ABSTRACT
Klotho is a beta-glucuronidase that reveals both anti-inflammatory and anti-oxidative properties that have been associated with mechanisms of aging. The study aimed to analyze the relationships between the serum concentration of soluble α-Klotho and cellular activity of two populations of lymphocytes; T and NKT-like cells corresponding to the level of cytokine secretion; i.e., IFN-γ, TNF-α, and IL-6. The studied population comprised three age groups: young individuals ('young'), seniors aged under 85 ('old'), and seniors aged over 85 ('oldest'). Both NKT-like and T cells were either non-cultured or cultured for 48 h and stimulated appropriately with IL-2, LPS or PMA with ionomycin to compare with unstimulated control cells. In all studied age groups non-cultured or cultured NKT-like cells revealed higher expressions of TNF-α, IL-6, and IFN-γ than T cells. α-Klotho concentration in serum decreased significantly in the process of aging. Intriguingly, only IFN-γ expression revealed a positive correlation with α-Klotho protein serum concentration in both non-cultured and cultured T and NKT-like cells. Since IFN-γ is engaged in the maintenance of immune homeostasis, the observed relationships may indicate the involvement of α-Klotho and cellular IFN-γ expression in the network of adaptive mechanisms developed during the process of human aging.
Subject(s)
Interferon-gamma , T-Lymphocytes , Aged , Humans , Aging , Anti-Inflammatory Agents/pharmacology , Cells, Cultured , Interferon-gamma/metabolism , Interleukin-6 , Tumor Necrosis Factor-alpha/metabolism , Klotho Proteins/metabolismABSTRACT
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
ABSTRACT
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
ABSTRACT
BACKGROUND: Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely 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 human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n = 30; 75.6 ± 0.9 years) and the young (n = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. RESULTS: The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. CONCLUSIONS: The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between expression of these cellular protective proteins both in stimulated and non-stimulated cells. These phenomena may contribute to the long lifespan of this group of elderly. Interestingly, in NK cells SOD2 revealed a distinct role in cellular stress response since it showed sensitivity to stimulation increasing with age of participants. These observations provide novel data concerning the role of NK cells in the process of ageing.
ABSTRACT
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
ABSTRACT
Introduction: Heme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation, and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors, and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation, that is, NOD-like receptor protein 3 (NLRP3), glutathione (GSH), GSH disulfide (GSSG), and interleukin 6 (IL-6). Methods: The study population comprised three age groups: young adults (age range, 19-23 years), older adults aged under 85 years (age range, 73-84 years), and older adults aged over 85 years (age range, 85-92 years). NLRP3, GSH, and GSSG concentrations were measured in serum, whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) with ionomycin. Results: The analysis of serum NLRP3, GSH, and GSSG concentrations revealed no statistically significant differences among the studied age groups. However, some typical trends of aging were observed, such as a decrease in GSH concentration and an increase in both GSSG level, and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover, statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells. Conclusions: These results showed that NK cells can express HO-1 at a basal level, which was significantly increased in activated cells, even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters.
Subject(s)
Aging , Heme Oxygenase-1 , Killer Cells, Natural , Oxidative Stress , Humans , Heme Oxygenase-1/metabolism , Aging/immunology , Aged, 80 and over , Aged , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Young Adult , Female , Glutathione/metabolism , Interleukin-6/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , AdultABSTRACT
PURPOSE: Transforming growth factor ß1 (TGF-ß1) plays a role in cell proliferation and differentiation, and it can modulate immune response. In this work, we asked whether levels of either TGF-ß1 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). METHODS: 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-ß1 levels in plasma and intestinal mucosa were estimated by ELISA and immunohistochemistry (IHC), respectively. Levels of TGF-ß1 mRNA were determined by reverse transcription and real-time PCR. RESULTS: In patients with IBD, and in subgroups with CD and UC, no significant differences in the TGF-ß1 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-ß1 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-ß1 plasma levels and TGF-ß1 mRNA amount in the intestinal mucosa was observed. CONCLUSIONS: The TGF-ß1 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.
Subject(s)
Inflammatory Bowel Diseases/diagnosis , Inflammatory Bowel Diseases/genetics , Transforming Growth Factor beta1/genetics , Adolescent , Case-Control Studies , Child , Child, Preschool , Female , Humans , Infant , Inflammatory Bowel Diseases/blood , Intestinal Mucosa/metabolism , Intestines/pathology , Male , Prognosis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transforming Growth Factor beta1/blood , Transforming Growth Factor beta1/metabolismABSTRACT
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.
ABSTRACT
INTRODUCTION: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Subject(s)
Aging/metabolism , Gene Expression Regulation , HSP70 Heat-Shock Proteins/biosynthesis , Natural Killer T-Cells/metabolism , Sirtuin 2/biosynthesis , Superoxide Dismutase/biosynthesis , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Natural Killer T-Cells/cytologyABSTRACT
INTRODUCTION: Aging causes morphological and functional changes in the thyroid gland. Free radicals play a key role in the pathology of normal aging. Vimentin and cytokeratin are cytoskeletal intermediate filaments that are often used as indirect indices of tissue injury. The aim of the study was to clarify the age-related alterations in the structure and function of the thyroid gland. The relationship between oxidative/antioxidative stress markers and cytoskeletal intermediate filaments (vimentin and cytokeratin) and oxidative/antioxidative stress markers as well as vascular endothelial growth factor (VEGF) during aging were elucidated. Finally, the role of Nigella sativa (NS) oil in ameliorating age-related alterations of the structure and function of the thyroid gland was studied. MATERIAL AND METHODS: Thirty Sprague-Dawley albino rats were divided into five groups: young adult control, young adult NS-treated, late adult control, late adult NS-treated, and senile. The age of young adult, late adult, and senile rats was nearly 7, 18 and 22 months, respectively. NS oil was added to food pellets and was administered at a daily dose of 0.1 g/kg body weight for one month. The thyroid gland was dissected and fixed immediately in 10% formalin saline. The assessment of thyroid structure was based on hematoxylin and eosin, and Masson's trichrome stainings, and histomorphometric analysis of the deparaffinized sections. Localization and distribution of vimentin and cytokeratin filaments was assessed by immunohistochemistry. Measurements of VEGF gene expression by qPCR and oxidative/antioxidative markers (malondialdehyde and glutathione content, superoxide dismutase activity) in thyroid gland homogenates were performed. Serum concentration of thyroid hormones (T3, T4) and TSH were assessed by radioimmunoassay. RESULTS: Follicles in the late adult control group were dilated and disrupted. Follicular cells showed cytoplasmic vacuolation. Follicles in the thyroids of senile rats were of irregular shape, often with cellular exfoliations. Many follicles were dilated and lined with flattened cells. A notable amelioration of these morphological alterations was observed in late adult NS-treated rats. Decrease in serum T3 and T4 levels and increase in TSH levels were observed in the late adult control and senile groups. A clear shift of the oxidative/antioxidative markers (MDA/ /GSH, SOD) was observed in the late adult control and senile groups in favor of oxidants. Administration of NS to late adult rats resulted in normalization of these parameters. Increased area of collagen fibers, immunoreactivity of vimentin and cytokeratin filaments and VEGF gene expression were observed in the thyroids of late adult and senile rat groups as compared to young animals. The mean number of follicular cells decreased in the late adult control and senile groups. Administration of NS to the late adult rats returned these parameters to the level of the young adult rats. CONCLUSIONS: Aging-related alterations in both structure and function of the rat thyroid gland that are associated with increased indices of oxidative stress might be abrogated by administration of antioxidative agents present in Nigella sativa oil.
Subject(s)
Keratins/metabolism , Nigella/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vimentin/metabolism , Aging , Animals , Antioxidants/metabolism , Malondialdehyde , Oxidative Stress/physiology , Rats, Sprague-Dawley , Thyroid Gland/metabolismABSTRACT
Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system engaged in rapid response against tumor or virus infected cells. After activation NK cells acquire enhanced cytotoxicity and are capable of producing cytokines to stimulate other immune cells. Quantitative PCR (qPCR) is a method of choice for gene expression analysis but the usage of reliable reference genes for the normalization process is critical. Commonly used reference genes may vary in expression level between different experimental conditions providing wrong quantitative results of the studied genes' expression levels. Fourteen potential endogenous control genes were analyzed by qPCR method in NK-92 cell line that shows characteristics of human natural killer cells and is often used in studies on biology of NK lymphocytes. NK-92 cells were stimulated with IL-2 or TNF for 2, 24 or 72 h. Results were analyzed with RefFinder, a program which enables evaluation and screening of reference genes and integrates the currently available major computational programs (Genorm, Normfinder, BestKeeper and Delta Ct). The most stable gene in activated and non-activated NK cells was B2M, followed by IPO-8 and GAPDH and the least stable were HPRT1, PPIA and RPL32. The normalization process was performed on SOD2 gene and the results of qPCR experiments were confirmed by flow cytometry. The flow cytometric data corresponded to the results of qPCR gene expression analysis performed for the reference genes qualified by RefFinder as the most stable.
Subject(s)
Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Polymerase Chain Reaction/methods , Cell Line , Cytokines/biosynthesis , Cytokines/genetics , Flow Cytometry , Gene Expression , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/genetics , Humans , Interleukin-2/immunology , Lymphocyte Activation/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reference Standards , Tumor Necrosis Factor-alpha/immunology , beta 2-Microglobulin/genetics , beta Karyopherins/geneticsABSTRACT
Almost all cases of hyperthyroidism in children result from Graves' disease (GD). Recent studies have confirmed a significant role of T regulatory cells (Tregs) in the development of autoimmune diseases. However, the interactions between T cell responses and Treg proliferation in GD are still poorly understood. The aim of this study was to assess the proliferation of Treg cells (Tregs) and CD3+ T lymphocytes isolated from 50 children with GD before and after treatment with the thyreostatic drug methimazole (MMI). The proliferation rates, measured by methyl-3H-thymidyne incorporation, of CD3+ cells and Tregs stimulated with mitogen phorbol 12-myristate 13-acetate (PMA) were compared with those of unstimulated cells. The proliferation rates of both PMA-stimulated and unstimulated CD3+ cells prior to treatment with MMI were significantly higher than after treatment. Simultaneously, the proliferation rates of both PMA-stimulated and unstimulated Tregs were significantly lower before MMI treatment. Moreover, we observed higher cell proliferation rates of unstimulated and PMA-stimulated Tregs before the initiation of MMI therapy and after treatment in patients who had no relapse of hyperthyroidism. There was a positive correlation between the CD3+ cells proliferation rate before MMI treatment and fT3, as well as fT4 concentration in peripheral blood. The proliferation rates of CD3+ T cells before and after MMI treatment positively correlated with the TSI index. Thus, children suffering from Graves' disease presented lower Tregs proliferative potential compared with CD3+ T cells. Cocultures of CD3+ T cells and Tregs showed that Tregs were not capable of efficiently inhibiting the proliferation of CD3+ T cells in GD patients. Conclusions. MMI treatment reduced the proliferative activity of CD3+ T cells in pediatric GD patients and increased the proliferation rate of Tregs. We suggest that Treg cells that are partly dysfunctional in GD disease are probably suppressed by CD3+ T cells and that methimazole exerts some immunomodulatory effects.
Subject(s)
Antithyroid Agents/therapeutic use , Cell Proliferation/drug effects , Graves Disease/drug therapy , Graves Disease/immunology , Methimazole/therapeutic use , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes/drug effects , Adolescent , CD3 Complex/metabolism , Case-Control Studies , Cells, Cultured , Child , Female , Humans , Lymphocyte Count , T-Lymphocytes/physiology , T-Lymphocytes, Regulatory/physiologyABSTRACT
INTRODUCTION: Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). MATERIAL AND METHODS: HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). RESULTS: Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. CONCLUSIONS: We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.
Subject(s)
Cell-Penetrating Peptides/toxicity , Galanin/metabolism , Galanin/toxicity , Gene Transfer Techniques/standards , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Tumor Suppressor Proteins/metabolism , Wasp Venoms/metabolism , Wasp Venoms/toxicity , Biological Assay , Cell Cycle/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Galanin/chemistry , HCT116 Cells , HT29 Cells , Humans , Lethal Dose 50 , Recombinant Fusion Proteins/chemistry , Wasp Venoms/chemistryABSTRACT
Previously published studies on levels of the transforming growth factor-ß1 (TGF-ß1) protein and mRNA of the corresponding gene in patients suffering from inflammatory bowel diseases (IBD) gave varying results, leading to contradictory conclusions. To solve the contradictions, we aimed to assess longitudinally TGF-ß1 protein and mRNA levels at different stages of the disease in children suffering from IBD. The study group consisted of 19 pediatric patients with IBD at the age between 3.5 and 18.4 years. The control group consisted of 42 children aged between 2.0 and 18.0 years. The plasma TGF-ß1 concentration was measured with ELISA. mRNA levels of the TGF-ß1 gene isolated from samples of the intestinal tissue were assessed by reverse transcription and real-time PCR. Levels of TGF-ß1 protein in plasma and corresponding mRNA in intestinal tissue were significantly higher in IBD patients than in controls. TGF-ß1 and corresponding transcripts were also more abundant in plasma and intestinal tissue, respectively, in patients at the active stage of the disease than during remission. In every single IBD patient, plasma TGF-ß1 level and mRNA level in intestinal tissue was higher at the active stage of the disease than during remission. Levels of TGF-ß1 and corresponding mRNA are elevated during the active stage of IBD but not during the remission. Longitudinal assessment of this cytokine in a single patient may help to monitor the clinical course of IBD.
Subject(s)
Inflammatory Bowel Diseases/genetics , RNA, Messenger/genetics , Transforming Growth Factor beta1/genetics , Adolescent , Child , Child, Preschool , Female , Humans , Inflammatory Bowel Diseases/blood , Inflammatory Bowel Diseases/pathology , Longitudinal Studies , Male , RNA, Messenger/blood , Transforming Growth Factor beta1/bloodABSTRACT
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 IC(90) 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.