Extracellular vesicles induce protective immunity against Trichuris muris.

Gastrointestinal nematodes, such as Trichuris trichiura (human whipworm), are a major source of morbidity in humans and their livestock. There is a paucity of commercially available vaccines against these parasites, and vaccine development for T. trichiura has been impeded by a lack of known host protective antigens. Experimental vaccinations with T. muris (murine whipworm) soluble Excretory/Secretory (ES) material have demonstrated that it is possible to induce protective immunity in mice; however, the potential for extracellular vesicles (EVs) as a source of antigenic material has remained relatively unexplored. Here, we demonstrate that EVs isolated from T. muris ES can induce protective immunity in mice when administered as a vaccine without adjuvant and show that the protective properties of these EVs are dependent on intact vesicles. We also identified several proteins within EV preparations that are targeted by the host antibodies following vaccination and subsequent infection with T. muris. Many of these proteins, including VWD and vitellogenin N and DUF1943-domain-containing protein, vacuolar protein sorting-associated protein 52 and TSP-1 domain-containing protein, were detected in both soluble ES and EV samples and have homologues in other parasites of medical and veterinary importance, and as such are possible protective antigens.

Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.

The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.

The role of TNF-alpha in Trichuris muris infection I: influence of TNF-alpha receptor usage, gender and IL-13.

Th1 and Th2 responses to the gut-dwelling nematode Trichuris muris have been well established in mouse models of infection, with Th2 responses clearly playing an important role in resistance. TNF-alpha has previously been shown to play an undefined role in resistance, although it is not a typical Th2 cytokine. However, the relative importance of the two TNF-alpha receptors, p55 and p75, has not previously been investigated. We demonstrate that p55 is the dominant TNF-alpha receptor during T. muris infection as p55-/- mice are more susceptible to infection than p75-/- mice. Moreover, p75 clearly plays a role in negatively regulating TNF-alpha. We also demonstrate that a gender difference influences the immune response of p55-/- and p75-/- mice in response to T. muris infection, with female mice fully expelling by day 35 post-infection (p.i.) and male mice harbouring chronic infections. Further, this gender difference can be reversed with recombinant IL-13 (rIL-13) in male gene-deficient mice or IL-13R2.Fc treatment in female gene-deficient mice.

The role of TNF-alpha in Trichuris muris infection II: global enhancement of ongoing Th1 or Th2 responses.

Host resistance to Trichuris muris is driven by Th2 responses. However, TNF-alpha has also been shown to play a role in protection. As TNF-alpha has a variety of actions, the exact role of TNF-alpha in immunity to T. muris is yet to be established. Here we demonstrate that although blocking TNF-alpha has been shown to abrogate resistance, rTNF-alpha treatment does not promote resistance. Further, we show that TNF-alpha functions to enhance the ongoing immune response. AKR animals that typically respond to infection with a polarized Th1 response produce greater levels of Th1 cytokines when treated with TNF-alpha and BALB/c animals that normally respond with a polarized Th2 response produce higher levels of Th2 cytokines. Crucially, blocking TNF-alpha in the strong Th2 responder strain BALB/c does not prevent expulsion of T. muris, thus supporting its role as a biological enhancer. TNF-alpha does increase transcription of both IFN-gamma and IL-13 in vitro but can also act synergistically with IL-13 in vitro to promote production of RELMbeta, which has also been shown to play a role in resistance to T. muris. Thus, this data demonstrates that TNF-alpha acts to enhance an ongoing immune response but is not necessary for a strong protective Th2 response.

The effect of challenge and trickle Trichuris muris infections on the polarisation of the immune response.

In the field, determination of mechanisms of immunity to geohelminths are problematic due to the variation in infection exposure, host genetics, nutrition and co-infection. This study uses a well defined laboratory model, Trichuris muris in the mouse to study immune responses to challenge and trickle infections. The rationale is thus to study parasite acquisition under more natural antigen dose exposure. Antigen dose has previously been shown in this system to affect the outcome of infection with low antigen doses favouring type 1 responses (and susceptibility) and high antigen doses favouring type 2 responses (and resistance). A high level challenge infection could be established in a normally resistant host but only following priming of the immune response by a low level infection. Once type 2 responses were initiated it was impossible to switch an ongoing type 2 response even using IL-12 which is a potent stimulus of type 1 responses. Trickle infections resulted in no clear polarisation of the immune response. It was possible to build up the level of infection to a threshold level beyond which type 2 responses and expulsion were initiated. This threshold level was dependent upon host genetic background. Our results reveal a complex spectrum of responses and demonstrate that resistance and type 2 responses can be built up with increasing parasite exposure. The data provide compelling evidence to support a role for acquisition of acquired immunity to gastro-intestinal nematodes under complex infection patterns such as those found in the field.

Mean platelet volume is a useful parameter: a reproducible routine method using a modified Coulter thrombocytometer.

The principal physiological function of platelets is to promote haemostasis but they also contribute to thrombosis and atherogenesis. Platelet volume is a marker and possibly a determinant of platelet function in that large platelets are more active than normal sized platelets. Mean platelet volume (MPV), a measure of platelet size, reflects changes in either the level of platelet stimulation or the rate of platelet production. For these reasons, we have developed a sensitive instrument to measure platelet volume, which we believe is more reliable and specific than previously used instruments. It is based on a computer-interfaced Coulter Thrombocytometer and a pulse analyser including a high-speed baseline restorer. We have developed a reproducible method to assay MPV and from the histogram derived the median (MED) and the skewness (SK) values. We have looked at the effects of anticoagulant used and time elapse prior to assay. A normal range has been established for MPV which correlates directly with MED and inversely with SK. The MPV decreases with age but there is no difference between genders. We have demonstrated a negative correlation between whole blood platelet number and MPV and MED, and a direct relationship with the SK of the histogram of the platelet volume.

Gastrointestinal nematode expulsion in IL-4 knockout mice is IL-13 dependent.

Female IL-4 knockout (KO) mice on a C57BL/6 background (F4KOC57) are susceptible to infection with the cecal-dwelling nematode Trichuris muris whereas wild-type C57BL/6 mice are resistant and expel the parasite. In this study we show that in sharp contrast, female IL-4 KO mice on a BALB/c background (F4KOB/c) are resistant to infection as are wild-type BALB/c mice. Although susceptible F4KOC57 make negligible levels of all type 2 cytokines, resistant F4KOB/c were capable of producing significant levels of antigen-specific IL-13 (a cytokine shown to be critical in resistance to T. muris). To examine if the IL-13 in F4KOB/c mice was of functional importance, it was neutralized in vivo using a fusion protein, A25 (sIL-13 R.Fc). The results presented here clearly demonstrate that neutralization of IL-13 in vivo did indeed prevent T. muris expulsion in normally resistant F4KOB/c mice. In addition, administration of recombinant mouse IL-13 to normally susceptible male IL-4KO BALB/c mice (M4KOB/c) caused an 87.85 % reduction in worm burden. Collectively, these data show that IL-13 is important in the poorly understood effector mechanisms resulting in the expulsion of T. muris from the gut. Moreover, the present data highlight the functional importance of gender and background strain in interpretation of studies using gene-targeted animals.

Vaccination against coccidiosis: host strain-dependent evocation of protective and suppressive subsets of murine lymphocytes.

BALB/c mice are normally more resistant than C57BL/6 (B6) mice to infection with Eimeria vermiformis, but these phenotypes can be reversed by oral or parenteral vaccination with a crude antigen prepared from the parasite. Treatment of mice with antibodies specific for CD4+ or CD8+ T cells showed that the increased susceptibility of vaccinated BALB/c mice was associated with the presence of CD4+ T cells. This finding was confirmed when the recipients of CD4+ T cells selected from the mesenteric lymph nodes (MLN) of vaccinated BALB/c mice produced more oocysts after challenge than the recipients of a similar population of cells from sham-vaccinated mice. The residual population of cells (presumably enriched for CD8+ T cells, 'CD8+'), on the other hand, conferred some protection and, in B6 mice, the findings were reversed. Thus, vaccination induced suppressive or protective CD4+ cells and protective or suppressive 'CD8+' cells, depending upon the normal resistance/susceptibility phenotype of the host. Examinations of the isotypes (IgG1, IgG2a) of specific serum antibodies, and of the levels of IFN-gamma and IL-5 cytokines released by MLN cells stimulated ex vivo, did not allow any further characterization of the mechanisms involved.

Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.

In vivo manipulation of cytokine and/or cytokine receptor expression has previously shown that resistance to infection with the caecum-dwelling helminth Trichuris muris is dependent on interleukin (IL)-4 and IL-13 while susceptibility is associated with a T helper cell type 1 (Th1) cytokine response. Using gene-targeted mice deficient in tumor necrosis factor (TNF) receptor signaling and anti-TNF-alpha monoclonal antibody treatment, we have extended these studies to reveal a critical role for TNF-alpha in regulation of Th2 cytokine-mediated host protection. In vivo blockade of TNF-alpha in normally resistant mice, although not altering IL-4, IL-5, or IL-13 production in the draining lymph node, significantly delayed worm expulsion for the duration of treatment. IL-13-mediated worm expulsion in IL-4 knockout (KO) mice was also shown to be TNF-alpha dependent, and could be enhanced by administration of recombinant TNF-alpha. Furthermore, TNF receptor KO mice failed to expel T. muris, producing high levels of parasite-specific immunoglobulin G2a and the generation of a predominantly Th1 response, suggesting that the absence of TNF function from the onset of infection dramatically alters the phenotype of the response. These results provide the first demonstration of the role of TNF-alpha in regulating Th2 cytokine-mediated responses at mucosal sites, and have implications for the design of rational therapies against helminth infection and allergy.

A critical role for IL-13 in resistance to intestinal nematode infection.

Mice in which either the IL-4 or the IL-13 gene has been disrupted (IL-4 KO and IL-13 KO) were susceptible to infection with the intestinal nematode Trichuris muris, whereas their wild-type littermates were highly resistant and expelled the parasite. IL-4 KO mice showed diminished Th2-type responses with T. muris infection and also failed to produce parasite-specific IgG1 Abs. Although IL-13 KO mice made reduced Th2-type responses early in infection, they were capable of generating strong Th2-type responses at later time points and were unable to regulate the magnitude of their Ab isotype response. These results confirm the importance of IL-4 in resistance to T. muris and provide the first demonstration of an important role for IL-13 in resistance to helminth infection. The IL-13 KO mouse had a separate phenotype to that of the IL-4 KO mouse, suggesting that both IL-4 and IL-13 play important yet different roles in mediating immunity to intestinal helminths.

Interleukin-12 promotes a chronic intestinal nematode infection.

Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively. The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role. Interleukin (IL)-12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)-gamma from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system. IL-12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania. It has also been proposed as an anti-tumor agent and for use in the treatment of HIV. Conversely, IL-12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity. Our studies demonstrate that by driving a strong Th1 response, IL-12 promotes chronic T. muris infection when given to normally resistant BALB/K mice. Parasite-specific IgG2a, a Th1 parameter of infection, was greatly up-regulated, whereas some Th2 parameters of infection were down-regulated. IL-12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response. The actions of IL-12 in promoting a chronic infection were IFN-gamma dependent as an anti-IFN-gamma mAb abrogated the effects of IL-12.

Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response.

Resistance and susceptibility to the intestinal nematode Trichuris muris has been shown to be dependent upon the induction of T helper type 2 (Th2) or Th1 cells, respectively. This study demonstrates that in a normally resistant strain of mouse, i.e. BALB/K which mounts a dominant Th2 response, sub-threshold levels of infection (< 40 eggs) can survive and become sexually mature adult worms (10-20 adults). The immunological basis of this phenomenon was found to be a dramatically altered polarization of the CD4 response. The Th2-response characteristic of this strain of mouse infected with T. muris was shown to be significantly down-regulated as assessed by in vitro cytokine production [interleukin (IL)-4, IL-5 and IL-9]. In contrast, Th1 parameters of infection such as in vitro interferon-gamma production and the presence of parasite-specific IgG2a were greatly up-regulated in these mice.

The role of CD4 cells in protective immunity to Brugia pahangi.

The BALB/c mouse immunized sub-cutaneously (s.c.) with 45 kRad attenuated third stage larvae (L3) of the lymphatic filarial nematode Brugia pahangi is strongly immune to a challenge infection (75-100% reduction in recovery at day six post challenge). Analysis of spleen cell supernatants from immunized mice re-stimulated in vitro, with parasite antigen or the non specific T cell mitogen Con-A reveals a cytokine profile (IL-4, IL-5 and IL-9) which indicates that the Th2 subset of CD4 cells has been expanded. In an attempt to formally prove a critical role for CD4 cells in immunity in this model system, immunized mice were given either anti-CD4 or anti-CD8 neutralizing antibodies. Administration of anti-CD4 antibody had a significant and detrimental effect on the immune response whereas anti-CD8 antibody had a negligible effect on immunity. The efficacy of antibody in neutralizing their target cells was determined by fluorescence activated cell sorting analysis (FACS). Spleen cells from anti-CD4 treated immunized mice, when re-stimulated with parasite antigen had a significantly reduced potential to secrete IL-4, IL-5 and IL-9 in vitro and serum from these mice had reduced levels of parasite specific IgG and IgE. These results demonstrate a critical role for CD4 T cells in host protective immunity to B. pahangi in vivo and strongly suggest that some component of the Th2 response plays an important role in the immune response elicited in this model system.

Cytokine production in BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode, Brugia pahangi.

BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode Brugia pahangi are strongly immune to challenge infection. Investigation of the profile of cytokines secreted by spleen cells from immune mice stimulated in vitro with either parasite Ag or with Con A revealed high levels of IL-5 and IL-9 and moderate levels of IL-4. In contrast, secretion of IFN-gamma by spleen cells from immune animals was negligible. Spleen cells from control mice secreted low levels of all cytokines assayed. Levels of parasite-specific IgE were significantly elevated in immune animals and a peripheral blood eosinophilia was observed, which exhibited a biphasic distribution. Our results are consistent with the preferential expansion of Th2 cells in immune animals and provide the basis for dissecting the means by which radiation attenuated larvae of filarial nematodes stimulate immunity.

Medication effects on one-carbon metabolism in schizophrenia, mania, and major depression.

Erythrocyte methionine adenosyltransferase (MAT) activity (Vmax) and phosphatidylcholine (PC) levels previously have been found increased in manic patients and decreased in depressive and schizophrenic patients. To evaluate whether these abnormalities were the result of medication effects, erythrocyte MAT activity (Vmax) was assayed for paired samples from 29 schizophrenic, 16 manic, and 12 depressive patients, an erythrocyte PC levels were obtained for paired samples from 13 schizophrenic, seven manic, and seven depressive patients. Patients were medication free for at least 3 weeks. Vmax was significantly increased in schizophrenic and depressive patients (p less than 0.01; p less than 0.01) and significantly decreased (p less than 0.01) in manic patients after 2 weeks of psychotropic medication. Similar trends were found in PC levels. The findings of those one-carbon metabolism tests following medication are generally opposite to those reported to be related to specific disorders and tend toward normalization. Moreover, in vitro preincubation of erythrocytes of three normal subjects with the most commonly used neuroleptics had no consistent effects of MAT Vmax. These findings confirm previous studies that showed similarities in one-carbon metabolism of schizophrenic and depressed patients as opposed to manic patients and suggest that medications tend to correct or minimize rather than induce such abnormalities.