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.

Targeting of neutrophil activation in the early phase of the disease for prevention of Coronavirus disease-19 severity.

The prevention of the disease severity seems critical for reducing the mortality of Coronavirus (CoV) disease-19. The neutrophils play a key role in the induction of severity. It is proposed here that inhibition of neutrophil activation and/or cascade reactions of complement, leading to this cell activation at the early phase of the disease, is a potential tool to inhibit aggravation of the disease. The need for appropriate timing in intervention is emphasized as follows. (1) Intervention at the very early stage of severe acute respiratory syndrome-CoV-2 infection may harm the defensive host response to the infection because of the critical function of neutrophils in this response, and (2) intervention at too late a stage will not stop the infiltration of fully activated neutrophils that produce large amounts of toxic substances.

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.

Structural divergence of GPI-80 in activated human neutrophils.

GPI-80 is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly expressed in human neutrophils. Previous studies using 3H9, a monoclonal antibody (mAb) against GPI-80, suggested that GPI-80 regulates leukocyte adherence and migration through Mac-1. GPI-80, which is anchored at the plasma membrane in resting neutrophils, moves into the pseudopodia and is released from activated human neutrophils. Here, we demonstrate that neutrophil activation affects GPI-80 dynamics using a new anti-GPI-80 mAb, designated 4D4, which is directed against the form of GPI-80 found on resting human neutrophils. Similar to 3H9, 4D4 influences Mac-1-dependent neutrophil adhesion. Treatment of purified GPI-80 with periodic acid and trypsin indicated that 3H9 and 4D4 recognize peptide and carbohydrate moieties, respectively. Stimulation with fMLP decreased the binding of 4D4 to GPI-80 on the neutrophil surface but increased the overall expression of GPI-80, as visualized by the 3H9 signal. Confocal laser microscopy revealed the 4D4 signal mainly on cell bodies and at a low level on pseudopodia during migration toward increasing concentrations of fMLP, whereas the 3H9 signal was observed in both areas. In addition, soluble GPI-80 released from activated neutrophils did not bind 4D4. These results suggest that there are two populations of GPI-80 that differ in the ability to bind 4D4. The 4D4-recognized form may regulate Mac-1-dependent neutrophil adhesion, and may subsequently be converted to a 4D4-unrecognized form during neutrophil activation.

Stent-induced neutrophil activation is associated with an oxidative burst in the inflammatory process, leading to neointimal thickening.

Activation of leukocytes plays an essential role in the mechanism of restenosis. Prior research has focused on monocytes and little is known about the role of neutrophils in this process. Neutrophils are known to contribute to tissue injury through oxygen-derived free radicals that nitrate tyrosine. This study was designed to elucidate clinically the role of neutrophil-mediated oxidative burst in the regulation of the post-stent inflammatory process. In 36 patients undergoing coronary stenting, we serially measured serum levels of glycosyl-phosphatidil-inositol-anchored protein (GPI)-80,a modulator of Mac-1 on the surface of neutrophils, in samples of coronary sinus as well as peripheral blood. We also simultaneously measured the serum 3-nitrotyrosine/tyrosine ratio as an index of oxidative stress. The GPI-80 level and the 3-nitrotyrosine/tyrosine ratio increased in the coronary sinus after coronary stenting in a time-dependent manner; with the maximum increase of GPI-80 level (3.1 +/- 2.9 to 8.6 +/- 4.3 ng/ml, P < 0.01) at 48 hours, and 3-nitrotyrosine/tyrosine ratio at 24 hrs (5.2 +/- 4.8 to 28.4 +/- 13.2 x 10(-4), P < 0.01), more strikingly than in the peripheral blood. In the coronary sinus blood, the 3-nitrotyrosine/tyrosine ratio was correlated with GPI-80 levels at 24 hr (R = 0.58, P < 0.001) and at 48 hr (R = 0.41, P < 0.01). Multiple regressions analysis showed that the maximum responses of GPI-80 level and 3-nitrotyrosine/tyrosine ratio were independent predictors of angiographic late lumen loss. Our results may support a hypothesis that Mac-1-dependent activation of neutrophils causes oxidative burst in the post-stent inflammatory process, possibly leading to restenosis.

Inhibition of cell spreading in CHO cells transfected with cDNA of a glycosylphosphatidyl inositol-anchored protein, GPI-80.

We have previously cloned a glycosylphosphatidyl inositol (GPI)-anchored protein, designated GPI-80 that associated with integrin and may modulate leukocyte adherence and migration. Recent studies have shown that GPI-80 belongs to a Vanin family that is related to pantetheinase, but the regulatory function of GPI-80 in cell adherence is still unclear. To clarify the possible functions of GPI-80, we transfected GPI-80 cDNA into Chinese hamster ovary (CHO) cells and observed adherence and morphological changes. Adherence of GPI-80 transfectants was significantly decreased when signal strength for the cell adhesion is weak, and the cell spreading of the transfectants was strongly inhibited. This inhibitory effect of GPI-80 expression was largely canceled by GPI-80 shedding with phosphatidy-linositol-specific phospholipase C. Interestingly, spreading of GPI-80 transfectants was temporarily recovered from the round shape but not maintained by stimulation with known activators of beta1 integrins, phorbol myristate acetate and manganese ions. Taken together, these results suggest that the expression of GPI-80 on CHO cells can influence cell spreading in weak adhesive signal conditions via extracellular matrix molecules.

The involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice.

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.

[Different impacts of DMSO and RA on GPI-80 expression].

Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. Dimethyl sulfoxide (DMSO) and all-trans-retinoic acid (RA) are two main inducers that can induce HL-60 cell to differentiate down the way of neutrophilic lineage. GPI-80 is a novel member of a human glycosylphosphatidylinositol (GPI)-anchored family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion, and is expressed mainly in human neutrophils. RT-PCR, flow cytometry and western-blot assays were used to detect the GPI-80 expression on HL-60 cells induced by DMSO or RA and to analyze the relationship between GPI-80 expression and CD11b or transferrin receptor (CD71) expression. The results showed GPI-80 was slightly expressed on the level of mRNA on RA-induced HL-60 cells by RT-PCR, while using flow cytometry and western-blot assays, We could not detect any GPI-80 expression on these cells, Furthermore, RA suppressed the induction of GPI-80 expression. However, DMSO could clearly derive the GPI-80 expression and this expression correlated well with the increasing dose and duration of DMSO stimulation. GPI-80 positive cells, which were included in CD11b-strongly positive and transferrin receptor-completely negative populations, were sufficiently differentiated cells suggesting that GPI-80 expression is tightly associated with the neutrophilic maturation and can be used as a mature neutrophilic marker. RA inhibits the GPI-80 expression on DMSO induced HL-60 cells suggests that different signal conduction has been activated by DMSO and RA when they induce the HL-60 cells to the neutrophilic differentiation.

Molecular proximity of complement receptor type 3 (CR3) and the glycosylphosphatidylinositol-linked protein GPI-80 on neutrophils: effects of cell adherence, exogenous saccharides, and lipid raft disrupting agents.

GPI-80, a novel glycosylphosphatidylinositol (GPI)-anchored protein on polymorphonuclear leukocytes, has been reported to cooperate with CR3 in several aspects of cell function including cell activation, adhesion and migration. The present study investigates the physical proximity of CR3 and GPI-80 on living cells using resonance energy transfer (RET) techniques, which gives positive results when the separation distance is < or = 7 nm. RET from donor-labeled anti-CR3 to acceptor-labeled anti-GPI-80 was detected on adherent neutrophils, but not observed for non-adherent cells. Furthermore, RET was not observed on cells treated with cell adhesion inhibitors 4-bromophenacyl bromide (BPB), N-ethylmaleimide (NEM) or cytochalasin D, suggesting dynamic interactions between CR3 and GPI-80. CR3-to-GPI-80 proximity was blocked by N-acetyl-D-glucosamine (NADG), but not by other monosaccharides such as D-mannose, fructose, fucose, glucose, sorbitol, or galactose; molecular proximity was also disrupted by the glycolipid raft depleting agents 2-OH-propyl-betaCD and MbetaCD. Thus, lipid rafts may be important for the physical and functional cooperation of CR3 and GPI-80.

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.

Localization of GPI-80, a beta2-integrin-associated glycosylphosphatidyl-inositol anchored protein, on strongly CD14-positive human monocytes.

Human monocyte/macrophage systems are extremely heterogeneous. Although many attempts have been made to define monocyte subpopulations, few antigens distinguish them. We previously reported that GPI-80, a novel glycosylphosphatidyl-inositol (GPI)-anchored protein that is expressed mainly on human neutrophils regulates neutrophil adherence and migration, and that GPI-80 is expressed on monocytes. In this study, we examined the precise distribution of GPI-80-positive monocytes using flow cytometry. Using anti-CD14 and anti-CD16 mAbs, almost all GPI-80-bearing monocytes belong to the strongly CD14-positive monocyte subpopulation. Furthermore, flow cytometric analysis of GPI-80 and other monocyte markers revealed that GPI-80 expression was high in CD11b-, CD32-, and CD64-positive monocytes. In contrast, GPI-80 expression was low in HLA-DQ-positive monocytes. These results suggest that almost all GPI-80 positive monocytes belong to a monocyte subpopulation that is superior in phagocytosis and reactive oxygen production, but inferior in antigen presentation. GPI-80 may be a useful antigen for classifying monocytes into subpopulations.

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.

GPI-80, a beta2 integrin associated glycosylphosphatidylinositol-anchored protein, concentrates on pseudopodia without association with beta2 integrin during neutrophil migration.

Previously, we identified a glycosylphosphatidylinositol (GPI)-anchored protein, designated GPI-80, present on human neutrophils and monocytes. GPI-80 is physically associated with beta2 integrin on the surface of human neutrophils and may be a regulator of neutrophil adherence and migration. However, it is not yet known how GPI-80 regulates cell adhesion and migration. To investigate the physiological role(s) of GPI-80, we examined the topological relationship of GPI-80 and the beta2 integrin subunit (CD18) on resting and migrating human neutrophils by confocal laser microscopy. On resting neutrophils, GPI-80 was evenly distributed on the cell surface and was associated with CD18. On the other hand, during the early phase of migration (5 - 30 minutes), GPI-80 was detected on cell bodies and also on pseudopodia, but CD18 was detected only on cell bodies, where it was associated with GPI-80. In the late phase of migration (60 minutes), GPI-80 was detected only on pseudopodia and its association with CD18 was hardly observed. Furthermore, some of the GPI-80 on pseudopodia of migrating neutrophils during the late phase was associated with urokinase-type plasminogen activator receptor (uPAR), a regulator of beta2 integrin-dependent adherence and migration. The distribution of GPI-80 on cell surfaces is similar to that of uPAR. These observations suggest that GPI-80 belongs to the beta2 integrin-associated GPI-anchored protein family, which has regulatory activity in cell adherence.

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.

Physical association of beta 2 integrin with GPI-80, a novel glycosylphosphatidylinositol-anchored protein with potential for regulating adhesion and migration.

We recently found a novel GPI-anchored protein, GPI-80, on human phagocytes that may regulate leukocyte adherence, locomotion, and extravasation. To clarify the mechanisms by which GPI-80 functions on leukocytes, we explored the possibility of its physical association with beta 2 integrin which is important for leukocyte adherence, locomotion, and extravasation. beta 2 integrin, detected by anti-CD18 mAb, was coprecipitated with GPI-80 from human neutrophil lysates by a mAb to GPI-80. In addition, GPI-80 was immunoprecipitated from human neutrophil lysates by anti-human CD18 mAb. These results clearly show that GPI-80 is physically associated with beta 2 integrin in human neutrophils.

Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin.

Previously, we described a novel glycosylphosphatidyl inositol (GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils and monocytes that may regulate beta(2) integrin-dependent neutrophil adherence and migration. However, the mechanism regulating beta(2) integrin remains to be clarified. To study this, we examined changes in beta(2) integrin expression and function caused by cross-linking GPI-80. GPI-80 cross-linking induced up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces and shedding of L-selectin, which depends on tyrosine phosphorylation and cytoskeleton remodeling. Furthermore, the cross-linking enhanced fMLP-induced human neutrophil adherence. These results suggest that GPI-80 may be a regulator of beta(2) integrin in neutrophils.

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