In vitro culture of Mesocestoides corti metacestodes and isolation of immunomodulatory excretory–secretory products

Cestode‐mediated diseases hold the interesting feature of persisting metacestode larvae dwelling within the host tissues, in the midst of the immune response. Excretory–secretory (ES) products of the metacestode larval stage modulate the host immune response and modify the outcome of the disease. Therefore, isolation and analysis of axenic metacestode ES products are crucial to study their properties. Here, we report the development of a system for long‐term in vitro cultivation of the metacestode of the parasitic cestode Mesocestoides corti (syn. Mesocestoides vogae). Although feeder cells and host serum supported the early growth of the parasite, long‐term survival was not dependent on host serum or host‐derived factors enabling the collection of parasite released products in serum‐free medium. Functionally, these axenic ES products recapitulated M. corti tetrathyridia's ability to inhibit LPS‐driven IL‐12p70 secretion by dendritic cells. Thus, our new axenic culture system will simplify the identification and characterization of M. corti‐derived immunomodulatory factors that will indirectly enable the identification and characterization of corresponding factors in the metacestode larvae of medically relevant cestodes such as Echinococcus multilocularis that are not yet amenable to serum‐free cultivation.     

Codelivery of DNA vaccination encoding LeIF gene and IL‐12 increases protection against Leishmania major infection in BALB/c mice

Previous studies have shown that Leishmania elongation initiation factor (LeIF) antigen causes a partial immunity against leishmaniasis. The antigen develops type I immunity by overexpression of inflammatory cytokines such as interleukin‐12 (IL‐12), IFN‐γ and TNF‐α. Therefore, We evaluated immune responses induced by the LeIF gene against Leishmania major infection. Immunization with LeIF gene alone or with IL‐12 induced Th1 response and produced higher IFN‐γ and lower IL‐4 levels by splenocytes than control groups (P < 0·05) and also ratios of IFN‐γ/IL‐4 were 11·68 to 18·53 times more in immunized groups than control groups after challenge. In addition, analysis of humoral immune response revealed that immunized mice had more IgG2a levels than both control groups (P < 0·05). On the other hand, lesion size was less for immunized animals than control groups from 4th week after challenge (P < 0·05). The percentage reduction in lesion size was 29·30% for LeIF and 51·98% for LeIF + IL‐12 than PBS at 12th week post‐infection. Spleen parasite burden decreased in all immunized groups in comparison with control groups (P < 0·05). The results indicated that LeIF gene induced partial immunity against L. major infection in BALB/c mice. However, LeIF plus IL‐12 group showed more potent immunity with smaller lesions than other groups.     

Cross‐protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania‐specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN‐γ, IL‐12 and GM‐CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti‐Leishmania Th1 response was maintained after infection, being the IFN‐γ production based mainly on CD4⁺ T cells. We described one conserved Leishmania‐specific protein that could compose a pan‐Leishmania vaccine.     

Analysis of iron superoxide dismutase‐encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection

The present work aimed to evaluate the immunogenicity of Leishmania amazonensis iron superoxide dismutase (SOD)‐encoding DNA experimental vaccine and the protective properties of this DNA vaccine during infection. The SOD gene was subcloned into the pVAX1 plasmid, and it was used to immunize BALB/c mice. Twenty‐one days after the last immunization, mice were sacrificed (immunogenicity studies) or subcutaneously challenged with L. amazonensis (studies of protection), and alterations in cellular and humoral immune responses were evaluated, as well as the course of infection. Mice only immunized with pVAX1‐SOD presented increased frequencies of CD4⁺IFN‐γ⁺, CD8⁺IFN‐γ⁺ and CD8⁺IL‐4⁺ lymphocytes; moreover, high levels of IgG2a were detected. After challenge, mice that were immunized with pVAX1‐SOD had increased frequencies of the CD4⁺IL‐4⁺, CD8⁺IFN‐γ⁺ and CD8⁺IL‐4⁺ T lymphocytes. In addition, the lymph node cells produced high amounts of IFN‐γ and IL‐4 cytokines. Increased IgG2a was also detected. The pattern of immunity induced by pVAX1‐SOD partially protected the BALB/c mice from a challenge with L. amazonensis, as the animals presented reduced parasitism and lesion size when compared to controls. Taken together, these results indicate that leishmanial SOD modulates the lymphocyte response, and that the elevation in IFN‐γ possibly accounted for the decreased skin parasitism observed in immunized animals.     

Prophylactic properties of a Leishmania‐specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads’ draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti‐Leishmania IgG2a antibodies and a predominant IL‐12‐driven IFN‐γ production (mainly produced by CD4⁺ T cells) against parasite proteins, whereas unprotected controls showed anti‐Leishmania IgG1 antibodies and parasite‐mediated IL‐4 and IL‐10 responses. Vaccinated mice showed an anti‐LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD‐specific IFN‐γ, IL‐12 and GM‐CSF cytokines before and after infection. The protection was correlated with the Leishmania‐specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.     

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice

Toxoplasmosis is a world‐wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL‐12‐inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1‐TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1‐TgCyP developed a high response with TgCyP‐specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1‐TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL‐2 and IFN‐γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1‐TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.     

Fasciola hepatica tegumental antigens indirectly induce an M2 macrophage‐like phenotype in vivo

The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory‐secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune‐modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2‐like phenotype in vivo that modulates cytokine production from CD4⁺ cells in response to anti‐CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym‐1/2 but not RELMα in FhTeg‐stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL‐13. FhTeg can induce IL‐13‐producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune‐modulatory source during F. hepatica infection and sheds further light on helminth–macrophage interactions.     

Brugia malayi soluble and excretory‐secretory proteins attenuate development of streptozotocin‐induced type 1 diabetes in mice

Understanding the modulation of the host‐immune system by pathogens‐like filarial parasites offers an alternate approach to prevent autoimmune diseases. In this study, we have shown that treatment with filarial proteins prior to or after the clinical onset of streptozotocin‐induced type‐1 diabetes (T1D) can ameliorate the severity of disease in BALB/c mice. Pre‐treatment with Brugia malayi adult soluble (Bm A S) or microfilarial excretory‐secretory (Bm mf ES) or microfilarial soluble (Bm mf S) antigens followed by induction of diabetes led to lowering of fasting blood glucose levels with as many as 57·5–62·5% of mice remaining nondiabetic. These proteins were more effective when they were used to treat the mice with established T1D as 62·5–71·5% of the mice turned to be nondiabetic. Histopathological examination of pancreas of treated mice showed minor inflammatory changes in pancreatic islet cell architecture. The therapeutic effect was found to be associated with the decreased production of cytokines TNF‐α & IFN‐γ and increased production of IL‐10 in the culture supernatants of splenocytes of treated mice. A switch in the production of anti‐insulin antibodies from IgG2a to IgG1 isotype was also seen. Together these results provide a proof towards utilizing the filarial derived proteins as novel anti‐diabetic therapeutics.     

Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post‐infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6‐8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti‐L. amazonensis IgG and increased production of IL‐10 and IL‐17. Because IL‐10 is a mediator of susceptibility to Leishmania infection, we blocked IL‐10 signalling in neutrophil‐depleted mice using anti‐IL‐10R. Interestingly, inhibition of IL‐10 signalling abrogated the increase in parasite loads observed in neutrophil‐depleted mice, suggesting that parasite proliferation is at least partially mediated by IL‐10. Additionally, we tested the effect of IL‐17 in inflammatory macrophages and observed that IL‐17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.     

Immunological interaction between Giardia cyst extract and experimental toxoplasmosis

Toxoplasmosis is mostly associated with other intestinal parasitic infections especially Giardia due to shared mode of peroral infection. Toxoplasma and Giardia induce a strong T‐helper 1‐ immune response. Our aim was to induce a protective immune response that results in significant impact on intestinal and extra‐intestinal phases of Toxoplasma infection. This study was conducted in experimental animals and assessment of Giardia cyst extract effect on Toxoplasma infection was investigated by histopathological examination of small intestine and brain, Toxoplasma cyst count and iNOS staining of the brain, measurement of IFN‐γ and TGF‐β in intestinal tissues. Results showed that the brain Toxoplasma cyst number was decreased in mice infected with Toxoplasma then received Giardia cyst extract as compared to mice infected with Toxoplasma only. This effect was produced because Giardia cyst extract augmented the immune response to Toxoplasma infection as evidenced by severe inflammatory reaction in the intestinal and brain tissues, increased levels of IFN‐γ and TGF‐β in intestinal tissues and strong iNOS staining of the brain. In conclusion, Giardia cyst extract generated a protective response against T. gondii infection. Therefore, Giardia antigen will be a suitable candidate for further researches as an immunomodulatory agent against Toxoplasma infection.     

Sambucus ebulus extract stimulates cellular responses in cutaneous leishmaniasis

This is the first study aiming to determine the therapeutic effects of the Sambucus ebulus aquatic extract as an antileishmanial herbal drug and evaluate the immune responses in Leishmania major major infected BALB/c mice. The antileishmanial activity of S ebulus aquatic extract was evaluated using MTT test as well as parasite rescue and transformation assay. Footpad swelling and parasite load of infected mice were measured by several techniques. The immune responses were evaluated by measuring the levels of IFN‐γ, IL‐4, nitric oxide and arginase. The results indicated that S. ebulus can significantly decrease L. major promastigotes and amastigotes viability, but it was not toxic to macrophages. The lesion size, parasite burden and the level of ARG decreased in the treated infected mice, while the IFN‐γ‐to‐IL‐4 ratio and the level of NO increased significantly. Altogether, the S. ebulus extract is an effective compound for killing Leishmania parasite without excessive toxicity to the host cells and can cure the CL by switching the host immune responses towards Th1 response. Thus, it may be a perfect therapeutic option for CL treatment.     

Early IL‐10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users

Summary. The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin‐10 (IL‐10) production (P = 0.048), in the relative absence of interferon‐gamma (IFN‐γ) and IL‐2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN‐γ (magnitude P < 0.001, breadth P = 0.004) and IL‐2 responses, in the relative absence of IL‐10. Early IL‐10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN‐γ and IL‐2 production was inversely correlated with HCV RNA level at baseline (IFN‐γP = 0.020, IL‐2 P = 0.050) and week 48 (IFN‐γP = 0.045, IL‐2 P = 0.026). Intracellular staining (ICS) indicated the HCV‐specific IFN‐γ response was primarily from CD8⁺ T cells and NK cells, whereas IL‐10 production was predominantly from monocytes, with a subset of IL‐10 producing CD8⁺ T cells present only in those who progressed to chronic infection. IL‐10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.     

Therapeutic efficacies of chitosan against Pneumocystis pneumonia of immunosuppressed rat

This study was designed to investigate the therapeutic efficacy of chitosan on Pneumocystis pneumonia (PCP) in immunosuppressed rats. The PCP rat model was established using intramuscular injections of dexamethasone sodium phosphate. To estimate treatment effects of chitosan on rat PCP, weight gain, lung weight, lung weight/body weight (LW/BW) ratio and per cent survival were measured and the HSP70 mRNA expression of Pneumocystis carinii was detected using real‐time PCR analysis. Rat lung tissues were stained with HE, and their pathological changes, inflammatory cells and alveolar macrophages were observed by light microscopy. Rat lymphocyte numbers and the concentrations of IL‐10, IFN‐γ and TNF‐α were measured by flow cytometry and ELISA analysis. Additionally, the ultrastructure of P. carinii was examined by electron microscopy to evaluate the effects of chitosan on the protist. Our results demonstrated that chitosan has some apparent treatment effects on rat PCP by reducing HSP70 mRNA expression and lung inflammation, increasing the concentrations of IL‐10 and IFN‐γ as well as CD4⁺ T‐lymphocyte numbers, reducing the CD8⁺ T‐lymphocyte numbers and the concentration of TNF‐α and inducing significant ultrastructural damage to P. carinii. Although its precise therapeutic mechanism has yet to be determined, these results lay a theoretical foundation for PCP chitosan therapy.     

Type I IFNs promote the early IFN‐γ response and the IL‐10 response in Leishmania mexicana infection

The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in humans and most mouse strains. We previously showed that STAT4‐deficient mice, but not IL‐12p40‐deficient mice, have more parasites and progressively growing lesions unlike those of wild‐type mice, the lesions and parasite burdens of which plateau by 10–12 weeks post‐infection. This demonstrates a STAT4‐dependent, IL‐12/IL‐23‐independent pathway of parasite control. Type I IFNs are important in viral and other infections and can activate STAT4. We found that IFN‐α/βR‐deficient mice have a nonpersistent, early IFN‐γ defect, and a persistent, early IL‐10 defect, without changes in serum IL‐12 or LN‐derived nitric oxide. We found less IL‐10 per cell in CD25⁺CD4⁺ T cells and possibly fewer IL‐10‐producing cells in the draining LN of IFN‐α/βR‐deficient vs. wild‐type mice. IFN‐α/βR‐deficient mice have chronic, nonprogressive disease, like wild‐type mice, suggesting that IL‐10 and IFN‐γ defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control of L. mexicana. Also, the lack of lesion resolution in IFN‐α/βR‐deficient mice despite lower IL‐10 levels indicates that other pathways independent of T cell IL‐10 help prevent an IL‐12‐driven clearance of parasites.     

Modulation of cytokine gene expression in spleen and Peyer's patches by feeding dahi containing probiotic Lactobacillus casei in mice

OBJECTIVE: In present study, feeding effect of probiotic dahi containing Lactobacillus casei on immune system in terms of cytokine gene expression in the spleen and Peyer's patches of mice was evaluated. METHODS: Animals were divided into three groups and fed with; synthetic diet [control group (CD)], dahi containing mixed dahi culture [control dahi‐fed group (CDF)]; and probiotic dahi fed group (PDF) for 28 days. The mRNA levels of IL‐2, IL‐4, IL‐6 and IFN‐γ were examined after 14 and 28 days. Total lactobacilli and lactococci counts were determined in the feces. RESULTS: The mRNA levels of IFN‐γ in both spleen and Peyer's patches was found to be significantly increased in PDF animals after 14 and 28 days (P < 0.05) compared with CD and CDF groups. The abundance of IL‐2 mRNA also increased significantly in the Peyer's patches of PDF‐fed animals. No significant changes were observed in mRNA levels of IL‐4 and IL‐6 in both spleen and Peyer's patches during whole experimental period. Further, total fecal lactobacilli and lactococci counts in the PDF group were significantly increased during first 10 days, then remained higher up to day 28 compared to other two groups. CONCLUSION: It is concluded that feeding probiotic dahi enhanced the expression of Th1 type cytokines (IFN‐γ and IL‐2), especially in the mucosal immune organ (Peyer's patches) rather than in systemic organs (the spleen). This indicates that feeding with probiotic dahi may strengthen the host immune system and protect against the progression of various immune‐mediated diseases.     

Schistosoma japonicum: susceptibility of neonate mice born to infected and noninfected mothers following subsequent challenge

This study was to investigate the differences between neonate mice born to Schistosoma japonicum‐infected mothers and those born to noninfected mothers in subsequent challenge. The intensity of infection (evidenced by worm burden and liver egg burden) and liver immunopathology (number and size of liver granulomas) were significantly reduced in neonates from infected mothers (I.M.) compared with neonates from noninfected mothers (N.M.). Anti‐soluble worm antigen of S. japonicum (SWA) IgG could be detected in sera of neonates from I.M. (N.N./I.M.) at 1 week after delivery, remained a plateau for 2 weeks and gradually decreased until 8 weeks of age. Parasite‐specific IgM was not detected in sera from N.N./I.M. at any time after delivery. At 6 weeks after infection, the level of anti‐SWA IgG in infected neonates from I.M. (I.N./I.M.) was significantly higher than that of infected neonates from N.M. (I.N./N.M.). In addition, production of IFN‐γ, IL‐12 and TGF‐β by cultured splenocytes from I.N./I.M. was significantly increased, while the level of IL‐4 was significantly decreased when compared to those from I.N./N.M.. These data demonstrate that congenital exposure to schistosomiasis japonica may render neonatal mice born to I.M. less susceptible to subsequent challenge and result in down‐regulation of both infection intensity and immunopathology.