No abstract

SummaryHuman monocyte-derived macrophages ingest diamide-treated red blood cells (RBC), anti-D immunoglobulin (Ig)G-opsonized RBC, or Plasmodium fakiparum ring-stage parasitized RBC (RPRBC), degrade ingested hemoglobin rapidly, and can repeat the phagocytic cycle. Monocytes fed with trophozoite-parasitized RBC (TPRBC), which contain malarial pigment, or fed with isolated pigment are virtually unable to degrade the ingested material and to repeat the phagocytic cycle. Monocytes fed with pigment display a long-lasting oxidative burst that does not occur when they phagocytose diamide-treated RBC or RPRBC. The phorbol myristate acetate-elicited oxidative burst is irreversibly suppressed in monocytes fed with TPRBC or pigment, but not in monocytes fed with diamide-treated or IgG-opsonized RBC. This pattern of inhibition of phagocytosis and oxidative burst suggests that malarial pigment is responsible for the toxic effects. Pigment iron released in the monocyte phagolysosome may be the responsible element. 3% of total pigment iron is labile and easily detached under conditions simulating the internal environment of the phagolysosome, i.e., pH 5.5 and 10/zM H202. Iron liberated from pigment could account for the lipid peroxidation and increased production of malondialdehyde observed in monocytes fed with pigment or in RBC ghosts and liposomes incubated at pH 6.5 in presence of pigment and low amounts of H202. Removal of the labile iron fraction from pigment by repeated treatments with 0.1 mM H202 at pH 5.5 reduces pigment toxicity. It is suggested that iron released from ingested pigment is responsible for the intoxication of monocytes. In acute and chronic falciparum infections, circulating and tissue-resident phagocytes are seen filled with TPRBC and pigment particles over long periods of time. Moreover, human monocytes previously fed with TPRBC are unable to neutralize pathogenic bacteria, fungi, and tumor cells, and macrophage responses decline during the course of human and animal malaria. The present results may offer a mechanistic explanation for depression of cellular immunity in malaria.

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Ultrasound-activated decafluoropentane/chitosan nanodroplets effectively release oxygen to the skin.

Matrix metalloproteinase-9 (MMP-9), secreted by activated monocytes, degrades matrix proteins, disrupts basal lamina, and activates TNF-α from its precursors. In turn, TNF-α enhances synthesis of MMP-9 in monocytes. We show here that trophozoite-parasitized RBCs/hemozoin-fed adherent human monocytes displayed increased MMP-9 activity and protein/mRNA expression, produced TNF-α time-dependently, and showed higher matrix invasion ability. MMP-9 activation was specific for trophozoite/hemozoin-fed monocytes, was dependent on TNF-α production, and abrogated by anti-TNF-α Ab and by a specific inhibitor of MMP-9/MMP-13 activity. Hemozoin-induced enhancement of MMP-9 and TNF-α production would have a 2-fold effect: to start and feed a cyclic reinforcement loop in which hemozoin enhances production of TNF-α, which in turn induces both activation of MMP-9 and shedding of TNF-α into the extracellular compartment; and, second, to disrupt the basal lamina of endothelia. Excess production of TNF-α and disruption of the basal lamina with extravasation of blood cells into perivascular tissues are hallmarks of severe malaria. Pharmacological inhibition of MMP-9 may offer a new chance to control pathogenic mechanisms in malaria.

Summary. Plasmodium falciparum-parasitized erythrocytes (RBCs) are progressively transformed into non-self cells, phagocytosed by human monocytes. Haemichromes, aggregated band 3 (Bd3) and membrane-bound complement fragment C3c and IgG were assayed in serum-opsonized stage-separated parasitized RBCs. All parameters progressed from control to rings to trophozoites to schizonts: haemichromes, nil; 0´64^0´12; 5´6^1´91; 8´4^2´8 (nmol/ ml membrane); Bd3, 1^0´1; 4´3^1´5; 23^5; 25^6 (percentage aggregated); C3c, 31^11; 223^86; 446^157; 620^120 (mOD 405 /min/ml membrane); IgG, 35^12; 65^23; 436^127; 590^196 (mOD 405 /min/ml membrane). All increments in rings versus controls and in trophozoites versus rings were highly significant. Parasite development in the presence of 100 mmol/l beta-mercaptoethanol largely reverted haemichrome formation, Bd3 aggregation, C3c and IgG deposition and phagocytosis. Membrane proteins extracted by detergent C 12 E 8 were separated on Sepharose CL-6B. Haemichromes, C3c and IgG were present exclusively in the highmolecular-weight fractions together with approximately 30% of Bd3, indicating the oxidative formation of immunogenic Bd3 aggregates. Immunoblots of separated membrane proteins with anti-Bd3 antibodies confirmed Bd3 aggregates that, in part, did not enter the gel. Immunoprecipitated antibodies eluted from trophozoites reacted preferentially with aggregated Bd3. Changes in parasitized RBC membranes and induction of phagocytosis were similar to oxidatively damaged, senescent or thalassaemic RBC, indicating that parasite-induced oxidative modifications of Bd3 were per se sufficient to induce and enhance phagocytosis of malaria-parasitized RBC.

No abstract

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 ofP 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.