The oldest, however, did not reveal an increased level of SOD2 expression, but it was significantly elevated in both NK cell subsets after stimulation.The pattern of expression of the studied cellular protective proteins in ageing process revealed the adaptation of NK cells to stress response in the oldest seniors which might accompany the immunosenescence and contribute to the long lifespan of this group of the elderly.
The role of the innate immunity during human ageing is not well understood. The aim of the study was to estimate reactivity of NK (natural killer) cells in the very old (mean age 91 years) and old subjects (mean age 78 years) compared to young individuals (mean age 26 years) in respect to the indices of the oxidative stress (telomere length of NK cells, serum content of -H groups), serum total antioxidant status and serum concentrations of interleukin 6 and tumor necrosis factor-α (TNF-α). The activation state of NK cells, reflected by telomerase activity and intracellular interferon γ (IFNγ) content, was also measured. We found that length of telomeres in NK cells and serum concentration of -SH groups decreased both in the old and the oldest subjects as compared to young individuals. The oldest seniors, on the contrary to the old ones, revealed similar level of serum antioxidant status as the young subjects. The serum level of IL-6, not detectable in the young subjects, did not differ in the oldest and old seniors. TNF-α serum concentrations progressively increased with age. After stimulation, NK cells of both old groups showed higher intracellular levels of IFNγ than young subjects. IL-2-activated NK cells of the oldest seniors showed the highest increase of telomerase activity as compared to the other age groups. Serum level of IL-6 correlated positively with activation markers of NK cells. Moreover, in seniors but not in young subjects, the number of active, IFNγ-expressing NK cells, correlated positively with the serum content of the -SH groups. These findings indicate that sensitivity of NK cells to activation is maintained during ageing and this phenomenon may be related to the oxidative and inflammatory status of the elderly.
NK cells are key effector lymphocytes of innate immunity provided with constitutive cytolytic activity, however, their role in human ageing is not entirely understood. The study aimed to analyze the expression of proteins involved in cellular stress response sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in non-stimulated NK cells of the oldest seniors (n = 25; aged over 85; mean age 88 years) and compare with NK cells of the old (n = 30; aged under 85; mean age 76 years) and the young (n = 32; mean age 21 years) to find potential relationships between the level of expression of these proteins in NK cells and longevity. The concentration of carbonyl groups and 8-isoprostanes in NK cell lysates reflecting the level of oxidative stress was also measured.The group of the oldest seniors differed from the other age groups by significantly higher percentage of NK cells expressing SIRT1, HSP70 and SOD2. The concentration of both carbonyl groups and 8-isoprostanes in NK cell extracts remained within the normal range in all age groups. The percentage of NK cells with the expression of, respectively, SIRT1, HSP70 and SOD2 correlated positively with age. Some correlations between expression levels of particular protective proteins SIRT1, HSP70 and SOD2 were observed in the study population.The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NK cells of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing NK cell homeostasis specific for healthy ageing process.
Transforming growth factor β1 (TGF-β1) plays a role in cell proliferation and differentiation, and it can modulate immune response. In this work, we asked whether levels of either TGF-β1 or mRNA of the corresponding gene in plasma or tissue can be useful in diagnosing and/or monitoring of the clinical course of inflammatory bowel diseases (IBD). The study group consisted of 104 pediatric patients with IBD: 36 with Crohn’s disease (CD) and 68 with ulcerative colitis (UC); 42 children represented the control group. TGF-β1 levels in plasma and intestinal mucosa were estimated by ELISA and immunohistochemistry (IHC), respectively. Levels of TGF-β1 mRNA were determined by reverse transcription and real-time PCR. In patients with IBD, and in subgroups with CD and UC, no significant differences in the TGF-β1 level in plasma and tissue were found relative to the control group. These variables were not dependent on the stage of the disease, its activity or severity of endoscopic and histopathological findings. TGF-β1 mRNA levels were significantly higher in tissue samples withdrawn during the relapse of the disease than in those taken during the remission or in the control group. However, no correlation between TGF-β1 plasma levels and TGF-β1 mRNA amount in the intestinal mucosa was observed. The TGF-β1 mRNA level, but not the amount of the gene product, was significantly increased in the pathologically changed tissue during the relapse of IBD. We suggest that this parameter might be considered as a potential prognostic value when assessing IBD in children.
'/%3e%3cpath fill='%23c09300' stroke-width='1.074' d='m453.16 315.06 9.613 43.755-3.158 3.155-3.3-2.439-5.451-39.018 2.296 39.018-3.157 4.016-3.157-4.016 2.296-39.018-5.45 39.018-3.303 2.44-3.154-3.156 9.611-43.755h6.314z'/%3e%3cg id='b' fill='none' stroke-linejoin='round' stroke-width='1.27'%3e%3cpath fill='%23fff' stroke-width='1.189' d='m428.48 295.84-19.08 67.705 26.255 4.018 11.188-50.924-18.363-20.8z'/%3e%3cpath d='m422.17 318.94 2.296 5.593 12.405-11.848'/%3e%3cpath d='m430.76 304.92 2.596 24.346 7.89-10.328m-3.156 4.012 4.31 14.875m1.69-5.465-8.718 13.241m2.72 13.216-2.791-13.198-2.436-13.423-5.84 7.91-2.562-9.12-8.174 8.385 4.157 15.25 5.738-9.434 3.155 9.612 5.89-9.162'/%3e%3cpath d='m414.99 361.97 5.3-7.444 3.452 11.457 4.735-8.032 3.157 9.614'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='matrix(-1 0 0 1 900 0)'/%3e%3cg stroke-linecap='round' stroke-linejoin='round' stroke-width='1.27'%3e%3cpath stroke-width='2.378' d='M450 393.81c19.943 0 39.024-1.578 50.213-4.734 4.735-.859 4.735-3.298 4.735-6.455 4.736-1.578 2.295-7.17 5.595-7.17-3.3.862-4.016-5.595-8.033-4.732 0-5.597-5.596-6.313-10.33-4.738-9.47 3.158-26.255 3.875-42.18 3.875-15.924-.717-32.567-.717-42.18-3.875-4.733-1.575-10.326-.859-10.326 4.738-4.018-.863-4.736 5.594-8.034 4.731 3.299 0 .86 5.593 5.594 7.17 0 3.158 0 5.597 4.878 6.456 11.045 3.156 30.128 4.734 50.068 4.734z'/%3e%3cpath d='M422.89 363.54c6.454.861 13.628 1.578 19.225.861 3.153 0 5.45 5.45-.863 6.313-5.593.715-14.345 0-19.08-.862-4.017-.718-12.768-2.295-18.364-3.876-5.594-2.436-1.576-7.168 1.578-6.452 4.879 1.577 11.192 3.157 17.505 4.016zm54.23 0c-6.457.861-13.628 1.578-19.081.861-3.3 0-5.596 5.45.717 6.313 5.596.715 14.347 0 19.078-.862 4.019-.718 12.77-2.295 18.364-3.876 5.597-2.436 1.58-7.168-1.577-6.452-4.878 1.577-11.19 3.157-17.501 4.016z'/%3e%3cpath d='M403.09 360.39c-4.877-.861-7.172 4.732-5.593 7.887.717-1.578 4.017-1.578 4.735-3.155.858-2.437-.718-2.437.858-4.732zm19.08 14.67c0-3.153 3.155-2.765 3.155-5.921 0-1.577-.86-4.018-2.437-4.018-1.579 0-3.158 1.578-3.158 3.156-.718 3.157 2.44 3.63 2.44 6.783zm22.71-9.08c4.73 0 4.262 6.314 1.966 9.47 0-2.295-4.017-3.156-4.017-4.732 0-2.442 3.626-2.442 2.051-4.738zm52.03-5.59c4.878-.861 7.174 4.732 5.595 7.887-.718-1.578-4.017-1.578-4.734-3.155-.86-2.437.718-2.437-.86-4.732zm-19.08 14.67c0-3.153-3.153-2.765-3.153-5.921 0-1.577.86-4.018 2.438-4.018 1.577 0 3.156 1.578 3.156 3.156.718 3.157-2.44 3.63-2.44 6.783zm-22.71-9.08c-4.732 0-4.263 6.314-1.967 9.47 0-2.295 4.015-3.156 4.015-4.732.001-2.442-3.626-2.442-2.048-4.738z'/%3e%3cpath stroke-width='.891' d='M404.67 361.97c1.58 0 4.018.718 4.735 1.577l-4.74-1.58zm7.88 2.44c.862 0 4.018.716 5.596 1.575l-5.6-1.58zm28.7 3.15c-1.578 0-4.734 0-5.593.715l5.59-.72zm-8.75 0c-.859-.862-4.016-.862-5.593 0h5.59zm62.84-5.59c-1.578 0-3.875.718-4.735 1.577l4.735-1.577zm-7.89 2.44c-.86 0-4.017.716-5.595 1.575l5.6-1.58zm-28.7 3.15c1.58 0 4.735 0 5.595.715l-5.6-.72zm8.75 0c.86-.862 4.018-.862 5.595 0h-5.6z'/%3e%3cg fill='%23c09300' stroke='none'%3e%3cpath d='M403.34 374.58c-.534-.102-.815-.525-.68-1.025.164-.609.651-.957 1.112-.794.294.104.83.645.832.839.003.222-.28.752-.402.754-.053 0-.135.046-.182.102-.1.118-.413.175-.68.124zm54.93 3.92c-.199-.074-.515-.518-.515-.724.002-.387.574-1.018.929-1.021.175-.001.655.248.834.434.325.338.254.893-.154 1.208-.201.155-.8.212-1.094.103zm.36 2.48c-.42-.142-.574-.348-.608-.814-.036-.485.012-.56.525-.84l.365-.2.347.155c.48.214.693.445.71.767.018.348-.24.704-.64.883-.352.158-.373.16-.699.05z'/%3e%3cpath d='M407.75 370.09c-.346-.01-.821.307-1.094.469-.649.152-1.404.55-2.031.125-.617-.191-1.438-.04-1.531.718.158.652 1.071 1.025 1.625.594.41-.537 1.531-.853 1.656.063-.457.692-.405 1.609-.781 2.344-.061.457-.252.892-.5 1.28-.462.047-.954-.007-1.344.282-.626.034-1.265.345-1.625.875-.403.578-.789 1.165-.906 1.875.107.725.937.933 1.562.969.667.183 1.335.424 2 .625 1.086.243 2.102.635 3.188.875 1.584.477 3.248.697 4.844 1.125.167.055.324.104.5.125.662.137 1.007-.583 1.062-1.125.329-1.16.58-2.34.906-3.5.225-.466.497-1.517-.406-1.281-.515.308-.952.773-1.594.812-.874.05-.375.945-.031 1.313.076.587-.11 1.288-.5 1.75-.52.33-1.172-.057-1.719-.188-.574.01-1.723-.283-1.187-1.062.19-.577.207-1.209.437-1.782.294-.614.336-1.285.469-1.937-.361-.777-.979.256-1.469.344-.338.234-1.605.334-.937.937.575.437.134 1.197.03 1.75-.036.77-.837.954-1.468.781-.628-.03-1.501-.497-.969-1.187.121-.575.322-1.14.438-1.719.237-.698.437-1.363.687-2.062a5.78 5.78 0 0 1 .625-1.75c.018-.686.478-1.276.47-1.97-.048-.338-.2-.461-.407-.468zm-3.594 7.25a.353.353 0 0 1 .219.031c.25.106.257.31 0 .531-.112.097-.24.191-.281.188-.468-.04-.606-.11-.625-.281-.017-.151.03-.195.343-.344a1.45 1.45 0 0 1 .344-.125zm-1.036 5.02c-.496-.466-.416-.806.317-1.353.314-.234.505-.208.863.12.553.504.575.823.087 1.255-.24.213-.346.257-.627.26-.293.003-.376-.034-.64-.282zm3.05.92a.86.86 0 0 1-.623-1.088c.129-.434.203-.477.84-.483.716-.007.854.112.861.741.004.355-.023.437-.2.6-.207.193-.605.297-.878.23zm89.11.08c-.227-.189-.273-.274-.275-.515-.003-.41.233-.712.746-.944.682-.308.96-.266 1.216.186.342.601.325.847-.084 1.252-.225.224-.268.237-.785.242-.507.003-.565-.01-.818-.221zm-85.06 1.15c-.364-.097-.552-.37-.557-.807-.004-.334.02-.387.253-.57.152-.118.39-.22.579-.245.267-.037.364-.013.58.138.55.387.667.77.348 1.14-.34.397-.662.488-1.203.344zm21.92.98c-.08-.1-.26-.16-.278-.287.032-.555.059-1.117.236-1.648.096-.523.012-1.07.194-1.579.187-.927.187-1.882.402-2.803.01-.475.055-.95.193-1.406.157-.722.013-1.476.226-2.19-.023-.267.285-.983.556-.559.394.577.912 1.045 1.421 1.516.438.252.036.624-.266.75-.416.163-.517.62-.487 1.022-.058.414-.225.806-.242 1.23-.087.622-.07 1.254-.151 1.876-.182.609-.113 1.26-.173 1.888.008.398-.182.76-.156 1.16-.048.406-.066.894-.407 1.172-.329.166-.804.138-1.068-.142zm29.7-9.8c-.482.365-.914.795-1.344 1.219-.592.449.487.71.625 1.094.157.586.148 1.21.188 1.812.179.552.258 1.138.218 1.719-.051.622-.863.491-1.218.844-.492.207-.688.69-1.063 1.03-.246.484-.312 1.077-.344 1.626.036.448-.302.95 0 1.344.04.055.086.132.125.187.102.145.343.03.5.063.504-.029 1.029.047 1.5-.157 1.21-.209 2.432-.226 3.657-.28.749.023 1.47-.238 2.218-.22.587.13.851-.506.844-.968-.332-.654-.045-1.429-.312-2.094-.235-.673-.143-1.428-.25-2.125-.015-.549-.711-.752-1.063-.344-.341.345-.893.414-1.156.844-.297.602.627.603.781 1.062.176.465.118.98.156 1.47.115.592-.529.625-.937.687-.537.2-1.29.336-1.594-.313-.19-.48-.165-.993-.219-1.5-.013-.892-.227-1.768-.28-2.656-.052-1.097-.242-2.187-.313-3.281-.01-.456-.071-1.198-.72-1.063zm-.625 8.25.313.031.03.375.032.375-.406.125a3.28 3.28 0 0 1-.407.094 2.55 2.55 0 0 1-.187-.125c-.22-.159-.164-.385.125-.688.163-.17.239-.203.5-.187zm-29.965-9.28c-.38.135-.862.693-1.25.688-.88.062-.757.919-.125 1.218.083.26.005.588.031.875.123.79-.375 1.425-.281 2.22-.044.87-.307 1.717-.344 2.593-.247.903-.25 1.832-.406 2.75-.104.823-.7.533-1.219.312-.004-.356-.027-.705-.031-1.062.216-.817-.441-1.056-1.094-1.156-.686.065-1.023-.523-.875-1.125.295-.351 1.036-.254 1.5-.313.981.204.942-1.112.438-1.562-.345-.636-1.199-.91-1.438-1.563.096-.866-.48-1.732-1.187-2.156-1.089-.076-1.91.863-2.313 1.781-.452.131-.904.267-1.344.438-.709.177-1.72 1.407-.75 1.875.545.15 2.2.528 1.47 1.28-.413.708-1.202.72-1.907.532-.724 0-1.53-.352-1.5-1.187-.148-.75-.123-1.558-.469-2.25-.148-.84-1.1-.643-1.156.125-.733.534-.636 1.397-.125 2.03.319.73.003 1.578-.313 2.25-.19.876-1.155.906-1.843 1.188-.338.131-1.629-.078-1.156.657.753.265 1.652.505 2.437.312.823-.102 1.538-.665 1.938-1.375.552-.56 1.502-.267 2.218-.281.773-.002 1.563.542 2.313.187.217-.564 1.222-1.495 1.531-.53-.053.862.71 1.325 1.5 1.218.864-.05.544.61.531 1.125.057.934.66 1.425 1.438 1.812.265.072.545.048.812 0 .748-.235 1.554-.568 1.781-1.406.304-.68.311-1.45.532-2.156.177-1.13.406-2.26.437-3.406.276-1.039.197-2.136.406-3.188.133-.782.231-1.563.313-2.344-.092-.417-.272-.487-.5-.406zm-6.781 4.156c.151.019.235.204.25.531.016.365.206.661.437.72.183.045.167.238-.03.374-.116.08-.322.127-.688.125-.578-.003-.937-.153-1.25-.469l-.188-.187.281-.188c.142-.108.399-.332.563-.5.285-.29.473-.424.625-.406zm66.031-7.876a8.365 8.365 0 0 0-1.656.25c-1.04-.065-1.563 1.027-.563 1.563.602 1.534 1.54-.317 2.5-.188 1.438.28 1.56 1.832 1.781 3 .081 1.157.415 2.282.75 3.375.963 1.07-.72 1.74-1.437.906-.611-.664-1.92-1.48-2.656-.562-.904.382-.94 1.59-1.688 1.937-1.209.368-1.361-1.119-2-1.75-.587-.824-1.71-.896-2.625-1.187-.429-.869-.205-2.346-1.094-2.938-.745.304-2.113 1.702-.968 2.344 1.117.96-.488 1.401-1 2.063-.732.814-.849 1.936-.969 2.968-1.26.696-1.504-.787-1.656-1.718-.098-1.112-1.036-.786-1.688-.375-.952.385-1.428 1.29-2.094 2-.08.647.038 1.356.032 2.03.216.812 1.202.431 1.812.376.979-.41 1.38.627.594 1.219-.532.676-2.22.322-2.063 1.406.544.063 1.111.043 1.657 0 1.09-.298 2.261-1.068 2.375-2.281.143-1.059 1.667-.866 2.5-1.094 1.04-.3 2.294-.377 2.53.937.71.745 2.25 1.584 3.032.563.795-.485 1.176-1.338 1.125-2.25-.162-.785 1.269-.813 1.625-.375.521.858 2.151.605 2.938.125.836-.617 1.017-1.705 2.218-1.719 1.783-.534 3.635-.841 5.375-1.5 1.41-.307-.21-1.198-.562-1.719-1.074-.53-2.008 1.642-3.219.563-.936-.716-.872-2.077-1.219-3.125-.237-1.358-.177-2.923-1.156-4-.672-.715-1.586-.887-2.531-.844zm-6.813 9.469c.126-.003.268.06.5.187.439.242.751.643.75.969 0 .213-.054.227-.375.375-.197.091-.42.189-.5.188-.18-.002-.624-.357-.625-.5 0-.06-.058-.287-.125-.47-.117-.32-.099-.316.063-.53.103-.138.187-.216.313-.22zm6.532.344c.372-.025.593.091.75.375.181.329.058.58-.407.78-.203.089-.407.16-.437.157-.03-.003-.198-.102-.375-.219-.27-.177-.31-.264-.313-.468-.003-.316.35-.597.782-.625zm-8.938.093c.11-.017.209.04.344.156.18.155.237.28.281.594a2.23 2.23 0 0 1 0 .625c-.048.226-.053.216-.562.219-.64.006-.7-.064-.688-.656.008-.377.012-.478.219-.688.16-.162.297-.233.406-.25zm-5.5 1.438a.57.57 0 0 1 .438.437c.065.318.008.462-.25.594-.222.113-.734.15-.875.063a.613.613 0 0 1-.188-.188c-.068-.121-.055-.18.031-.25.06-.05.126-.12.125-.156 0-.037.068-.163.157-.281a.543.543 0 0 1 .562-.22zm9.25 1.187a.651.651 0 0 1 .438.156c.185.163.179.4-.031.563-.136.104-.872.095-1.125 0-.113-.042-.156-.099-.157-.219-.002-.14.038-.227.281-.344.205-.097.416-.155.594-.156zm-17.171 4.819c-.096-.063-.238-.117-.095-.222.173-.302.518-.407.825-.52a3.436 3.436 0 0 0 1.373-1.118c.05-.333.424-.513.422-.861.003-.475.032-.962-.084-1.426-.129-.426-.452-.752-.827-.974-.256-.202-.674-.258-.768-.614-.053-.343.253-.604.36-.91.223-.421.411-.863.651-1.275.26-.288.659-.015.793.263.19.277.357.576.45.897.303.332.571.721.68 1.162.148.31.383.602.369.966 0 .416.235.787.22 1.206.014.56.013 1.145-.191 1.674-.14.294-.421.47-.607.733-.319.295-.67.579-1.08.736-.289.092-.457.426-.819.345-.514.009-1.037.079-1.547-.004.032.054-.156-.092-.125-.058zm-6.85.45c-.243-.25-.295-.355-.298-.6-.003-.248.045-.346.282-.586.439-.443.653-.453 1.41-.07.722.365.967.399 1.032.14.062-.25.478-.48.874-.481a.82.82 0 0 1 .592.203c.251.196.263.227.27.68.004.463-.002.48-.29.736-.278.244-.323.259-.666.224-.437-.043-.7-.244-.77-.588-.052-.25-.14-.31-.206-.14-.021.055-.129.122-.239.15-.204.051-.549.268-.794.5-.093.087-.255.128-.519.131-.355.003-.405-.018-.678-.299zm-19.82-8.9c-.665.007-1.38.566-1.469 1.219.152.752.777 1.49.375 2.281.278 1.013-.761 1.397-1.469.813-.427-.972-.643-2.036-1.28-2.907-.805-.227-1.155 1.057-1.72 1.5.28.755 1.096 1.433 1.188 2.375.195.915-.361 1.93-1.156 2.406-.686.655-1.626.421-2.47.563-1.023.599.753.818 1.22.844 1.047.106 2.135-.032 2.906-.813.769-.367.71-1.697 1.625-1.844 1.588-.07 3.163.244 4.75.282.85.203 2.163-.101 2.75.53.002 1.108.94 1.791 1.844 2.22.498.138 1.024-.48 1.53-.594.966-.308.673-1.502 1.25-1.969 1.651-.09 3.317-.07 4.97-.093.314.007.407-.44.625-.626-.037-.638-.06-1.272-.157-1.906-.318-.806.052-1.844-.53-2.53-.83-.227-1.537.533-2.376.593-.926.412-1.706 1.268-1.75 2.312-.548.897-1.427-.16-1.125-.906-.034-.325.07-.704-.031-1-.507-.608-1.395-.41-2.094-.5-1.018.04-2.012-.18-3.031-.156-.893-.068-1.822.078-2.688-.156-1.031-.03-.89-1.039-1.062-1.75a1.018 1.018 0 0 0-.625-.188zm2 3.688c.343.006.667.051 1 .093.43.071.89-.148 1.313 0 .976.166 1.953.127 2.937.219.26-.004.446.185.625.344.002.683 0 1.379 0 2.062-.03.258.076.598-.156.781a.7.7 0 0 1-.25.125c-.27-.07-.558-.247-.688-.5-.041-.357.018-.704.031-1.062-.007-.093.04-.237 0-.313a1.248 1.248 0 0 0-.718-.25c-.85-.029-1.682-.091-2.531-.125-.707-.044-1.434.06-2.125-.125-.269-.097-.618-.064-.782-.343-.234-.273.104-.597.344-.72a2.28 2.28 0 0 1 1-.187zm11.281.25c.159-.003.21.05.281.343.046.188.1.417.125.5.037.118-.022.156-.187.219-.494.186-.986.168-1.156-.063-.078-.104.012-.434.156-.562.29-.259.61-.437.781-.438z'/%3e%3c/g%3e%3c/g%3e%3cpath stroke-width='1.15' d='M449.98 327.23c32.642-25.106 29.843-61.883 29.843-61.883a12.72 12.72 0 0 1-2.54.258c-6.848 0-23.179-3.917-26.989-8.892-4.081 4.503-20.77 8.892-27.574 8.892-.861 0-1.722-.086-2.54-.258 0 0-2.843 36.776 29.8 61.883z'/%3e%3cpath stroke-width='.916' d='M477.22 268.04a13.55 13.55 0 0 1-.848.026c-6.205 0-20.619-3.183-26.175-7.957-5.82 4.41-20.47 7.957-26.576 7.957a4.7 4.7 0 0 1-.85-.086c-.014 1.39.059 2.832.145 4.144.323 4.9 1.183 9.819 2.488 14.552 4.108 14.895 12.516 27.553 24.572 37.155 12.066-9.61 20.484-22.28 24.6-37.186 1.308-4.732 2.17-9.65 2.496-14.55.086-1.285.157-2.692.148-4.055z'/%3e%3cpath fill='%23c09300' stroke='none' d='M439.38 265.03c-5.872 1.845-12.229 3.031-15.75 3.031a4.7 4.7 0 0 1-.844-.093c-.014 1.39.07 2.845.156 4.156a72.77 72.77 0 0 0 2.47 14.53c2.764 10.026 7.506 19.033 13.968 26.782V265.03zm20.62-.09v49.25c6.794-7.921 11.733-17.206 14.594-27.562a72.952 72.952 0 0 0 2.469-14.531c.085-1.284.166-2.7.156-4.063-.28.018-.56.032-.844.032-3.73 0-10.42-1.17-16.375-3.125z'/%3e%3cg stroke-width='1.27'%3e%3cpath stroke-width='1.218' d='M462.31 253.09c.667.04-.902-3.568-.902-3.568 1.765 1.804 8.43 2.235 8.43 2.235-4-1.766-7.998-15.096-7.528-25.721.432-10.664-1.528-14.86-3.097-16.428-2-2-8.43-3.764-12.664-4-2.392-.117-2 1.804-2 1.804-4.43-1.137-8.86-1.568-10.86-.235-1.883 1.255-2.275 7.528-.902 6.43 3.332-2.666 6.234-.235 8.233 2.666 1.764 2.549 1.647 9.763-.902 18.192-2.666 8.861-9.959 17.722-9.959 17.722 3.96 0 9.528-3.528 9.528-3.528l-1.333 5.528c4.195-2 7.528-5.097 7.528-5.097l3.999 4.195c1.333-1.764 4-4.195 4-4.195s3.332 3.529 8.429 4z'/%3e%3cpath fill='none' d='M446.12 227.57s-2.235 16.428-6.43 21.094m9.96-21.524s-.863 16.623-3.764 21.956m6.894-21.286s0 18.192 1.098 21.289m2.862-20.389s.902 15.291 4.666 20.819'/%3e%3cpath fill='%23c09300' stroke-width='.354' d='M442.08 219.61c-.196-1.45-.549-2.588-1.059-3.333-2-2.901-4.9-5.332-8.233-2.666 0 0 1.137-3.529 3.568-3.646 1.882-.118 6.155 1.411 9.92 7.841 0 0-2.784-.627-3.45-.039-1.256 1.098-.746 1.843-.746 1.843z'/%3e%3cpath fill='%23c09300' stroke-width='.354' d='M432.44 209.26c.274-.902.706-1.725 1.255-2.078 2-1.333 6.43-.902 10.86.235 0 0-.392-1.921 2-1.803 4.234.235 10.664 2 12.663 3.999.47.51 1.02 1.255 1.49 2.391h-.078c-.98-1.372-3.764-1.293-4.431-1.215-1.059.118-1.725.078-3.137.431-.666.157-1.686.353-2.235.784-.43.353-.784 1.647-1.45 1.647-1.059 0-.98-.274-1.255-.588-.353-.431-.549-1.059-.902-1.02-1.097.197-2.862-.666-5.096-2.43-2.235-1.765-3.098-2.196-5.999-2-2.862.235-3.764 1.843-3.764 1.843l.079-.196z'/%3e%3ccircle cx='448.824' cy='210.672' r='1.176' stroke='none'/%3e%3c/g%3e%3c/g%3e%3c/svg%3e)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)