Dihydroartemisinin Disrupts Zinc Homeostasis in Plasmodium falciparum To Potentiate Its Antimalarial Action via Pyknosis.

Artemisinins have been used as first-line drugs worldwide to treat malaria caused by Plasmodium falciparum; however, its underlying mechanism is still unclear. This study aimed to identify the factors inducing growth inhibition via pyknosis, a state of intraerythrocytic developmental arrest, when exposing the parasite to dihydroartemisinin (DHA). Changes in the expression of genome-wide transcripts were assessed in the parasites treated with antimalarials, revealing the specific downregulation of zinc-associated proteins by DHA. The quantification of zinc levels in DHA-treated parasite indicated abnormal zinc depletion. Notably, the zinc-depleted condition in the parasite produced by a zinc chelator induced the generation of a pyknotic form and the suppression of its proliferation. The evaluation of the antimalarial activity of DHA or a glutathione-synthesis inhibitor in the zinc-depleted state showed that the disruption of zinc and glutathione homeostasis synergistically potentiated the growth inhibition of P. falciparum through pyknosis. These findings could help further understand the antimalarial actions of artemisinins for advancing malaria therapy.

Profiling molecular factors associated with pyknosis and developmental arrest induced by an opioid receptor antagonist and dihydroartemisinin in Plasmodium falciparum.

Malaria continues to be a devastating disease, largely caused by Plasmodium falciparum infection. We investigated the effects of opioid and cannabinoid receptor antagonists on the growth of intraerythrocytic P. falciparum. The delta opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) and the cannabinoid receptor antagonists rimonaband and SR144528 caused growth arrest of the parasite. Notably BNTX and the established antimalarial drug dihydroartemisinin induced prominent pyknosis in parasite cells after a short period of incubation. We compared genome-wide transcriptome profiles in P. falciparum with different degrees of pyknosis in response to drug treatment, and identified 11 transcripts potentially associated with the evoking of pyknosis, of which three, including glutathione reductase (PfGR), triose phosphate transporter (PfoTPT), and a conserved Plasmodium membrane protein, showed markedly different gene expression levels in accordance with the degree of pyknosis. Furthermore, the use of specific inhibitors confirmed PfGR but not PfoTPT as a possible factor contributing to the development of pyknosis. A reduction in total glutathione levels was also detected in association with increased pyknosis. These results further our understanding of the mechanisms responsible for P. falciparum development and the antimalarial activity of dihydroartemisinin, and provide useful information for the development of novel antimalarial agents.

Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells: implication of inflammation-associated carcinogenesis and tumor progression.

QR-32 tumor cells, a clone derived from a murine fibrosarcoma, are poorly tumorigenic and nonmetastatic when injected into syngeneic C57BL/6 mice. However, they are converted to highly malignant ones once they have grown in vivo after being co-implanted in a subcutaneous site with a foreign body, a gelatin sponge. Early phase of inflammation induced by the gelatin sponge participates in the conversion and histological analysis shows predominant infiltration of neutrophils. The objective of this study was to determine whether the depletion of the infiltrating neutrophils has any effect on the tumor progression. Intraperitoneal administration of a monoclonal anti-granulocyte antibody, RB6-8C5 (RB6), depleted neutrophils from both the peripheral blood circulation and the local inflamed site in mice with co-implantation of QR-32 tumor cells and gelatin sponge. The RB6 administration did not inhibit either tumor development or growth of QR-32 tumor cells. In contrast, tumor cell lines established from RB6-administered mice showed a significant decrease in metastatic incidence as compared with the tumor cell lines obtained from the mice with administration of control rat IgG or saline. Metastatic ability was significantly suppressed when RB6 had been administered in the early phase (from day -2 to day 6 after implantation); however, the administration in the middle (from day 6 to day 14) or late (from day 14 to day 22) phase did not affect the metastatic ability. We confirmed the phenomena by using integrin beta(2) knockout mice that had impaired neutrophil infiltration into inflamed sites. In the knockout mice, neutrophils hardly infiltrated into the gelatin sponge and the tumors showed dramatically suppressed metastatic phenotype as compared with those in wild-type mice or nude mice. Immunohistochemical analysis demonstrated that expressions of 8-hydroxy-2'-deoxyguanosine and nitrotyrosine were parallel to those in the presence of neutrophils. These results suggested that inflammation, especially when neutrophils infiltrate into tumor tissue, is primarily important for benign tumor cells to acquire metastatic phenotype.

Successful elimination of memory-type CD8+ T cell subsets by the administration of anti-Gr-1 monoclonal antibody in vivo.

During investigating the expression of Gr-1 antigen on various subsets of mouse spleen cells, we found that Gr-1 was expressed on memory-type CD8(+)CD44(high)CD62L(high) T cells in addition to granulocytes. Intraperitoneal administration of anti-Gr-1 mAb caused almost complete elimination of Ly-6C(+) memory-type CD8(+) T cells as well as Ly-6G(+) granulocytes. Anti-Gr-1 mAb-treated mouse spleen cells exhibited greatly reduced IFN-gamma production in response to anti-CD3 mAb both in vitro and in vivo. This reduced cytokine production appeared to be derived from elimination of IFN-gamma-producing Gr-1(+)CD8(+) T cells. Indeed, CD8(+) T cells with IFN-gamma-producing activity and cytotoxicity were generated from isolated Gr-1(+)CD8(+) cells but not from Gr-1(-)CD8(+) T cells. We also demonstrated that therapeutic effect of MBL-2 tumor-immunized spleen cells was greatly reduced by anti-Gr-1 mAb-treatment. Thus, we initially demonstrated that anti-Gr-1 mAb might become a good tool to investigate a precise role for memory-type CD8(+) T cells in vivo.

Glycosylphosphatidyl inositol-anchored protein (GPI-80) gene expression is correlated with human thymoma stage.

Thymoma is one of the most common solid tumors in the mediastinum. Because there is no typical cell line for human thymoma, the development and use of molecular-based therapy for thymoma will require detailed molecular-genetic analysis of patients' tissues. Recent reports showed that genetic aberrations in thymoma were most frequently seen in chromosome 6q regions. We investigated the use of oligonucleotide arrays to monitor in vivo expression levels of genes in chromosome 6 regions in early- (stage I or II) and late- (stage IVa) stage thymoma tissues from patients. These in vivo gene expression profiles were verified by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) using LightCycler for 48 thymoma patients and sandwich ELISA for 33 thymoma patients. Using both methods, a candidate gene was identified which was overexpressed in stage IV thymoma. This was a known glycosylphosphatidyl inositol (GPI)-anchored protein (GPI-80), which is highly homologous with Vanin-1, a mouse thymus homing protein. Serum level of GPI-80 was confirmed to be elevated in stage IV thymoma compared with in stage I thymoma by using sandwich ELISA. The combined use of oligonucleotide microarray, real-time RT-PCR, and ELISA analyses provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of thymoma.

Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

Pharmacological analysis for mechanisms of GPI-80 release from tumour necrosis factor-alpha-stimulated human neutrophils.

1 GPI-80, a glycosylphosphatidylinositol (GPI)-anchored protein initially identified on human neutrophils, plays a role(s) in the regulation of beta2 integrin function. Previous studies have shown that GPI-80 is sublocated in secretory vesicles. It is also found in soluble form in the synovial fluid of rheumatoid arthritis patients, and in the culture supernatant of formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils. To understand the behaviour of GPI-80 under conditions of stimulation, we investigated the effects of tumour necrosis factor (TNF)-alpha on its expression and release. We also probed the mechanism of its release with various pharmacologic tools. 2 TNF-alpha induced the release of GPI-80 from human neutrophils in a concentration- and time-dependent manner (in the range of 1-100 u ml(-1) and 30-120 min, respectively), but did not affect surface GPI-80 levels. 3 Cytochalasin B, genistein, and SB203580 but not PD98059 inhibited TNF-alpha-stimulated GPI-80 release and neutrophil adherence at the same concentration. In addition, TNF-alpha-induced GPI-80 release was inhibited by blocking monoclonal antibodies specific to components of Mac-1 (CD11b and CD18). 4 Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-L-cysteine) inhibited GPI-80 release by TNF-alpha stimulation, but superoxide dismutase did not. Antioxidants but not superoxide dismutase reduced an intracellular oxidation state. 5 These findings indicate that TNF-alpha-stimulated GPI-80 release from human neutrophils depends upon adherence via beta2 integrins. They also suggest that cytochalasin B, genistein, and SB203580 inhibit GPI-80 release by suppressing signals for cell adherence, rather than by a direct effect on its secretion. Finally, we suggest that GPI-80 release involves an intracellular change in a redox state.

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-gamma 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.

Pruba de anglutinación indirecta de partículas de gelatina, un medio simple y sensible para el serodiagnóstico de la de la enfermedad de Chagas / Annual Reports 1992 / The gelatine praticle indirect agglutination test, a means of simple and sensitive serodiagnósis of Chagas' disease

An indirect agglutination test using Trypanosoma cruzi antigen-coated gelatin particles was employed to Trypanosomiasis in Paraguay. Results with this test were quite comparable to those obtained with enzyme-linked immusorbent assay (Elisa). Furthermore, nonspecific reaction to the gelatin particles alone was not found in either acute or chronic infection. This method is more convenient than the ELISA, since the antigen-conjugate particles are stable for at least 1 year at 47 ºC and the test itself is short and simple to perform and does not require specialized equipment

Caracterización de un anricuuerpo monoclonal espicífico para el Trypanosoma cruzi / Annual Reports 1992 / Characterization of a monoclonal antibody specific to Trypanosoma cruzi

Amonoclonal antibody (MoAb), designated TCY-3, against Trypanosoma cruzi was obtained from spleen cells of BALB/c mice immunized with crude soluble antigens of T. curzi trypomastigotes. This MoAb Showed no cross-reactivity with 12 other heterologous parasite antigens, including promastigotes of Leishmania amazonensis and L. panamensis. Moreover, this MoAb failed to react with various organs of mouse and rat, including brain and heart. The molecular wieght to the molecule recognized by this MoAb is approximately 53 kD. Its determinant is likely protein nature, since it is sensitive to trypsin but not to periodate. Sera from mice chronicaly infected with T. cruzi reacted with the 53 kD molecule, which suggests that TCY-3 may be useful in the purification of T. cruzi- Specific antigen

Precencia de receptores de células rojas sangínea de cabral en células mononucleares de mono Cebus apella / Annual Reports 1991 / Presence of receptorst to sheep red blood cells in mononucleares cells from Cebus apella monkeys

The configuration of E Rosettes with sheep red blood cells and the expression of the epitope detected for the monoclonal antibody anti-CD2 was detected in mononuclear cells from 9 Cebus apella monkeys. Nine human controls were employed, and also the results demostrated the existence of inhibition in the formation of rosettes when anti-CD2 antiboides were used to a maximumdilution of 1/1000