Thymic Atrophy Development in a Non-Lethal Plasmodium Infection.

Effects of estrogen receptor subtype-selective agonists on immune functions in ovariectomized mice

Gastrointestinal nematode infections, highly prevalent in malaria endemic areas, are known to modulate host immune responses to unrelated pathogens. However, the complex relationship between worms and malaria and the impact on malarial incidence vs. severity are unclear. Previously, we observed that C57BL/6 (B6) mice infected with the gastrointestinal nematode Heligosomoides polygyrus (Hp) for 2 wks before blood-stage Plasmodium chabaudi AS (Pc) infection developed significantly higher parasitemia compared to mice infected with Pc alone. Despite this, malaria-induced body weight loss, anemia and hepatosplenic damage were not exacerbated in co-infected mice. Indeed, there was less severe hypothermia and hypoglycemia and more robust reticulocytosis in co-infected mice. Co-infection with Hp suppressed type 1 immune responses induced by P. chabaudi infection with significantly reduced plasma levels of IFN-γ, TNF-α and nitric oxide, but increased IL-6 and IL-10 levels. Co-infected STAT-6−/− mice, which have disrupted Th2 responses, experienced only slightly more severe hypothermia, hypoglycemia, and anemia than co-infected WT B6 mice while in vivo administration of anti-IL-10R (1B1.2) mAb resulted in higher mortality in co-infected WT mice. Although concurrent nematode infection resulted in increased parasitemia due to impaired anti-malarial type 1 immunity, our data suggest that malaria-associated pathology is alleviated by a complex immunoregulatory network, possibly involving IL-10, and, to a lesser extent, Th2 cytokines.

Tumour necrosis factor (TNF) plays important roles in the pathogenesis of severe malaria, as well as in the generation of immune responses against malaria parasites. However, far less is known about the role of the closely related TNF family member lymphotoxin alpha (LTalpha) during malaria. We have used mice deficient in either TNF or LTalpha, as well as chimeric mice generated using donor bone marrow from these animals, to study the roles of these cytokines following Plasmodium chabaudi chabaudi AS infection. TNF and LTalpha were not required for the resolution of P. chabaudi chabaudi AS blood-stage infection. However, LTalpha, but not TNF, was necessary for early IFNgamma production and the regulation of IFNgamma production later in infection. A similar delay to that found for IFNgamma production was also observed for TNF production in LTalpha-deficient mice, compared with control mice. These results identify divergent roles for TNF and LTalpha in the regulation of host immune responses during P. chabaudi chabaudi AS infection.

The effects of 3,3′,4,4′-tetrachloroazoxy-benzene (TCAOB) on the immune system of mice was examined and compared with cyclophosphamide (CY). This chemical can be produced as a result of microbial degradation of commonly used chloroaniline herbicides. Herbicides of the acylan-iline class generally have a low mammalian toxicity while remaining relatively inexpensive. Therefore, TCAOB could be formed anywhere in the environment where choroaniline pesticides are used. TCAOB treatment caused thymic atrophy and a decrease in white blood cell (WBC) count after sheep red blood cell (SRBC) immunization. A decline in the number of Lyt-1+ cells (T-helper lymphocytes) was seen in all TCAOB animals, with unimmunized mice also showing a decreased number of Lyt-2.2+ cells (T-suppressor lymphocytes). No change was found in the ratio of these two cell types. TCAOB did, however, result in a severe reduction in the number of plaque-forming-cells (PFCs) and in serum antibody concentration. CY caused a decrease in thymus weight, WBC count, the number of cells recovered per spleen, and the relative percentages of Lyt-l+ and Lyt-2.2+ cells recovered. The mice exhibited a lower lymphocyte blastogenic response to lipopolysaccharide (LPS) than control animals, but no change in Concanavalin A (Con-A) responsiveness. CY also resulted in a severe drop in the number of PFCs and the quantity of antibody produced following SRBC immunization.

Thymic Atrophy Caused by Ethanol in a Mouse Model for Binge Drinking: Involvement of Endogenous Glucocorticoids

Sodium methyldithiocarbamate (SMD) is an agricultural fumigant-type pesticide commonly used as a pre-plant biocide. SMD is currently listed by the US Environmental Protection Agency (EPA) as the third most commonly used conventional pesticide. Previously, SMD has been shown to be immunotoxic in mice. Initial immunotoxicological studies indicated that thymocytes are major targets of SMD or its breakdown products in mice. The purpose of the present study was to determine if this effect was mediated by an SMD-induced stress response. The decrease in thymus weight correlated to a decrease in all thymocyte subpopulations. However, as seen in earlier studies, the double positive (CD4+CD8+) thymocyte subpopulation was more selectively decreased than the other subpopulations. The double negative (CD4−CD8−) and single positive (CD4+CD8− or CD4−CD8+) thymocyte subpopulations decreased in absolute numbers while increasing in percentage of the remaining cells in the thymus after SMD intoxication. In the current study, SMD caused an increase in serum corticosterone, a stress-related hormone. Blocking corticosterone either by 1) adrenalectomy or by 2) chemically blocking synthesis of nascent corticosterone in adrenal-competent animals abrogated the thymocyte atrophy caused by SMD. However, an additional stressor (restraint) did not act additively or synergistically to increase atrophy. Therefore, it is likely that SMD causes thymic atrophy by increasing serum corticosterone.

Sodium methyldithiocarbamate (SMD) is a widely used agricultural agent that causes immunological changes in B6C3F1 mice. The most prominent effects of SMD include a decrease in thymus weight and percentage of CD4+CD8+ thymocytes, an increase in spleen weight, an increase in the percentage of neutrophils in the blood, a decrease in the percentage of lymphocytes in the blood, and a decrease in natural killer (NK) cell activity in the spleen. The mechanism by which SMD causes these changes is unknown, and the relative importance of the parent compound and its decomposition products is not known. In addition, it is not known if these effects are unique to mice, or if other mammals are affected similarly. This prompted the present investigation of the major decomposition product of SMD, methylisothiocyanate (MITC), and two minor products, methylamine and carbon disulfide, in mice. Equimolar dosages of methylamine and carbon disulfide caused minimal immunological changes, and these changes were not characteristic of those noted for SMD. In contrast, MITC significantly decreased thymus weight and cellularity and changed peripheral white blood cell populations in a manner similar to that noted for an equimolar dosage of SMD. However, MITC did not significantly affect NK cell activity or increase spleen weight. Thus, MITC is probably responsible for some of the immunological changes noted in SMD-treated mice. The remaining changes are not produced by MITC, methylamine, or carbon disulfide. Thus, it is likely that the parent compound or a synergistic action of the parent compound with one or more of the decomposition products is responsible for these remaining changes (increased spleen weight and decreased splenic NK cell activity). Data are also presented that indicate that SMD-induced thymic atrophy occurs in rats as well as mice and that the dosage required to decrease thymus weight by 50% is lower for rats than for mice. Investigations of other mammals are needed to indicate SMD's potential as a human immunotoxicant and to compare the role of MITC in the immunotoxic effects of SMD in different species.

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Comparison of murine Epo ELISA and Epo bioassays in detecting serum Epo levels during anemia associated with malaria infection

JP-8 jet fuel is handled extensively by personnel in the military and commercial airlines, despite the paucity of information regarding its potential human health effects. JP-8 is a complex mixture primarily consisting of kerosene plus aliphatic and aromatic hydrocarbons. Recent reports indicate that acute JP-8 exposure via inhalation or dermal routes can overtly and persistently impair immune function in mice. Data from preliminary studies in this laboratory assessing the immunotoxicity of JP-8 indicated that oral JP-8 exposure caused an increase in liver weight, a decrease in thymus weight, and a decrease in the PFC response. As these results were similar to classic effects elicited by TCDD, a strong AhR ligand, it was hypothesized that JP-8 may exert immunosuppression via a similar mechanism. To test this hypothesis, an Ah-responsive mouse strain (B6C3F1) and a classically non-responsive mouse strain (DBA/2) bearing a lower affinity AhR were gavaged with JP-8 for 7 days. The results suggest that both mouse strains were equally sensitive to JP-8's toxicity at several endpoints including thymus weight and cellularity, liver weight, and specific IgM antibody responses. Furthermore, JP-8 did not induce CYP1A1 or promote down regulation of the AhR when evaluated by Western blot in either B6C3F1 or DBA/2 mice. In vitro studies corroborated these findings as JP-8 did not induce CYP1A1, promote down regulation of the AhR, or activate an XRE-driven reporter gene in murine Hepa-1 cells. These results suggest that JP-8 may exert its toxicity via an AhR-independent mechanism.

Toxicity of 3,3′,4,4′-Tetrachloroazobenzene in Rats and Mice

Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lymphoblasts

DW/J dwarf mice have a defect in their anterior pituitary and are deficient in growth hormone (GH) and prolactin (PRL). These mice have been demonstrated previously to have a deficiency in CD4/CD8 double- positive thymocytes, which could be corrected by treatment of these mice with recombinant human GH. Since PRL has been implicated in T cell function and human GH can interact with the PRL receptor, DW/J dwarf mice were treated with either ovine GH (ovGH) (20 micrograms/d) or ovine PRL (ovPRL) (20 micrograms/d). The ovine hormones can only bind their own specific receptors in the mouse. After several weeks of treatment, it was found that these two hormones produced markedly contrasting effects on T cells. Phenotypic analysis of the lymphoid organs was performed by flow cytometry and the functional capability of the peripheral T cells was assessed by immunizing the mice and determining the extent of antigen-specific proliferation of T cells obtained from the draining lymph nodes or by determining splenic mitogen responses. The results indicated that ovGH administration to dwarf mice resulted in significant increases in thymic cellularity yet had little effect on peripheral T cell responses. In contrast, the administration of ovPRL resulted in a further decrease in thymic cellularity when compared with untreated dwarf mice. No thymic effects of either ovGH or ovPRL administration were detected on the normal +/? counterparts. However, ovPRL administration resulted in a significant increase in the number and function of antigen-specific peripheral T cells in both immunized dwarf and +/? mice. The adjuvant effects of PRL occurred even though the mice also received complete Freund's adjuvant. These results suggest that neuroendocrine hormones may act in concert in T cell development. GH appears to promote thymocyte proliferation, while PRL appears to decrease thymus size and yet augment the number and function of antigen-specific T cells in the periphery.

BackgroundThe potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis.MethodsThe ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals.ResultsCys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite.ConclusionThese findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1317-3) contains supplementary material, which is available to authorized users.

The requirement for an architecturally intact spleen in the afferent and efferent arms of immunity to the murine malaria parasite Plasmodium chabaudi AS was analyzed. C57BL/6 mice with intact spleens develop a single, patent parasitemia and resolve the infection. In contrast, surgically splenectomized mice experience persistent waves of patent parasitemia interrupted briefly by periods of parasitologic crises. Transfer of spleen cells from immune donors, but not transfer of spleen cells from normal mice, into splenectomized mice enabled the recipients to resolve the infection similar to mice with intact spleens. B-cell depletion, but not T-cell depletion, of spleen cells prior to transfer abrogated the ability of splenectomized recipients to resolve the infection. Compared with mice with intact spleens, splenectomized mice exhibited a delayed antibody response whereas all groups of immune cell recipients had an accelerated antibody response. Nevertheless, splenectomized mice and recipients of B-cell-depleted cells failed to resolve infections, despite the development of high-titer antibodies late during the course of infection. Analysis of immunoglobulin G isotype responses showed a lower representation of immunoglobulin G2a in mice which failed to resolve infections. The latter mice had characteristic histopathologic changes in the liver. These observations indicate a unique role of the splenic microenvironment for the induction and development of an effective B-cell-dependent response against malarial parasites.