CpG stimulates protective immunity in Balb/cJ mice infected with larval Taenia crassiceps.

Balb/cJ mice infected in the peritoneal cavity with larval Taenia crassiceps fail to mount a protective immune response. In mice, inflammatory immune responses are believed to control larval reproduction, whereas antibody-mediated responses are believed to be permissive. In the present study, mice were treated with CpG-oligodeoxynucleotides (CpG) to determine whether stimulation of the innate inflammatory response would confer increased resistance to larval growth. Female mice treated with CpG displayed a decrease in mean parasite burden by 54%, while male mice displayed a 73% reduction. Moreover, 5 of 12 CpG-treated male mice completely eliminated all larvae by 9 wk post-infection. In contrast, no female animals were found to be infection free. CpG treatment induced an increase in the transcript levels of tumor necrosis factor-α and inducible nitric oxide synthase (iNOS) from splenocytes and resulted in elevated levels of the proinflammatory molecules monocyte chemotactic protein (MCP)-1, MCP-3, and interleukin-6 at the site of infection. Additionally, CpG administration induced the enhanced recruitment of neutrophils and macrophages to the site of infection. The finding that both neutrophils and macrophages were recruited in significantly higher numbers in the male host as compared to the female host may explain the increased level of protection realized in male animals in response to CpG treatment.

Balb/Cj male mice do not feminize after infection with larval Taenia crassiceps.

Balb/cJ mice fail to mount an immune response capable of clearing infection with larval Taenia crassiceps. Additionally, male Balb/cJ mice display a lag in larval growth of approximately 3 wk as compared to growth in female mice. It has been reported that male Balb/ cAnN mice generate a protective immune response early in infection, and become permissive to larval growth after they feminize (200-fold increase in serum estradiol and 90% decrease in serum testosterone). To determine if a different strain of Balb/c mice (Balb/cJ) also feminize, serum was collected from infected male mice for 16 wk and levels of 17-beta-estradiol and testosterone were measured via ELISA. In addition, the mounting responses of 12- and 16-wk infected male mice, as well as uninfected control mice, were determined after isolation with a female mouse. The results of these experiments show that male Balb/cJ mice do not feminize during infection with larval T. crassiceps. There was no significant change in serum levels of either 17-beta-estradiol or testosterone during the course of infection (> 16 wk). Moreover, there was no significant decrease in the number of times infected male mice mounted the female mouse as compared to uninfected controls. These results suggest that there may be variances between the substrains of Balb/c mice that lead to the phenotypic differences reported for male Balb/cJ and Balb/cAnN mice.

The initial immune response during experimental cysticercosis is of the mixed Th1/Th2 type.

The immunological events that occur during the initial stages of experimental cysticercosis are not known. The studies presented here examined the cytokines produced by peritoneal exudate cells (PECs), splenocytes and mesenteric lymph node (MLN) cells during the first week of infection with larval Taenia crassiceps in BALB/cJ mice. Proliferation assays determined that the earliest time when antigen-specific responses could be measured was 5 days post-infection. Concanavalin A (ConA) stimulation of host cells elicited an initial burst of IL-4 production at 24 h of infection and ConA-stimulated Th2-type cytokine production is predominant by 7 days post-infection. Thus, there are responses at day 1 of infection that seem to promote a Th2-type response. Stimulation of MLN cells, splenocytes and PECs with larval antigens supported previous reports of mixed Th1/Th2-type cytokine production with increases in interleukin (IL)-4, IL-10 and interferon (IFN)-gamma. Ex vivo IFN-gamma production by PECs from infected mice was increased at 3, 5 and 7 days post-infection, whereas at these times reduced ex vivo IL-10 production was observed. This ex vivo IFN-gamma response preceded an increasing IL-10 production by PECs between 3 and 7 days post-infection in parasite-specific and ConA-induced proliferation assays. Thus, infection with larval T. crassiceps results in an initial response mediated by IFN-gamma that is quickly followed by an increase in IL-10 production and subsequent reduction in the amount of IFN-gamma being produced.

Larval Taenia crassiceps secretes a protein with characteristics of murine interferon-gamma.

Previous studies have shown that excretory/secretory products of larval Taenia crassiceps have immunomodulatory activities. We report here that one of these products, termed p66, possesses activities that mimic some characteristics of murine interferon-gamma (IFN-gamma). Purified p66 cross-reacts with anti-murine IFN-gamma on immunoblots and increases concanavalin-A-induced splenic T-cell proliferative responses of normal and chronically infected mice. It has also been shown that p66 induces enhanced IFN-gamma and interleukin-10 (IL-10) production in cells from infected or normal mice. Adherent and non-adherent peritoneal exudates cells were stimulated with p66 and showed that the adherent cells were induced to produce IL-10 and that the non-adherent cells were induced to produce IFN-gamma. p66 was shown as well to upregulate nitric oxide production in macrophages and two T-cell lines, IEL and YAC-1, were shown to be stimulated to produce IFN-gamma and IL-10, respectively, by p66. Although the significance of p66 in immunoregulation is not known, we show here that this molecule mimics characteristics of IFN-gamma.

Suppressed cytotoxic T lymphocyte responses in experimental cysticercosis.

Mitogen-induced T cell responses are suppressed in mice infected with larvae of Taenia crassiceps. The effects of experimental infection on specific T cell responses, however, have not been examined. In the present study, we demonstrate that larval-infected mice exhibit suppressed ability to develop anti-virus specific cytotoxic T lymphocyte (CTL) responses while maintaining apparently normal natural killer (NK) cell responsiveness.

Epidemiologic analysis of an urban, public emergency department's frequent users.

OBJECTIVES: To determine how the demographic, clinical, and utilization characteristics of emergency department (ED) frequent users differ from those of other ED patients. METHODS: A cross-sectional and retrospective cohort study was performed using a database of all 348,858 visits to the San Francisco General Hospital ED during a five-year period (July 1, 1993, to June 30, 1998). A "frequent user" visited the ED five or more times in a 12-month period. RESULTS: Frequent users constituted 3.9% of ED patients but accounted for 20.5% of ED visits. The relative risk (RR) of frequent use was high among patients who were homeless (RR = 4.5), African American (RR = 1.8), and Medi-Cal sponsored (RR = 2.1). Frequent users were more likely to be seen for alcohol withdrawal (RR = 4.4), alcohol dependence (RR = 3.4), and alcohol intoxication (RR = 2.4). Frequent users were also more likely to visit for exacerbations of chronic conditions, including sickle cell anemia (RR = 8.0), renal failure (RR = 3.6), and chronic obstructive pulmonary disease (RR = 3.3). They were less likely to visit for all forms of trauma (RR = 0.43). Survival analysis showed that only 38% of frequent users for one year remained frequent users the next year. However, 56% of frequent users for two consecutive years remained frequent users in the third year. CONCLUSIONS: Frequent use of the ED reflects the urban social problems of homelessness, poverty, alcohol abuse, and chronic illness. Frequent use of the ED shows a high rate of decline from one year to the next. This rate of decline slows after the first year and suggests the existence of a smaller group of chronic frequent users.

Parasite-secreted products regulate the host response to larval Taenia crassiceps.

Parasite-induced immunosuppression is believed to play a significant role in the pathology of cysticercosis, a disease caused by the larval stage of cestode parasites. The biochemical basis for immunoregulation by Taenia crassiceps in experimental cysicercosis is unknown. In order to determine whether or not excretory/secretory (E/S) products from the parasite have the ability to regulate host immune function, the activity of these products was examined. Excretory/secretory products from larvae early in the infection were found to suppress T cell proliferative responses in vitro as well as the production of IFN-gamma and IL-4. In contrast, E/S products secreted from larvae harvested late in infection were not suppressive. Electrophoretic analysis of E/S products revealed both qualitative and quantitative differences in the pattern of proteins produced by larvae taken from an early infection versus those taken from a chronic infection. The viability of parasites taken from an early infection was greatly reduced compared to those taken from chronically infected mice, suggesting a change in the nature of the host immune response to the parasite during the course of the infection. The proliferative activity and cytokine profiles of host immune cells were examined. Both mesenteric lymph node cells and peritoneal exudate cells were found to produce high levels of both IFN-gamma and IL-4, consistent with the high levels of these cytokines in sera of chronically infected animals. Chronic infection with Taenia crassiceps therefore is characterized by high levels of production of both Th1 and Th2 cytokines by host cells.

Immune destruction of larval taenia crassiceps in mice.

Immune destruction of larval Taenia crassiceps was examined by first injecting BALB/cJ mice subcutaneously with larval buds and 30 to 60 days later challenging the mice with larvae injected into the peritoneal cavity. The larvae injected intraperitoneally (i.p.) secondarily are killed by host cells that completely encase the larvae in a thick sheath. The peritoneal exudate cells and the cytokines they produced were characterized by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and reverse transcription PCR (RT-PCR). No changes in percentage of CD4(+) T cells, CD8(+) T cells, B1 cells, or macrophages were detected in the peritoneal cavities of mice that were killing larvae compared to mice with a primary 7-day infection i.p. Both RT-PCR and ELISA demonstrated a decrease in cytokines including gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 in mice that were killing the larvae compared to control mice infected for 30 to 60 days i.p. alone, although there was little difference compared to mice infected for 7 days i.p. alone. Serum cytokine levels in mice that were killing the larvae showed a decrease in IFN-gamma and IL-4, an increase in IL-10 when compared to mice infected for 30 to 60 days i.p. alone, and increases in all cytokines compared to mice infected for 7 days i.p. alone. Inhibition of nitric oxide production did not significantly affect the number or the viability of larvae in the peritoneal cavity of mice that were killing larvae during secondary infection.

The symbiotic water mite Unionicola formosa (Acari: Unionicolidae) ingests mucus and tissue of its molluscan host.

Unionicola formosa is a symbiotic water mite that passes most of its life cycle in the mantle cavity of freshwater mussels. Although mites of this genus are often referred to as parasitic, little is known about their nutritional biology. A few species reportedly pierce the gill of a host mussel and ingest tissue or hemolymph. The present study was undertaken to identify possible sources of nutrition for U. formosa. To determine if mites ingested particulate matter in the mucous strand produced by a mussel during feeding, mussels with resident mites were exposed to a suspension of fluorescent microspheres. There was no evidence that U. formosa ingested the beads. Histochemical staining did, however, indicate a mucous material present in the midgut of the mites. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays revealed a high molecular weight component, consistent with a mucopolysaccharide, present both in the mussel gill and the mites. Results from western blots and an immunoaffinity binding assay with antibodies against mussel gill tissue and hemolymph also indicated that mites ingested host tissue. Whereas U. formosa probably does not ingest particulate material acquired by its host's suspension feeding, it is apparent that this mite utilizes host mucus, gill tissue, or hemolymph for at least part of its nutrition.

gamma delta T cells do not play a major role in controlling infection in experimental cysticercosis.

Protective immunity against larval Taenia crassiceps has been shown to rely on T cells; however, the roles of the specific subsets of T cells during infection are not known. To investigate a possible role for gamma delta T cells, this study investigated larval infection in delta-chain knock-out C57BL/6 (deltaKO) and wild-type C57BL/6 mice. It was found that deltaKO mice and C57BL/6 mice were equally susceptible to infection suggesting gamma delta T cells do not play a major role in protective immunity. Cytokine production by concanavalin A (ConA)-stimulated spleen cells from infected deltaKO mice and C57BL/6 mice were determined. All infected mice demonstrated an increased IL-10 production suggesting a Th1-inhibitory function. Cells from infected deltaKO mice and C57BL/6 mice did not show increases in IL-4 production. Heavily-infected C57BL/6 mice showed a decrease in IFN-gamma production compared to deltaKO mice. These observations suggest that an increase in IL-10 production best correlates with a non-protective immune response. To make comparisons between in vitro cytokine production and systemic immune responses, cytokine levels in serum were determined. C57BL/6 mice and deltaKO mice showed increases in serum levels of IL-4 and IFN-gamma at 52 days post-infection. The systemic immune response of these mice, therefore, is a mixed Th1/Th2-type response and gamma delta T cells are apparently not responsible for the systemic increases in these cytokines.

The systemic immune response of BALB/c mice infected with larval Taenia crassiceps is a mixed Th1/Th2-type response.

The subsets of lymphocytes and cytokines regulating the site-specific immune response in experimental cysticercosis (Taenia crassiceps) are not known. This study investigated the cells present at the site of infection (PECs) using flow cytometry and measured the cytokines produced by these cells through 50 days of infection. The results showed an expansion of B220+CD5+, B220+CD5-, alpha beta TCR+CD4+ and CD8+ cells coincident with a transient increase in IL-10 production. After the initial increase, the percentage of B220+CD5- and helper T cells decreased with a concomitant decrease in IL-10 production. CD8+ T cells continued to increase throughout infection and gamma delta TCR+ cells increased after 10 days of infection. PECs demonstrated an increased IFN-gamma and IL-4 production throughout infection when stimulated with larval antigens. Because a Th2-type polarization has been shown for spleen cells from infected BALB/c mice, cytokine profiles of spleen cells and PECs in response to ConA and larval antigens were compared. ConA and antigen-specific stimulation of spleen cells from 50-day-infected mice produced increased amounts of IL-10 while PECs showed a decreased IL-10 production suggesting that anatomically distinct lymphoid populations produce different cytokines and promote different types of responses. Surprisingly, late in infection the levels of IL-4 and IFN-gamma in serum increased substantially (460-fold and 100-fold, respectively). The systemic immune response of BALB/c mice during experimental cysticercosis, therefore, is a mixed Th1/Th2-type response.

Trypanosoma cruzi does not induce apoptosis in murine fibroblasts.

The intracellular cycle of Trypanosoma cruzi in mammalian host cells involves the differentiation of dividing amastigote forms into flagellated trypomastigote forms. The mechanism(s) regulating the growth and differentiation of the intracellular parasites is (are) not known. The number of parasites in infected cells can be several hundred and may be enough to induce apoptosis, a suicide-like death programme, generating products (e.g. nuclear proteins) that could function as signals to initiate the differentiation of amastigotes into trypomastigotes. Murine fibroblasts infected with T. cruzi were examined during a 5-day course of infection for evidence of apoptosis. However, characteristics of apoptosis, including degeneration of nuclear structure, condensation of chromatin, loss of plasma membrane integrity, or the cleavage of DNA into nucleosomal fragments, were not observed. Therefore, it is unlikely that products resulting from host cell apoptosis function to induce parasite differentiation. The possibility that T. cruzi might inhibit host cell apoptosis by increasing intracellular levels of Bcl-2, an endogenous inhibitor of apoptosis, was then investigated. Analysis of infected cells by flow cytometry did not demonstrate a significant amount of intracellular Bcl-2. This suggests that if the parasite is inhibiting host cell apoptosis, it is by a method that does not involve increasing levels of Bcl-2.

Mice infected with the larvae of Taenia crassiceps exhibit a Th2-like immune response with concomitant anergy and downregulation of Th1-associated phenomena.

Infection of intermediate hosts with eggs of taeniid parasites results in a larval infestation known as cysticercosis. A number of studies have indicated that cysticercosis is associated with immunosuppression, although little is known about the mechanisms involved. In the present study, mice infected with the larvae of Taenia crassiceps were found to exhibit a pronounced energy, which preferentially affected T-cells located anatomically close to the parasite. This anergy was linked to late events in the T cell activation pathway; that is, stimulation through the T cell receptor(TCR)/CD3 complex by Concanavalin-A, or plate-bound monoclonal antibodies (mAb) to TCR alpha beta or CD3 epsilon, or combinations of phorbol ester and ionomycin (all of which can bypass early membrane-related events), failed to fully activate T lymphocytes. The relative proximity of T cells to the parasite was directly related to upregulation of IL-4 and downregulation of IL-2 production. In addition, the profiles of parasite-specific Abs showed an exclusive increase of serum IgG1 during infection. Taken together, the data suggest that infection of mice with larvae of T. crassiceps alters the balance of CD4+ Th cells by upregulating Th2 and downregulating Th1 cells located in close proximity to the parasite.

Trypanosoma cruzi affects nitric oxide production by murine peritoneal macrophages.

Macrophages from mice that are infected with various intracellular pathogens including Leishmania major, Trypanosoma cruzi, and Salmonella typhimurium are stimulated to produce large quantities of nitric oxide (NO). Both viable and heat-treated L. major amastigotes have been shown to be effective co-signals for NO production in vitro. NO produced by macrophages has anti-microbial and immunosuppressive functions in an immune response. We have shown previously that NO plays a complicated role in T. cruzi infections since macrophages are important both in mediating an immune response against the parasite as well as in mediating immunosuppression. In this study we examined how T. cruzi affects NO production by macrophages from C3HeB/FeJ and C57BL/6 mice in vitro. We found that live trypomastigotes neither stimulate nor decrease NO production by interferon (IFN)-gamma-activated macrophages. However, heat-treated or glutaraldehyde-fixed trypomastigotes of T. cruzi significantly decrease NO production by IFN-gamma-activated macrophages and as a result decrease macrophage-mediated trypanocidal and immunosuppressive activity. We have determined that this decrease in NO production by T. cruzi is not due to stimulation of transforming growth factor-beta production and involves tumor necrosis factor-alpha only in C3HeB/FeJ macrophages. This study demonstrates the complexity of the T. cruzi-macrophage interaction as well as confirms previously demonstrated differences between macrophages from 2 strains of mice.

Macrophages in experimental Chagas' disease.

The foregoing provides a basis for considering that macrophages are important in basically every aspect of the host-parasite relationship in experimental Chagas' disease. The myriad of activities of macrophages and the diverse responses of these cells to T. cruzi and various stimulatory and inhibitory cytokines as measured both in vitro and in vivo are suggestive of the complexity of the cell-cell interactions of the host during the course of infection and the evasive activities of the parasite.

Interleukin-2 receptors in experimental Chagas' disease.

Mammals infected with the protozoan parasite Trypanosoma cruzi develop suppressed cellular and humoral immune responses. This immunosuppression has been correlated with reduced T-cell responses involving deficient interleukin-2 (IL-2) production and is apparently mediated primarily by suppressor macrophages. Various forms of immunosuppression in other systems have been associated with increased levels of soluble IL-2 receptors (sIL-2R), and in the present study levels of sIL-2R in the sera of T. cruzi-infected mice during the course of infection were examined in enzyme-linked immunosorbent assays. It was found that serum levels of sIL-2R were elevated only during the third week of acute infection, a time of intense immunosuppression. In addition, IL-2R on the surface of T cells were examined by flow cytometric analyses to determine whether there is an alteration in the number of IL-2R-positive cells and whether there is a change in expression of these receptors as infection progresses. The results revealed no significant change in the percentage of cells expressing IL-2R, nor did T cells become suppressed in their ability to express IL-2R in response to concanavalin A during the course of infection.

Binding of complement to trypomastigotes of a Brazil strain of Trypanosoma cruzi: evidence for heterogeneity within the strain.

Binding of the complement components C3 and C5 to epimastigote and trypomastigote stages of the Brazil strain of Trypanosoma cruzi was examined using radioligand binding and flow cytometric assays. Fibroblast-derived trypomastigotes bound approximately 40% fewer molecules of [125I]C3 per parasite than did epimastigotes. The predominant molecular species of C3 deposited on fibroblast-derived trypomastigotes was the inactive form iC3b. Addition of parasite-specific antisera failed to enhance the number of molecules of [125I]C3 per parasite or the proportion of active to inactive C3b. Flow cytometric studies revealed that only 50% of trypomastigotes (fibroblast-derived or blood-form) bound C3. In contrast to results of the [125I]C3 binding studies, flow cytometric analysis showed that the percentage of trypomastigotes binding C3 actually increased upon incubation with parasite-specific antisera. C5 was found also to bind to only a percentage of trypomastigotes.

Changes in humoral responses to Trypanosoma cruzi during the course of infection in mice held at elevated temperature.

A parasite-specific, enzyme-linked immunosorbent assay and immunoblot analysis were used to examine the development of humoral immunity in Trypanosoma cruzi-infected C3H mice that survive acute infection when held at elevated environmental temperature. Both parasite-specific antibody levels and numbers of antigens identified increased during infection in mice held at 36 C, with the greatest reactivity measured in sera from mice that had resolved parasitemias. Heat shock of culture forms of T. cruzi resulted in production of different antigens, but there was no strong difference in the antigens recognized by sera from mice held at room temperature and those recognized by sera from mice held at 36 C. Immunoblot analysis using blood-form trypomastigote antigens identified a band of approximately 61 kDa produced by trypomastigotes in mice held at 36 C that was strongly detected by sera from mice held at 36 C. Little if any reactivity to this antigen was observed using sera from mice held at room temperature.

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.

Effect of antigen-specific T helper cells or interleukin-2 on suppressive ability of macrophage subsets detected in spleens of Trypanosoma cruzi-infected mice as determined by limiting dilution-partition analysis.

Trypanosoma cruzi, a protozoan parasite and the causative agent of Chagas' disease, induces a state of lymphocyte hyporesponsiveness to both mitogenic and antigenic stimuli in mice during the acute phase of infection. Addition of spleen cells from T. cruzi-infected mice (SCinf) to microcultures of spleen cells from noninfected mice (SCn) suppresses the responsiveness of such cultures to antigenic challenge and to mitogenic stimulation. We analyzed the regulatory cell populations in SCinf by limiting dilution-partition analysis and found a complex regulatory circuit in T. cruzi-infected mice consisting of two suppressive macrophage subsets and an enhancing T-cell population. This T-cell population was able to abrogate or escape the suppressive ability of one suppressor macrophage subset, yet was suppressed by the other macrophage subset. To further study the cellular interactions of this regulatory circuit and analyze the suppressive abilities of the two suppressor macrophage subsets, we examined the effect of adding either primed T helper cells of known specificity or interleukin-2 to the limiting dilution-partition analysis microcultures. The results of these experiments suggest that one suppressor macrophage subset, which is abundant and, therefore, detected with low doses of SCinf, is able to suppress both mitogen- and primary antigen-specific responses but is unable to inhibit cells once they are already activated or primed. The other macrophage subset, which is presumably a less abundant or less active population (since high doses of SCinf are required to detect it), is able to suppress the response of activated or primed T cells by the inhibition of interleukin-2 production.