A pilot serosurvey of Babesia microti in Chinese blood donors.

BACKGROUND AND OBJECTIVES: Babesia spp. are tick-borne, intraerythrocytic protozoan parasites, several of which are transfusion-transmissible. Transfusion-transmitted babesiosis poses serious risk to a diverse patient population, including neonates, patients aged >50 years, the asplenic and the immunocompromised that are over-represented among transfusion recipients. Despite reports of B. microti and B. venatorum in People's Republic of China (PRC), no surveillance of blood donors for Babesia has previously been undertaken. We sought to determine the rates of B. microti seroreactivity in a sample of blood donors in the PRC. MATERIALS AND METHODS: A pilot serosurvey was conducted of community blood donors (n = 1000) who donated July-August 2016 at Mudanjiang Blood Center (Heilongjiang Province) using indirect fluorescent antibody testing for antibodies against B. microti. The slides were prepared using B. microti-infected hamster blood. Samples that were initially positive to a titre of 64 were subjected to repeat IFA testing. Final seroreactivity was based on repeat reactivity to ≥64. RESULTS: A total of 1000 individual donor samples were evaluated, comprising 888 whole blood and 112 platelet donations. Thirteen of 1000 (1·3%) donors were seroreactive for B. microti [8 (0·8%) and five (0·05%) at titres of 64 and 128, respectively]. CONCLUSION: Our preliminary findings support the need for expanded Babesia surveillance in Chinese blood donors, replete with molecular evaluation, to evaluate the risk to the blood supply.

Gene therapeutic approaches to induction and maintenance of tolerance.

Tolerance induction would be an ideal way to treat autoimmune diseases, especially if achievable in primed individuals. Moreover, specific tolerance would preclude the need for immunosuppressive treatment with its side effects. In this review, we will revisit the historical concepts of tolerance, and introduce a novel approach to tolerance via gene therapy. Our model system is based on the tolerogenicity of IgG carriers and B-cell antigen presentation. Results with this model demonstrate that it is simple and effective even in primed recipients, and has proven efficacy in three autoimmune models. We discuss the mechanisms of tolerance with fusion IgG's and analyze how our model of gene therapy approached can be utilized to fit in the future treatment of autoimmune conditions.

B-cell receptor and Fas-mediated signals for life and death.

A series of B-cell lymphoma lines with an immature phenotype has been used as a model system to study molecular events associated with receptor ligation induced death. B-cell receptor (BCR) cross-linking with antibodies to membrane IgM (but not with anti IgD) induces c-Myc downregulation via nuclear factor kappaB inactivation and p27(Kip1) accumulation in these B lymphomas. Anti-mu-treated cells then undergo G1 arrest and die by apoptosis independent of Fas. Steroids and retinoids similarly downregulate c-Myc and induce apoptosis in these B cells and synergize with anti-mu. Rescue from apoptosis induced by anti-mu or steroids occurs with T-cell signals, like CD40L, or a broad-range caspase inhibitor, but only CD40L prevents the loss of c-Myc, p27 accumulation and growth arrest. Both IgM and IgD signaling lead to modulation of phosphatidylinositol 3-kinase (PI3K) signals, including the activation of p70(S6K), but this pathway recovers under anti-IgD treatment. Blockade of the PI3K pathway augments anti-mu-induced death and converts anti-delta to an apoptotic signal. Resistance to Fas-mediated death may be an important factor in B-cell transformation in vivo. Many of our panel of lymphomas are insensitive to Fas-mediated death signals, although all can form a death-inducing signaling complex (DISC). Additional studies suggest that some lymphomas can be blocked at the DISC complex by anti-apoptotic proteins, whereas others are inhibited downstream of caspase 8 activation. Anti-Ig treatment of a Fas-sensitive line, A20.2J, activated a number of genes whose products may block apoptosis proximally (like FLICE-inhibitory protein (FLIP1)) or at late points, such as bcl-2-family members. Our data suggest that B lymphomas develop multiple pathways of resistance to Fas-mediated signals during lymphomagenesis, in part via signaling through the BCR.

A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.

TGF-beta is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans.

Resistance and susceptibility of mice to systemic infection with the fungus Candida albicans are associated with the preferential expansion of Th1 and Th2 cells, respectively. In this study, endogenous production of TGF-beta was found to be increased soon after infection of healer mice with a live vaccine strain of the fungus, but down-regulated in nonhealer mice with virulent yeast challenge. Although not affecting the outcome of primary challenge, serologic ablation of TGF-beta in the former animals abrogated development of acquired resistance and resulted in impaired production of IL-12/IFN-gamma and higher expression of IL-4/IL-10 at the time of reinfection with virulent yeast. A CD4+ population expressing the memory phenotype, CD44highMEL-14low, which appeared to be expanded by yeast infection of nonhealer mice, was similarly increased in the healer mice by anti-TGF-beta treatment. In vitro rTGF-beta impaired the candidacidal function of IFN-gamma-activated macrophages. Yet in nonhealer mice infected with virulent C. albicans, administration of rTGF-beta delayed progression of the disease, which was concomitant with the detection of lower levels of IL-4. In addition to previous evidence for an obligatory role of IFN-gamma and IL-12 in Candida-driven Th1 cell differentiation in vivo, the present data establish TGF-beta as a third cytokine, the presence of which may be required for optimal Th1 development leading to long-lived anticandidal resistance.

Interleukin-4 and -10 exacerbate candidiasis in mice.

Neutralization of endogenous interleukin (IL)-4 or IL-10 in mice with Candida albicans infection initiates or accelerates development of a T helper (Th)1-associated protective response. Here, we report the effect of IL-4 and IL-10 administration on the course of systemic or gastrointestinal (GI) candidiasis and on the development of Th immunity using yeast/host combinations that result either in Th1-associated self-limiting infection (healer mice) or in Th2-associated progressive disease (nonhealer mice). Treatment with IL-4 or IL-10 greatly exacerbated the course of systemic infection in nonhealer mice and rendered healer mice, inoculated with attenuated yeast cells, susceptible to infection. Under the latter conditions of yeast challenge and IL-4/IL-10 administration, the development of a fatal disease was associated with inhibition of IL-12 production and detection of progressive Th2 cell dominance. In contrast, in healer mice allowed to resolve their infections and to develop long-lived anti-candidal resistance, the expression of this acquired resistance was not impaired by IL-4 and/or IL-10, as shown by the outcome of reinfection with virulent yeast cells. In the GI model of infection, both IL-4 and IL-10 were found to exacerbate the course of infection and to induce the appearance of CD4+ T cells producing high levels of IL-4 and IL-10 in Peyer's patches. These findings demonstrate that exogenous IL-4 and IL-10 may greatly affect the development of Th responses to C. albicans in vivo, but do not modify the expression of established and predominant Th1 cell reactivity.

T helper cell type 1 (Th1)- and Th2-like responses are present in mice with gastric candidiasis but protective immunity is associated with Th1 development.

The relative contributions of local T helper cell type 1 (Th1)- and Th2-like responses to the course of primary and secondary gastrointestinal (GI) candidiasis were examined in adult immunocompetent BALB/c mice. Both Th1 cytokines, such as interferon-gamma (IFN-gamma), and the Th2 cytokines, interleukin (IL)-4 and IL-5, were produced by CD4+ cells from Peyer's patches (PP) and mesenteric lymph nodes at a time when the fungus was cleared from the stomach and intestine. Augmentation of antigen-specific Th2-like responses by treatment with cholera toxin did not modify the course of disease. In contrast, treatment with soluble IL-4 receptor, which increased Th1 cells, was associated with enhanced yeast clearance. In addition, IFN-gamma but not IL-4 mRNA was present in PP and spleen CD4+ cells in mice resistant to subsequent GI inoculation. Activation of Th1- but not Th2-like responses may be responsible locally for controlling GI candidiasis and generating protective immunity.

IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis.

In murine models of systemic candidiasis, healing and nonhealing patterns of disease are associated with preferential expansion of Th1 and Th2 cells, respectively. As previous studies have shown IL-12 to be expressed transcriptionally in healer mice and to be required for Th1 development in vitro, this cytokine might play a role in Candida-driven Th1 cell differentiation in vivo. In the present study, IL-12-neutralizing Abs or recombinant IL-12 were administered to mice with healing or progressive candidiasis, respectively, and the animals were monitored for mortality, resistance to reinfection, serum levels of specific Abs, and IFN-gamma, IL-4, and IL-10 message/protein expression by CD4+ cells. In self-limiting infection by a yeast vaccine strain, neutralization of IL-12 ablated development of acquired anticandidal resistance and led to appearance of Candida-specific IgE and IL-4-producing cells. In mice with progressive systemic disease as well as in a mucosal infection model, administration of IL-12 did not result in therapeutic activity under conditions of yeast infection that would instead be resolved by serologic ablation of IL-4 or IL-10. Yet, in systemically infected mice cured by anti-IL-4 or anti-IL-10 therapy, the emergence of a Th1 response correlated with the detection of high levels of circulating IL-12 and splenic IL-12 transcripts. Although exogenous IL-12 may not be sufficient for Th conversion in the presence of an overwhelming IL-4/IL-10 response, endogenous production of IL-12 may be both required and prognostic for Th1 differentiation in vivo.

Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection.

Neutralization of IL-10 up-regulates nitric oxide production and protects susceptible mice from challenge with Candida albicans.

In contrast to several inbred strains of mice that develop Th1-associated anticandidal protection, DBA/2 mice are highly susceptible to systemic infection with Candida albicans cells of the attenuated variant PCA-2, and fatal outcome is observed in concurrence with sustained CD4+ cell production in vitro of IL-4 and IL-10. These Th2 cytokines were previously shown to inhibit nitric oxide (NO) production and yeast killing by activated macrophage cultures. We now show that macrophages from DBA/2 mice, either intact or infected with PCA-2, have lower capacity than resistant strains to synthesize NO in response to IFN-gamma. However, when treated with anti-IL-10 Abs at the time of infection, DBA/2 mice survived challenge and displayed increased production of NO in vitro after IFN-gamma activation. Cure was associated with the onset of footpad responses and durable protection, and higher frequencies of IFN-gamma-secreting cells were found in splenic CD4+ lymphocytes that expressed lower levels of IL-4 and IL-10 mRNA. Therefore, in DBA/2 mice, IL-10 contributes significantly to the selection of a Th2 response and lethality after PCA-2 challenge. An IL-10-induced defect in the activation and/or expansion of IFN-gamma-producing Th1 cells, IL-10 suppression of yeast killing, and the relative inability of DBA/2 macrophages to produce adequate levels of candidacidal NO may all contribute to the abnormal susceptibility of these mice to candidiasis.

Interleukin-12 but not interferon-gamma production correlates with induction of T helper type-1 phenotype in murine candidiasis.

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-gamma (IFN-gamma), IL-4, and IL-10 in CD4+ and CD8+ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection ("healer mice") or in a Th2-associated progressive disease ("nonhealer mice"). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-gamma or IL-10 mRNA in CD4+ and CD8+ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4+ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4+ and CD8+ cells was assessed in vitro, while IFN-gamma-producing cells were enumerated in CD4- CD8- cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-gamma protein in vitro by CD4+ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4+ cells would expand in nonhealer mice in the face of high levels of circulating IFN-gamma, likely released by CD4- CD8- lymphocytes; (iii) a finely regulated IFN-gamma production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-gamma-producing CD4+ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-gamma by early CD4+ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-gamma production may be an indicator of Th1 differentiation.

Natural killer cells do not play a dominant role in CD4+ subset differentiation in Candida albicans-infected mice.

The effects of in vivo administration of monoclonal antibodies against NK-1.1-bearing cells on the early production of gamma interferon (IFN-gamma) in vitro and development of Th1-associated immunity were studied in mice infected with a live vaccine strain of Candida albicans. At 1 and 4 days postinfection, natural killer (NK) cell-enriched fractions from the spleens of antibody-treated mice displayed a dramatic reduction in 5E6+ lymphocytes and negligible anti-YAC-1 cytotoxic activity in vitro. Nevertheless, the frequency of IFN-gamma-producing cells in those fractions was reduced by less than half, on average, by anti-NK-1.1 treatment in vivo. In addition, the antibody-treated and infected mice demonstrated unchanged T helper cell responses, as measured by yeast-specific footpad reactions, resistance to reinfection, occurrence of antibodies of different isotypes, and production in vitro of interleukin-2 (IL-2), IFN-gamma, IL-4, and IL-10 by CD4+ cells. Therefore, although NK cells may contribute to early IFN-gamma production in Candida-vaccinated mice, these cells apparently do not play a dominant role in the qualitative development of yeast-specific T helper responses.

Low-dose streptozotocin-induced diabetes in mice. II. Susceptibility to Candida albicans infection correlates with the induction of a biased Th2-like antifungal response.

We have previously found that the development of fatal disseminated candidiasis correlates with the detection of a strong Th2 response, while protective antifungal immunity is associated with a predominant Th1 response. In the present study we verified the hypothesis that an altered antifungal Th response could be responsible for the high susceptibility of diabetic mice to systemic Candida albicans infection. Outbred CD1 mice rendered diabetic with multiple low doses of the pancreatic islet beta-cell toxic, streptozotocin, develop a fatal systemic infection when injected with low-virulence C. albicans cells. Progressive disease was found to be associated with the presence in the serum of IgA, IgE, and IgG1 Candida-reactive specific antibodies, absent footpad reactions, and elevated production in vitro of the Th2 cytokines IL-4, IL-6, and IL-10 but not the Th1 cytokine IFN-gamma. Both the Th2 and Th1 (IL-2 and IFN-gamma) cytokines were produced in vitro by CD4+ lymphocytes from noninfected diabetic mice that, in addition, showed a noticeable footpad reaction to Candida antigens. Thus, it appears that a perturbation in the anticandidal T helper responses resulting in the induction of a biased Th2-like antifungal response renders diabetic mice highly susceptible to systemic C. albicans infection.