Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis.

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

In vivo studies of the early, peritoneal, cellular and free radical response in rats infected with Fasciola hepatica by flow cytometric analysis

Early recruitment of the peritoneal cell population was observed during migration of newly excysted juvenile flukes. The peritoneal lavages were examined for T cells, cytotoxic NK cells (CNK) and free radicals production of rats at an early stage of infection by Fasciola hepatica. Male Sprague–Dawley rats were infected with 50 metacercariae of F. hepatica and non-infected controls were euthanized 2, 4 and 7 days post infection (d.p.i.), respectively. The peritoneal fluid of experimental animals was analyzed by flow cytometry to estimate cell phenotypes. The peritoneal areas were infiltrated by inflammatory cells, particularly from numerous neutrophils, eosinophils and CD4+ lymphocytes, which were significantly higher for infected rats than non-infected. CNK cells dominated in the peritoneal fluid of infected rats as early as 2 d.p.i. However, after 4 d.p.i. there was a decreased level of CNK cells which may indicate a change from a cytotoxic natural killer (NK) to a regulatory NK response. The challenged group generated very high in vivo levels of inducible nitric oxide (NO) from eosinophils. Superoxide expression was very high in macrophages and neutrophils compared to the uninfected control. In conclusion, our studies suggest that early F. hepatica infection could directly affect lymphoid cells and generate a high in vivo NO production by eosinophils in the peritoneal cavity. Moreover juvenile flukes could stimulate the macrophages and neutrophils to generate H₂O₂ radicals. The host parasite interactions resulting from immune response regulation by effector cells and immune evasion are discussed.     

Alteration in density of eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis

The density of eosinophils in the cerebrospinal fluid (CSF) and blood of male ddY mice infected with Angiostrongylus cantonensis was examined on days 14, 20 and 27 post-infection (p.i.) with discontinuous Percoll gradient centrifugation. Normal blood eosinophils had a density of between 1.070 and 1.080 g ml-1. No significant changes in density in blood eosinophils were noted during the course of the observations. CSF eosinophils began to become hypodense (defined as density less than 1.070 g ml-1) on day 20 p.i., and 88% of eosinophils were hypodense on day 27. Our results suggest therefore that eosinophils probably become hypodense in the CSF and brain tissues, but not in the blood.     

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.     

Trichinella spiralis: infection reduces airway allergic inflammation in mice

In an effort to define the mechanism underlying the host immune downregulation inherent to Trichinella spiralis infection, we compared the levels of Th1, Th2, and regulatory cytokines and CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ T (T_reg) cell recruitment, as well as cellular pathology in the airway between T. spiralis infected and uninfected asthma-induced mice. After the induction of allergic airway inflammation, we noted influxes of inflammatory cells into the peribronchial tree. However, in the T. spiralis infection groups, cellular infiltration was minimal around the bronchial tree, with only a smattering of inflammatory cells. In the OVA-challenged group after T. spiralis infection, the numbers of macrophages and eosinophils in the bronchial alveolar lavage fluid were reduced by 23% and 52%, respectively, as compared to those of the OVA-challenged group. Airway hyperresponsiveness of OVA-challenged mice after T. spiralis infection was significantly suppressed as compared to the OVA-only challenged mice. The T. spiralis-infected mice exhibited a significant reduction in IL-5 concentrations relative to that noted in the OVA-challenged group (p < 0.01). Nevertheless, the regulatory cytokines IL-10 and TGF-β levels were increased significantly as the result of T. spiralis infection, and we verified the recruitment of T_reg cells in lung draining lymph nodes via T. spiralis infection. Therefore, T_reg cells, which were recruited by T. spiralis infection, might ameliorate lung function and reduce allergic airway inflammation.     

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 °C for three days of culture (dc), 28 °C for seven dc or 18 °C for seven dc. On days 1, 3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and BALF of infected mice. Larvae at 28 °C/3dc induced earlier eosinophils in the PCF and BALF as opposed to larvae at 28 °C/7dc and 18 °C/7dc. Larvae at 28 °C/7dc induced higher synthesis of IL-4, IL-5 and IL-10 on days 5 and 7 post-infection. Larvae at 28 °C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-γ after larval migration had ceased and only adult worms were present. Larvae at 28 °C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture.     

Eosinophil-mediated destruction of Schistosoma mansoni eggs III. lymphokine involvement in the induction of eosinophil functional abilities

Granulomas isolated from the livers of CBA/J mice infected for 8 weeks with Schistosoma mansoni produced a chemotactic activity for eosinophils, in a manner which correlated with the production of the lymphokine eosinophil stimulation promoter (ESP). ESP and chemotactic activities were also produced when eosinophilrich peritoneal exudative cells from S. mansoni-infected mice were cultured with S. mansoni eggs. These S. mansoni-related eosinophils destroyed approximately 20% of the eggs whereas eosinophils from normal (uninfected) mice did not have this ability. However, normal cells exposed to ESP-containing fluids in the co-cultivation system actively participated in egg destruction. Eosinophil-rich peritoneal exudative cells obtained from Trichinella spiralis-infected mice were incapable of destroying S. mansoni eggs during the normal 24 hr co-cultivation period, but did achieve destruction if the incubation period was extended to 48 hr. Marginal levels of chemotactic activity for eosinophils were detected in the co-cultivation fluids from T. spiralis-related cells and S. mansoni eggs, although these fluids did not contain demonstrable levels of ESP. Together, these data indicate that ESP/chemotactic factor-containing culture fluids can induce in normal, unreactive eosinophils the functional ability to destroy S. mansoni eggs in vitro. This may account for the ability of T. spiralis-related eosinophils to do so upon extended incubation.     

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P < 0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2′-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.     

Evidence of Infectious Asthma Phenotype: Chlamydia-Induced Allergy and Pathogen-Specific IgE in a Neonatal Mouse Model

Asthma is a chronic respiratory disease whose etiology is poorly understood. Recent studies suggest that early-life respiratory infections with atypical bacteria may play an important role in the induction or exacerbation of chronic respiratory disease. The current study utilized a neonatal mouse ovalbumin (OVA) sensitization model of asthma to determine the course of early-life respiratory tract infection by Chlamydia. Neonatal (day 1) and adult (6 wks) BALB/c mice were infected intranasally with Chlamydia (MoPn) and 7 weeks later were sensitized and challenged with ovalbumin. Allergic airway disease was characterized by examination of serum and bronchoalveolar lavage fluid (BAL) cellularity, cytokine production and antibody response. The presence of Chlamydia was determined by PCR and culture. Ova-specific IgE was quantified by ELISA and Chlamydia-specific IgE was determined via Western blot analysis. Chlamydial infection in neonatal mice induced increased production of Th₂ cytokines (IL-4, 5, 10, and 13) in both BAL and serum, while infected adult mice produced increased Th₁ cytokines (IL-2, IFN-γ). The BAL from infected neonates contained significantly elevated levels of eosinophils compared to infected adult mice. Although adult mice cleared the infection ∼30 days post infection (pi), neonates were still infected 66 days after initial infection. Chlamydia-specific IgE was detected in both the BAL and serum of neonatal mice beginning 28 days post infection, however, infected adult mice did not produce Chlamydia-specific IgE antibodies over the course of the study. When allergic airway was induced using Ova, infected neonatal mice increased their production of IL-4, IL-5 and IL-13 by >2 fold compared to uninfected controls and infected adult groups. Our findings demonstrate that early-life Chlamydia infection induces a Th₂-dominant cytokine response in the airways of neonatal mice, leading to chronic infection. More significantly, early life respiratory colonization with Chlamydia elicits pathogen-specific IgE production, which further supports an infectious asthma phenotype.     

MicroRNA miR-155-5p knockdown attenuates Angiostrongylus cantonensis-induced eosinophilic meningitis by downregulating MMP9 and TSLP proteins

Angiostrongylus cantonensis infection is a major cause of eosinophilic meningitis (EM). Severe cases or cases that involve infants and children present poor prognoses. MicroRNAs (miRNAs), which are important regulators of gene expression in many biological processes, were recently found to be regulators of the host response to infection by parasites; however, their roles in brain inflammation caused by A. cantonensis are still unclear. The current study confirmed that miR-155-5p peaked at 21 days after A. cantonensis infection, and its expression was positively correlated with the concentration of excretory and secretory products (ESPs). We found that miR-155-5p knockdown lentivirus successfully ameliorated brain injury and downregulated the expression of major basic protein (MBP) in vivo, and the number of eosinophils in CSF (and the percentage of eosinophils in peripheral blood were also decreased in the miR-155-5p knockdown group. Moreover, the expression of several eosinophilic inflammation cytokines such as CCL6/C10, ICAM-1, and MMP9, declined after the miR-155-5p knockdown. SOCS1 protein, which is an important negative regulator of inflammation activation, was identified as a direct miR-155-5p target. We further detected the effect of miR-155-5p knockdown on phosphorylated-STAT3 and phosphorylated-p65 proteins, which were found to be negatively regulated by SOCS1 and play an important role in regulating the inflammatory response. We found that miR-155-5p knockdown decreased the activity of p-STAT3 and p-p65, thereby leading to lower expression of MMP9 and TSLP proteins, which were closely related to the chemotaxis and infiltration of eosinophils. Interestingly, the inhibition of p-STAT3 or p-p65 was found to induce the downregulation of miR-155-5p in an opposite manner. These observations suggest that a positive feedback loop was formed between miR-155-5p, STAT3, and NF-κB in A. cantonensis infection and that miR-155-5p inhibition might provide a novel strategy to attenuate eosinophilic meningitis.     

Meningitis patients with Angiostrongylus cantonensis may present without eosinophilia in the cerebrospinal fluid in northern Vietnam

BackgroundEosinophilic meningitis (EM) is a rare clinical syndrome caused by both infectious and noninfectious diseases. In tropical pacific countries, Angiostrongylus cantonensis is the most common cause. However, the EM definition varies in the literature, and its relation to parasitic meningitis (PM) remains unclear.Methodology/Principal findingsAdult and adolescent patients of 13 years old or above with suspected central nervous system (CNS) infections with abnormal CSF findings were prospectively enrolled at a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. Patients with EM or suspected PM (EM/PM) were defined by the presence of either ≥10% eosinophils or an absolute eosinophil cell counts of ≥10/mm³ in the CSF or blood eosinophilia (>16% of WBCs) without CSF eosinophils. In total 679 patients were enrolled: 7 (1.03%) had ≥10% CSF eosinophilia, 20 (2.95%) had ≥10/mm³ CSF eosinophilia, and 7 (1.03%) had >16% blood eosinophilia. The patients with ≥10% CSF eosinophilia were significantly younger (p = 0.017), had a lower body temperature (p = 0.036) than patients with ≥10/mm³ CSF eosinophilia among whom bacterial pathogens were detected in 72.2% (13/18) of those who were tested by culture and/or PCR. In contrast, the characteristics of the patients with >16% blood eosinophilia resembled those of patients with ≥10% CSF eosinophilia. We further conducted serological tests and real-time PCR to identify A. cantonensis. Serology or real-time PCR was positive in 3 (42.8%) patients with ≥10% CSF eosinophilia and 6 (85.7%) patients with >16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm³ CSF eosinophilia.ConclusionsThe etiology of PM in northern Vietnam is A. cantonensis. The eosinophil percentage is a more reliable predictor of parasitic EM than absolute eosinophil count in the CSF. Patients with PM may present with a high percentage of eosinophils in the peripheral blood but not in the CSF.     

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12^−/− and wild-type C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12^−/− and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12^−/− mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12^−/− mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection.     

Disturbances in the production of inteleukin-1 tumor and necrosis factor in disseminated murine sporotrichosis

Production of Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) by adherent peritoneal cells from BALB/c mice was measured at week 2, 4, 6, 8 and 10 after intravenous inoculation with 10⁶ Sporothrix schenckii yeasts. As compared with age-matched controls, IL-1 and TNF production by adherent peritoneal cells fromS. schenckii-infected mice was reduced severely at week 4 and 6 of infection and greater than normal at week 8 and 10. Moreover, between week 4 and 6 of infection there was a depression of delayed type hypersensitivity response to a specific whole soluble antigen, and an increase in fungal multiplication in the livers and spleens of infected mice. Thus, the deficits of cell-mediated immunity in mice with systemicS. schenckii infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL-1 and TNF.     

IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity

Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.     

Modulation of P2X7 purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination

The purinergic P2X₇ receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X₇R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X₇R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X₇R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X₇R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X₇R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis.     

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism

Purpose: Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. Methods: To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [⁵¹Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. Results: Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. Conclusions: These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism. Received: 28 January 2001 / Accepted: 25 April 2001     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

Induction of Interleukin 2-Responsiveness in Thymocytes of the Transgenic Mice Carrying lck-Transgene

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2K^b and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56^lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4⁺8^– single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2Rα and IL-2Rβ of these CD4⁺8^– thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2Rβ was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56^lck of transgenic thymocytes was not down-reguated at 4 hr after stimulaion with IL-2, whereas p56^lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56^lck in the thymocytes from transgenic mice: the kinase activities of p56^lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56^lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56^lck may play a role in the IL-2R-mediated signaling system in CD4⁺8^– thymocytes.     

Influenza Infection Neutralizes the Attractiveness of Male Odour to Female Mice (Mus musculus)

This study aimed to determine if female house mice, Mus musculus domesticus, are able to assess a male's infection status from odour cues. We collected urine from male mice before, during, and after they were experimentally infected with influenza, a respiratory virus. Females spent more time investigating urine collected from males while they were uninfected than when they were infected. Also 70 % of females released into a large enclosure preferred to nest in boxes containing urine collected from uninfected rather than infected males. This is the first evidence that mice can discriminate virally infected individuals through chemical signals and the first evidence that infection causes odour changes in the urine. To determine if the odour of infected males is repulsive, we presented females with urine samples and neutral water blanks. Normal urine collected from uninfected males was more attractive, whereas urine collected during infection was as attractive as water. This indicates that rather than being aversive, influenza infection abolishes the attractiveness of a male's odour. A similar effect also occurs when male mice are infected with coccidian gut parasites (Kavaliers & Colwell 1995, Proc. R. Soc. Lond. 261B, 31–35). One proximate reason for the neutralization of the attractiveness of a male's odour may be a decrease in serum androgen concentrations during infection.