Maria Doligalska

Antibacterial and antiparasitic activity of oleanolic acid and its glycosides isolated from marigold (Calendula officinalis).

The antibacterial and antiparasitic activities of free oleanolic acid and its glucosides and glucuronides isolated from marigold (Calendula officinalis) were investigated. The MIC of oleanolic acid and the effect on bacterial growth were estimated by A600 measurements. Oleanolic acids influence on bacterial survival and the ability to induce autolysis were measured by counting the number of cfu. Cell morphology and the presence of endospores were observed under electron and light microscopy, respectively. Oleanolic acid inhibited bacterial growth and survival, influenced cell morphology and enhanced the autolysis of Gram-positive bacteria suggesting that bacterial envelopes are the target of its activity. On the other hand, glycosides of oleanolic acid inhibited the development of L3 Heligmosomoides polygyrus larvae, the infective stage of this intestinal parasitic nematode. In addition, both oleanolic acid and its glycosides reduced the rate of L3 survival during prolonged storage, but only oleanolic acid glucuronides affected nematode infectivity. The presented results suggest that oleanolic acid and its glycosides can be considered as potential therapeutic agents.

Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus.

To analyze the potential of RNA interference (RNAi) in the intestinal nematode Heligmosomoides polygyrus, we delivered double-stranded RNA (dsRNA) of tropomyosin to various life stages of the parasite. Three different methods were examined for their potential use. First, feeding of recombinant bacteria that expressed dsRNA did neither result in phenotypical changes of H. polygyrus nor in a significant reduction of tropomyosin mRNA levels. In contrast, feeding of such bacteria to Caenorhabditis elegans elicited the expected phenotypes. Quantification of bacteria ingested by C. elegans and H. polygyrus larvae (L1) revealed that the parasitic worms took up only a fraction of the bacteria ingested by C. elegans. Second, electroporation of L1 failed to transport siRNA through the cuticle and was lethal to the larvae. However, the cuticle of adult worms was penetrated by dye-labeled RNA, but no systemic spreading was observed. Third, soaking of adult H. polygyrus in tropomyosin dsRNA led to a higher proportion of worms showing symptoms of ageing, such as a disintegrated gut and ovaries, but did not induce reduction of tropomyosin mRNA levels. Database analysis revealed that orthologous proteins involved in dsRNA-uptake and -systemic spread in C. elegans are missing in the parasitic nematodes Brugia malayi and Haemonchus contortus, whereas proteins responsible for dsRNA-processing, -amplification and mRNA-regulation are present. Thus, our data indicate that the study of gene function by RNAi in H. polygyrus is limited, possibly due to deficiencies of genes involved in RNA-uptake and spread.

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 role of TGF-β in mice infected with Heligmosomoides polygyrus

Hyporesponsiveness induced by Heligmosomoides polygyrus was quantified and the relationship between TGF‐β and inflammation was identified in BALB/c mice. The immune response and pathological changes modified by neutralization of TGF‐β were characterized in vivo. Nine and twelve days following infection, BALB/c mice were injected intraperitoneally with anti‐TGF‐β (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‐α 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‐β 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‐β 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‐β production or activity and that the Th2 related immune response was not affected by neutralization.

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.

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 β‐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‐1β, TNF‐α, IL‐6, myeloperoxidase (MPO) concentration, macrophages infiltration and MOR1, POMC and β‐endorphin increased expression in the small intestine and inhibition of those in the colon.

Heligmosomoides polygyrus: EAE remission is correlated with different systemic cytokine profiles provoked by L4 and adult nematodes.

Primary exposure of mice to gastrointestinal nematode infection with Heligmosomoides polygyrus reduces inflammation in an experimental model of multiple sclerosis. In this study, we aimed to evaluate the ability of H. polygyrus L4 larvae and adults infection to reduce the symptoms of ongoing experimental autoimmune encephalomyelitis (EAE) in female C57Bl/6 mice. EAE was induced by myelin oligodendrocyte glycoprotein MOG(p35-55) and after 21 days mice were orally infected with 200 infective larvae (L3) of H. polygyrus. Reduction in EAE symptoms was observed from 2 days post infection and the symptoms were almost completely inhibited at 6 days post infection. This effect was associated with limited total protein content in the cerebrospinal fluid; CSF, and significant decreased pro-inflammatory IL-12p40 concentration and increased concentration of the regulatory cytokines IL-10, TGF-β and IL-6 in the CSF and in the serum. The reduction of EAE symptoms in the enteral phase was associated with higher IL-12p40 concentration in the CSF and very low concentrations of IL-17A and IL-2 in the serum. The fourth stage of gastrointestinal nematode can reverse systemic inflammation in animal models of multiple sclerosis by reducing IL-12 and promoting regulatory cytokines production. The mechanism induced by adult nematodes which sustained EAE inhibition can be provoked by regulatory mechanism connected with reduce IL-17A concentration.

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‐γ)‐, Th2 (IL‐4, IL‐5)‐ and Treg (IL‐10)‐related cytokines as well as IL‐6 and TNF‐α 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.

Colitis Promotes Adaptation of an Intestinal Nematode: A Heligmosomoides Polygyrus Mouse Model System

The precise mechanism of the very effective therapeutic effect of gastrointestinal nematodes on some autoimmune diseases is not clearly understood and is currently being intensively investigated. Treatment with living helminths has been initiated to reverse intestinal immune-mediated diseases in humans. However, little attention has been paid to the phenotype of nematodes in the IBD-affected gut and the consequences of nematode adaptation. In the present study, exposure of Heligmosomoides polygyrus larvae to the changed cytokine milieu of the intestine during colitis reduced inflammation in an experimental model of dextran sulphate sodium (DSS)- induced colitis, but increased nematode establishment in the moderate-responder BALB/c mouse strain. We used mass spectrometry in combination with two-dimensional Western blotting to determine changes in protein expression and changes in nematode antigens recognized by IgG1 in mice with colitis. We show that nematode larvae immunogenicity is changed by colitis as soon as 6 days post-infection; IgG1 did not recognize highly conserved proteins Lev-11 (isoform 1 of tropomyosin α1 chain), actin-4 isoform or FTT-2 isoform a (14-3-3 family) protein. These results indicate that changes in the small intestine provoked by colitis directly influence the nematode proteome. The unrecognized proteins seem to be key antigenic epitopes able to induce protective immune responses. The proteome changes were associated with weak immune recognition and increased larval adaptation and worm growth, altered localization in the intestine and increased survival of males but reduced worm fecundity. In this report, the mechanisms influencing nematode survival and the consequences of changed immunogenicity that reflect the immune response at the site colonized by the parasite in mice with colitis are described. The results are relevant to the use of live parasites to ameliorate IBD.

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‐α, IL‐12p70, IFN‐γ, IL‐6, IL‐10 and TGF‐β synthesis, but lowered IL‐4 and IL‐2 levels. In the chronic phase of infection an increasing production of IFN‐γ, monocyte chemotactic protein‐1 (MCP‐1), IL‐10 and TGF‐β with decreasing concentrations of other cytokines resulted in restored apoptosis. The cytokine response in serum showed moderate production of TNF‐α, temporary involvement of IL‐12p70, induction of IFN‐γ 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.