Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis.

Infection of cattle and sheep with the parasite Fasciola gigantica is a cause of important economic losses throughout Asia and Africa. Many of the available anthelmintics have undesirable side effects, and the parasite may acquire drug resistance as a result of mass and repeated treatments of livestock. Accordingly, the need for developing a vaccine is evident. Triton-soluble surface membrane and tegumental proteins (TSMTP) of 60, 32, and 28 kDa previously shown to elicit protective immunity in mice against challenge F. gigantica infection were found to be strongly immunogenic in sheep eliciting vigorous specific antibody responses to a titer>1:16,000 as assessed by enzyme-linked immunosorbent assay. Furthermore, the 60 kDa fraction induced production of antibodies able to bind to the surface membrane of newly excysted juvenile flukes and mediate their attrition in antibody-dependent complement- and cell-mediated cytotoxicity assays, and significant (P<0.05) 40% protection of sheep against F. gigantica challenge infection. Amino acid micro sequencing of the 60 kDa-derived tryptic peptides revealed the fraction predominantly consists of F. gigantica enolase. The cDNA nucleotide and translated amino acid sequences of F. gigantica enolase showed homology of 92% and 95%, respectively to Fasciola hepatica enolase, suggesting that a fasciolosis vaccine might be effective against both tropical and temperate liver flukes.

Papain-Based Vaccination Modulates Schistosoma mansoni Infection-Induced Cytokine Signals.

We have previously shown that immunization of outbred rodents with cysteine peptidases-based vaccine elicited type 2-biased immune responses associated with consistent and reproducible protection against challenge Schistosoma mansoni. We herein start to elucidate the molecular basis of C57BL/6 mouse resistance to S. mansoni following treatment with the cysteine peptidase, papain. We evaluated the early cytokine signals delivered by epidermal, dermal, and draining lymph node cells of naïve, and S. mansoni -infected mice treated 1 day earlier with 0 or 50 µg papain, or immunized twice with papain only (10 µg/mouse), papain-free recombinant S. mansoni glyceraldehyde 3-phosphate dehydrogenase and 2-Cys peroxiredoxin peptide (10 and 15 µg/mouse, respectively = antigen Mix), or papain-adjuvanted antigen Mix. Schistosoma mansoni infection induced epidermal and lymph node cells to release type 1, type 2 and type 17 cytokines, known to counteract each other. Expectedly, humoral immune responses were negligible until patency. Papain pretreatment or papain-based vaccination diminished or shut off S. mansoni infection early induction of type 1, type 17 and type 2 cytokines except for thymic stromal lymphopoietin and programmed the immune system towards a polarized type 2 immune milieu, associated with highly significant (P < 0.005 - <0.0001) resistance to S. mansoni infection.

Adjuvant selection for vaccination against murine schistosomiasis.

Schistosoma mansoni cercariae penetrate mouse epidermis, detach the glycocalyx and transform into schistosomula, triggering innate immune responses by host keratinocytes and Langerhans cells. Schistosomula leave the dermis and enter blood capillaries, releasing excretory/secretory products (ESP), which induce readily detectable primary adaptive immunity responses, dominated by T helper (Th) 1 and 17 cytokines. Partial protection against murine schistosomiasis may be achieved using subunit antigens and Th1 cytokine-inducing adjuvants. Conversely, resistance to primary and/or secondary schistosomiasis in rats, mice and humans is associated with production of Th2 cytokines. Accordingly, we reasoned that effective vaccination against murine primary schistosomiasis might be achieved provided selection of an adjuvant capable of skewing the S. mansoni larval ESP-mediated Th1/Th17 immune responses towards a Th2 profile. In an aim to select such an adjuvant, we administered the prototypical Th1 and Th2, respectively, C57BL/6 and BALB/c mice with polyinosinic-polycytidylic acid (Poly I/C), peptidoglycan (PGN), or thymic stromal lymphopoietin (TSLP) before exposure to S. mansoni cercariae. Serum antibody reactivity and ex vivo spleen cells (SC) immune responses to larval ESP, in a recombinant or multiple antigen peptide form, were assessed 1 week after infection. Injection with Poly I/C failed to increase interleukin (IL)-4 and led to elevated gamma interferon (IFN-γ) levels released by unstimulated or ESP-stimulated SC. Treatment with PGN triggered hightened amounts of IL-4, IL-17 and IFN-γ released by unstimulated or ESP-stimulated C57BL/6 SC. In contrast, TSLP succeeded in directing the ESP-mediated immune responses towards a Th2-biased profile in prototypical Th1 and Th2 mice.

Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis.

Excretory-secretory products (ESP) products of ex vivo Fasciola gigantica adult worms were used for immunodiagnosis of sheep experimental infection with F. gigantica and natural infection with Fasciola spp. by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Specific IgG antibody binding to native or denatured ESP was detected as early as 2 weeks after experimental sheep infection with 100 or 200 metacercariae. No specific IgG antibody binding was displayed by sera obtained from 192 sheep considered to be Fasciola- and other parasite-free by microscopic examination of bile and feces. Additionally, sera from 200 apparently Fasciola-free sheep, yet infected with other parasites, were all negative. The data, thus, indicated that ESP-based ELISA reached nearly 100% sensitivity and specificity in immunodiagnosis of sheep fasciolosis. As expected, the ESP molecules were immunogenic in sheep eliciting interleukin-12p40 mRNA response and considerable amounts of antibodies, which were able to bind to the surface of newly excysted juvenile worms as judged by membrane indirect immunofluorescence, and mediate their attrition via antibody-dependent cell-mediated cytotoxicity. The ESP-induced cellular and humoral immune responses were associated with a modest reduction in worm count, yet with a highly significant (P<0.0001) decrease in size of recovered worms, thus suggesting that ESP immunization might be a safe and cost-effective strategy for reducing transmission of the infection.

Evaluation of cholesterol content and impact on antigen exposure in the outer lipid bilayer of adult schistosomes.

Developing and adult Schistosoma mansoni and S. haematobium intact worms do not bind specific antibodies, likely because of structural and biochemical modifications of the outer lipid bilayer. We have estimated the amount of cholesterol in the apical membrane of adult schistosomes via extraction with the membrane-impermeable, cholesterol-binding drug, methyl-beta-cyclodextrin (MBCD), followed by filipin staining of the worms, and evaluation of the amount of cholesterol released in the medium by a commercially available, enzymatic colorimetric assay. Positive correlations between amount of released cholesterol, MBCD concentration, and worm number and age provided evidence for the sensitivity and validity of the newly developed method. Treatment with 40 mm MBCD for 2 h at 37 degrees C led to total loss of cholesterol from the worm outer membrane, as assessed by filipin staining, and the released cholesterol values were used to estimate the amount of cholesterol per worm and per an approximate surface area unit. Additionally, total depletion of outer membrane cholesterol was associated with exposure of surface membrane antigens to specific antibody binding in 50% and 70% of S. haematobium and S. mansoni worms, respectively. These findings together suggest that cholesterol is an essential, but not the sole, factor in sequestration of surface membrane antigens in schistosomes.

Immunogenicity and vaccine potential of dipeptidic multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase.

Six peptides, A, B1, B, C, D and E, derived from the primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) were selected based on lowest homology to human glyceraldehyde 3-P dehydrogenase (G3PDH), multimerized in dipeptidic multiple antigen peptide (D-MAP) constructs and used for the immunization of BALB/c mice. Tetrabranched D-MAPs A-B, B1-C and D-E and the bis-D-MAP B-E elicited strong cell-mediated and antibody responses against immunogen, unit peptides and their cognate sequences in the native and denatured protein. D-MAP A-B induced protection against challenge infection. Immunization with D-MAP B1-C failed to affect the challenge worm parameters, probably because peptides B1 and C, previously shown to elicit immune responses associated with increase and decrease in challenge worm fertility, respectively, induced immune responses with opposing effects when combined in a D-MAP construct. A similar suggestion may explain the failure of D-MAP D-E to protect the host against challenge infection. In contrast, immunization with D-MAP B-E resulted in robust protection of the host, possibly because it contains peptides known to evoke immune responses associated with a significant decrease in challenge worm burden and fertility. The data together suggest that the specificity, not the quantity, of the induced immune responses is the determining factor for the efficacy of synthetic peptide-based vaccine for schistosomiasis.

[Electromyographic changes in bruxism after auricular stimulation. A randomized controlled clinical trial]. / Variazioni elettromiografiche nel bruxismo dopo stimolazione auricolare. Uno studio clinico controllato e randomizzato.

AIM: The aim of this study was to verify in bruxism patients the possible efficacy of auricular stimulation in reducing the hypertonicity of some masticatory muscles. METHODS: Forty-three bruxism patients were randomly allocated to 3 groups: acupuncture, needle contact for 10 seconds, no treatment (control). Helkimo's clinical dysfunction index (CDI) and anamnestic dysfunction index (ADI) were used to assess the functional state of the masticatory system. The resting electrical activity of the anterior temporalis (AT), masseter (MM), digastric (DA) and sternocleidomastoid (SCM) muscles was measured, according to Jankelson, with surface electrodes at baseline, after stimulation and continually for 30 minutes (120 measurements in total). The electromyographical variations in the 3 groups were studied with t test for independent samples. RESULTS: Acupuncture and needle contact were superior to control in reducing the muscle hypertonicity of all muscles except SCM. In the comparison between acupuncture and needle contact the former showed better results only for the right TA and left DA (p = 0.000). CONCLUSION: In this study it was possible to measure the efficacy of the stimulation of only one point or area, which is an ideal model for research in acupuncture. The auricular area we chose for stimulation was never used before for the purpose of relaxing masticatory muscles. Acupuncture and needle contact for 10 seconds showed similar effects.

Human and murine humoral immune recognition of multiple peptides from Schistosoma mansoni glyceraldehyde 3-P dehydrogenase is associated with resistance to Schistosomiasis.

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) is a target of cellular and humoral immune responses of Brazilian and Egyptian subjects putatively resistant to reinfection with S. mansoni. In an aim to develop a safe, stable and effective vaccine based on this promising molecule, six peptides derived from its primary sequence were selected based on the lowest homology to human G3PDH. The synthetic peptides were tested by ELISA against plasma of humans putatively susceptible or resistant to reinfection with S. mansoni or S. haematobium following chemotherapeutic cure of previous infection. Repeat experiments indicated that the six peptides bear human B-cell epitopes that bind immunoglobulin (Ig)M, IgG1 and IgG3 antibodies. Resistance to reinfection appeared to be significantly associated with humoral immune responses to multiple peptides. This contention was supported by studies in the murine model, whereby we examined the B cell immune responses of Swiss and inbred BALB/c and C57BL/6 mice immunized with recombinant SG3PDH (rSG3PDH) to the six SG3PDH-derived peptides. The serum antibodies of rSG3PDH-immunized Swiss mice were directed to only one of the six peptides tested by ELISA. Antibodies from rSG3PDH-immunized C57BL/6 and BALB/c mice bound, respectively, to four and six out of six peptides. In contrast to Swiss mice, immunization of C57BL/6 and BALB/c mice with rSG3PDH induced protection against challenge cercariae which reached the level of significance (P < 0.05) for BALB/c mice. The data together indicate that host recognition of multiple peptides of a candidate vaccine antigen is necessary for the expression of its ability to contribute to protective immunity against Schistosomiasis.

Role of IgE in primary murine Schistosomiasis mansoni.

Schistosoma mansoni infection proceeds in normal mice in the absence of detectable levels of polyclonal or specific immunoglobulin (Ig)E until worms mature and deposit eggs. Hence, the course of a primary S. mansoni infection is not expected to vary appreciably in mice with defects in the IgE production. Experimental increase of IgE production early after infection may, however, influence worm development. In the first approach towards this goal, BALB/c mice were injected with interleukin(IL)4 to raise the level of endogenously synthesized IgE. A significant increase in serum polyclonal IgE and antischistosome IgG1 during the prepatent period was not associated with significant changes in worm and egg burden or liver pathology. During the second approach, mice were injected with IgE which was affinity purified from serum of BALB/c mice infected for 16 weeks with S. mansoni. The purified IgE bound to carbohydrate-independent epitopes of soluble antigens from 3 h larvae, adult worms and eggs and recognized the schistosomular surface membrane. No differences in worm and egg load or granuloma number and size were noted between untreated and exogenous IgE-injected mice. Together, the data demonstrate that by itself IgE does not influence the outcome of infection in primary murine S. mansoni.

Human T- and B-cell responses to Schistosoma mansoni recombinant glyceraldehyde 3-phosphate dehydrogenase correlate with resistance to reinfection with S. mansoni or Schistosoma haematobium after chemotherapy.

Recently we reported that human T- and B-cell recognition of a 42-kDa protein (p42) in soluble extracts of adult Schistosoma mansoni worms correlates with resistance to reinfection with S. mansoni or S. haematobium. Amino acid microsequencing of p42 revealed that it consists predominantly of schistosome glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). We have expressed SG3PDH in Escherichia coli and purified the recombinant protein in a soluble and enzymatically active form. Recombinant SG3PDH (rSG3PDH) reacted with human monospecific antibodies to p42. Lymphoproliferation and production of interleukin-4 and gamma interferon (IFN-gamma) after in vitro stimulation with rSG3PDH and serum isotype responses to rSG3PDH were examined in individuals with extremes of resistance and susceptibility to reinfection after treatment of previous S. mansoni or S. haematobium infection. Lymphoproliferation and IFN-gamma production in response to rSG3PDH and the presence of serum immunoglobulin G1 (IgG1), IgG3, and IgA antibodies to rSG3PDH generally characterized individuals who are resistant to reinfection after chemotherapy. The data indicate that T- and B-cell immune reactivity to rSG3PDH correlates with resistance to reinfection, confirming previous studies identifying SG3PDH as a target of protective immunity in humans, and suggest that SG3PDH should be investigated as a possible vaccine for human schistosomiasis.

A Schistosoma mansoni 62-kDa band is identified as an irradiated vaccine T-cell antigen and characterized as calreticulin.

The Schistosoma mansoni soluble adult worm antigen (SAWA) bands of 62/60 kDa were found to contain immunodominant T-cell immunogen(s) in irradiated cercariae-immunized Swiss and C57BL/6 mice. In the present study, spleen T cells of BALB/c mice immunized twice with ultraviolet light-irradiated cercariae proliferated and produced interleukin 4 in response to the 62/60-kDa SAWA bands in T-cell western assays. To characterize the 62/60-kDa bands, an adult S. mansoni worm cDNA expression library constructed in lambdagt11 was immunoscreened with serum of mice immunized with the 62/60-kDa antigens, and the immunoreactive cDNA inserts were sequenced. Purified 62- and 60-kDa proteins were used for amino acid microsequencing and for immunization studies in Swiss, C57BL/6, and BALB/c mice and rabbits. Taken together, the data indicated that the 60-kDa molecules are poorly immunogenic in mice and rabbits, whereas the 62-kDa species identified as S. mansoni calreticulin, is a good T- and B-cell antigen and represents a potential vaccine candidate.

Parasite-specific antibody profile in human fascioliasis: application for immunodiagnosis of infection.

The antibody isotype response to an adult Fasciola worm antigen preparation (FWAP) was examined in sera from 60 Egyptians with parasitologically confirmed fascioliasis by an ELISA. The FWAP-specific IgG1 and IgG4 antibodies were found in 97-100% of the patients. The ratio of the mean absorbance values between infected patients and healthy controls was 9.7 and 29.7 for IgG1 and IgG4 antibodies, respectively. The IgM, IgA, IgG2, and IgG3 antibodies were less dominant. In contrast to IgG1 antibodies, which were often detected in sera from patients infected with Schistosoma, Echinococcus granulosus, Ascaris lumbricoides, Ancylostoma duodenale, or Hymenolepis nana, FWAP-specific IgG4 antibodies were detected exclusively in the sera of patients with fascioliasis. The data thus support the conclusion that an IgG4/ELISA with crude FWAP as antigen may be used for sensitive and accurate immunodiagnosis of human fascioliasis.

Tissue engineering with HaCaT cells and a fibroblast cell line.

Most skin models consist of primary cells. Our aim was to develop a highly reproducible skin model consisting only of cell lines to investigate irradiation effects. The spontaneously immortalized human keratinocyte line HaCaT is known for its capacity for epidermal differentiation. As an organotypic coculture, HaCaT cells were grown air-exposed on top of a dermis equivalent consisting of a murine fibroblast cell line (L929) in collagen. The technique for the preparation of this coculture system is described. After 3 weeks a multilayered epithelium with signs of differentiation developed. The expression of several markers for differentiation and basal membrane formation were compared with those of healthy human skin by immunohistochemical staining. In the epithelium of the skin model several cytokeratins, especially keratin 10, and involucrin were expressed comparable to normal skin. Laminin expression was found along the basal zone of the epithelium. BrdU labeling indicating proliferation was mainly found in the basal parts of the epithelium. Differentiated cells showing DNA fragmentation were detected in the upper parts of the epithelium by the TUNEL assay. Fluorescence in situ hybridization was used to discriminate between HaCaT and L929 cells in the coculture. Some L929 cells growing on top of the epithelium could be detected. This might have been due to an invasion of highly proliferating L929 cells and might be one of the limits of tissue engineering with cell lines. In conclusion, the organotypic coculture used as a skin model is a promising additional tool for addressing specific research questions.

T and B cell reactivity to a 42-kDa protein is associated with human resistance to both schistosomiasis mansoni and haematobium.

Egyptian subjects living in areas endemic for Schistosoma mansoni or Schistosoma haematobium were selected on the basis of their apparent extremes of resistance or susceptibility to schistosomiasis and examined for T and B cell responses against the major electrophoretically resolved protein species from soluble adult worm extracts. A 42-kDa band was specifically recognized by a significant majority of subjects resistant to schistosomiasis. The 42-kDa species (p-42) from S. mansoni and S. haematobium were immunologically cross-reactive and induced significant protection in mice and hamsters against infection with cercariae. Amino acid sequence analysis of S. mansoni p-42 showed that it consists predominantly of glyceraldehyde 3-P dehydrogenase (G3PDH), which has been shown to be preferentially recognized by the sera of Brazilian subjects resistant to schistosomiasis mansoni. The present data extend the previous findings and imply that S. mansoni-derived G3PDH represents a target of protective T and B cell-mediated antischistosomiasis immunity in humans.

Schistosoma mansoni infection in IgE-producing and IgE-deficient mice.

The immunoglobulin E (IgE) response, a hallmark of helminthic infection, is generally considered a major host defense against schistosomiasis mansoni. In support, it was reported that mice with a null mutation of the Ce gene, which are thus incapable of making IgE, developed Schistosoma mansoni worm burdens 2-fold greater than wild-type mice. However, in another study, reduction of the IgE response in mice to a primary S. mansoni infection by anti-IgE treatment resulted in decreased worm burden and fecundity, suggesting that IgE plays a detrimental, rather than beneficial, role for the host in schistosomiasis. In a third study, S. mansoni worm burden and egg production in normal and in IL-4-deficient mice that produce negligible IgE levels did not differ significantly, and it appeared that IgE did not affect parasite survival or fecundity. In an attempt to resolve these controversies, we examined hepatic worm load and egg production in the liver and small intestine of IgE-deficient (SJA/9) and control IgE-producing (SJL/J) mice, 8 wk after S. mansoni infection. No differences were observed in worm burden, total egg production, and number of eggs produced per female worm in the 2 mouse strains, confirming the data that imply that IgE does not play an essential role in primary S. mansoni infection.

The myofibroblast markers α-SM actin and ß-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin.

To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: α-smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and ß-actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed α-SM actin. Furthermore, similar amounts of ß-actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed α-SM actin. They expressed ß-actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing α-SM actin and overexpressing ß-actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts.

Preferential recognition of human myocardial antigens by T lymphocytes from rheumatic heart disease patients.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune sequelae of upper respiratory infections with group A streptococci (GAS). To gain a better understanding of the pathogenesis of these diseases, we examined the in vitro proliferative responses of peripheral blood mononuclear cells (PBMC) from RHD patients to human myocardial proteins in a T-cell Western assay. A number of myocardial proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were recognized by PBMC from both patients and controls. However, PBMC from a significant percentage of RHD patients (40%) responded to a discrete band of myocardial proteins migrating with an apparent molecular mass of 50 to 54 kDa while none of the control subject PBMC responded to this protein band (P < or = 0.0001). To further investigate the link between infections with GAS and autoimmune carditis, we studied the proliferative responses of PBMC from patients and controls to myocardial proteins before and after in vitro stimulation of the cells with opsonized GAS isolated from ARF patients. Priming of PBMC with rheumatogenic GAS caused the percentage of RHD patients responding to the 50- to 54-kDa myocardial proteins to increase from 43 to 90% (P < or = 0.0284). By contrast, PBMC from control subjects failed to recognize the 50- to 54-kDa myocardial proteins even after stimulation with the opsonized streptococci (P < or = 0.0001). The assay sensitivity was increased from 40 to 90% after priming of a patient's cells with opsonized GAS, but the positive predictive value was 100% in both unprimed and primed cultures. Antibodies generated to partially purified 50- to 54-kDa myocardial proteins did not cross-react with either streptococcal homogenates, purified M protein, myosin, laminin, or vimentin, suggesting a lack of cross-reactivity at the humoral level. This study suggests that the 50- to 54-kDa myocardial proteins contain a putative antigen that is preferentially recognized by T cells from RHD patients and demonstrates that exposure to streptococcal antigens enhances the ability of patients to recognize these proteins.

Immunization of mice with ultraviolet-attenuated cercariae of Schistosoma mansoni transiently reduces the fecundity of challenge worms.

In the present study cohorts of ICR and BALB/c mice were immunized with u.v.-irradiated cercariae of S. mansoni and challenged 5 weeks later, in parallel with unimmunized control mice, with approximately 100 cercariae. Total worm burdens at 5, 6, 7 and 8 weeks after challenge were significantly reduced by 27-65% in immunized mice. The total number of eggs and the number of eggs/female worm trapped in liver and small intestine were reduced significantly at 6 and 7 weeks post challenge in immunized, as compared to unimmunized mice. Decrease in tissue egg load could be achieved in BALB/c mice passively transferred with spleen cells from u.v.-attenuated cercaria-immunized mice. The proportion of female worms laying eggs in vitro was diminished only in worms recovered from highly resistant mice. The reduction in worm oviposition in immunized mice was no longer apparent at 8 weeks. The data taken together indicate that highly effective immunization of outbred and inbred mice with attenuated cercariae leads to significant, but transient, impairment in challenge worm egg production.