Helminth Therapy: Advances in the use of Parasitic Worms Against Inflammatory Bowel Diseases and its Challenges.

Development of modern medicine and better living conditions in the 20th century helped in reducing a number of cases of infectious diseases. During the same time, expansion of autoimmunological disorders was noticed. Among other are Inflammatory Bowel Diseases (IBD) including ulcerative colitis and Crohn's disease which are chronic and relapsing inflammation of the gastrointestinal tract. Absence of effective treatment in standard therapies effects the search for alternative opportunities. As per hygienic hypothesis increasing number of cases of autoimmune diseases is as a result of reduced exposure to pathogens, especially parasites. Thus, one of the promising remedial acts against IBD and other allergic and autoimmune disorders is "helminth therapy". Cure with helminths seems to be the most effective therapy of IBD currently proposed. Helminth therapy focuses on advantageous results that have been obtained from the clinical trials, but its mechanisms are still unclear. Explanation of this phenomenon would help to develop new drugs against IBD based on helminth immunomodulatory molecules.

The intestinal nematode inhibits T-cell reactivity by targeting P-GP activity.

Host immunosuppression occurs during chronic nematode infection, partly due to effector T-cell hyporesponsiveness. The role of P-glycoprotein (P-gp), a member of the ABC transporter family, has been assessed in T-cell activity. This study assesses the possible role of P-gp in T-cell activity during nematode infection. Our findings indicate that blockade of P-gp in vivo increased protection against Heligmosomoides polygyrus nematode infection and was associated with the enhanced T-cell activity. Three P-gp-inhibitors, verapamil (VRP), cyclosporine (CsA) and tariquidar (XR9576), were used to determine the influence of nematode infection on the P-gp function of T cells. The influence of the nematode on the uptake, efflux and kinetics of extrusion in T-cell subsets CD4+ and CD8+ was assessed by the accumulation of Rho123 dye. The results indicate that H. polygyrus infection contributes to the inhibition of T-cell function by elevating P-gp activity. The blockade of P-gp in the T cells of infected mice led to an impressive increase in T-cell proliferation and IL-4 cytokine release through the upregulation of NF-κB activation. These results provide the first evidence that the P-gp function of T cells is altered during nematode infection to open the way for further studies aiming to explore the role of P-gp in host-parasite interactions.

The antiapoptotic activity of Heligmosomoides polygyrus antigen fractions.

Our study identified Heligmosomoides polygyrus antigen factors with potential activity for regulation of T-cell proliferation and surviving of CD4(+) CD25(-) , CD4(+) CD25(hi) and CD3(+) CD8(+) cell populations. The antiapoptotic activity of antigenic fractions separated by HPLC was evaluated in vitro after exposure of cells to DEX and rTNF-α. Different populations of cells responded to antigen fractions in distinct pattern; the most sensitive population of cells to H. polygyrus products were CD4(+) CD25(hi) after exposure to DEX and CD3(+) CD8(+) T cells after exposure to rTNF-α. H. polygyrus antigens may influence survival of CD8(+) T cells by regulation of c-FLIP rather than Bcl-2, which affects survival of CD4(+) CD25(hi) Treg cells and CD4(+) T cells. Activation of NF-κB subunits, for example, p50 and p65 was essential for resistance of cells to apoptosis, and antigenic fractions F9 and F17 exerted different effect to F13. The most active fraction in inhibition of apoptosis was F9, which includes Hsp-60, calumenin, ferritin, galectin and thrombospondin. This study may provide new clues for recognition of factors that regulate the immune response during infection and which engage the TNF-α receptor-mediated and the mitochondria-mediated death pathway.

Heligmosmoides polygyrus fourth stages induce protection against DSS-induced colitis and change opioid expression in the intestine.

Primary exposure of mice to the nematode Heligmosomoides polygyrus infection reduces inflammation in an experimental model of colitis. The aim of the present investigation was to evaluate whether the reduced inflammation provoked by H. polygyrus L4 larvae in BALB/c mice treated with dextran sulphate sodium is associated with changed expression of opioids in the small intestine and colon. Colitis was induced by 5% Dextran sulphate sodium (DSS) oral administration for 3 days before oral infection with 200 infective larvae (L3) H. polygyrus until the end of the experiment, 6 days post-infection. Clinical disease symptoms were monitored daily. The expressions of proopiomelanocortin POMC1, MOR1 (Oprm1) - opioid receptor and ß-endorphin were determined by RT-PCR, Western blot and immunoassay, respectively, in the colon and small intestine of mice. RT-PCR analysis of colon tissues showed up-regulation of the expression of POMC and MOR1 opioid-dependent genes in mice with DSS-induced colitis. H. polygyrus L4 larvae inhibited DSS-induced colitis symptoms that were correlated with increased IL-1ß, TNF-α, IL-6, myeloperoxidase (MPO) concentration, macrophages infiltration and MOR1, POMC and ß-endorphin increased expression in the small intestine and inhibition of those in the colon.

Comparative ultrastructural studies of the alterations to mouse lung parenchyma during Trichinella spiralis or Toxocara canis infection.

Trichinella spiralis and Toxocara canis larvae migrated through the lung and induced many alterations in the lung parenchyma. Using electron microscopy, we identified and described the histopathological changes. These changes resulted from mechanical damage or from local inflammatory reactions provoked by larvae. The pattern of changes was described between 6 and 12 days post-infection (DPI) with T. spiralis larvae, and between 21 and 28 DPI with T. canis. The ultrastructural studies demonstrated that T. spiralis larvae migrating through the lungs evoked mainly destruction of type I epithelial cells, destruction of lamellar bodies of epithelial cells or extracellular alveolar lining layer. The severity of these changes was dependent on the number of infective larvae (400 or 800 T. spiralis larvae) and possibly the result of mechanical damage in the lung parenchyma. In contrast, infection with T. canis larvae initiated mainly eosinophilic perivasculitis and vasculitis as well as macrophage accumulation in the lung, which were additionally impacted by numerous crystalloid inclusions in macrophages. Trichinella spiralis larvae and T. canis larvae induced different pathological changes in the lungs of infected mice.

Triterpenoid saponins affect the function of P-glycoprotein and reduce the survival of the free-living stages of Heligmosomoides bakeri.

We studied the effect of triterpenoid saponins on the development of free-living stages of Heligmosomoides bakeri, a parasitic nematode of the mouse intestine. We evaluated the effectiveness of oleane-type glucuronides (GlcUAOA) isolated from Calendula officinalis and Beta vulgaris. The rhodamine 123 retention assay was used to detect dysfunctions of the major membrane transporter for xenobiotics, P-glycoprotein (Pgp). Both C. officinals and B. vulgaris GlcUAOA affect the development of the free living stages and function of Pgp in H. bakeri. The GlcUAOA inhibits egg hatching and moulting of larvae and also changes their morphology. These saponin fractions reversed the toxic effect of thiabendazole on the nematode; the function of Pgp was also inhibited.

Heligmosomoides bakeri antigen rescues CD4-positive T cells from glucocorticoid-induced apoptosis by Bcl-2 protein expression.

Heligmosomoides bakeri infection in mice is associated with a dominant CD4(+) T-cell response and with the activity of natural Treg cells with CD4(+) CD25(+) phenotype. The polarization of Th2 T-cell phenotype and the increase in the CD4(+) CD25(+) T cell population are regulated by glucocorticoids that induce apoptosis in CD4(+) CD25(-) T cells and inhibit apoptosis in CD4(+) CD25(+) T cells. However, exposure of mice to H. bakeri antigen induces a high glucocorticoid concentration in serum and a reduction in the number of CD4-positive; CD4(+) CD25(-) and CD4(+) CD25(+) apoptotic T cells in mesenteric lymph node cells. In this study to evaluate the in vitro effect of the anti-apoptotic property of H. bakeri antigen on T cells, apoptosis of these cells was induced by glucocorticoids-dexamethasone (Dex). Excretory-secretory (ES) antigen of the nematode prevented Dex-induced apoptosis in CD4-positive T cells with CD4(+) CD25(-) and CD4(+) CD25(High) phenotype by Bcl-2 protein expression. Contrary to the effect on CD4-positive T cells, survival of CD8(+) T cells was not connected with expression of Bcl-2 protein. This suggest that H. bakeri antigen modulates CD4-positive T cell sensitivity to glucocorticoid-induced apoptosis by induction of Bcl-2 protein.

Selenium supplementation enhances the protective response to Toxocara canis larvae in mice.

The effect of oral and intraperitoneal supply of sodium selenite on the immune response to, and the course of T. canis larvae infection in mice were determined. The number of worms in the host tissue was reduced but the migratory route of larvae was not affected. Selenite (Se) supplementation influences Se retention in the liver, enhanced IL-5 and eosinophil responses and evoked IL-6 production in mice infected with T. canis. The enhanced protection in mice given Se intraperitoneally was associated with high levels of parasite-specific IgE, and enhanced concentration of Th1-related cytokines such IL-12p70, TNF-α and IFN-γ. In mice given Se orally, the predominant cytokines produced were IL-10, MCP-1 and IL-6 and these mice had lower protection. In conclusion, Se supplementation increases production of specific cytokines in mice infected with T. canis and increases protection against infection.

Heligmosomoides polygyrus infection down-regulates eotaxin concentration and CCR3 expression on lung eosinophils in murine allergic pulmonary inflammation.

There is growing evidence that helminth infections might suppress allergic responses by mechanisms potentially involving regulatory T lymphocytes, cytokines, helminth molecules and polyclonal IgE. Heligmosomoides polygyrus infection in mice is associated with reduced local and systemic immune responses, thus providing an excellent model to study the mechanisms of immune regulation. In this research, we examined the way that nematode infection modulates the influx of eosinophils into the airways of asthmatic mice. We observed a reduction in the total number and percentage of lung eosinophils that coincided with decreased levels of eotaxin in bronchoalveolar lavage fluid (BALF), lower expression of the CCR3 receptor on eosinophils and impaired chemotaxis of these cells toward eotaxin. We conclude that allergen-induced immune response was down-regulated as production of Th1 (IFN-gamma)-, Th2 (IL-4, IL-5)- and Treg (IL-10)-related cytokines as well as IL-6 and TNF-alpha was diminished upon nematode infection. We postulate that attenuation of allergic inflammation during H. polygyrus infection is a consequence of the dichotomy of the immune response in the face of concurrent antigenic challenge.

Heligmosomoides polygyrus: decreased apoptosis in fast responder FVB mice during infection.

Primary infection with Heligmosomoides polygyrus in some strains of mice is chronic although fast responder mouse strains eliminate the parasite in a short period of time. The reason for the differences is unknown. In this study apoptosis, proliferation, IL-2 and IL-6 production of mesenteric lymph node (MLN) and spleen cells in vitro from fast (FVB) and slow (C57Bl/6) responder mice were compared during H. polygyrus infection. FVB cells showed decreased apoptosis, more proliferation and more cytokine production than cells from C57Bl/6 mice during infection. At the beginning of infection in C57Bl/6 mice the apoptosis of CD4(+) but not CD8(+) cells significantly increased in MLN and spleen cell cultures. Apoptosis, when the first immune signal is given by infective larvae, might play an important role in the modulation of the response in slow responder mice.

Reduced apoptosis in BALB/c mice infected with Heligmosomoides polygyrus.

We evaluated levels of apoptosis and the immune response ex vivo in BALB/c mice infected with Heligmosomoides polygyrus. Cell proliferation, apoptosis and cytokine production were measured in mesenteric lymph nodes (MLN) without exposure to H. polygyrus antigens in culture. The inhibited apoptosis and cytokine production reported might reflect a state of cell hyporesponsiveness in the prepatent phase of infection. These changes were accompanied by changes in the percentage of CD4+ cells in MLN and popliteal lymph nodes (PLN). The prolonged reduction in apoptosis coexisted with induced cell proliferation, elevated TNF-alpha, IL-12p70, IFN-gamma, IL-6, IL-10 and TGF-beta synthesis, but lowered IL-4 and IL-2 levels. In the chronic phase of infection an increasing production of IFN-gamma, monocyte chemotactic protein-1 (MCP-1), IL-10 and TGF-beta with decreasing concentrations of other cytokines resulted in restored apoptosis. The cytokine response in serum showed moderate production of TNF-alpha, temporary involvement of IL-12p70, induction of IFN-gamma and IL-10 synthesis, as well as growing IL-6 and MCP-1 production. It is suggested that a synchronized synthesis of distinct cytokines is accompanied by different levels of inhibited apoptosis during the prepatent and chronic phases of H. polygyrus infection in BALB/c mice. We suggest that immunosuppression provoked by the nematode is not the outcome of parasite-induced apoptosis, but rather results from a hyporesponsiveness experienced by cells during H. polygyrus infection.

Alternatives to anthelmintics for the control of nematodes in livestock.

Efficient and welfare-friendly livestock production demands the control of nematode infection. Current control measures rely upon anthelmintic treatment but are threatened by the widespread evolution of drug-resistance in parasite populations. Several methods have been advocated to control nematodes without relying on effective anthelmintics. These include grazing management, biological control, nutritional supplementation, vaccination, and genetic approaches. Each method has its advantages and disadvantages. There are several grazing management schemes that can reduce the severity of infection but they are insufficient on their own to control infection. Biological control includes the use of predatory fungi to control nematode populations and the use of pasture species that can reduce the intensity of infection. Fungi can control nematodes but the current requirement for daily feeding means that this approach will be most useful for animals that are handled daily. Feeding supplementary protein can control nematode infection. The method is simple but can be expensive and may not be cost-effective for some marginal enterprises. Genetic approaches include the use of resistant breeds and selective breeding. Some breeds will thrive in conditions that kill animals from other breeds but substitution of resistant breeds is not always feasible. Selective breeding is effective and inexpensive but requires a high level of expertise. The most appropriate method or set of methods to minimize the adverse consequences of nematode infection may vary among farms.

The role of TGF-beta in mice infected with Heligmosomoides polygyrus.

Hyporesponsiveness induced by Heligmosomoides polygyrus was quantified and the relationship between TGF-beta and inflammation was identified in BALB/c mice. The immune response and pathological changes modified by neutralization of TGF-beta were characterized in vivo. Nine and twelve days following infection, BALB/c mice were injected intraperitoneally with anti-TGF-beta (1,2,3) antibodies, isotype control antibodies or isosmotic solution. We assessed both Th1 and Th2 related cytokines production ex vivo and in vitro, IgA, the number of CD4+ cells, and eosinophils in the lamina propria and the villus : crypt ratio in the small intestine 6 weeks after infection. The pattern of cytokine production differed in the intestine, peritoneal fluid and serum. In mice infected with H. polygyrus the concentrations of IL-5, IL-12, TNF-alpha and IL-10 were raised in the intestine, but in serum the level of cytokines was diminished below the value observed in uninfected mice. The neutralization of TGF-beta converted the pattern of immune response induced by H. polygyrus. The elevation of cytokines in serum coincided with the reduction of cytokine concentration in the intestine or peritoneum. Neutralization of TGF-beta restored infiltration of eosinophils into the lamina propria of the intestine despite the low level of IL-5. We conclude that H. polygyrus infection suppresses the immune response through pathways involving TGF-beta production or activity and that the Th2 related immune response was not affected by neutralization.

Screening for immunomodulatory proteins of the intestinal parasitic nematode Heligmosomoides polygyrus.

Parasitic nematodes are constantly exposed to the immune effector mechanisms of their hosts. One strategy of the worms to cope with these defence reactions is the secretion of modulatory proteins that down-regulate cell-mediated immune responses. We analysed the proliferation of mesenteric lymph node cells of mice infected with Heligmosomoides polygyrus and showed that cellular proliferation was strongly suppressed in the chronic phase of infection. To identify proteins of H. polygyrus that are involved in parasite-induced immunomodulation, worm extract and culture supernatant of adult H. polygyrus were fractionated by gel chromatography and activity of each fraction was determined. One of the fractions (fraction 9) of worm extract as well as worm secretory products inhibited the antigen-specific cellular proliferation by about 40%. This reduced cellular reactivity coincided with a down-regulation of inducible nitric oxide production of mouse macrophages by 57%. Furthermore, fraction 9 contained antigens that were recognized by IgE antibodies of H. polygyrus-infected mice and induced degranulation of an IgE-sensitized basophil cell line. Single proteins of fraction 9 were analysed by mass spectrometry. These data suggest that antigens that are recognised by IgE antibodies might play an important role in immunomodulation exerted by nematode infections.

The mucosal mast cell and IgA plasma cell responses to primary Trichinelia spiralis infection in BALB/c mice treated with ketotifen.

The effect of ketotifen on inflammation in the intestine during T. spiralis infection in BALB/c mice was examined. The lack of enteropathy in treated mice was accompanied to affected the size of worms and the number of worms, but did not prevent the retardation of nematodes in the intestine. Fecundity of females in vitro and the number of muscle larvae were not statistically changed after treatment. Ketotifen reduced the number of mast cells and IgA plasma cells in the intestine. As inflammatory influx, epithelial damage and IgA response are related to the action of T cells, ketotifen affected T dependent reactions in the gut of T. spiralis infected mice.

[Changes in the immune response of BALB/c mice coinfected with Heligmosomoides polygyrus and Trichinella spiralis]. / Zmiana odpowiedzi immunologicznej u myszy BALB/c w koinwazji Heligmosomoides polygyrus i Trichinella spiralis.

The influence of Heligmosomoides polygyrus on infection with Trichinella spiralis was studied in BALB/c mice. Mice coinfected with T. spiralis and previously given H. polygyrus harboured both nematode species till day 34. The number of T. spiralis muscle larvae was greater in mice coinfected with H. polygyrus/T. spiralis or T. spiralis/H. polygyrus than after infection with T. spiralis alone. Infection with H. polygyrus did not enhance eosinophil and IL-5 levels induced by T. spiralis. Additionally, the production of IgG1 specific to L1 T. spiralis was inhibited by co-infection. Changes in the levels of IFN-gamma and IgG2a implicated a disturbance in Th2 cell activation during protective response and resulted in the greater number of T. spiralis muscle larvae in coinfected mice.

Inhibition of the protective IgA response by ketotifen is related to the inflammatory reaction in the peritoneal cavity and intestinal mucosa of BALB/c mice infected with Trichinella spiralis.

Interleukin-5 (IL-5) production, eosinophilia, and IgA responses of BALB/c mice infected with Trichinella spiralis were measured in the peritoneal cavity and intestinal mucosa. Ketotifen, an anti-allergic compound, was used to control the inflammatory response. IgA responses differed against adult and muscle stages of the parasite and between the intestine and the peritoneal cavity. IL-5 and eosinophil production also differed between the intestine and the peritoneal cavity. The occurrence of parasite-specific IgA antibodies in the peritoneal cavity may reflect the migration of cells from the lamina propria. Following ketotifen treatment there was a reduction in the IL-5, eosinophilic, and IgA responses; these responses affected both the size of worms and the number of worms.

Immune response to Trichinella spiralis larvae after treatment with the anti-allergic compound ketotifen.

Ketotifen was used as an anti-allergic agent to study the relationship between eosinophil-related responses and IgG1 and IgG2a antibody responses in BALB/c mice infected with Trichinella spiralis. The results showed that leukocyte and eosinophil numbers and interleukin-5 (IL-5) concentrations in the peritoneal fluid increased after exposure to nematodes and the increases were slightly greater in animals treated with ketotifen. A decreased concentration of eosinophil peroxidase and an elevation in IgG1 accompanied the muscle phase of infection. In mice treated with ketotifen, antibody-mediated recognition of muscle larvae was delayed. The retardation of IgG1 and IgG2a responses may have been responsible for the ineffective immune response against larvae migrating into the muscle. The activation of eosinophils was accompanied by changes in IL-5 concentration, but these changes were not associated with differences in protection against infection.

[Effect of ketotifen on the immune response in BALB/c mice infected with Trichinella spiralis]. / Wplyw ketotifenu na odpowiedz obronna myszy BALB/c zarazonych Trichinella spiralis.

Infection with Trichinella spiralis in mice generates Th-2 mediated response, which controls effector mechanism operating in the intestine. It is associated with a pronounced intestinal mastocytosis, eosinophilia and destruction of intestinal epithelial layer during expulsion of parasite from the gut. It is believed that protection is dependent on non-specific inflammatory reaction mediated by mast cells. Furthermore, the higher serum levels of parasite specific IgG1 and IgG2a and also mucosal IgA response were related to the course of infection. Inhibition of humoral and cellular immune responses using ketotifen as anti-allergic compound, resulted in the greater worm burden and worm size, but not in the significant prolongation of intestinal phase. Moreover, in treated mice epithelial layer of the gut was protected from destruction provoked by the nematode. As interaction between effector leukocytes and antibodies were not effective it was proposed that other mechanisms, not related to hypersensitivity or conventional inflammatory response regulated the level of infection. The immunological and physiological phenomenons are discussed in terms of events associated with protection to the parasite. Possibly, immunoregulatory capasity of the nematode is involved in the induction of multiple mechanisms operated during infection.

[Regulation of defence and allergic reaction in infections with parasites]. / Regulacja reakcji obronnej i alergicznej w inwazjach pasozytów.

Cytokines regulate development, differentiation and expression of effector function of the immune system. The profile of immune reactions depends on cytokine contents at the recognition of parasite. However, parasites themselves can modify immunological events and favourite these, which allow them to survive. The host immune defence can be transformed into the chronic reaction. Inflammatory reactions result from the recruitment of different cells, to the site where worms are localised. The eosinophil and mast cell migration preferentially is induced. These cells play also a major role in immediate allergic responses. Mast cells can bind allergen-specific IgE to their FcepsilonRI, the high affinity receptor for IgE. Eosinophils, through their receptors for IgG1, IgG2, IgA and IgE are mainly involved in the cytotoxic reaction directed against parasites. Crosslinking of these receptors by antigen binding will lead to subsequent release of stored mediators and cytokines. Granular materials released from mast cell accelerate inflammatory reaction and in the case of intestinal worm parasites may be involved in the expulsion phenomenon. However these cells may also induce Th2 related immunological response because they produce and release of IL-4. Eosinophils are required into the tissue and release cytotoxic and stress proteins including reactive oxygen species. Parasites are destroyed but accelerated reaction results in the destruction of host proteins and cells. Antibodies, cytokines, chemokines and adhesion molecules are essential for elevation of defence against parasites. The role of cytokines, emphasizing IL-5, and function of eosinophils, mast cells and IgE are discussed in terms of induction and effector mechanisms during parasite infections.