Biomedical subjects

A J Verhoeven

Publications and source records attributed to A J Verhoeven.

At least 19 recordsLinked to original sources

PubMed2006

Differential sensitivities of pathogens in red cell concentrates to Tri-P(4)-photoinactivation.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) with the cationic porphyrin, mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride [Tri-P(4)], has previously been shown to be effective at inactivating vesicle stomatitis virus (VSV) in red cell concentrates (RCC) with limited damage to red blood cells (RBC). The aim of this study was to determine the pathogen-inactivating capacity of PDT with Tri-P(4) for a broader range of pathogens and to establish the associated effect on in vitro RBC quality. MATERIALS AND METHODS: A series of viruses and bacteria was spiked into 60% RCC. Pathogen inactivation was determined after PDT with 25 microm Tri-P(4) and red light up to 360 kJ/m2. Human immunodeficiency virus (HIV)-infected cells were evaluated for cell death induction, and RCC were analysed for the induction of haemolysis and ATP content. RESULTS: For the lipid-enveloped viruses bovine viral diarrhoea virus, HIV and pseudorabies virus, and for the Gram positive bacterium, Staphylococcus aureus, and the Gram-negative bacteria, Pseudomonas aeruginosa and Yersinia enterolitica, inactivation of > or = 5 log10 was measured after 60 min of PDT with Tri-P(4). The required treatment time to achieve this level of inactivation was four times longer than required for VSV. For cell-associated HIV, only 1.7 log10 of inactivation was found, despite clear induction of cell death of HIV-infected cells. The non-enveloped virus, canine parvovirus, was completely resistant to the treatment. PDT of RCC with Tri-P(4) for 60 min, and subsequent storage in AS-3, resulted in 4% haemolysis after 35 days of storage. The ATP content of untreated and treated RBC declined with similar kinetics during storage. CONCLUSION: PDT of RCC with Tri-P(4) for 60 min inactivates a wide range of pathogens, but not cell-associated HIV and a non-enveloped virus, and compromises RBC quality. This reduces the suitability of PDT with Tri-P(4) for red cell sterilization. Therefore, further improvements in the treatment procedures to potentiate pathogen inactivation and to preserve RBC integrity will be required to generate an effective treatment for sterilizing RCC.

Anti-Bacterial Agents↗

PubMed2006

Prolonged storage of red blood cells affects aminophospholipid translocase activity.

BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). MATERIALS AND METHODS: Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. RESULTS: PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. CONCLUSION: This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.

Adenosine Triphosphate↗

PubMed2006

Mitochondria in neutrophil apoptosis.

Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis and contribute to caspase activation, but they also act as sensors of the metabolic and redox state of the cell and as scavengers of free Ca2+. The balance of the expression and activity of the proapoptotic and antiapoptotic members of the Bcl-2 family of proteins determines the life span of neutrophils, because these proteins are essential for the formation of a permeability transition pore in the mitochondria and also seem to control the release of Ca2+ from the endoplasmic reticulum and thereby mitochondrial energy metabolism.

Animals↗

PubMed2005

The effect of storage time of human red cells on intestinal microcirculatory oxygenation in a rat isovolemic exchange model.

OBJECTIVE: To determine whether the storage time of human leukodepleted red blood cell concentrates compromises intestinal microvascular oxygen concentration oxygen (muPo(2)) during isovolemic exchange transfusion at low hematocrit. DESIGN: Prospective, randomized, controlled study. SETTING: University research institute laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Intestinal muPo(2) was determined by Pd-porphyrin phosphorescence life-time measurements. MEASUREMENTS AND MAIN RESULTS: Rats were brought near to a state of oxygen supply dependency by hemodilution with a pasteurized plasma protein solution to a hematocrit of 14.3 +/- 1.1% (n = 24). Subsequently, an isovolemic exchange transfusion with human leukodepleted red blood cells, stored for 2-6 days (fresh, n = 8), 2-3 wks (intermediate, n = 8), or 5-6 wks (old, n = 8), was performed to determine whether intestinal muPo(2) would be preserved. Immunologic reactions were avoided by washing the red blood cell concentrates three times before use. Isovolemic exchange with fresh and intermediate red blood cells maintained muPo(2) whereas old cells decreased muPo(2) with 26%. Subsequent transfusion with red blood cells (hematocrit approximately 60%) until reaching a hematocrit of 32.4 +/- 2.1 % (n = 24) increased intestinal muPo(2) in all three groups to the same extent between 28% and 32%. No changes in red blood cell deformability, as determined by a Laser-assisted Optical Rotational Cell Analyzer, could be demonstrated during 5 wks of storage. CONCLUSION: This study shows that at low hematocrit, the oxygen-delivering capacity of human red blood cells stored 5-6 wks is reduced compared with fresh cells and red blood cells stored for an intermediate period. Although red blood cells stored for 2-3 wks are completely devoid of 2,3-diphosphoglycerate, their oxygen-delivering capacity to the intestines was the same as fresh red blood cells. Our study showed that red blood cell deformability was preserved during storage, suggesting that other mechanisms may account for the observed decrease in oxygen delivery by red blood cells stored 2-3 wks.

Animals↗

PubMed2005

The age of stored red blood cell concentrates at the time of transfusion.

In the past decade, studies suggesting a reduced oxygen delivery by stored red blood cell concentrates (RBCCs) have initiated a discussion about the use of fresh versus old blood. We determined whether old RBCCs represent a significant part of the total of RBCCs issued. The age of RBCCs at the time of transfusion was determined in 74 084 units during a 5-year period in the Academic Medical Center, a main Dutch University Hospital. The mean (+/-SD) storage time of the total number of transfused RBCC was 19.4 +/- 7.0 days, and 37% were older than 3 weeks. As more than one-third of the transfused RBCC units are stored for longer than 3 weeks, the research examining differences in oxygen delivery between fresh and stored RBCC is relevant for packed RBC transfusion practice.

Blood Preservation↗

PubMed2004

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.

4-Chloro-7-nitrobenzofurazan↗

PubMed2004

Quality determinants of erythrocyte destined for transfusion.

An overview is given of a series of standard assays to evaluate the quality of red cell concentrates for transfusion. These are visual inspection, assessment of hemolysis, quantitation of 2,3-DPG and nucleotide levels (especially ATP) and evaluation of morphology. These parameters, relatively easy to measure, are main determinants of in vivo recovery after transfusion. In addition, some other assays are described, which should give more information about the function of red blood cells after transfusion. These assays include plasma-induced hemolysis, binding of annexin-V, deformability measurements and a rat model to judge oxygen delivery by human red blood cells (RBC). Especially in judging new protocols for the preparation of red cell products, involving e.g. improved additive solutions or pathogen inactivation methods, these quality parameters should not be compromised.

2,3-Diphosphoglycerate↗

PubMed2003

Additive effects of dipyridamole and Trolox in protecting human red cells during photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. MATERIALS AND METHODS: Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). RESULTS: Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. CONCLUSIONS: Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.

Antioxidants↗

PubMed2003

Composition of the additive solution affects red blood cell integrity after photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.

Blood Preservation↗

PubMed2003

Comparison of various dimethylsulphoxide-containing solutions for cryopreservation of leucoreduced platelet concentrates.

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression (<35%), low CD63 expression (<20%) and a swirling effect of about 2, at 24 h after thawing. At the same time-point, platelet recovery was >80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was <200 and the CD62P expression was >40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant.

Blood Platelets↗

PubMed2002

Diversion of first blood volume results in a reduction of bacterial contamination for whole-blood collections.

BACKGROUND AND OBJECTIVES: In a previous study we established a reliable setpoint for the prevalence of bacteria in whole blood. In the present study we investigated the possible preventive effect, of diversion of the first 10 ml of a blood donation, on the bacterial contamination rate. MATERIALS AND METHODS: To divert the first 10 ml of a whole-blood donation, we used a special five-bag system equipped with a Composampler device. After venepuncture, the first 10 ml of a donation was sampled into a vacutainer tube. This was followed by the collection of the whole-blood unit. The extra bag allowed direct sampling of the final donation in a closed system for BacT/Alert. Whole-blood samples were taken after storage (2-14 h at 20 degrees C) and subsequent mixing. BacT/Alert culture bottles were incubated until positive, or for 7 days if negative. Confirmation and identification of positive cultures was performed according to internationally recognized standard reference methods. RESULTS: The prevalence of bacteria in whole blood, as determined by using standard collection techniques, was 0.35% (95% confidence interval 0.27-0.44%, n = 18 257). After diversion of the first 10 ml this value was significantly lower: 0.21% (P < 0.05, 95% confidence interval 0.12-0.35%, n = 7087). Most strikingly, a reduction in the frequency of staphylococcal species was observed (P < 0.02, reduction from 0.14 to 0.03%). CONCLUSIONS: Diversion of the first 10 ml of blood was shown to contribute significantly to a reduction in the prevalence of superficial skin bacteria in whole-blood units. In our opinion, blood collection systems should be adapted to use the first 10-30 ml of a whole-blood donation for testing purposes.

Bacterial Infections↗

PubMed2002

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2001

Hepatic lipase promoter activity is reduced by the C-480T and G-216A substitutions present in the common LIPC gene variant, and is increased by Upstream Stimulatory Factor.

The common -216G-->A and -480C-->T substitutions in the promoter region of the human hepatic lipase (LIPC) gene show high allelic association, and are correlated with decreased hepatic lipase activity and increased high-density lipoprotein cholesterol levels. To test the functionality of these substitutions, CAT-reporter assays were performed in HepG2 cells. LIPC (-650/+48) but not (-650/+61) promoter constructs showed transcriptional activity. LIPC (-650/+48) constructs with both -216A and -480T exhibited significantly lower promoter activity (-45%) than the wild-type form. Activities of -289/+48 constructs were not significantly affected by the -216G-->A substitution. The -480C/T site lies within a binding region for Upstream Stimulatory Factor (USF). Gel-shift assays showed that the binding affinity of USF protein for HL specific oligonucleotides was decreased four-fold by the -480C-->T substitution. However, promoter activity of the -650/+48 constructs was not significantly affected by the -480C-->T substitution alone. Co-transfection of HepG2 cells with USF(43) cDNA yielded a similar dose-dependent increase in activity of all -650/+48 constructs; the absolute difference in promoter activity increased but the relative difference between the variant promoter forms was maintained. Our studies demonstrate that the common LIPC promoter variation is functional, which explains the association of the -480T allele with a lower hepatic lipase activity in man.

Bacterial Proteins↗

PubMed2001

Neutrophils enhance eosinophil migration across monolayers of lung epithelial cells.

During the late-phase asthmatic response eosinophils and neutrophils infiltrate the lungs and cause severe damage. In this study, we investigated in vitro the migration of eosinophils, in the absence and presence of neutrophils, across a monolayer of lung H292 epithelial cells. The migration of eosinophils towards the complement fragment 5a (C5a) was increased when neutrophils were added to the upper compartment of the Transwells, and decreased when neutrophils were added to the lower compartment. Moreover, neutrophils exclusively stimulated eosinophil migration towards C5a, and not towards other chemoattractants such as RANTES, IL-8 or PAF. Neutrophils and eosinophils differed in that neutrophils, but not eosinophils, rapidly inactivated C5a, suggesting that neutrophils in the upper compartment remove part of the active C5a that has diffused into the upper compartment. Indeed, we found that the addition of other C5a-degrading agents, such as human serum or carboxypeptidase B, also enhanced eosinophil migration when added to the upper compartment and decreased migration when added to the lower compartment. Taken together, these results indicate that the presence of neutrophils influences the migratory behaviour of eosinophils in vitro. The neutrophils presumably maintain a proper C5a chemotactic gradient in the transmigration model, which results in enhanced eosinophil chemotaxis.

Cell Communication↗

PubMed2001

Improved platelet compatiblity of water vapour glow discharge treated non-woven poly(ethylene terephthalate) leukocyte-reduction filters for different types of platelet concentrates.

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.

Biocompatible Materials↗

PubMed2001

The effect of the transfusion of stored RBCs on intestinal microvascular oxygenation in the rat.

BACKGROUND: Although it is known that the transfusion of stored RBCs does not always improve tissue O(2) consumption under conditions of limited tissue oxygenation, the efficiency of O(2) delivery to the microcirculation by stored RBCs has never been determined. STUDY DESIGN AND METHODS: In a rat hemorrhagic shock model, the effects of resuscitation with fresh or 28-day-old RBCs stored in CPD plasma, saline-adenine-glucose-mannitol, and CPDA-1 plasma were investigated. Systemic hemodynamic and intestinal oxygenation measures were monitored. Intestinal microvascular PO(2) was determined with the O(2)-dependent quenching of palladium-porphyrin phosphorescence, and the RBC deformability was measured with a Laser-assisted optic rotational cell analyzer. RESULTS: Hemodynamic and oxygenation measures were significantly decreased during hemorrhagic shock. Intestinal oxygen consumption and mesenteric venous pO(2) were restored with the transfusion of both fresh and stored RBCs, except for CPD-stored RBCs. The intestinal microvascular pO(2) improved only with the transfusion of fresh RBCs. Deformability of the stored RBCs was significantly decreased. CONCLUSION: In contrast to that of fresh RBCs, the transfusion of stored RBCs did not restore the microcirculatory oxygenation, possibly because of impaired O(2) unloading, but, except for CPD-stored RBCs, the storage-induced changes were not enough to impair intestinal VO(2) and mesenteric venous pO(2).

Animals↗

PubMed2000

Intracellular activation of rat hepatic lipase requires transport to the Golgi compartment and is associated with a decrease in sedimentation velocity.

Hepatic lipase (HL) is an N-glycoprotein that acquires triglyceridase activity somewhere during maturation and secretion. To determine where and how HL becomes activated, the effect of drugs that interfere with maturation and intracellular transport of HL protein was studied using freshly isolated rat hepatocytes. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), castanospermine, monensin, and colchicin all inhibited secretion of HL without affecting its specific enzyme activity. The specific enzyme activity of intracellular HL was decreased by 25-50% upon incubation with CCCP or castanospermine, and increased 2-fold with monensin and colchicin. Glucose trimming of HL protein was not affected by CCCP, as indicated by digestion of immunoprecipitates with jack bean alpha-mannosidase. Pulse labeling experiments with [(35)S]methionine indicated that conversion of the 53-kDa precursor to the 58-kDa form, nor the development of endoglycosidase H-resistance, were essential for acquisition of enzyme activity. In sucrose gradients, HL protein from secretion media sedimented as a homogeneous band of about 5.8 S, whereas HL protein from the cell lysates migrated as a broad band extending from 5.8 S to more than 8 S. With both sources, HL activity was exclusively associated with the 5.8 S HL protein form. We conclude that glucose trimming of HL protein in the endoplasmic reticulum is not sufficient for activation; full activation occurs during or after transport from the endoplasmic reticulum to the Golgi and is associated with a decrease in sedimentation velocity.

Animals↗

PubMed2000

Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase.

Previously, we observed in a rat model that intravenous administration of intramuscular immunoglobulin preparations induced a long-lasting hypotension, which appeared to be associated with the presence of IgG polymers and dimers in the preparations, but unrelated to complement activation. We found evidence that this hypotensive response is mediated by platelet-activating factor (PAF) produced by macrophages. In this study, we compared the vasoactive effects of 16 intravenous immunoglobulin (IVIG) products from 10 different manufacturers, in anesthetized rats. Eight of the IVIG preparations showed no hypotensive effects (less than 15% decrease), whereas the other 8 had relatively strong effects (15%-50% decrease). The hypotensive effects correlated with the IgG dimer content of the preparations. Pretreatment of the rats with recombinant PAF acetylhydrolase completely prevented the hypotensive reaction on IVIG infusion, and administration after the onset of hypotension resulted in normalization of the blood pressure. We also observed PAF production on in vitro incubation of human neutrophils with IVIG, which could be blocked by anti-Fcgamma receptor antibodies. This indicates that induction of PAF generation may also occur in a human system. Our findings support the hypothesis that the clinical side effects of IVIG in patients may be caused by macrophage and neutrophil activation through interaction of IgG dimers with Fcgamma receptors. Because phagocyte activation may also lead to the release of other inflammatory mediators, recombinant PAF acetylhydrolase (rPAF-AH) provides a useful tool to determine whether PAF plays a role in the clinical side effects of IVIG. If so, rPAF-AH can be used for the treatment of those adverse reactions. (Blood. 2000;95:1856-1861)

1-Alkyl-2-acetylglycerophosphocholine Esterase↗