Tatsuru Hara

AFRICAN TRYPANOSOME INTERACTIONS WITH AN IN VITRO MODEL OF THE HUMAN BLOOD–BRAIN BARRIER

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood–brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell™ inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly–infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstream-form T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.

The 14-3-3 proteins of Trypanosoma brucei function in motility, cytokinesis, and cell cycle

The cDNAs for two isoforms (I and II) of the 14-3-3 proteins have been cloned and functionally characterized in Trypanosoma brucei. The amino acid sequences of isoforms I and II have 47 and 50% identity to the human τ isoform, respectively, with important conserved features including a potential amphipathic groove for the binding of phosphoserine/phosphothreonine-containing motifs and a nuclear export signal-like domain. Both isoforms are abundantly expressed at approximately equal levels (1–2 × 106 molecules/cell) and localized mainly in the cytoplasm. Knockdown by induction of double-stranded RNA of isoform I and/or II in both bloodstream and procyclic forms resulted first in a reduction of cell motility and then significant reduction in cell growth rates and morphological changes; the changes include aberrant numbers of organelles and abnormal shapes and sizes that mimic phenotypes produced by various cytokinesis inhibitors. Morphological and fluorescence-activated cell sorting analysis of the cell cycle suggested that isoforms I and II might play important roles in nuclear (G2-M transition) and cell (M-G1 transition) division. These findings indicate that the 14-3-3 proteins play important roles in cell motility, cytokinesis, and the cell cycle.

Schistosoma japonicum egg granuloma formation in the interleukin-4 or interferon-gamma deficient host.

The roles of interleukin (IL)‐4 and interferon (IFN)‐γ in Schistosoma japonicum egg granuloma formation were investigated in cercariae‐infected (infection model) or after implantation of laid parasite eggs (egg implantation model) in cytokine deficient mice. Two weeks after hepatic egg‐implantation, a markedly decreased mononuclear cell infiltration and lack of multinuclear cell formation were characteristic features in IL‐4 deficient mice. By 4 weeks (late stage), the cellular reactions around the eggs were negligible in the deficient mice. Compared to the controls, there was a drastic reduction in the production of the Th2 cytokines, IL‐4, IL‐5 and IL‐13. MCP‐1 levels were also significantly lowered. In mice experimentally infected with cercariae, granuloma cellularity in both the wild‐type and IL‐4 deficient mice at 45 days and 10 weeks postinfection was analogous to the egg implantation model at 2 and 4 weeks. Overall, the effects of IFN‐γ deficiency on granuloma induction differed markedly from the IL‐4 results. Two weeks after egg implantation, IFN‐γ deficient mice showed suppressed neutrophil response and hepatic necrosis with confluent mononuclear cell infiltration along the outer layer of granulomas. By 4 weeks, there was a decrease in cell infiltration, fibrosis and MCP‐1 production while IL‐10 production increased. While these early characteristic features for IFN‐γ deficiency were common to both the egg implantation (at 2 and 4 weeks) and cercariae infection model (at 45 days), there was a surprising difference, i.e. marked fibrosis was found in the late stages (at 10 weeks postinfection) of cercariae‐infected mice, but not in parasite egg implanted mice. Furthermore, while IL‐13 levels were unchanged, both MCP‐1 and IL‐4 production were significantly lower at 10 weeks in comparison with wild‐type. The present study clearly demonstrates the importance of both Th1 and Th2 cytokine responses in S. japonicum egg‐induced granuloma formation.

Effect of nitric oxide synthase inhibition on Schistosoma japonicum egg‐induced granuloma formation in the mouse liver

Nitric oxide (NO) plays diverse roles in a variety of pathological processes. We investigated the role of NO in Schistosoma japonicum egg‐induced granuloma formation in a mouse hepatic model. Immunohistological analysis revealed that there is the most intense and extensive inducible nitric oxide (iNOS) expression 2 weeks after egg implantation, and thereafter it decreased considerably with time. Treatment with nitric oxide synthase inhibitors, NIL (lN6‐ (iminoethyl)‐lysine) or Nω‐nitro‐l‐arginine methyl ester (l‐NAME), resulted in two different types of unusual granulomas at 2 weeks. One type showed suppressed fibrosis, while another showed foreign body‐type multinuclear cell formation which frequently appeared particularly when 50 µg/ml NIL was given. At 3 weeks following treatment, fibrotic granulomas with scanty peripheral cellularity was obvious. However, there were no apparent changes after this period (at 4 weeks). Cytokine analysis in NIL‐treated mice showed a significant increase of IL‐4 and IL‐13 production at 2 weeks. These findings indicated that nitric oxide contributes to granuloma development during the early stages, probably through the regulation of Th2 cytokine production.

Neutropenia augments experimentally induced Schistosoma japonicum egg granuloma formation in CBA mice, but not in C57BL/6 mice

The present study was designed to investigate the role of neutrophils during the development of Schistosoma japonicum egg granulomas, in C57BL/6 and CBA mice. Laid eggs were implanted into the liver and monoclonal antibody, RB6‐8C5, was used to eliminate neutrophils. After daily antibody treatment between days 9 and 13 of egg implantation, both strains of mice showed a marked decrease in neutrophil infiltration and coagulative hepatocyte necrosis at 2 weeks. At 4 weeks, after antibody administration every other day between days 16 and 26, granuloma formation in C57BL/6 mice was not affected by the treatment, whereas CBA mice exhibited a significant increase of reactions. Neutropenia augmented the Th2 cytokine response (IL‐4, IL‐13 and IL‐5), but not for IFN‐γ at any time point examined and in either strain of mice. Higher levels of IL‐4 and IL‐13 were noted in CBA mice at early and late stages of granuloma formation, compared to C57BL/6 mice. There was also a striking difference in IL‐13 production between the two strains. Our results indicate that neutropenia is associated with a significant augmentation of S. japonicum egg‐induced granuloma formation in CBA mice, probably through increase in Th2 cytokines, however, the effects differ between early and late stages and between high and low responders.

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.

Inhibitory effect of circulating egg antigens on Schistosoma japonicum egg-induced granuloma formation

Schistosoma japonicum produces an enormous quantity of eggs during infection. This study was conducted to examine the effect of egg-derived antigens on the development of granuloma formation around S. japonicum eggs in the livers of mice. When soluble egg antigen (SEA) (75 micrograms/mouse/day) was injected 3-4 times via a vein into mice implanted with laid eggs, the magnitude of tissue lesion was drastically inhibited when assessed at maximal occurrence (14 days after implantation of eggs), whereas adult worm antigen (AWA), rabbit hyperimmune serum against SEA, or bovine serum albumin (BSA) did not show any effect on either the cellularity or the magnitude. In contrast to intravenous injection, there was no effect from subcutaneous injections of SEA. When serum taken from heavily infected mice or rabbit was transferred, there was a considerable extent of inhibition. In addition, an immune complex fraction of infected rabbit serum was found to have a stronger inhibitory effect than the supernatant fraction. This study indicates that the amount of egg-derived circulating antigens has a crucial effect on the development of schistosome granuloma formation.