Biomedical subjects

Mariano Rodríguez

Publications and source records attributed to Mariano Rodríguez.

At least 19 recordsLinked to original sources

PubMed2006

On-line hemodiafiltration reduces the proinflammatory CD14+CD16+ monocyte-derived dendritic cells: A prospective, crossover study.

It is not known whether high convective transport may have a role in modulating the chronic inflammation of hemodialysis (HD) patients. The aim of this study was to evaluate the effect of on-line hemodiafiltration (OL-HDF) on proinflammatory peripheral monocytes: Percentage of CD14+CD16+ cells and their telomere length and spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6). In a prospective, crossover study, 31 patients who were on high-flux HD (HF-HD) were evaluated. Patients underwent the following sequence of treatments (4 mo each): HF-HD (basal), OL-HDF (period 1), HF-HD (period 2), OL-HDF (period 3), and HF-HD (period 4). The dialysis characteristics were similar in the two modalities; the only difference was a higher convective transport in the OL-HDF than in the HF-HD. All patients who were on OL-HDF periods showed a significantly lower number of CD14+CD16+ cells than on HF-HD (18.5 +/- 2.3 basal versus 13.6 +/- 2.9 period 1 and 13.9 +/- 2.3 period 3; P = 0.001). By contrast, HF-HD restored the number of CD14+CD16+ cells to the basal values (19.2 +/- 2.8 and 18.6 +/- 1.4, periods 2 and 4, respectively; NS). During OL-HDF periods, the reduction of CD14+CD16+ was paralleled by a decreased number of short telomere cells. Spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6) was increased in HF-HD as compared with OL-HDF. In conclusion, these results demonstrate that as compared with HF-HD, OL-HDF markedly reduces the number of proinflammatory CD14+CD16+ cells and the production of TNF-alpha and IL-6. Future studies are needed to assess the possible therapeutic effect of convective transport on chronic inflammation that is associated with HD.

Adult↗

PubMed2006

Microinflammation in hemodialysis is related to a preactivated subset of monocytes.

Increased percentage of monocytes with low CD14 expression and that co-express CD16 (CD14+/CD16+) have been reported in hemodialysis (HD) patients. We sought to determine whether CD14+/CD16+ monocytes in HD therapy are sensibilized cells to a proinflammatory activity. Cells from 32 HD patients, and from 9 Systemic Lupus Erythematosus (SLE), 9 individuals with human immunodeficiency virus (HIV)-1- and 15 healthy controls were studied. Cells were analyzed by means of flow cytometry for CD14/CD16 expression and immune function (cytokine, chemokines, and sialoadhesin expression), and phagocytosis. Increased percentage of CD14+/CD16+ monocytes was observed in HD patients. Compared with CD14++ monocytes, the CD14+/CD16+ monocytes exhibited increased expression of proinflammatory cytokines and markers of differentiated cells. In addition, these monocytes showed an increased phagocytic activity. Similarly, CD14+/CD16+ monocytes from SLE and HIV patients showed increased inflammatory activity as compared with CD14++ cells. These results support that CD14+/CD16+ monocytes from HD patients evidence characteristics of primed prestimulated proinflammatory cells, similar to data observed in SLE and HIV.

Adult↗

PubMed2005

Evaluation of supercritical fluid extraction/aminopropyl solid-phase "in-line" cleanup for analysis of pesticide residues in rice.

Supercritical fluid extraction (SFE) and the use of aminopropyl solid-phase material for "in-line" cleanup was evaluated for residue analysis of 22 GC-amenable pesticides in wild- and white-rice samples with a fat content of 1.9 and 0.4%, respectively. After optimizing the extraction conditions on glass beads as inert material and evaluating the fat amount extracted from rice by SFE, the use of Florisil, Celite, Extrelut, Hydromatrix, and an aminopropyl material as fat-retention materials for SFE "in-line" cleanup was assessed, aminopropyl being the most suitable material for this cleanup of fat. Pesticide mean recoveries obtained from rice samples, at fortification levels around 0.5 mg/kg, by means of the SFE/in-line cleanup method finally proposed (15-mL CO2 volume, 50 degrees C temperature, 200 atm pressure, 200 muL of methanol static modifier, and a 1-cm layer of aminopropyl at the bottom of the extraction vessel), ranged between 74 and 98%, except for captafol and dimethoate for which mean recoveries lower than 21% were determined.

Chromatography, Supercritical Fluid↗

PubMed2005

Calcium-sensing receptor expression and parathyroid hormone secretion in hyperplastic parathyroid glands from humans.

In uremic patients, severe parathyroid hyperplasia is associated with reduced parathyroid calcium-sensing receptor (CaR) expression. Thus, in these patients, a high serum Ca concentration may be required to inhibit parathyroid hormone (PTH) secretion. This study compares the magnitude of reduction in CaR expression and the degree of the abnormality in Ca-regulated PTH release in vitro. A total of 50 glands from 23 hemodialysis patients with refractory hyperparathyroidism were studied. Tissue slices were incubated in vitro to evaluate (1) the PTH secretory output in a normal Ca concentration (1.25 mM) and (2) the PTH secretory response to high (1.5 mM) and low (0.6 mM) Ca concentration. Tissue aliquots were processed for determination of CaRmRNA expression. The results showed that, corrected for DNA, parathyroid tissue with lowest CaR expression secreted more PTH than that with relatively high CaR expression (146 +/- 23 versus 60 +/- 2 pg/microg DNA; P < 0.01). Furthermore, glands with low CaR expression demonstrated a blunted PTH secretory response to both the inhibitory effect of high Ca and the stimulatory effect of low Ca. The study also showed that the larger the gland, the lower the CaRmRNA expression. Thus, large parathyroid glands produce a large amount of PTH not only as a result of the increased gland size but also because the parathyroid tissue secretory output is increased. These abnormalities in PTH regulation are related to low CaR expression.

Female↗

PubMed2005

Lymphocyte apoptosis: role of uremia and permeability of dialysis membrane.

BACKGROUND: Uremia is associated to host defense mechanism disorders. Lymphocyte apoptosis, which may cause alteration of the immune system, is increased in uremic patients. The aim of the present study was to determine if, in addition to uremia, dialysis membranes with different biocompatibility and permeability have an effect on lymphocyte apoptosis. METHODS: Cell apoptosis and Fas expression were assessed using flow cytometry in four groups of patients: (1) uremic non-dialyzed (Non-D) patients; (2) hemodialysis (HD) patients on hemophan; (3) low-flux polysulfone, and (4) high-flux polysulfone membrane. Ten healthy volunteers were used as controls. RESULTS: At baseline, lymphocytes from patients on hemophan showed an increase in apoptosis (18.4 +/- 6.9%) as compared with Non-D (7.2 +/- 2.8%; p < 0.001), low-flux (6.4 +/- 2.4%; p < 0.001), high-flux (2.6 +/- 1.2%; p < 0.001) and controls (2.0 +/- 1.0%; p < 0.001). Fas expression was similar in lymphocytes from Non-D and hemophan dialyzed patients (40.5 +/- 5% vs. 40.4 +/- 6%), and in both groups it was greater than low-flux (30%+/-7%; p < 0.001), high-flux (11 +/- 4%; p < 0.001) and controls (12.6 +/- 3%; p < 0.001). When lymphocytes were cultured for 48 h, apoptosis was similar in Non-D and hemophan (27.0 +/- 4.3% vs. 27.1 +/- 6.9%); apoptosis of lymphocyte from patients on low-flux (14.1 +/- 3.5%) was greater than on high-flux polysulfone membrane (7.0 +/- 2.0%; p < 0.001). CONCLUSION: These findings suggest that in dialysis patients lymphocyte apoptosis is influenced not only by the biocompatibility but also by the permeability of the dialysis membrane.

Adult↗

PubMed2005

Nutritional secondary hyperparathyroidism in rabbits.

The present study was designed to document the effect of a low (0.6%) calcium-high (1.2%) phosphorus (LCaHP) diet on the development of parathyroid gland hyperplasia in rabbits and to describe the dynamics of parathyroid function (PTH-Ca2+ curves) in rabbits with nutritional secondary hyperparathyroidism (N2HPT). Parathyroid gland weight, parathyroid cell proliferation (measured as percentage of cells in S-phase), and parathyroid calcium (CaRmRNA) and Vitamin D (VDRmRNA) receptor expression were measured in normal rabbits and in rabbits with N2HPT. The PTH-Ca2+ curve was studied in normal rabbits (Group I) and in rabbits with N2HPT at two stages: 2-3 weeks (Group IIA) and 5-6 weeks (Group IIB) after being fed LCaHP diet. An increase in parathyroid gland weight and percentage of cells in S-phase was detected in the course of N2HPT. After receiving a LCaHP diet for 6 weeks rabbits had decreased levels of CaRmRNA but VDRmRNA remained unchanged. A progressive increase in the concentrations of plasma PTH (Group IIA=167+/-14 pg/ml and Group IIB=377+/-54 pg/ml, P<0.05 versus Group I=27+/-3 pg/ml) was detected in the rabbits fed a LCaHP diet. This was accompanied by an increase in maximal and minimal PTH, reductions in plasma Ca2+ and calcitriol and elevations in plasma phosphate and creatinine. In conclusion, feeding a LCaHPD results in a rapid induction of N2HPT in rabbits. After 6 weeks on the LCaHPD rabbits develop parathyroid hyperplasia characterized by increases in PTH secretion, glandular weight and proliferation and by a decrease in CaRmRNA.

Animals↗

PubMed2005

Stress-induced premature senescence in mononuclear cells from patients on long-term hemodialysis.

BACKGROUND: Repeatedly stimulated mononuclear cells may become senescent prematurely as a result of frequent activation-induced replication. In hemodialysis patients, mononuclear cells are activated repeatedly with each hemodialysis procedure. Characteristics of senescent mononuclear cells include telomere length shortening, increased p53 expression, CD14dim/CD16bright expression, and interleukin overproduction. METHODS: Peripheral mononuclear cells from 15 hemodialysis patients and 15 age-matched controls were evaluated. Telomere length was assessed by means of fluorescence in situ hybridization in flow cytometry. Expression of p53, CD14/CD16, and intracellular cytokine production (interleukin-1beta [IL-1beta], IL-6, and IL-4) was evaluated by means of flow cytometry using specific antibodies. RESULTS: Features of senescence were found in a subpopulation of mononuclear cells: (1) accelerated telomere shortening, (2) increased p53 expression, (3) CD14dim/CD16bright expression, and (4) cytokine overproduction (IL-1beta, IL-6, and IL-4). Telomere length shortening was present in 40% +/- 6% of cells from hemodialysis patients compared with less than 5% from age-matched controls. Percentage of cells with short telomeres correlated positively with serum C-reactive protein level, which reflects inflammation. p53 expression was increased in mononuclear cells from hemodialysis patients. Mononuclear cells from hemodialysis patients with decreased telomere length mainly showed the CD14dim/CD16bright phenotype; conversely, cells with normal telomeres presented the CD14bright/CD16dim phenotype. Finally, mononuclear cells from hemodialysis patients, but not controls, spontaneously produced the proinflammatory cytokines IL-1beta and IL-6. CONCLUSION: This study shows the presence of a prematurely senescent subpopulation of peripheral mononuclear cells in hemodialysis patients. These senescent cells probably result from repeated activation and may have a pathophysiological role in the chronic inflammation described in hemodialysis patients.

Adult↗

PubMed2005

Replicative senescence in patients with chronic kidney failure.

BACKGROUND: Chronic activation of immunocompetent cells may lead to stress-induced premature senescence (SIPS); these senescent cells are characterized by a decrease in telomere length. The present study evaluates SIPS in circulating immunocompetent cells from predialysis patients, patients on hemodialysis, and in renal transplant patients with normal renal function. METHODS: Determination of telomere length by flow-fluorescence in situ hybridization (FISH), expression of surface molecules, and evaluation of apoptosis was performed by flow cytometry. RESULTS: In uremic predialysis patients, we observed a subpopulation of lymphocytes with short telomeres. However, in this population of patients we did not observe SIPS mononuclear cells. In hemodialysis patients, we found a subpopulation of SIPS mononuclear cells that also showed phenotypic changes of proinflammatory activity. Finally, transplant patients with normal renal function also exhibited a subpopulation of SIPS lymphocytes, which can be attributed to chronic lymphocyte activation induced by the major histocompatibility complex. CONCLUSION: In chronic kidney disease patients, immunocompetent cells undergo SIPS, a process associated with chronic cell activation and induced by numerous stimuli including uremia, hemodialysis membranes, and bacterial products. Because SIPS immunocompetent cells are activated cells with proinflammatory features and live longer in peripheral blood, it is likely that SIPS cells contribute significantly to the chronic inflammatory state of patients with advanced renal failure.

Apoptosis↗

PubMed2004

Role of acidosis-induced increases in calcium on PTH secretion in acute metabolic and respiratory acidosis in the dog.

Recently, we showed that both acute metabolic acidosis and respiratory acidosis stimulate parathyroid hormone (PTH) secretion in the dog. To evaluate the specific effect of acidosis, ionized calcium (iCa) was clamped at a normal value. Because iCa values normally increase during acute acidosis, we now have studied the PTH response to acute metabolic and respiratory acidosis in dogs in which the iCa concentration was allowed to increase (nonclamped) compared with dogs with a normal iCa concentration (clamped). Five groups of dogs were studied: control, metabolic (clamped and nonclamped), and respiratory (clamped and nonclamped) acidosis. Metabolic (HCl infusion) and respiratory (hypoventilation) acidosis was progressively induced during 60 min. In the two clamped groups, iCa was maintained at a normal value with an EDTA infusion. Both metabolic and respiratory acidosis increased (P < 0.05) iCa values in nonclamped groups. In metabolic acidosis, the increase in iCa was progressive and greater (P < 0.05) than in respiratory acidosis, in which iCa increased by 0.04 mM and then remained constant despite further pH reductions. The increase in PTH values was greater (P < 0.05) in clamped than in nonclamped groups (metabolic and respiratory acidosis). In the nonclamped metabolic acidosis group, PTH values first increased and then decreased from peak values when iCa increased by > 0.1 mM. In the nonclamped respiratory acidosis group, PTH values exceeded (P < 0.05) baseline values only after iCa values stopped increasing at a pH of 7.30. For the same increase in iCa in the nonclamped groups, PTH values increased more in metabolic acidosis. In conclusion, 1) both metabolic acidosis and respiratory acidosis stimulate PTH secretion; 2) the physiological increase in the iCa concentration during the induction of metabolic and respiratory acidosis reduces the magnitude of the PTH increase; 3) in metabolic acidosis, the increase in the iCa concentration can be of sufficient magnitude to reverse the increase in PTH values; and 4) for the same degree of acidosis-induced hypercalcemia, the increase in PTH values is greater in metabolic than in respiratory acidosis.

Acidosis↗

PubMed2004

The imbalance in the ratio of Th1 and Th2 helper lymphocytes in uraemia is mediated by an increased apoptosis of Th1 subset.

BACKGROUND: In uraemia there is a reduction in the total number of T lymphocytes and an imbalance in the ratio of Th1/Th2 T-helper (Th) lymphocytes. A higher rate of apoptosis in T lymphocytes has been reported in haemodialysis patients. The aims of the present study were to assess the Th1/Th2 pattern in uraemia and to evaluate whether a relative increase in Th1 apoptosis may explain the Th1/Th2 imbalance observed in uraemic patients. METHODS: Seventeen non-dialysed uraemic patients were evaluated; eight healthy volunteers served as controls. Intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured by direct intracellular immunofluorescence and flow cytometry. Apoptosis was determined by flow cytometry using annexin V or TUNEL. Mechanisms of apoptosis were assessed by determination of Fas and Bcl-2 expression. RESULTS: Cell production of cytokines is significantly higher in uraemic patients than in controls. In addition, in uraemic patients only 5.1+/-2.1% of the T lymphocytes contained IFN-gamma (Th1 cells) while 61.9 +/- 14.8% contained IL-4 (Th2 cells) (P < 0.0001). The percentage of apoptosis was 29.6 +/- 6.3% and 4.7 +/- 1.6% in Th1 and Th2 lymphocytes, respectively (P < 0.001). Fas expression was higher in Th1 than in Th2 cells and the expression of Bcl-2 was lower in Th1 than in Th2 cells. The apoptosis induced by anti-Fas antibodies was similar in both types of lymphocytes. CONCLUSIONS: In uraemia there is a reduction in the proportion of Th1 lymphocytes due to a higher rate of apoptosis in this subset of lymphocytes. Th1 from uraemic patients show a higher expression of Fas and a lower expression of Bcl-2 than Th2. This makes uraemic Th1 cells more susceptible to apoptosis. The Th1/Th2 imbalance may contribute to alterations in cellular immunity observed in chronic kidney disease patients.

Adult↗

PubMed2003

Supercritical fluid extraction of pesticides from a table-ready food composite of plant origin (gazpacho).

Supercritical fluid carbon dioxide extraction (SFE) has been evaluated for the extraction of 17 organohalogen and organophosphate pesticides in gazpacho (a table-ready food composite containing crude vegetables, white bread, vegetable oil, water, and other minor components) using anhydrous magnesium sulfate as drying agent. The effects of different parameters, such as fat content in gazpacho composites, magnesium sulfate/gazpacho ratio, supercritical fluid volume, pressure, temperature, and static modifier additions, on SFE recoveries from spiked gazpacho samples have been studied. Analyses were performed by gas chromatography (GC) with flame photometric (FPD), electron capture (ECD), and mass spectrometry (MSD) detectors. In most experiments, recoveries obtained for the nonpolar organohalogen pesticides were lower than those obtained for the most polar organophosphate pesticides, but overall pesticide recoveries determined by using the optimal SFE conditions indicate that SFE could be used to determine pesticide residue levels in gazpacho.

Chromatography, Gas↗

PubMed2003

Effects of uremic ultrafiltrate on the regulation of the parathyroid cell cycle by calcitriol.

BACKGROUND: Calcitriol (CTR) is used in the treatment of hyperparathyroidism secondary to renal failure because it decreases parathyroid hormone (PTH) synthesis and parathyroid cell proliferation. Previous studies in tissues other than parathyroids have demonstrated that uremic factors affect the action of CTR on the target cells. We questioned whether the uremic milieu interferes with the inhibition of parathyroid cell proliferation by CTR. METHODS: Studies were performed in vitro using freshly excised normal dog parathyroid tissue incubated for 24 hours with and without CTR and in the presence of either total uremic ultrafiltrate (UUF) from uremic patients or high-pressure liquid chromatography (HPLC)-derived fractions (hydrophilic compounds eluting early and hydrophobic compounds eluting late) of this UUF (F1 to F4). Parathyroid cell proliferation was assessed by flow cytometry. RESULTS: The addition of CTR 10-8 and 10-7 mol/L to parathyroid tissue produced an inhibition of the proliferation that was prevented in the presence of UUF. In a medium containing CTR 10-8 mol/L, the addition of F1, F2 and F3, but not F4, prevented the CTR-induced inhibition of parathyroid cell proliferation. With CTR 10-7 mol/L, the inhibition of proliferation was observed even in the presence of F1, F2 and also F4, but was prevented by F3. Uric acid (7 mg/dL), indoxyl sulfate (5 mg/dL) and p-cresol (1.4 mg/dL), which coeluted with F1, F2 and F4, respectively, did not interfere with the inhibitory action of CTR 10-7 mol/L; however, the addition of phenol (0.14 mg/dL), which coeluted with F3, prevented the CTR-induced inhibition of parathyroid cell proliferation. CONCLUSIONS: The presence of uremic toxins prevents the inhibition of parathyroid cell proliferation induced by calcitriol.

Animals↗

PubMed2003

Dynamics of secretion and metabolism of PTH during hypo- and hypercalcaemia in the dog as determined by the 'intact' and 'whole' PTH assays.

BACKGROUND: Recent evidence has shown that the assay for 'intact' parathyroid hormone (I-PTH) not only reacts with 1-84 PTH but also with large non-1-84 PTH fragments, most of which is probably 7-84 PTH. As a result, an assay specific for 1-84 PTH named 'whole' PTH (W-PTH) has been developed. The present study was designed: (i) to determine whether the W-PTH assay reliably measures PTH values in the dog; (ii) to evaluate differences between the W-PTH and I-PTH assays during hypo- and hypercalcaemia; and (iii) to assess the peripheral metabolism of W-PTH and I-PTH. METHODS: In normal dogs, hypocalcaemia was induced by EDTA infusion and was followed with a 90 min hypocalcaemic clamp. Hypercalcaemia was induced with a calcium infusion. RESULTS: I-PTH and W-PTH values increased from 36+/-8 and 13+/-3 pg/ml (P=0.01) at baseline to a maximum of 158+/-40 and 62+/-15 pg/ml (P=0.02 vs I-PTH) during hypocalcaemia. The W-PTH/I-PTH ratio, 38+/-4% at baseline, did not change during the induction of hypocalcaemia, but sustained hypocalcaemia increased (P<0.05) this ratio. During hypercalcaemia, maximal suppression for I-PTH was 2.0+/-0.5 and only 5.7+/-0.6 pg/ml for W-PTH, due to a decreased sensitivity of the W-PTH assay at values <5 pg/ml. The disappearance rate of PTH was determined in five additional dogs which underwent a parathyroidectomy (PTX). At 2.5 min after PTX, W-PTH was metabolized more rapidly, with a value of 25+/-2% of the pre-PTX value vs 30+/-3% for I-PTH (P<0.05). CONCLUSIONS: (i) The W-PTH/I-PTH ratio is less in the normal dog than in the normal human, suggesting that the percentage of non-1-84 PTH measured with the I-PTH assay is greater in normal dogs than in normal humans; (ii) the lack of change in the W-PTH/I-PTH ratio during acute hypocalcaemia is different from the situation observed in humans; and (iii) the dog appears to be a good model to study I-PTH and W-PTH assays during hypocalcaemia.

Animals↗

PubMed2002

Pesticide residue levels in peppers grown in a greenhouse after multiple applications of pyridaben and tralomethrin.

Residue levels of pyridaben and tralomethrin were determined in peppers grown in an experimental greenhouse, during a 4 week period in which up to four successive treatments with both pesticides were applied. In all cases, plants were sprayed with a mixture of pyridaben and tralomethrin at application rates of active ingredients of 140 and 36 g/ha, respectively. Sampling was carried out at 1, 3, 7, 8, 10, or 14 days after each multiple application, simulating the typical harvesting practices in greenhouse plantations. Residue levels of pyridaben and tralomethrin were determined by using ethyl acetate extraction and GC-ECD. During the study, residue levels in the plantation ranged between 0.22 and 0.04 mg/kg for pyridaben and between 0.09 and 0.02 mg/kg for tralomethrin, with median values of 0.14 and 0.06 mg/kg, respectively. These values represent 28 and 600%, respectively, of the corresponding maximum reside limits currently established in Spain. The application of an intensive washing process to the pepper samples did not lead to a significant reduction of the residue level of either pesticide. Likewise, no significant differences were found between the residue levels in the "edible" and "inedible" parts of the peppers.

Capsicum↗

PubMed2002

What are we determining using gas chromatographic multiresidue methods: tralomethrin or deltamethrin?

The analytical behaviour of the relatively new pyrethroid insecticide tralomethrin has been evaluated by using gas chromatography (GC) with electron-capture and mass spectrometry (MS) detectors, and liquid chromatography (LC)-atmospheric pressure ionization mass spectrometry with electrospray interfacing. Under the GC conditions commonly used in pesticide residue analysis, it was found that tralomethrin is transformed into deltamethrin (in a reproducible way) in the injector port of the GC system. Results obtained in this work indicate that the GC multiresidue methodologies routinely applied in the analysis of pyrethroid pesticides in foods cannot distinguish between these two pesticides, and the chromatographic signal obtained at the retention time of deltamethrin/tralomethrin can be really quantified as either deltamethrin or tralomethrin, including when it is confirmed as deltamethrin by MS. Under the LC-MS conditions assessed in this work, deltamethrin and the two diasteroisomers of tralomethrin were well separated and identified.

Chromatography, Liquid↗

PubMed2002

Cell apoptosis and hemodialysis-induced inflammation.

Hemodialysis patients exhibit a defective immune response leading to an increased susceptibility of infections and neoplasms. Far from being helpful, dialytic therapy per se also may be responsible for this acquired immunodeficiency. Dialysis membranes and bacterial products present in dialysis water may trigger and even perpetuate an abnormal mononuclear cell activation. Upon contact with cellulosic dialysis membranes, monocytes display an increased expression of surface markers of cell activation, such as adhesion molecules CD18, CD49, CD54 and the lipopolysaccharide (LPS) ligand (CD14). Moreover, proinflammatory cytokines as IL-1beta and TNF-alpha are released both in vivo and in vitro when monocytes are exposed to cellulosic membranes. Of special interest is the fact that end-stage renal disease patients undergoing hemodialysis exhibit an increased mononuclear cell apoptosis. This apoptosis is directly related to the degree of biocompatibility of the dialysis membrane. Apoptosis is activated when monocytes enter in contact with the cellulosic dialysis membrane through cell surface receptors linked to G-proteins. In early steps of apoptosis signaling, pertussis toxin-sensitive G proteins are coupled to protein kinase C (PKC)-dependent phosphorylative mechanisms. Furthermore, recent evidence support that the execution phase of apoptosis is mediated by a caspase-3 dependent pathway. Finally, very recent available data support that monocytes subjected to repeated activation suffer a process of accelerated senescence, as demonstrated by the senescent phenotype (CD14 and CD32) expressed and their shortened telomeric length. This senescent profile may generage a defective cellular response in acute stress situations, explaining (at least in part) the altered immune response observed in hemodialysis patients.

Acute-Phase Reaction↗

PubMed2002

Caspase-3-dependent pathway mediates apoptosis of human mononuclear cells induced by cellulosic haemodialysis membranes.

BACKGROUND: Mononuclear cells from patients dialysed with cellulosic membranes undergo rapid apoptosis in vitro. The aim of the present study was to determine whether the apoptosis associated with cellulosic haemodialysis membrane shares similar features with the spontaneous apoptosis described in normal monocytes. Thus, we determined whether apoptosis is dependent on caspase-3 activity and is inhibited by lipopolysaccharide (LPS), which are two features of spontaneous apoptosis in normal monocytes. METHODS: We examined mononuclear cells from healthy subjects and from 14 end-stage renal failure patients on haemodialysis with cellulosic membranes (n=7) and non-cellulosic membranes (n=7). Isolated mononuclear cells were cultured for 48 h. To determine the effect of haemodialysis membrane exposure on caspase-3 activity, on mononuclear apoptosis, or both, cells from healthy subjects were cultured in mini-dialysers with the same membrane types that were used in the haemodialysis patients. Caspase-3 active form was determined by flow cytometric analysis using anti-human-active caspase-3 antibodies. The effect of LPS and Ac-DEVD-CHO, a specific inhibitor of active caspase-3, was also evaluated. Cell apoptosis was assessed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling method. RESULTS: After 48 h of culture, the percentage of mononuclear cells expressing the active form of caspase-3 was greater in patients dialysed with cellulosic membranes than in patients using non-cellulosic membranes and in healthy subjects. This increase in caspase-3 activity was associated with a high rate of apoptosis, which was prevented by Ac-DEVD-CHO, an inhibitor of caspase-3 activity. LPS decreased both apoptosis and caspase-3 activity in mononuclear cells from patients dialysed with cellulosic membranes. Finally, in cells from healthy subjects, both caspase-3 activation and apoptosis were induced after incubation with cellulosic membranes. In contrast, the active form of caspase-3 was not increased in cells cultured with non-cellulosic membranes and was significantly lower than with cellulosic membranes. CONCLUSION: These findings suggest that the apoptosis of mononuclear cells induced by cellulosic haemodialysis membranes occurs through a pathway that is similar to the spontaneous apoptosis of normal monocytes. They additionally suggest that LPS regulates the proteolytic activation of caspase-3.

Adult↗

PubMed2002

The effect of LPS, uraemia, and haemodialysis membrane exposure on CD14 expression in mononuclear cells and its relation to apoptosis.

BACKGROUND: Both uraemia and bioincompatible haemodialysis membranes induce mononuclear cell apoptosis. Recent reports demonstrate that spontaneous apoptosis in normal monocytes is associated with the down-regulation of CD14 molecules, whereas LPS which prevents the down-regulation of CD14 favours monocyte survival. The aim of the present study was to evaluate a possible association between mononuclear cell apoptosis and low expression of CD14 molecules. This study also investigated whether LPS affects mononuclear cell CD14 expression and the apoptosis induced by uraemia and exposure to Cuprophan (CU) membrane. METHODS: The study was performed in vitro examining the effects of CU membrane and LPS on mononuclear cells from normal subjects and from end-stage renal failure patients. Cells were analysed by flow cytometry with fluorescent monoclonal antibodies to determine CD14 expression and with Annexin-V labelling to determine apoptosis. RESULTS: In mononuclear cells from uraemic patients cultured for 48 h, there was a subset of cells with low CD14 expression; this subset of cells was not observed in normal monocytes cultured for the same period of time. Cells with low CD14 expression were also observed when normal or uraemic mononuclear cells were cultured in the presence of CU membrane. Simultaneous measurement of apoptosis and CD14 expression revealed that cells with low CD14 expression underwent apoptosis. The addition of LPS to the medium markedly reduced the number of mononuclear cells with low CD14 expression and also reduced the rate of apoptosis in these cells. CONCLUSION: Our data suggest that mononuclear cell apoptosis induced by uraemia and the CU membrane is associated with low CD14 expression. Furthermore, LPS prevented the decrease in CD14 and reduced the rate of apoptosis.

Adult↗