Biomedical subjects

Jacek M Witkowski

Publications and source records attributed to Jacek M Witkowski.

15 recordsLinked to original sources

PubMed2007

Klotho--a common link in physiological and rheumatoid arthritis-related aging of human CD4+ lymphocytes.

Human CD4(+) T lymphocytes undergo aging-related changes leading to decreased immunity to infections and neoplasms, and to increased frequency of autoimmune diseases including rheumatoid arthritis (RA). Certain changes, observed in the CD4(+) cells of RA patients, resemble those observed during physiological aging, but occur at earlier age. Underlying cellular mechanism(s) of these similarities are so far largely unknown. Here we show that KLOTHO, a beta-glucuronidase gene whose activity changes are associated with aging phenotype, is down-regulated at the mRNA, protein, and enzymatic (beta-glucuronidase) activity levels both in the healthy elderly and especially in RA CD4(+) lymphocytes. Although the exact role of Klotho activity for CD4(+) cell function is unknown, we propose here that it might be involved in anti-inflammatory processes occurring in the young and healthy individuals, but reduced in both healthy elderly and RA patients. To support this hypothesis, we show here that the reduction of Klotho expression and activity in both elderly and patients' lymphocytes occurs in concert with the down-regulation of T cell costimulatory molecule CD28, the latter known to be dependent on increased levels of TNF-alpha. Thus, a common mechanism of KLOTHO down-regulation, but executed at various times in life, may underlie both physiological and disease-related T cell aging. Klotho activity might become a target of anti-RA drug development as well as a tool to help increase the immune system efficiency in the elderly.

Adult↗

PubMed2006

Induction of G2/M phase arrest and apoptosis of human leukemia cells by potent antitumor triazoloacridinone C-1305.

In this study, we show that triazoloacridinone derivative C-1305, a potent antitumor compound, in human lymphoblastic (MOLT4) and promyelocytic (HL60) leukemia cells induces G2/M arrest followed by apoptosis. In both type of cells, C-1305 at biological relevant concentrations corresponding to EC(90) value, induced a significant increase in the fraction of G2/M cells. The cell cycle perturbations were accompanied by the appearance of sub-G1 fraction, which can be considered as the apoptotic cells population. In both human leukemia cells apoptosis was additionally proved by appearance of DNA fragmentation, activation of caspase-3, PARP cleavage, externalization of phosphatydilserine as well as decrease of the mitochondrial transmembrane potential DeltaPsi(m) and ATP depletion. Treatment of lymphoblastic MOLT4 cells with the C-1305 at EC(90) concentration, caused massive death by apoptosis. Compared to MOLT4 cells, the capacity of HL60 cells to execute apoptosis after C-1305 treatment at equitoxic dose was significantly weaker, but very effective at high concentration (4x EC(90)). These differences could originate from different sensitivity of both cell types to cytotoxic action of C-1305 (EC(50) value for MOLT4 cells was 8 times lower than for HL60 cells and the EC(90) value was 14 times lower, respectively). Collectively, these results show that C-1305 is a novel and potent compound which induces G2/M arrest and subsequent apoptosis of human leukemia cells. This strong ability to induce apoptosis of tumor cells support the view that C-1305 could be consider as a new potent and promising antitumor agent.

Acridines↗

PubMed2005

Modulation of CD28 expression with anti-tumor necrosis factor alpha therapy in rheumatoid arthritis.

OBJECTIVE: The immune system of patients with rheumatoid arthritis (RA) is characterized by the accumulation of CD4+ T cells deficient in CD28 expression and the up-regulation of tumor necrosis factor alpha (TNFalpha). Previous in vitro studies have shown that TNFalpha induces transcriptional silencing of the CD28 gene. Because reduced expression of CD28 in T cells compromises immunocompetence, we examined whether CD28 expression is reduced in patients with RA in vivo and whether the reduction is related to TNFalpha. METHODS: Patients with RA and age-matched individuals were recruited. Peripheral blood mononuclear cells were stained for CD3, CD4, CD8, CD28, TNF receptor I (TNFRI), and TNFRII, and analyzed by quantitative flow cytometry. The number of CD28 and TNFR molecules was monitored in a subgroup of patients with RA undergoing treatment with anti-TNFalpha. RESULTS: In addition to higher frequencies of CD28null T cells, patients with RA had significantly reduced numbers of CD28 and TNFRI molecules on CD4+,CD28+ T cells. Normal expression could be restored in vitro by overnight culture, suggesting that CD28 in patients was modulated by exogenous factors. In contrast, treatment with TNFalpha in vitro resulted in further down-regulation. CD28 expression was normalized in patients undergoing TNFalpha-neutralizing therapy. CONCLUSION: Overproduction of TNFalpha in RA induces a global down-regulation of CD28 in CD4+ T cells and may cause reduced sensitivity to costimulatory signals in T cell responses.

Adolescent↗

PubMed2005

Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes.

BACKGROUND: Recent studies show that mechanical stress modifies both morphology and protein expression in podocytes. Ambient glucose is another factor modulating protein synthesis in these cells. In diabetes, podocytes experience elevated glucose concentrations as well as mechanical strain generated by high intracapillary pressures. Both these factors are responsible for podocyte injury, leading to impairment of kidney glomerular function. In the present study, we examined the effects of glucose concentration and mechanical stress on glucose uptake in podocytes. METHODS: Following a 24 h pre-incubation in low (2.5 mM, LG), normal (5.6 mM, NG) or high (30 mM, HG) glucose media, cultured rat podocytes were exposed to 4 h mechanical stress. We used the labelled glucose analogue, [3H]2-deoxy-D-glucose, to measure glucose uptake. The distribution of facilitative glucose transporters GLUT2 and GLUT4 was assessed by flow cytometry. RESULTS: In the control (static) cells, glucose uptake was similar in the three glucose groups. In mechanically stressed podocytes, glucose uptake increased 2-fold in the LG and NG groups but increased 3-fold in the HG group. In the NG cells, mechanical load increased the membrane expression of GLUT2 and reduced the membrane-bound GLUT4. In stretched HG cells, the membrane expression of both GLUT2 and GLUT4 was decreased. High glucose decreased the plasma membrane GLUT2 content in the stretched cells, whereas both static and stretched podocytes showed an elevation in GLUT4. CONCLUSION: Mechanical stress potentiated glucose uptake in podocytes and this effect was enhanced by high ambient glucose. The decreased expression of GLUT2 and GLUT4 on the surface of stretched cells suggests that the activity of other glucose transporters may be regulated by mechanical stress in podocytes.

Animals↗

PubMed2005

Expression of scavenger receptor CD36 in chronic renal failure patients.

BACKGROUND: Patients with chronic renal failure (CRF) are at increased risk of atherosclerosis development. One of the major steps in pathogenesis of atherosclerosis is formation of foam cells. Scavenger receptor CD36 is among the major receptors for oxidized low density lipoproteins (oxLDL) and therefore it plays a crucial role in foam cell formation. The aim of the present study was to investigate the expression of CD36 on blood monocytes of CRF patients. METHODS: Expression of CD36 on blood monocytes of CRF patients treated with hemodialysis (HD), peritoneal dialysis (PD), those not yet on dialysis (predialysis), and controls was assessed with the use of flow cytometry. Additionally, the major lipid peroxidation markers, malondialdehyde (MDA) and 4-hydroxyalkenals (HAE), were measured. Further, impact of treatment with HMG-CoA reductase inhibitors (statins) on CD36 expression in CRF patients was evaluated. RESULTS: Expression of monocyte CD36, measured as mean fluorescence intensity (MFI) was significantly higher in HD and PD patients, when compared to controls without renal insufficiency (respectively: 1011 +/- 288 and 1000 +/- 309 vs. 710 +/- 313; P < 0.01 for both groups). This was not the case in predialysis group (828 +/- 363 vs. 710 +/- 313). Higher concentrations of lipid peroxidation indicators, MDA and HAE were observed in all three subgroups of CRF patients (2.1 +/- 0.51, 2.02 +/- 0.27, and 1.81 +/- 0.53 microm in HD, PD, and predialysis group, respectively, vs. 1.13 +/- 0.59 microm in controls; P < 0.01). Patients treated with statins showed significantly lower CD36 expression than patients without statin therapy. CONCLUSIONS: The present study, for the first time, demonstrates increased expression of CD36 scavenger receptor in CRF patients. This may be a possible risk factor for accelerated atherogenesis observed in this group of patients.

Atherosclerosis↗

PubMed2005

[Flow cytometric analysys of Enterococcus faecalis pAD1(-) strains response to cAD1 pheromone].

Conjugative plasmids transfer in Enterococcus faecalis is inducted by sex pheromones. The pheromone is excreted by recipient cells and induces expression of aggregation protein AS in donor cells. This protein is involved in formation of matting aggregates. Use of flow cytometry and anti-As monoclonal antibodies allowed collect of interesting data pheromone response. However, according to our knowledge, no study focused on unspecific influence on particular pheromone for plasmid-free recipient strains. Six pAD1 (-) and tree pAD1 (+) Enterococcus faecalis stains were cultivated for 18h in BHI, with and without cAD1 pheromone (Sigma, Germany), respectively. The bacteria were washed, stained with carboksyfluorescein (FCDA, and analyzed by flow cytometry in FACS BD scan cytometr. Relative fluorescence and size of aggregation was used to compare influence on particular strains. Surprisingly, the results shows divergence in fluorescence, size of aggregates and degree of correlation between fluorescence of aggregates and their sizes among pAD1(-) strains, allowing for distinguish of two groups. Three of studied strains have higher fluorescence than pAD (+) stains. Correlation between fluorescence and size of aggregates, significant higher than in pAD1(+) stains, decrease from r = 0.88 to r=0.74 in reaction to cAD1. The strains if other group fluorize with lower intensity than pAD1 (+). Furthermore, 30.4% pAD1 (-) of second group have no detectable fluorescence. In contrast to pAD1 (-) ) strains of the first group and pA1 (+) strains, low (r=0.55) correlation between fluorescence and size of aggregates of group II increase up to r=0.74 after incubation with cAD1 pheromone. Previous study of these pAD1 (-) strains, currently assigned to group II, shown their low frequency of collecting aph2" gene encoded on other conjugative plasmid, pMG. According to these results, such flow cytometric analysis may be used to predict ability of strain to collect unrelated conjugative plasmid.

Enterococcus faecalis↗

PubMed2004

Characterization of glucose uptake by cultured rat podocytes.

The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.

Animals↗

PubMed2004

Paradoxical age-related cell cycle quickening of human CD4(+) lymphocytes: a role for cyclin D1 and calpain.

Precise determination of cell cycle length and G(0)-->G(1) transition time of CD4(+) lymphocytes in relation to age was never done before. We show that the cell cycle of healthy elderly donors' CD4(+) cells is significantly shorter, while time to the first division (G(0)-->G(1)) extended compared to cells of young people. The G(0)-->G(1) time inversely correlates with cycle length and the number of CD28 molecules. Quickening of elderly CD4(+) cell divisions depends on overexpression of cyclin D1, possibly related to lowered proteolytic degradation by calpain. Apoptosis eliminates most of responding cells after only one or two divisions, especially in older donors.

Adult↗

PubMed2004

Decreased proliferative capability of CD4(+) cells of elderly people is associated with faster loss of activation-related antigens and accumulation of regulatory T cells.

Decreased proliferation of CD4(+) lymphocytes of elderly people is at least in part due to lowered proportion of cells that are capable of dividing and producing viable progeny (effective precursors). We show that age-dependent reduction in effective precursor numbers depends on the one hand, extensive, activation-dependent apoptosis occurring from the very onset of stimulation and, on the other, the accumulation of non-dividing, regulatory (suppressor) CD4(lo)CD25(+)CD28(lo) T cells. In addition, analysis of changes in surface expression of activation-related antigens, including CD25, CD69, and PCNA in consecutive generations of dividing CD4(+) cells traced by carboxyfluorescein diacetate succinimidyl ester staining showed variable patterns of these changes that may relate to various aspect of impaired division of these cells in elderly.

Adult↗

PubMed2004

CD3 receptor modulation in Jurkat leukemic cell line.

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.

Alkaloids↗

PubMed2003

Modulation of CD40L antigen expression in Jurkat cells: involvement of protein kinase C activity.

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.

Alkaloids↗

PubMed2003

Viral strategies in modulation of NF-kappaB activity.

Activation of the nuclear factor (NF)-kappaB transcription factor family in response to different stimuli, such as inflammatory cytokines, stress inducers or pathogen products, results in host innate and adaptive immunity. NF-kappaB plays a central role in promoting the expression of genes involved in inflammatory, immune and apoptotic processes, including those encoding cytokines, chemokines, cytokine receptors and proteins involved in antigen presentation. Although the main function of NF-kappaB is to activate specific genes in the cells of the immune system, its role in controlling the host cell cycle makes NF-kappaB an interesting target for pathogenic viruses. Some viruses take advantage of the anti-apoptotic properties of NF-kappaB to escape host defence mechanisms, others use apoptosis to spread. This review describes the role of the NF-kappaB family in immune responses, the mechanism of NF-kappaB activation, and different strategies that viruses have developed to modulate the NF-kappaB pathway in order to facilitate and enhance viral replication and avoid host immune responses.

Apoptosis↗

PubMed2002

Modulation of the activity of calcium-activated neutral proteases (calpains) in chronic lymphocytic leukemia (B-CLL) cells.

Decreased susceptibility to apoptosis and impaired proliferative control are thought to be responsible for prolonged life span and accumulation of chronic lymphocytic leukemia (B-CLL) cells. The activity of calpains (calcium-dependent, neutral proteases, active in the cells responding to signals inducing a rise of cytoplasmic Ca(++)) is involved in the regulation of apoptosis of some cell types by interaction with caspase-3. This work verifies the hypothesis of the abnormal activity of calpains and its role in reduced apoptosis of the B-CLL cells. Casein zymography, reverse transcriptase-polymerase chain reaction, and Western blotting were used for identification and quantification of the activity and expression of calpains in B-CLL cells and purified normal B lymphocytes. The activity and expression of mu-calpain (requiring micromolar Ca(++) for activation) are significantly higher in the leukemic than in nonmalignant cells. Contrarily, the activity and expression of m-calpain (requiring millimolar Ca(++)) as well as the expression of calpastatin (an endogenous inhibitor of calpains) are unchanged or reduced in the B-CLL lymphocytes. Correspondingly, the activity of caspase-3 is many times lower in the B-CLL cells than in normal B lymphocytes. Inhibition of overexpressed mu-calpain in living B-CLL cells in vitro results in doubling of the proportion of the cells undergoing spontaneous apoptosis. This observation suggests a possible role for calpains in longer survival of the B-CLL cells and may open new therapeutic possibilities.

Adult↗

PubMed2002

Heterogeneous susceptibility to spontaneous and induced apoptosis characterizes two related transplantable melanomas with different biological properties.

A family of phenotypically and biologically different transplantable hamster melanomas was derived from a single tumor more than 40 yr ago. In this work, we were seeking the differences between the abilities of the cells from two biologically heterogeneous (melanotic and amelanotic) members of this family to undergo spontaneous or camptothecin-induced apoptosis. We studied these differences by looking at three important features of the apoptotic process, i.e. binding of annexin V, DNA fragmentation and caspase-3 activity. Of these, annexin binding and DNA fragmentation were more pronounced in the parental, melanotic line while the activity of caspase-3 was stronger in the amelanotic tumor cells. We concluded that a spontaneous alteration of the original, melanotic melanoma line into an amelanotic one, associated with more aggressive tumor progression, was accompanied by significant decrease in ability to undergo spontaneous and camptothecin-induced apoptosis, and that apoptosis of these two cell types may not depend on the activity of caspase-3.

Animals↗

PubMed2002

Effect of angiotensin II on ANP-dependent guanylyl cyclase activity in cultured mouse and rat podocytes.

The presence of a well-developed contractile apparatus is the feature determining major roles of podocytes in the renal glomeruli. Receptors for a variety of vasoactive hormones are expressed in these cells; however, most of the signaling pathways are still unknown and remain to be elucidated. Angiotensin II (Ang II) and atrial natriuretic peptide (ANP), due to their opposite action, are the major modulators of glomerular filtration. In podocytes, Ang II induces rise in intracellular calcium concentration, whereas ANP stimulates generation of cGMP. The present study was designed to check whether ANP-stimulated cGMP synthesis in podocytes might be affected by Ang II. Cultured rat (RP) and mouse (MP) podocytes were stimulated with ANP, in the absence or presence of Ang II and cyclic GMP was determined by RIA method. Co-incubation of podocytes with ANP and Ang II caused significant (p < 0.01) suppression of ANP-dependent cGMP generation. The effect was prevented by saralasin, an inhibitor of angiotensin receptors. Phorbol-12-myristate-13-acetate (PMA) mimicked, whereas chelerythrine reversed inhibitory effect of Ang II. In conclusion, angiotensin II counteracts ANP-stimulated cGMP synthesis in cultured podocytes. It seems likely that the protein kinase C pathway is involved in this effect.

Angiotensin II↗