Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals.

This multicentre study aimed at evaluating the reliability (reproducibility) and relevance (predictivity) of a new commercially available human corneal epithelial (HCE) model (SkinEthic Laboratories, Nice, France) to assess acute ocular irritation. A prevalidation approach (protocol optimisation, transfer and performance) was followed and at each of the four participating laboratories, 20 coded reference chemicals, covering the whole range of irritancy, were tested. The compounds were applied topically to the HCE cultures and the level of cytotoxicity (tissue viability and histological analysis) was determined. Once a standardised protocol was established, a high level of reproducibility between the laboratories was observed. In order to assess the capability of the HCE model to discriminate between irritants (I) and non-irritants (NI), a classification prediction model (PM) was defined based on a viability cut-off value of 60%. The obtained in vitro classifications were compared with different in vivo classifications (e.g. Globally Harmonised System) which were calculated from individual rabbit data described in the ECETOC data bank. Although an overall concordance of 80% was obtained (sensitivity = 100% and specificity = 56%), the predictivity of the HCE model substantially increased when other sources of in vivo and in vitro data were taken into account.

Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency.

In population-based studies it has been established that inherited deficiency of erythrocyte (E) glucose-6-phosphate dehydrogenase (G6PD) confers protection against severe Plasmodium falciparum (P falciparum) malaria. Impaired growth of parasites in G6PD-deficient E in vitro has been reported in some studies, but not in others. In a systematic analysis, we have found that with five different strains of P falciparum (FCR-3, KI, C10, HB3B, and T9/96), there was no significant difference in either invasion or maturation when the parasites were grown in either normal or G6PD-deficient (Mediterranean variant) E. With all of these strains and at different maturation stages, we were unable to detect any difference in the amount of P falciparum-specific G6PD mRNA in normal versus deficient parasitized E. The rate of 14C-CO2 production from D-[1-14C] glucose (which closely reflects intracellular activity of G6PD) contributed by the parasite was very similar in intact normal and deficient E. By contrast, in studies of phagocytosis of parasitized E by human adherent monocytes, we found that when the parasites were at the ring stage (ring-stage parasitized E [RPE]), deficient RPE were phagocytosed 2.3 times more intensely than normal RPE (P = .001), whereas there was no difference when the parasites were at the more mature trophozoite stage (trophozoite-stage parasitized E [TPE]). Phagocytic removal markers (autologous IgG and complement C3 fragments) were significantly higher in deficient RPE than in normal RPE, while they were very similar in normal and deficient TPE. The level of reduced glutathione was remarkably lower in deficient RPE compared with normal RPE. We conclude that impaired antioxidant defense in deficient RPE may be responsible for membrane damage followed by phagocytosis. Because RPE, unlike TPE, are nontoxic to phagocytes, the increased removal by phagocytosis of RPE would reduce maturation to TPE and to schizonts and may be a highly efficient mechanism of malaria resistance in deficient subjects.

Plasmodium falciparum glutathione metabolism and growth are independent of glutathione system of host erythrocyte.

Plasmodium falciparum parasites grew normally in glutathione (GSH)-depleted normal and G6PD-deficient (Mediterranean variant) erythrocytes (RBC). Growth inhibition was observed only at less than approximately 6-12% residual GSH. Parasites studied separately with the Sendai virus technique synthesized GSH de novo and regenerated reduced GSH 10-20 times faster than non-parasitized RBC. Electron spin resonance measurement of Tempol reduction indicated that the ability to reduce free radicals was restricted to the parasite. The marked efflux of oxidized GSH was mainly derived from the parasite. In conclusion, parasites are endowed with powerful and host-independent mechanisms which de novo synthesize or regenerate GSH and allow undisturbed parasite development in GSH-depleted RBC.

Role of hemichrome binding to erythrocyte membrane in the generation of band-3 alterations in beta-thalassemia intermedia erythrocytes.

Nine splenectomized, hematologically well-compensated beta-thalassemia intermedia patients randomly chosen from a pool of 60 similar patients were studied. Membrane proteins solubilized with nondenaturing detergent C12E8 were gel filtered on Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden). Fractions containing higher than 4,000-kD molecular-weight aggregates were isolated and analyzed. Four patients had remarkably increased amounts of membrane-bound hemichromes and Igs. In those patients, band 3 underwent oxidative modifications such as aggregation and a decrease in sulfhydryl groups. The other five patients had low amounts of membrane-bound hemichromes and less modifications of band 3. The same band-3 modifications could be reproduced by challenging normal membranes with artificially generated hemichromes or with hemolysates prepared from thalassemic erythrocytes of the high-hemichrome group. Addition of reduced glutathione to the challenged membranes did not hinder hemichrome binding, but prevented oxidative modifications of band 3 and Ig binding to high-molecular-weight band-3 aggregates. Hemichrome binding to band 3, hemichrome-mediated oxidation of band-3 cytoplasmic domains, generation of high-molecular-weight band-3 aggregates, and enhanced opsonization by anti-band-3 antibodies is a possible sequence of events leading to phagocytic removal of erythrocytes in thalassemia.

Cloning of the glucose 6-phosphate dehydrogenase gene from Plasmodium falciparum.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the human genetic traits that confer relative resistance against malaria caused by Plasmodium falciparum. It has been previously shown that this organism, during its intraerythrocytic development, produces its own G6PD, which has properties different from those of human G6PD. In order to investigate the role of this enzyme in parasite-host cell interactions, we have isolated the G6PD gene from Plasmodium falciparum as a set of overlapping lambda gt11 clones. By sequence analysis we have found a single open reading frame, uninterrupted by introns, coding for a protein of 910 amino acids, almost twice as long as any previously sequenced G6PD molecule. The P. falciparum G6PD mRNA is 5.1 kb in size and has an exceptionally long 5' untranslated region of some 1000 nucleotides. We have mapped the G6PD gene to chromosome 14. The C-terminal portion of the predicted protein, from amino acid 310-910 (except for an 'insert' of 62 amino acids), has 39% homology to human G6PD, with a number of characteristic, fully conserved peptides. The N-terminal portion of the predicted protein has no homology to G6PD, but it contains a peptide in which 7 out of 12 amino acids are identical to the putative glutathione binding site of human glutathione S-transferase.

Binding of naturally occurring antibodies to oxidatively and nonoxidatively modified erythrocyte band 3.

Both oxidative clustering (elicited by diamide treatment) and nonoxidative clustering (elicited by zinc/BS3 (bis[sulfosuccinimidyl]suberate) treatment) of erythrocyte integral membrane proteins induce binding of autologous antibodies with anti-band 3 specificity, followed by complement deposition and phagocytosis. Autologous antibodies eluted from nonoxidatively clustered erythrocytes bind to and stimulate phagocytosis of oxidatively damaged erythrocytes. Those eluted antibodies bind specifically to disulfide-crosslinked band 3 dimers generated by diamide treatment. Band 3 dimerization and antibody binding are abrogated by cleavage of band 3 cytoplasmic domain. Thus, disulfide-crosslinked band 3 dimers are the minimal band 3 aggregate with enhanced affinity for anti-band 3 antibodies. The eluted antibodies do not bind to band 3 dimers generated nonoxidatively by BS3 treatment but bind avidly to larger band 3 clusters generated nonoxidatively by zinc/BS3 treatment. Possibly, disulfide crosslinking of cytoplasmic domain cysteines induces reorientation of intramembrane domains as to expose putative anti-band 3 epitopes and allow bivalent binding of anti-band 3 antibodies. Extensive nonoxidative band 3 clustering appears to disrupt the native band 3 conformation and generate reoriented dimers which expose putative anti-band 3 epitopes in the proper distance and orientation as to allow bivalent antibody binding.

Phagocytosis of P. falciparum malarial pigment hemozoin by human monocytes inactivates monocyte protein kinase C.

Hemozoin (malarial pigment) is a ferriprotoporphyrin IX-rich hemoglobin degradation product present in parasitized RBC. Avidly phagocytosed hemozoin abolishes phagocyte TPA-induced oxidative burst. Membrane-associated PKC increased transiently in hemozoin-fed monocytes by 50% after 30 min and decreased irreversibly to 20% of initial value within 5 h after phagocytosis. Control RBC-fed monocytes showed transient decay of membrane-associated PKC followed by complete recovery 12 h after phagocytosis. Cytosolic PKC was not impaired within 12 h and diminished drastically 24 h after phagocytosis of hemozoin. Results are compatible with increased degradation of membrane-translocated PKC, possibly by iron/H2O2-mediated damage of cysteine-rich regulatory domains of PKC.

Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation.

The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.