Biomedical subjects

S Sundaram

Publications and source records attributed to S Sundaram.

At least 37 records · Page 2Linked to original sources

PubMed1999

The vitamin D3 analog EB 1089 enhances the response of human breast tumor cells to radiation.

Previous studies from this laboratory as well as others have demonstrated that breast tumor cells fail to undergo primary apoptosis in response to agents which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor adriamycin. Similarly, the primary response of breast tumor cells to vitamin D(3) [1,25-(OH)(2)-D(3)] and its analogs such as EB 1089 is growth inhibition, with apoptosis occurring in only a small fraction of the cell population. The possibility that the combination of vitamin D(3) compounds with radiation might promote cell death (i.e. through a differentiation stimulus plus DNA damage) was investigated by exposing both TP53 (formerly known as p53) wild-type and TP53 mutated breast tumor cells to 1,25-(OH)(2)-D(3) or EB 1089 for 48 h prior to irradiation. This combination resulted in enhanced antiproliferative effects in the TP53 wild-type MCF-7 cells based on both a clonogenic assay and the determination of numbers of viable cells. The combination of EB 1089 with radiation increased DNA fragmentation based on both the terminal transferase end-labeling (TUNEL) and bisbenzamide spectrofluorometric assays, suggesting the promotion of apoptosis. The observed increase in DNA fragmentation was not due to an enhancement of the extent of initial DNA damage induced by radiation. These findings suggest that vitamin D compounds may be useful in combination with radiation in the treatment of breast cancer.

Breast Neoplasms↗

PubMed1999

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix.

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterized and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.

Adsorption↗

PubMed1998

Progression of hepatic neoplasms is severely retarded in mice lacking the bisecting N-acetylglucosamine on N-glycans: evidence for a glycoprotein factor that facilitates hepatic tumor progression.

The glycosyltransferase termed GlcNAc-TIII is dedicated to the transfer of a single N-acetylglucosamine (GlcNAc) residue (the bisecting GlcNAc), to a subset of N-glycans in glycoproteins. The addition of this GlcNAc is differentially regulated during development and is induced in certain cancers, particularly in hepatic tumorigenesis. To investigate a functional role for the bisecting GlcNAc in the development of liver cancer, the Mgat3 gene that codes for GlcNAc-TIII, was inactivated by targeted gene disruption, and the susceptibility of Mgat3-/- mice to tumor induction was tested. After a single injection with diethylnitrosamine and subsequent treatment with phenobarbitol for 6 months, Mgat3+/+ and Mgat3+/- mice had grossly enlarged livers that contained numerous tumors. By stark contrast, Mgat3-/- mice had livers of normal size, and only 50% of mice had one to four small tumors. However, histological examination showed that Mgat3-/- livers had significant numbers of basophilic foci, and by 10-12 months after diethylnitrosamine injection, tumors had developed in Mgat3-/- mice. Therefore, initiation occurred in Mgat3-/- mice but progression was severely retarded. Assays for Mgat3 gene expression in tumor tissue gave an unexpected result. In contrast to the situation in the rat, hepatic tumor formation in the mouse was not accompanied by a dramatic increase of GlcNAc-TIII activity nor of glycoproteins with a bisecting GlcNAc, nor of Mgat3 gene expression in tumor tissue from wild-type mice. The data suggest that a glycoprotein factor with the bisecting GlcNAc facilitates tumor progression in liver. In the absence of the bisecting GlcNAc in Mgat3-/- mice, the factor is reduced in activity, and tumor progression is severely retarded.

Acetylglucosamine↗

PubMed1998

Effect of glucose concentration, osmolality, and sterilization process of peritoneal dialysis fluids on cytokine production by peripheral blood mononuclear cells and polymorphonuclear cell functions in vitro.

We sought to investigate the effects of high glucose concentration, osmolality, and heat sterilization of peritoneal dialysis fluids on tumor necrosis factor-alpha (TNF-alpha) production by peripheral blood mononuclear cells (PBMC) and polymorphonuclear cell (PMN) functions. Blood samples were obtained from eight healthy volunteers. PBMCs and PMNs were harvested by centrifugation with Ficoll-Hypaque (Sigma, St Louis, MO). PBMC were incubated with an equal volume of test fluids and RPMI for 4 hours (pH equilibrated), followed by incubation for 20 hours in RPMI with or without endotoxin (10 ng/mL). Total TNF-alpha production was measured by radioimmunoassay. PMNs were incubated with pH-adjusted test fluids for 30 minutes. After incubation, phagocytosis was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrouscytochrome C on stimulation with phorbol myristate acetate, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing factor. To study the effects of increasing glucose concentration and osmolality on PBMC and PMN functions, we compared conventional 1.5% Dianeal (1.5%D), (Baxter Healthcare Corp, Deerfield, IL) 2.5% Dianeal (2.5%D), 4.25% Dianeal (4.25%D), and control (RPMI for PBMCs and Hank's balanced salt solution for PMNs). PMNs exposed to 4.25%D exhibited an inhibition of phagocytosis, phorbol myristate acetate (PMA)-stimulated oxidative burst, and bactericidal/permeability increasing factor release compared with control, 1.5%D, or 2.5%D. To study the effects of increased osmolality when controlled for glucose concentration, we compared 1.5%D with 1.5%D in which osmolality was increased to that of 4.25%D with the addition of either sodium chloride (1.5%D+NaCl) or mannitol (1.5%D+M). High osmolality induced higher TNF-alpha production by unstimulated PBMCs and decreased TNF-alpha production by endotoxin-stimulated PBMCs. PMN functions were also inhibited by high osmolality. To study the effects of increased glucose concentration when controlled for osmolality, we compared 4.25%D with 1.5%D+NaCl and 1.5%D+M. High glucose concentration induced an increase in TNF-alpha production by unstimulated PBMCs, a decrease in TNF-alpha production by endotoxin-stimulated PBMCs, and an inhibition of PMN functions. Finally, to investigate the effects of heat sterilization, we compared 4.25%D (heat sterilized) to a filter-sterilized 4.25%D (4.25%D-F). The filter-sterilized fluid induced less changes in PBMC and PMN functions compared with the heat-sterilized fluid. These data suggest that the high glucose concentration, high osmolality, and heat sterilization of peritoneal dialysis fluids adversely affect PBMC and PMN functions. These effects could predispose continuous ambulatory peritoneal dialysis patients to peritonitis, compromise host defense during infection, and jeopardize long-term survival of the peritoneal membrane.

Biocompatible Materials↗

PubMed1998

Effect of formaldehyde/bleach reprocessing on in vivo performances of high-efficiency cellulose and high-flux polysulfone dialyzers.

Among the several disadvantages of reprocessed dialyzers is the concern that reuse could decrease the clearance of uremic toxins, leading to a decrease in the delivered dose of dialysis. To examine this possibility in the clinical setting, the clearances of small molecular weight solutes (urea and creatinine) and middle molecular weight substances (beta 2 microglobulin) were compared during dialysis with "high-efficiency" cellulose (T220L) and "high-flux" polysulfone (F80B) dialyzers reprocessed with formaldehyde and bleach. In a crossover study, six chronic hemodialysis patients were alternately assigned to undergo 21 dialysis treatments with a single T220L dialyzer or F80B dialyzer. Each patient was studied during first use (0 reuse), 2nd reuse (3rd use), and 5th, 10th, 15th, and 20th reuse of each dialyzer. Urea, creatinine, and beta 2 microglobulin clearances were measured at blood flow rates of 300 ml/min (Qb 300) and 400 ml/min (Qb 400). Total albumin loss into the dialysate was measured during each treatment. Urea or creatinine clearance of new T220L dialyzers was not significantly different from that of new F80B dialyzers at either Qb. Urea clearance of F80B dialyzers at Qb 300 decreased from 241 +/- 2 ml/min for new dialyzers to 221 +/- 5 ml/min after 20 reuses (P < 0.001), and Qb 400 from 280 +/- 4 ml/min for new dialyzers to 253 +/- 7 ml/min after 20 reuses (P = 0.001). Similarly, with reuse, creatinine clearance of F80B dialyzers also decreased at Qb 300 (P = 0.07) and Qb 400 (P = 0.03). In contrast, urea or creatinine clearance of T220L dialyzers did not decrease with reuse at either Qb. Urea clearance of T220L dialyzers was significantly higher than that of F80B at Qb 300 at the 5th, 10th, 15th, and 20th reuse (P < 0.001, = 0.005, = 0.004, and = 0.006, respectively), and Qb 400 at the 2nd, 5th, 10th, 15th, and 20th reuse (P = 0.04, 0.008, 0.03, 0.02, and 0.008, respectively). Beta 2 microglobulin clearance of T220L dialyzers was < 5.0 ml/min across the reuses studied. Beta 2 microglobulin clearance of F80B was < 5.0 ml/min for new dialyzers, but increased to 21.2 +/- 5.3 ml/min (Qb 300) and 23.6 +/- 3.3 ml/min (Qb 400) after 20 reuses (P < 0.001). Throughout the study, albumin was undetectable in the dialysate with T220L dialyzers. With F80B dialyzers, albumin was detected in the dialysate in four instances (total loss during dialysis, 483 mg to 1.467 g). In summary, the results of this study emphasize the greater need for information on dialyzer clearances during clinical dialysis, especially with reprocessed dialyzers. A more accurate knowledge of dialyzer performance in vivo would help to ensure that the dose of dialysis prescribed is indeed delivered to the patients.

Albumins↗

PubMed1998

Polymyxin-B stimulates tumor necrosis factor-alpha production by human peripheral blood mononuclear cells.

Gram-negative bacterial lipopolysaccharide (LPS) is a well known stimulus for cytokine production, particularly interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). Polymyxin B (PMX-B) is a cationic polypeptide that binds to LPS, neutralizing its biological effects. PMX-B also disrupts gram-negative bacterial cell membrane phospholipids but is highly toxic to mammalian cells, therefore is of limited use. PMX-B is used as additive to media, as a way to handle LPS contamination. To derive benefit from the ability of PMX-B to neutralize lipid A in vivo while avoiding its systemic toxicity, PMX-B was covalently bound to polystyrene-derivative fibers, creating a hemoperfusion column (PMX-F) for the selective removal of circulating ET. In vitro PMX-F hemoperfusion studies have demonstrated effective ET removal, using either the Limulus amebocyte lysate assay or TNF alpha production by peripheral blood mononuclear cells (PBMC) as an index of ET removal. However, the question whether PMX-B itself could stimulate human PBMC to produce cytokines has not been adequately addressed. We examined the effect of increasing concentrations of PMX-B on cytokine production by PBMC in vitro. PBMC harvested from healthy volunteers were incubated for 24 hours at 37 degrees C with control (tissue culture media RPMI), or 5 microg/ml, 10 microg/ml, 20 microg/ml or 100 microg/ml PMX-B. At the end of 24 hours, PBMC were subjected to three freeze-thaw cycles, and total TNF alpha production (pg/2.5x10(6) PBMC) was measured by radioimmunoassay. Total TNF alpha production by PBMC was 163 +/- 3 pg, 171 +/- 9 pg, 164 +/- 4 pg, 323 +/- 63 pg and 331 +/- 58 pg, in the control, PMX-B 5 microg/ml, 10 microg/ml, 20 microg/ml and 100 microg/ml conditions, respectively. Compared to controls (RPMI), the percentage increase in TNF alpha production by PBMC was 5 +/- 6% (P=0.23), 1 +/- 3% (P=0.45), 99 +/- 40% (P=0.03) and 103 +/- 36% (P=0.02) in the presence of 5 microg/ml, 10 microg/ml, 20 microg/ml and 100 microg/ml of PMX-B, respectively. Furthermore, total TNF alpha production correlated significantly with increasing concentrations of PMX-B (R=0.53, P=0.007). We conclude that the use of PMX-B in in vitro studies as an LPS-neutralizing agent, or in the experimental treatment of endotoxic or septic shock can lead to erroneous interpretations of cytokine production by PBMC, and should be used cautiously in in vitro systems at high concentrations.

Anti-Bacterial Agents↗

PubMed1998

Popliteal artery thrombosis associated with trampoline injuries and anterior knee dislocations in children.

Recent reports have emphasized the benign nature of trampoline-associated injuries. However, this study describes the limb-threatening problem of popliteal artery thrombosis occurring in association with anterior knee dislocation and trampoline injuries. Three children (ages 11, 13, and 17) were referred to the emergency room within the past 12 months with anterior dislocations of the knee, which occurred while jumping on trampolines. All patients had reduction of their dislocations at outside facilities and were referred within 6 to 12 hours after their injuries, with pulse, motor, and sensory deficits. All patients were taken directly to the operating room, where arteriography confirmed thrombosis of the popliteal artery below the knee. One patient had transection of the artery, whereas two patients had stretch injuries with intimal separation. Each patient required interposition grafting with reversed saphenous vein and underwent concomitant four-compartment fasciotomy. All patients had persistent sensory and motor deficits postoperatively, which were presumed to be a combination of ischemic injury and neuropraxia. All patients have functioning grafts with an average follow-up of 1 year (range, 9-15 months). One patient required a second interposition graft to treat an area of intimal hyperplasia, which developed at the proximal anastomosis, at 6 months postoperatively. Eighty per cent of trampoline injuries are associated with minor injuries with minimal long-term complications. However, dislocations of the knee may be associated with significant arterial injury and amputation rates of up to 30 per cent in many blunt trauma series. Based on our experience, physicians should recognize the possibility of significant arterial injuries occurring in children with anterior knee dislocations while jumping on trampolines.

Adolescent↗

PubMed1997

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro.

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH approximately 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 x 10(6) cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, pelleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated PBMC was not significantly different (P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher (P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNF-alpha production compared with 4.25%D (P = 0.02), but was not significantly different from 4.25%D-B (P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 x 10(6) cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis (P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D (P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D (P < 0.001) or 4.25%D-B (P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F (P = 0.01). GDP's acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial affect on peritoneal defenses.

Bicarbonates↗

PubMed1997

Necrosis and apoptosis of polymorphonuclear cells exposed to peritoneal dialysis fluids in vitro.

Conventional peritoneal dialysis (PD) fluids are known to inhibit polymorphonuclear cells (PMN) phagocytosis, oxidative burst and enzyme release. However, the relative contributions of apoptosis and/or necrosis to this dysfunction have not been examined. We investigated the effects of osmolality, glucose concentration and heat-sterilization of PD fluids on necrosis and apoptosis of PMN. Polymorphonuclear cells were isolated from 8 healthy volunteers and exposed to different PD fluids for four hours. PMN were then double-stained with Hoechst 33342 and propidium iodide to study the proportion of viable, apoptotic and necrotic cells. Transmission electron microscopy (TEM) was performed to confirm the results obtained with flow cytometry. The fluids studied were conventionally heat-sterilized 1.5% Dianeal (1.5% D), conventionally heat-sterilized 4.25% Dianeal (4.25% D), 1.5% D in which the osmolality was increased to that of 4.25% D by adding mannitol (1.5% D + M), a filter-sterilized version of 4.25% D (4.25% D-F) and a 1.1% amino acid PD fluid (AA) (Nutrineal PD4). All PD fluids had their pH equilibrated (pH = 7.4) by the addition of sodium bicarbonate. Compared to PMN exposed to culture medium, a significantly higher proportion of necrosis was observed in PMN exposed to 1.5% D (P = 0.04). The 4.25% D induced greater necrosis than 1.5% D (P = 0.001), and the 4.25% D also induced significantly more necrosis (P = 0.002) compared to 4.25% D-F. These data suggest that the consequences of heat-sterilization, rather than high glucose concentration are responsible for the necrosis observed. Indeed, the proportion of necrotic PMN with 4.25% D-F was not significantly different from 1.5% D. The 1.5% D + M and AA induced significantly more apoptosis compared to 1.5% D (P = 0.006 and P < 0.05, respectively), suggesting that apoptosis can be induced by the high osmolality of PD fluids. However, 1.5% D +/- M also induced significantly more apoptosis (P = 0.007) compared to 4.25% D-F. This suggests that the apoptosis effect is specific for the osmolyte present in PD fluids, and that mannitol and amino acids induce more apoptosis than glucose. In summary, the different non-physiological components of conventional PD fluids evaluated in this study had a differential effect on PMN survival. Heat sterilization of high glucose-containing PD fluids was associated predominantly with necrosis of PMN, and high osmolality with apoptosis.

Apoptosis↗

PubMed1997

Ionizing radiation and teniposide increase p21(waf1/cip1) and promote Rb dephosphorylation but fail to suppress E2F activity in MCF-7 breast tumor cells.

Ionizing radiation and the topoisomerase II inhibitor, teniposide (VM-26) both increase levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1) and promote dephosphorylation of the retinoblastoma tumor suppressor protein, Rb, in MCF-7 breast tumor cells, perturbations associated with suppression of the activity of the transcription factor, E2F. However, studies using an E2F binding site-luciferase reporter plasmid transfected into MCF-7 cells failed to demonstrate a reduction in E2F activity in response to VM-26 or to ionizing radiation. In contrast, E2F activity (both basal and E1A stimulated) could be suppressed by transfection with a plasmid expressing Rb, indicating that the capacity of E2F to bind to Rb and to be inactivated by Rb is functionally intact in MCF-7 cells. These findings in MCF-7 breast tumor cells suggest that E2F activity may not be directly susceptible to modulation by endogenous p21(waf1/cip1) and Rb.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Lipopolysaccharide-binding protein and bactericidal/permeability-increasing factor during hemodialysis: clinical determinants and role of different membranes.

The host response to the presence of lipopolysaccharide (LPS) is complex and varied. Two closely related endogenous serum proteins, LPS-binding protein (LBP) and bactericidal/permeability-increasing factor (BPI), regulate delivery of LPS to CD14 antigen on effector cell surfaces and modulate the host response to LPS. In the study presented here, plasma levels of LBP and BPI were measured, predialysis, 15 min into dialysis and postdialysis in patients dialyzed with cellulose, cellulose-tri-acetate (CTA), and polysulfone dialyzers. Further, the association between LBP levels and BPI release during hemodialysis and clinical and laboratory characteristics of patients, complement activation represented by plasma C3a levels, and monocyte cytokine production represented by interleukin-1 receptor antagonist (IL-1Ra) synthesis was also studied. Predialysis plasma levels of LBP were 14,459 +/- 544, 13,889 +/- 1362 and 12,622 +/- 6305 ng/mL, respectively, with cellulose, CTA, and polysulfone dialyzers, and postdialysis levels were 17,834 +/- 861, 20,979 +/- 8485 and 18,177 +/- 1656 ng/mL, respectively. Postdialysis plasma levels of LBP were consistently higher than predialysis levels with all three dialyzers (P < 0.05). However, plasma LBP levels were not significantly different between the three dialyzers either predialysis (P = 0.28) or postdialysis (P = 2.8). There were no significant differences in predialysis BPI levels between the three dialyzers (P = 0.21). BPI levels at 15 min of dialysis with CTA (10.91 +/- 3.65 ng/mL) and polysulfone (10.73 +/- 2.24 ng/mL) dialyzers were significantly greater (P < 0.05) than that observed with cellulose (5.49 +/- 0.66 ng/mL). Similarly, postdialysis levels with CTA and polysulfone were significantly greater (P < 0.05) than that observed with cellulose dialyzers. The percentage change in BPI levels between predialysis and 15 min was 1341 +/- 243%, 2935 +/- 1033%, and 3790 +/- 1151% for cellulose, CTA, and polysulfone dialyzers, respectively. The changes in BPI levels from predialysis to 15 min and between pre- and postdialysis samples were statistically significant for all three dialyzers (P < 0.05). Postdialysis LBP:BPI ratios were 50 +/- 6%, 18 +/- 4%, and 22 +/- 6% of predialysis ratios for cellulose, CTA, and polysulfone dialyzers, respectively. These changes were statistically significant (P < 0.05) for all three dialyzers. There was no significant correlation between baseline clinical or laboratory characteristics and predialysis LBP levels. Similarly, the correlation between BPI levels at 15 min of dialysis with the clinical and laboratory characteristics was also poor, with the exception of serum albumin (r = 0.43, P = 0.008). The correlation between BPI levels at 15 min of dialysis with plasma LBP levels (r = -0.29; P = 0.08), plasma C3a levels (r = -0.1; P = 0.55), peripheral blood mononuclear cells (PBMC) content of IL-1Ra (r = 0.01; P = 0.94), and IL-1Ra production by unstimulated (r = 0.13; P = 0.45), and endotoxin-stimulated PBMC (r = 0.32; P = 0.06) was not statistically significant. The results of this study demonstrate that dialysis with cellulose, CTA, and polysulfone dialyzers results in a significant increase in LBP and BPI levels. BPI release is probably mediated by non-complement factors and may be related to the nutritional status of the patient. The release of BPI during HD and consequent lowering of the LBP:BPI ratio could potentially afford some protection against endotoxin in the dialysate.

Acute-Phase Proteins↗

PubMed1997

Modeling and optimising alcohol production by fermentation of dextrose-xylose mixed feed using a fluorosensor.

Dextrose with differing amounts of xylose (mixed substrate medium) has been fermented at 28 degree Celsius with sacchromyces cerevisiae (Baker's Yeast) as seeding. The progress of the reaction was recorded by measuring the fluorescent signal due to intracellular reduced nicotinamide adenine di nucleotide (NADH) present in the cells with a Dr. Ingold (Switzerland) fluorosensor which has an excitation wavelength of 360 nm and measurement wavelength of 450 nm. The concentration of xylose in the xylose-dextrose feed was varied from 0.7% to 5.0% by weight. The optimum concentration of xylose at which the production of alcohol was a maximum was found to be 3.4 percent xylose. The fluorescent voltage data for different concentration of xylose fitted a first order model with an average absolute deviation of less than one percent. Development of this model is useful in design of model predictive controllers.

Ethanol↗

PubMed1996

A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A Chinese hamster ovary glycosylation mutant.

The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N-linked glycans with GlcNAcbeta(1,6)Manalpha(1,6) branches that are initiated by the transferase termed GlcNAc-TV. Detergent extracts of Lec4 cells have no detectable GlcNAc-TV activity, but Lec4A extracts have activity equivalent to that of parental Chinese hamster ovary cells. This discrepancy occurs because Lec4A GlcNAc-TV activity co-localizes with membranes of the endoplasmic reticulum (ER) instead of with Golgi membranes (Chaney, W., Sundaram, S., Friedman, N., and Stanley, P. (1989) J. Cell. Biol. 109, 2089-2096). cDNAs from the coding region of the GlcNAc-TV gene have now been isolated from each mutant line. Lec4 GlcNAc-TV cDNA was found to possess two insertions, the first of which shifts the open reading frame and codes for a truncated transferase missing 585 amino acids from the catalytic domain. By contrast, Lec4A GlcNAc-TV cDNA possesses a single point mutation from T to G, which results in a change from Leu to Arg at position 188. When transfected into Lec4 cells, both cDNAs gave the appropriate phenotype; Lec4 cDNA was unable to restore GlcNAc-TV activity, whereas Lec4A cDNA converted Lec4 cells to the Lec4A phenotype, with an active GlcNAc-TV mislocalized to ER membranes. Moreover, Lec4A cDNA cured of its mutation restored a functional, Golgi-localized GlcNAc-TV to Lec4 cells. The results demonstrate that a single change in the 740 amino acids of GlcNAc-TV serves to functionally inactivate the transferase in an intact cell by causing it to localize to the ER instead of the Golgi compartment. The mislocalized transferase retains full enzyme activity, showing that it is well folded and stable and suggesting that the L188R mutation either prevents association with exit complexes from the ER or causes retrograde transport from a Golgi compartment.

Amino Acid Sequence↗

PubMed1996

Determination of contact phase activation by the measurement of the activity of supernatant and membrane surface-adsorbed factor XII (FXII): its relevance as a useful parameter for the in vitro assessment of haemodialysis membranes.

We investigated hemodialysis membrane biocompatibility with respect to contact phase activation by determination of FXII-like activity (FXIIA) on the membrane surface and in the supernatant phase, during plasma contact with various hemodialysis membranes using an in vitro incubation test cell. The results were compared to the influence of these membranes on the activation of purified FXII. A time course for the generation of activated FXII using purified FXII solution at physiologic concentrations on two similar negatively charged polymers was performed. The membranes assessed were regenerated cellulose (Cuprophan; Akzo Faser AG, Germany), modified cellulosic (Hemophan; Akzo Faser AG), acrylonitrile-sodium methallyl copolymer-based membrane AN69S (Hospal, France), and SPAN, a new polyacrylonitrile-based copolymer (akzo Nobel AG). The plasma FXIIA at the membranes surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, activation of purified FXII in a plasma-free system with respect to supernatant activity indicated significant differences between the materials. A similar finding for the membrane-bound factor XIIA was also observed when purified factor XII was used. The membrane-bound FXIIA values observed in the plasma system containing heparin were significantly greater than in citrated plasma. This demonstrated the strong influence of heparin and the interaction of other plasma components to the membrane surface on the activation of contact phase of coagulation.

Adsorption↗

PubMed1996

Role of leucocytes in coagulation induced by artificial surfaces: investigation of expression of Mac-1, granulocyte elastase release and leucocyte adhesion on modified polyurethanes.

Thrombus formation on artificial surfaces can be viewed as the sequential and concomitant involvement of protein adsorption, platelet reactions, activation of the coagulation system, participation of complement, fibrinolytic and kallikrein-kinin systems, and the interaction of cellular elements. This study examines the activation of leucocytes on a series of well-characterized polyurethanes with different ionic groups [sulphonate groups (negatively charged); quaterinary amine groups (positively charged)], in terms of adhesion, degranulation and cell surface integrin receptor expression. Leucocyte adhesion was monitored with radiolabelled neutrophils and scanning electron microscopy (SEM), degranulation by measurement of human neutrophil elastase using an indirect enzyme-linked immunosorbent assay and cell surface expression of the integrin receptor Mac-1, using fluorescent-activated cell sorting (FACS). Our results indicate a trend towards enhanced adhesion and degranulation with respect to the negatively charged polyurethane. Similar results were observed with respect to the integrin Mac-1 from recovered adherent cells. The findings of enhanced adhesion and spreading, Mac-1 up-regulation and granulocyte elastase release from the negatively charged sulphonated polyurethane indicate the potential of leucocytes to contribute towards thrombus formation on such surfaces.

Biocompatible Materials↗

PubMed1996

Transmembrane passage of cytokine-inducing bacterial products across new and reprocessed polysulfone dialyzers.

The widespread use of bicarbonate dialysate and reprocessed high-efficiency and "high-flux" dialyzers has raised concerns about the increased risk of reverse-transfer of dialysate contaminants into the blood compartment. To evaluate this concern, the reverse-transfer of bacterial products from contaminated bicarbonate dialysate into the blood compartment was compared during in vitro dialysis with new or reprocessed high-flux polysulfone dialyzers. In vitro dialysis was carried out at 37 degrees C by use of a counter-current recirculating loop dialysis circuit with either new high-flux polysulfone dialyzers or dialyzers reprocessed once or 20 times with formaldehyde (0.75%) and bleach (< 1%) with an automated system. Heparinized whole blood from healthy volunteers was circulated through the blood compartment, and bicarbonate dialysate was circulated in the dialysate compartment. The dialysate was challenged sequentially by 1:1000 and 1:100 dilutions of a sterile Pseudomonas aeruginosa culture supernatant (bacterial challenge). Samples were drawn from the blood and dialysate compartments 1 h after each challenge. Peripheral blood mononuclear cells (PBMC) were harvested by Ficoll-Hypaque separation from whole blood in the blood compartment and a 5 x 10(6) PBMC/mL cell suspension was prepared. Likewise, dialysate samples (0.5 mL) were added to 0.5 mL suspension of 5 x 10(6) PBMC/mL drawn at baseline. All PBMC suspensions were incubated upright in a humidified atmosphere at 37 degrees C with 5% CO2 for 24 h, and total interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha) cytokine production (cell-associated and secreted) was measured by radioimmunoassay. Eight experiments were performed for each arm of the study with the same donor for each arm. One hour after contaminating the dialysate with a 1:1000 dilution of the bacterial challenge, IL-1 alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 171 +/- 11, and 270 +/- 35 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.004). One hour after challenging the dialysate with 1:100 dilution, IL-1 alpha production by PBMC harvested from the blood compartment was 188 +/- 20, 228 +/- 35, and 427 +/- 67 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.006). IL-1 alpha production by PBMC from dialyzers reprocessed 20 times was significantly greater than both new and dialyzers reprocessed once. However, there were no significant differences between new dialyzers and dialyzers reprocessed once. Similarly, after the 1:1000 challenge, TNF alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 160 +/- 0, and 213 +/- 22 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.008). After the 1:100 challenge, TNF alpha production was 168 +/- 8, 188 +/- 20, and 225 +/- 32 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.20). These results demonstrate that reprocessing of high-flux polysulfone dialyzers with bleach increases the risk of reverse-transfer of bacterial products from contaminated dialysate, and this risk appears to increase with the number of reuses. Consequently, units that reprocess membranes with bleach and have suboptimal water quality might subject their patients to a higher risk of cytokine-related morbidity.

Anti-Bacterial Agents↗

PubMed1996

Plasma lipopolysaccharide binding protein and bactericidal/permeability increasing factor in CRF and HD patients.

The recent characterization of lipopolysaccharide binding protein (LBP) and bactericidal/permeability increasing factor (BPI) have provided the opportunity to examine the natural factors that regulate cytokine production in response to endotoxin in patients on hemodialysis (HD). Whole blood was collected in EDTA from 28 undialyzed patients with chronic renal failure (undialyzed CRF), 36 patients on chronic HD (HD) and 15 healthy controls, and plasma levels of LBP and BPI were measured by a sandwich ELISA. Plasma LBP levels in undialyzed patients with CRF (P = 0.04) and patients on HD (P = 0.01) were significantly higher than those in healthy controls, but not significantly different from each other. Plasma BPI levels in undialyzed patients with CRF and patients on HD were not significantly different from those in healthy controls. There was no correlation between serum creatinine and plasma levels of either LBP or BPI. Peripheral blood mononuclear cells (PBMC) were harvested from healthy volunteers by FLcoll-Hypaque separation, and 0.125 mL of 10 x 10(6)/mL suspensions were incubated with 0.125 mL of test plasma (containing different LBP/BPI ratios) and 0.25 mL of RPMI, containing 1 ng/mL of endotoxin, for 24 h at 37 degrees C. Samples were subjected to three freeze-thaw cycles, and total interleukin-1 receptor antagonist (IL-1Ra) or interleukin-1 alpha (IL-1 alpha) production was measured by a specific non-crossreactive RIA. The results of this study showed: (1) IL-1Ra production by endotoxin-stimulated PBMC incubated with pooled plasma from HD patients with LBP/BPI ratios of 11 x 10(2), 167 x 10(2), 379 x 10(2), and 778 x 10(2), respectively was 1466 +/- 195 pg, 3105 +/- 462 pg, 8179 +/- 1020 pg, and 4770 +/- 1185 pg (P < 0.001); (2) Paired plasma collected before dialysis (predialysis) and at 15 min after the start of dialysis (15 minute) with cellulose membranes showed a negligible change in plasma LBP levels (-3 +/- 5%), but a 6681 +/- 1788% increase in plasma BPI levels. Consequently, compared with predialysis plasma, there was a 35 +/- 6% decrease in endotoxin-stimulated IL-1 alpha production by PBMC incubated with plasma drawn at 15 min (P = 0.001); (3) Compared with the PBMC incubated with predialysis plasma from HD patients, there was a 39 +/- 5%, 53 +/- 5%, and 60 +/- 5% decrease in endotoxin-stimulated IL-1 alpha production in the presence of 1 ng/ mL, 10 ng/mL, or 1 microgram/mL of recombinant BPI, respectively (P < 0.003). These results suggest that the plasma LBP:BPI ratio could influence cytokine production in response to bacterial endotoxin; the high LBP:BPI ratios observed in patients with chronic renal failure probably imparts an increased susceptibility to endotoxin-stimulated cytokine production; and natural or pharmacological increases in plasma BPI levels and the consequent decrease in LBP:BPI ratios could attenuate this susceptibility to endotoxin-stimulated cytokine production.

Acute-Phase Proteins↗

PubMed1996

Impact of single use versus reuse of cellulose dialyzers on clinical parameters and indices of biocompatibility.

Hemodialysis with reprocessed dialyzers has been associated with an increased mortality in patients on chronic dialysis, but the causes for this increased mortality have not been identified thus far. The aim of this study was to compare the qualitative and/or quantitative differences in activation of cellular and plasma elements, intradialytic signs and symptoms, adequacy of dialysis, and serum biochemistry and hematology in patients dialyzed with new or reprocessed cellulose dialyzers. This study measured the plasma levels and production of interleukin-1 receptor antagonist (IL-1Ra) by peripheral blood mononuclear cells (PBMC), indices of cytokine synthesis; plasma C3a levels, an index of complement activation; plasma levels of lipopolysaccharide binding protein (LBP), an acute phase reactant; and plasma levels of bactericidal-permeability increasing factor (BPI), a neutrophil primary granule protein, in 37 patients on chronic hemodialysis with glutaraldehyde and bleach-reprocessed cellulose dialyzers after random assignment to 12 wk of dialysis with new (single use) or reprocessed (reuse) cellulose dialyzers. These indices were studied before dialysis, 15 min after the start of dialysis, and at the conclusion of dialysis in both groups. Intradialytic clinical symptoms and signs, urea reduction ratios, monthly blood chemistry, and hematology were also studied during the 12-wk period. Before randomization, clinical and laboratory characteristics and IL-1Ra production by PBMC were similar in the two groups. During the 12-wk study, the mean number of dialyzer reuses was 7 +/- 1 in the reuse group and there were no breaks in protocol in the single-use group. At the end of the study, plasma levels of IL-1Ra, cell content and production of IL-1Ra by unstimulated, endotoxin-stimulated, and lgG-stimulated PBMC among patients assigned to reuse were not significantly different from those in the single-use group either before dialysis, at 15 min, or at the conclusion of dialysis. Similarly, plasma levels of C3a, LBP, and BPl were not significantly different between groups at any of the three time points. During the 12-wk study, none of the patients in either arm of the study experienced chills, rigors, or fever, and there were no differences in the number of episodes of symptomatic hypotension in patients on reused dialyzers (11 +/- 3) compared with patients on single-use dialyzers (8 +/- 2). The mean monthly urea reduction ratio during the 3 months of the study was 63 +/- 2% and 65 +/- 2% for reuse and single-use dialyzers, respectively (not significant). Similarly, the hematocrit, white blood cell count, serum calcium, phosphorus, cholesterol, triglycerides, total protein, and albumin levels were also not significantly different between the two groups at the end of the 12-wk study period. These results suggest that the reprocessing of cellulose dialyzers with glutaraldehyde and bleach does not affect indices of blocompatibility, intradialytic symptoms and signs, adequacy of dialysis, or serum biochemistry and hematology.

Acute-Phase Proteins↗