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.     

Osteoid resorption by mononuclear cells in vitro

For the first time, mononuclear cell-mediated ingestion of osteoid in cultures of long bones of fetal rats is described and characterized. The mononuclear cells, located at sites of osteoid deposition, ingest collagen fibrils and clumps of mineral crystals which are segregated within cytoplasmic vacuoles or multivesicular bodies. The ingestion of osteoid continues in cultures treated with agents that normally inhibit osteoclastic bone resorption. Morphologically, the osteoid-containing cells are characterized by a moderate number of mitochondria and short-stranded rough endoplasmic reticulum, a modest Golgi apparatus and variable numbers of vesicles, vacuoles, and multivesicular bodies. The morphologic appearance of the mononuclear cell is consistent with that of a macrophage.     

Hydroxyl radical scavengers inhibit TNF-alpha production in mononuclear cells but not in polymorphonuclear leukocytes

The hydroxyl radical (HO*) scavengers dimethylthiourea (DMTU), tetramethylthiourea (TMTU), dimethylsulfoxide (DMSO) and deferoxamine (DFX), the latter being an iron chelator which prevents HO* formation by blocking the Fenton reaction, were found to inhibit TNF-alpha production in LPS-stimulated human PBMC but not in PMN. Furthermore, this effect was not LPS-specific, as TNF-alpha production was reduced by HO* radical scavengers to a similar extent upon stimulation of PBMC with immune complexes (IC), concanavalin A (Con A) and phorbol myristate acetate (PMA). Other scavengers such as glutathione (GSH), N-acetylcysteine (NAC), ascorbic acid (ASC) and mannitol (MAN) do not have effect on the production of TNF-alpha either in PBMC or PMN. These results provide evidence that the participation of ROI in the regulation of TNF-alpha production differ in different cell types. Particularly, the data presented in this work indicate that HO* radicals have a central role in the production of this inflammatory cytokine by human PBMC.     

In vitro effect of indomethacin on polymorphonuclear leukocyte function in preterm infants

During 1984 to 1988, the U.S. Occupational Safety and Health Administration (OSHA) investigated 10 incidents, with 11 fatalities, involving the inadvertent connection of air-line respirators to inert gas supplies. Seven deaths resulted from connecting an air-line respirator supply hose to a line which normally carried inert gas. Four deaths were caused by leakage or backfill of inert gas into a line which normally carried breathable air. Ten of the deaths were from nitrogen and one from argon. The circumstances of the 11 deaths indicated that coupling compatibility and supervisory oversight were major factors in the inappropriate supply of irrespirable gas to the respirators worn by these workers. Conscientiousness among safety personnel to the hazards of asphyxiation by inert gas, and compliance with current OSHA regulations, the ANSI Z88.2 standard, and NIOSH respirator certification approval regulations would have prevented these fatalities.     

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.     

Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in immunoblot tests. The 74 kDa and perhaps the 82 kDa components may thus represent equine 'Mxanalogous proteins'. The 74 kDa protein was only detected in PBMC of ten out of 20 horses examined. The induction of Mx protein in the horse by Type 1 interferon may therefore resemble that in the mouse, where Mx protein is involved in selective resistance to influenza virus. The influence of rEqIFN-beta 1 on protein expression in equine PBMC and PMN was monitored by metabolic labeling and 2-D gel electrophoresis. Proteins of 82, 74, 58 and 40 kDa were induced in PBMC following exposure to rEqIFN-beta 1. A constitutively expressed 35 kDa protein, however, was no longer demonstrable upon treatment with interferon. None of the proteins induced within PBMC was found in highly purified PMN treated with interferon. PMN exposed to rEqIFN-beta 1 synthesized four proteins in the range of 25 to 27 kDa. These proteins have not been described in interferon-treated PMN of any other species.     

Interaction of dexamethasone and Salmonella enteritidis immune lymphokines on Salmonella enteritidis organ invasion and in vitro polymorphonuclear leukocyte function

OBJECTIVE: To evaluate the effects of gemeprost on utero-placental and luteal circulation and on the embryo/fetus in normal first trimester pregnancies. STUDY DESIGN: Sixty-seven women with a normal first trimester pregnancy requesting termination of pregnancy for psychosocial reasons were randomly allocated to pre-operative treatment with vaginal suppositories containing placebo or gemeprost. The women underwent transvaginal color and spectral Doppler ultrasound examination before the application of the suppository, 4 h after the application of the suppository but before the abortion, and on the seventh post-operative day. Blood flow velocities in the uterine and subchorionic arteries, the intrachorionic area and arteries in the wall of the corpus luteum and the embryonic/fetal heart rate were measured. RESULTS: The median value for pulsatility index (PI) in the dominant uterine artery was 2.4 before treatment with gemeprost and 8.5 4 h after treatment (P = 0.0006); the corresponding values for time-averaged maximum velocity (TAMXV) being 27 cm/s and 10 cm/s (P = 0.0006). Four (14%) of 28 embryos/fetuses in the gemeprost group were dead 4 h after treatment with gemeprost and the median heart rate of those still alive was significantly lower than before treatment (130 vs. 163 bpm; P = 0.003). In the placebo group, the results for the uterine arteries and the embryonic/fetal heart rate did not differ significantly between the first and second ultrasound examinations. The median values for PI and TAMXV in the arteries of the corpus luteum wall at the first ultrasound examination were 0.71 and 18 cm/s, respectively, in the placebo group and 0.71 and 20 cm/s, respectively, in the gemeprost group. These values remained almost unchanged at the second and third ultrasound examinations in both groups. CONCLUSION: Gemeprost has profound effects on utero-placental circulation in the first trimester and can induce embryonic/fetal bradycardia and sometimes embryonic/fetal demise. It has no unequivocal effect on luteal circulation.     

Simultaneous flow cytometric measurement of Streptococcus suis phagocytosis by polymorphonuclear and mononuclear blood leukocytes

A simple flow cytometric method was used to study simultaneously the phagocytosis of Streptococcus suis serotype 2 by polymorphonuclear and mononuclear blood leukocytes from swine and humans. Using this method with a bacteria-to-leukocytes ratio of 10:1 and after 60 min of incubation, 80.2 +/- 2.8% of swine granulocytes and 77.0 +/- 2.8% of swine monocytes were shown to contain FITC-labelled S. suis serotype 2 strain 735. Using the same strain, FITC-labelled bacteria were found in 95.5 +/- 3.2% of human granulocytes and in 92.8 +/- 3.6% of human monocytes. The phagocytosis rates of avirulent and virulent strains of S. suis were not significantly different.     

In vitro effect of disodium cromoglycate on phagocytic activity of polymorphonuclear cells of healthy asthmatic patients

Disodium chromoglicate interferes cellular membrane transport, inhibits mediators release and activation of polymorfonuclear cells (PMN) by blocking intracellular free calcium increment. In order to assess if DC reduces in vitro phagocytic activity of PMN, these cells obtained from allergic asthmatic patients and healthy subjects were incubated with DC or Hanks solution (HS). Their phagocytic activity was measured with chemoluminiscence technique. There were not significant differences in phagocytic activity between the cells incubated with DC and those incubated with HS, nor between cells from asthmatic patients and healthy subjects. We conclude that DC has not in vitro effect on phagocytic activity of PMN from healthy and asthmatic subjects. There are no difference in PMN phagocytic capacity between healthy and asthmatic subjects.     

Immunoglobulin-secreting cells in healthy peripheral blood mononuclear cells

Immunoglobulin-secreting cells were measured in healthy uncultured peripheral blood mononuclear cells (PBMCs) in vitro. The percentage of IgM-, IgG- and IgA-secreting cells in adult PBMCs was 0.053, 0.099 and 0.065%, respectively. The percentage of IgM-, IgG- and IgA-secreting cells was 0.73, 5.2 and 3.8% of surface IgM-, IgG- and IgA-bearing cells, respectively. The numbers of IgM-, IgG- and IgA-secreting cells increased with age in childhood. However, the numbers of all three classes were slightly decreased in adults compared with children aged 9-15 years. These results may explain the difference in immunity in vivo.     

Antioxidant function of fungal melanin

Polyphenols have been implicated in the virulence and oxidant resistance of Cryptococcus neoformans. Although monomeric polyphenols did not protect against the prooxidant, plumbagin, polymeric dopamine-melanin conferred resistance both to hypochlorite and to permanganate. The physiologic antioxidant capacity conferred by melanin was found to be 21.3 x 10(-15) mole-equivalents per cell, a value which approximates oxidant production by stimulated macrophages.     

Tumor necrosis factor alpha enhances antifungal activities of polymorphonuclear and mononuclear phagocytes against Aspergillus fumigatus

Invasive aspergillosis is a serious complication in immunocompromised patients. The effects of recombinant human tumor necrosis factor alpha (TNF-alpha) on antifungal activities of human neutrophils (polymorphonuclear leukocytes [PMNs]), human monocytes (MNCs), and rabbit pulmonary alveolar macrophages (PAMs) against Aspergillus fumigatus were studied. The percentage of PMN-induced hyphal damage was increased after 30 min of incubation of PMNs with 0.1 ng of TNF-alpha per ml at 37 degreesC (P = 0.043). At 0.1 to 10 ng/ml, TNF-alpha also increased superoxide anion (O2-) produced by PMNs in response to phorbol myristate acetate, N-formylmethionyl leucyl phenylalanine, and unopsonized hyphae (P < 0.01) but did not exert any effect on PMN phagocytosis of conidia in the presence of serum. By comparison, TNF-alpha induced only a slight increase in O2- production by MNCs in response to phorbol myristate acetate (P = 0.05) and no concomitant increase in the percentage of MNC-induced hyphal damage. Incubation of MNCs with TNF-alpha at 0.001 to 10 ng/ml for 2 days had no effect on phagocytosis or conidiocidal activity. By contrast, incubation of PAMs with TNF-alpha at 0.1 to 10 ng/ml for 2 days increased phagocytosis of conidia (P = 0.03). Thus, TNF-alpha augments the capacity of PMNs to damage Aspergillus hyphae, possibly through enhanced oxidative mechanisms, and increases PAM phagocytic activity against conidia. As such, TNF-alpha may have an important role in host defense against aspergillosis, and neutralization of its activity may be complicated by increased susceptibility to aspergillosis.     

The effects of retinol on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells

In this study we examined the effects of retinol (ROH), a metabolic precursor of retinoic acid (RA), on Staphylococcus aureus Cowan I (SAC)-induced immunoglobulin synthesis of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). ROH augmented SAC-induced IgM synthesis of CBMC by 5.9 +/- 1.5-fold (n = 7, mean +/- s.d.), and IgG synthesis of adult PBMC by 16.3 +/- 5.1-fold (n = 3) at optimal concentrations of 10(-6) M and 10(-11) M, respectively. No augmenting effects could be demonstrated for the other immunoglobulin isotypes. Time-course studies showed that the synthesis of IgM by CBMC was accelerated with detectable immunoglobulin in supernatant fluids starting on day 3. ROH augmented immunoglobulin synthesis of CBMC stimulated by Epstein-Barr virus (EBV), a T cell-independent polyclonal activator, and of EBV-transformed B cell clones (2.5 +/- 0.2 and 4.1 +/- 1.5-fold increase, respectively), which suggests that ROH can act directly on B cells to enhance immunoglobulin synthesis. In contrast, when ROH was preincubated with cord blood T cells, washed and added to the B cell-enriched fraction with SAC, no increase (0.9-1.8-fold) in IgM synthesis was obtained. Thus, the principal mechanism(s) by which ROH augments immunoglobulin synthesis is by acting on B cells. This is in contrast to the immunoglobulin-enhancing effects of RA which is mediated by T cells, or T cell products, e.g. cytokine. Our studies suggest that RA and ROH may have different pathways of immunoglobulin-enhancing effects, perhaps mediated by different retinoid binding proteins resulting in gene activation and immunoglobulin synthesis.     

Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to produce a differential toxicity in the nigrostriatal and mesolimbic dopaminergic pathways with the nigrostriatal pathway being more vulnerable. We, therefore, investigated whether oxidative stress and the antioxidant system play a role in this phenomenon. Balb/c mice were treated with either saline or MPTP (30 mg/kg/d) for 7 d, and were sacrificed on the next day. Results revealed that MPTP increased lipid peroxidation in the striatum (ST) and decreased glutathione concentration in the substantia nigra (SN) without markedly affecting these measures in the nucleus accumbens (NAc) and ventral tegmental area (VTA). Further, MPTP produced approximately twofold increases in both manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities in the VTA while it only increased MnSOD activity in the SN. Both catalase and glutathione peroxidase (GPx) activities were not markedly altered by MPTP in both systems. However, the basal levels of catalase and GPx activities were higher in the VTA and NAc than in the SN and ST. These results together suggest that a lesser degree of oxidative damage and a more inducible CuZnSOD activity observed in the mesolimbic dopaminergic pathway may partially explain the differential toxicity MPTP produced in these two dopaminergic systems.     

Restoration of polymorphonuclear leukocyte function in elderly subjects by thymomodulin

Senescence is a specific physiological evolution of human beings associated with a reduction in the functionality of several apparatuses, including the immune system. Thymomodulin (TMD) contains thymus polypeptides (< 10,000 D) and it has been used in a variety of disorders associated with defective immunological functions. The aim of this study was to investigate the effect on polymorphonuclear leukocyte (PMNs) phagocytosis and oxidative burst of a 6-week treatment with 160 mg/day TMD orally in elderly subjects (85.5 +/- 9.7 years). Elderly subjects have impaired PMN phagocytosis and the following release of oxidant radicals. Treatment with TMD for 6 weeks had a restoring effect; phagocytosis and the phagocytic index were significantly improved, with increases of 132.6% and 112.5%. These findings indicate that TMD might be given to enhance the immunodefenses of immunocompromised elderly subjects. Luminol-dependent chemiluminescence was increased by 15.6%, which was not significant, indicating a different response between phagocytosis and release of oxidant radicals.     

Isotopic survival criteria of transfused polymorphonuclear cells

The enthalpies of interaction of urea with five globular proteins, ribonuclease A, trypsin, beta-lacto-globulin, ovalbumin and bovine serum albumin have been measured in aqueous solution at pH 7.0, I=0.005 M and 25 degrees C over a range of urea molality m from 0-15 mmol g-1 (where a 1 molal solution contains 1 mmol g-1). For all the proteins the interaction is exothermic, and there is an appreciable heat evolution at low urea concentrations, m less than 5 mmol g-1, which increases sharply at higher urea concentrations when the proteins undergo unfolding. If account is taken of the endothermic enthalpies of unfolding of the native proteins, the enthalpies of interactions of urea per unit mass denatured protein lie in the range -45 to -75 J g-1, corresponding to an average binding enthalpy of -23 kJ mol-1 bound urea.     

Effects of parathyroid hormone and serum calcium on the phenotype and function of mononuclear cells in patients with primary hyperparathyroidism

The University of Pennsylvania Smell Identification Test (UPSIT) and a smell ability questionnaire were administered to 167 Japanese volunteers ranging in age from 20 to 59 years. Of these subjects, 80 also received the T&T olfactometer threshold test. Of the latter subjects, 36 were patients tested before endoscopic nasal surgery for sinusitis and polyposis. The patients exhibited decreased smell function, as measured by the T&T olfactometer, the UPSIT, and a 30-item version of the UPSIT in which the 10 least familiar items were removed (ps < 0.001). Spearman correlations ranging from 0.53 to 0.70 were found between (i) scores on the 30- and 40-item UPSITs and (ii) the T&T detection and recognition threshold values. Significant correlations were found between scores on the smell ability questionnaire and the olfactory test measures (UPSIT30 r = 0.56; UPSIT40 r = 0.58; T&T detection r = 0.56; T&T recognition r = 0.69, p < 0.001), indicating that subjects are relatively accurate in assessing their olfactory ability. This study suggests that the 30 and 40-item UPSITs correlate well with measures derived from the T&T olfactometer, and that all three tests are sensitive to the smell loss of Japanese sinusitis/polyposis patients.     

Antioxidant activity of the drug distinol in vitro

Distinol, ionol and dimethylsulphoxide were investigated in model reaction of thermal oxidation of methyl oleate for their antioxidant activity and for their influence on the rate of malonic dialdehyde formation in the liver tissue of chicken with E-hypovitaminosis in vitro. It has been found that the level of antioxidant activity in the homogeneous system of methyl oleate oxidation permits arranging the preparations studied in a series: ionol > distinol > dimethylsulphoxide. Distinol inhibits peroxidation of lipids in the liver homogenates of chicken most efficiently. Its inhibitory effect is 4.6 times higher than that of dimethylsulphoxide and 1.3 times higher than that of ionol. No correlation was found between antioxidant activity of distinol in the homogeneous model system and its influence on peroxidation of lipids in biomembranes.     

Identification of monocytes in suspensions of mononuclear cells

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.