Search

This article analyzes data from scientific publications (mainly reviews) concerning the link between human neurocysticercosis and epilepsy. Along with data from our own studies on experimental hippocampal sclerosis induced by a Taenia crassiceps metacestode factor in mice, it explores the connection between mechanisms that likely favor the development of epilepsy in cases of human neurocysticercosis. The data from both sources suggest the idea that the T. solium metacestode factor causes hippocampal sclerosis and later epilepsy in humans with neurocysticercosis.

Objectives: Enterococcus faecalis has been associated with root canal infections, while Streptococcus mutans has a central role in the etiology of dental caries. One of the main reasons of endodontic failure has been associated to the presence of E. faecalis and the formation of biofilms. S. mutans inhabits the oral cavity, specifically the dental plaque, which is a multispecies biofilm formed on the hard surfaces of the tooth. The biofilm formation is the main factor determining the pathogenicity of numerous bacteria. Natural antimicrobial peptides in the saliva protect against pathogenic bacteria and biofilms. The aim of this study was to assess the ultrastructural damage induced by salivary peptides in bacteria involved in biofilms has not been previously studied., Material and Methods: Enterococcus faecalis and S. mutans incubated with cystatin C, chromogranin A, or histatin 5 were morphologically analyzed and counted. The ultrastructural damage was evaluated by transmission electron microscopy (TEM)., Results: A decrease in bacterial numbers was observed after incubation with cystatin C, chromogranin A, or histatin 5, compared to the control group (P <  0.001). Ultrastructural damage in E. faecalis and S. mutans incubated with salivary peptides was found in the cell wall, plasma membrane with a decreased distance between the bilayers, a granular pattern in the cytoplasm, and pyknotic nucleoids., Conclusions: This study demonstrated that salivary peptides exert antibacterial activity and induce morphological damage on E. faecalis and S. mutans. Knowledge on the ultrastructural damage inflicted by salivary antimicrobial peptides on two important bacteria causing dental caries and root canal infections could aid the design of new therapeutic approaches to facilitate the elimination of these bacteria., (© 2021 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

An experimental Taenia crassiceps mouse model was used to assess the role of Taenia solium metacestode factor (Fac) in human neurocysticercosis. Intraperitoneal infection with T. crassiceps metacestodes or subcutaneous inoculation with a T. crassiceps metacestode factor (Fac) produced significant impairment of performance (learning) in the Barnes maze and induced bilateral hippocampal sclerosis in mice. Several staining techniques revealed important cell dispersion, extensive apoptosis and cell loss in the dentate gyrus, hilus and CA1-CA3 regions of both hippocampi, as well as intense deterioration of the adjacent cortex. An outstanding disruption of its histoarchitecture in the surrounding tissue of all these regions and apoptosis of the endothelial cells were also observed.

Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes.

Seizures, headache, depression and neurological deficits are the signs and symptoms most frequently reported in human neurocysticercosis. However, the cause of the associated learning and memory deficits is unknown. Here, we used Taenia crassiceps infection in mice as a model of human cysticercosis. The effects of T. crassiceps metacestode infection or T. crassiceps metacestode factor (MF) treatment on mouse hippocampal cells were studied; control mice were included. At 45 days after infection or treatment of the mice with MF, all mice were anaesthetized and perfused transcardially with saline followed by phosphate-buffered 10% formalin. Then the brains were carefully removed. Coronal sections stained using several techniques were analysed. Extensive and significant apoptosis was found in the experimental animals, mainly in the dentate gyrus, CA1, CA2, CA3 and neighbouring regions, in comparison with the apparently intact cells from control mice (P < 0.01). These results suggest that neurological deficits, especially the learning and memory deficits, may be generated by extensive apoptosis of hippocampal cells.

This study was undertaken to determine whether a parasite substance produces structural pathology in the mouse spleen. A low-molecular-weight Taenia crassiceps metacestode factor (MF) isolated from the peritoneal fluid of female mice infected with T. crassiceps metacestodes induced pathological and immunological changes in mouse spleen cells in vivo. Electron microscopy and confocal microscopy revealed severe changes in the spleen histoarchitecture of T. crassiceps-infected and MF-treated mice. Apoptotic degenerated spleen cells were observed in the white and red pulps and were more conspicuous in the white pulp of the spleen from the T. crassiceps-infected mice than in that of the MF-treated mice. Flow cytometry analysis revealed that the numbers of spleen CD4+T cells were significantly lower in both experimental groups than in control mice. The ex vivo expression of transforming growth factor (TGF)-β and factor Foxp3 were significantly higher in splenocytes of the experimental mice than the basal expression observed in the control cells. These findings may have potential applications for a better understanding of the host-parasite relationship in human neurocysticercosis.

The histopathological effects of Taenia crassiceps infection or T. crassiceps metacestode factor inoculation on the mouse ovary were determined using six female mice in three groups: infected mice, mice inoculated with the metacestode factor and control mice. The control group was subcutaneously inoculated with healthy peritoneal fluid. The infected group was intraperitoneally inoculated with 40 T. crassiceps metacestodes, and the metacestode factor group was subcutaneously inoculated with T. crassiceps metacestode factor (MF). Light and electron microscopy and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling) assays revealed a significant increase in ovarian follicular atresia (predominantly in antral/preovulatory stages of development), oocyte degeneration (P< 0.05), and a decrease in the amount of corpus luteum in follicles of mice infected and inoculated with MF compared with the control group. Significant abnormalities of the granulosa cells and oocytes of the primordial, primary and secondary ovarian follicles occurred in both treated mouse groups (P< 0.05) compared with no degeneration in the control group. These pathological changes in female mice either infected with T. crassiceps metacestodes or inoculated with T. crassiceps MF may have consequences for ovulation and fertility.

The present research was performed to isolate and study the effects of a low molecular weight (<1300Da) parasite-associated substance, obtained from peritoneal fluids of female mice infected with Taenia crassiceps cysticerci, on seminiferous epithelium cells of male mice testis. The results showed an intense disruption of Sertoli cells and germ cells within the seminiferous tubules of experimental mice, along with the destruction of their gap junction (GJ). Significant generalized apoptosis of germ cells within seminiferous tubules was determined by TUNEL staining (P=0.0159). In addition, a significant number of infiltrating macrophages were found in the luminal space of these seminiferous tubules (P<0.0001). Finally, electron microscopy studies revealed structural and morphological abnormalities in the somatic cells (Sertoli and Leydig cells) and in the germ cells, primarily in the round and elongate spermatids., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This research was carried out to study the effects of infection with Taenia crassiceps cysticerci on the seminiferous epithelium histoarchitecture in the testes of male mice. Our results showed a severe disruption of the histoarchitecture of the testis epithelium in infected mice. In these animals, a significant infiltration of macrophages within seminiferous tubules was observed (P < 0.001). Generalized apoptosis of germ cells within the seminiferous tubules was observed, as assessed by TUNEL assay and apoptotic nuclei were quantified. The total number of fluorescent objects (DNA) (including clusters, singles, and objects in clusters) was significantly higher in the infected cells than in the control group (P = 0.0286). Observation of the interstitial tissue showed disorder and deterioration of many Leydig cells of infected mice, as well as intense vacuolization and destruction of their inter-cellular junctions. Several ultrastructural abnormalities were observed through electron microscopy as well. The observed pathology could lead to a state of infertility., (Copyright © 2010 Elsevier Inc. All rights reserved.)

After an intraperitoneal infection of mice with Taenia crassiceps metacestodes, peritoneal inflammatory cells labeled with fluoresceinated MoAb anti-mouse were analyzed by flow cytometry. Apoptosis was studied by annexin A/PI, TUNEL assays, DNA laddering, caspase-3 activity, and electron microscopy. An important continuous decrease of CD4+, CD8+ and CD19+ lymphocytes, and an increase of eosinophils and macrophages throughout the observation time were found. Apoptosis of eosinophils was quantified during the observation period with a peak at 6 days post-infection (67.27%). In an additional experiment at 12 days post-infection using TUNEL staining, a high level of apoptosis of eosinophil (92.3%) and a significant decrease of CD4+, CD8+, and CD19+ lymphocytes were confirmed. Caspase-3 activity in peritoneal fluid, peritoneal cells' DNA fragmentation, and apoptosis of eosinophils and monocytes were found. The dramatic decrease of peritoneal inflammatory T and B cells and the high level of apoptosis of inflammatory eosinophils induced in mice by infection with T. crassiceps cysticerci may be important factors of the immunosuppression observed in cysticercosis.

The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.

In the current research, we report apoptosis of lymphocytes in the inflammatory reaction around metacestodes in muscle tissue from cysticercotic pigs. Two events, high metacestode viability (100%) and high cysteine protease activity were found to be closely related to a high phosphatydilserine expression by inflammatory lymphocytes (56%). Testing the RPMI medium used for washing away inflammatory cells from metacestodes with 100% viability, with the fluorescent substrate Z-Phe-Ala-AFC for measuring cysteine protease activity, significant fluorescent values were found. In contrast, tests performed with RPMI medium used for washing away inflammatory cells from metacestodes with 90% viability or less, showed low fluorescence values. Flow cytometry analyses of inflammatory cells obtained from four naturally cysticercotic pigs, and stained with Annexin-V/PI, showed lymphocytes expressing phosphatidylserine with values of 0, 6, 41 and 56% on their outer surfaces. Electron microscopy studies of inflammatory cells from metacestodes with 100% viability, showed lymphocytes with strangled and fragmented nuclei, and heterochromatin displaced to the nuclear periphery. In addition, DNA from these cells showed fragmentation in electrophoresis assays. Apoptosis of lymphocytes in the inflammatory reaction around Taenia solium metacestodes, might have been induced by the parasite cysteine protease, and may be involved in impairing cell-mediated immune responses in human and porcine cysticercosis.

Infection of the central nervous system by Taenia solium cysticerci is the cause of human neurocysticercosis, a major neurological infection in the Third World and an emerging infectious disease in the United States. We previously isolated a cysteine proteinase from cysticerci of Taenia crassiceps and demonstrated that it degrades human IgG in vitro. We have now isolated a 48 kDa thiol-dependent proteinase from T. solium. The T. solium enzyme also degrades human IgG, but does not significantly degrade albumin. IgG degradation was inhibited by cysteine proteinase inhibitors, but not significantly by inhibitors of aspartic, serine, or metalloproteinases. The peptide substrate specificity and pH optimum resemble cathepsin L. The Km for the peptide substrate Z-Phe-Arg-AFC was calculated to be 7.0 x 10(-6) M, the Kcat was 1.98 x 10(-5) s(-1), and the Kcat/Km 2.84 x 10(9) M(-1) s(-1), a value which is within the diffusion control limit for highly catalytic enzymes. We propose that immunoglobulin degradation by the T. solium cysteine proteinase may play a key role in the host-parasite interface and could be employed as a target for chemotherapy.

The purpose of this study was to determine the effect of the implantation of Taenia solium metacestodes and the treatment with suppressive metacestode factor (F1) on the ability of spleen cells from Balb/c mice to produce cytokines. Cytokine production was estimated 12 days following the implantation or 4 days after the last dose of F1 (five doses) by RT-PCR and flow cytometry analyses. Spleen cells were obtained from metacestode-implanted, F1-treated and control mice. They were stimulated with concanavalin A (ConA) ex vivo and used for RT-PCR studies and for CD25 expression and intracellular cytokine production estimations using specific monoclonal antibodies labeled with phycoerithrin or fluorescein. Results of the RT-PCR showed that all cells expressed IFN-gamma, IL-2 and IL-4 mRNAs. IL-10 mRNA was not expressed in any case. Flow cytometry analyses showed that both spleen CD4+ and CD8+ cells from metacestode-implanted or treated-F1 mice expressed significantly diminished percentages of CD25 when compared with control cells (P<0.05). The estimation of intracellular cytokines showed that the production of IL-2 and IL-4 in CD8+ cells, and of IFN-gamma in CD4+ cells from mice implanted with metacestodes was significantly impaired when compared with the values from control cells (P<0.05).

In double-blind, immunological assays, samples of cerebrospinal fluid (CSF) from 141 patients with neurocysticercosis (NCC) or other neurological disorders were tested for IgG reacting with the excretory/secretory (E/S) antigens of Taenia solium metacestodes. The results for the cases of NCC were then correlated with the developmental stage of the metacestodes present in each case, as assessed by computerized tomography and magnetic-resonance imaging. In the ELISA first used, the samples of CSF from most (88%) of the patients with the vesicular stage of NCC (some of whom also had the degenerate and/or calcified metacestodes) were found to contain the specific IgG. In electro-immunotransfer blot (EITB) assays, three of the E/S antigens, of 95, 49 and 29 kDA, were recognized by 86%-100% of the ELISA-positive CSF. When these three antigens were isolated and tested, as a pool, against all the CSF samples in double-blind ELISA, almost all (96.6%) of the CSF samples from patients with metacestodes at the vesicular stage were recognized. In the detection of individuals with vesicular metacestodes, the assay based on the three isolated antigens was significantly more sensitive than that based on the crude extract of E/S antigens (P < 0.05). In EITB assays based on the three antigens, the isolated proteins were again recognized by IgG in the CSF samples from those with vesicular metacestodes, but without the background 'noise' seen with the crude extract. In every assay employed, none of the CSF samples from NCC cases who only harboured degenerative and/or calcified metacestodes and none of those from patients who had other neurological disorders gave a positive result. The use in ELISA and EITB of antigens purified from crude extracts of metacestode E/S proteins could improve the immunodiagnosis of the vesicular stage of NCC, and allow better evaluation of NCC cases both pre- and post-treatment.

Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.

The purpose of this study was to determine the Th1 and Th2 cytokine responses induced by Taenia solium metacestode antigens in mice and correlate them with the immune responses elicited in vivo. To assess this aim, mice were inoculated with metacestode antigens. RNA was obtained from spleen cells of immunized or control mice incubated with metacestode antigens and used to determine the cytokine profile. Peripheral blood eosinophilia was measured daily in each mouse and specific serum antibody levels were determined. Results showed that metacestode antigens induce the synthesis of IL-4, IL-5 and IFN-gamma mRNAs in spleen cells. They also induced peripheral blood eosinophilia and elicited specific IgE and IgG antibodies, especially IgG1. Three antigens were recognized by all IgG subclasses and by IgE (104, 88 and 7 kDa), and a 57-kDa protein was recognized by IgG1, IgG2a, IgG2b, and IgE. IgG1 and IgG2b recognized 52, 30 and 20 kDa antigens. Immune responses elicited in vivo and the cytokine profile showed good correlation.

The immune responses of indigenous naked neck (NaNa and Nana) and normally feathered (nana) chickens against a Salmonella Gallinarum (SG) infection were evaluated and compared with those of a commercial line (B-380). Groups of 28-day-old chickens (NaNa, Nana, nana, and B-380) were immunized orally and subcutaneously with 50 microg of SG antigens. Control non-immunized animals were inoculated with sterile saline solution. All chickens were challenged with 1 LD(50) of SG and mortality was recorded daily for 20 days. Antibodies to SG were measured in sera before immunization, before the challenge, 10 days after the challenge, and at sacrifice. Peripheral blood lymphocyte proliferation assays were performed using concanavalin A and SG antigens. Results showed that non-immunized Nana chickens exhibited the best natural resistance to Salmonella infection, since only 30% of them died. In contrast, all control B-380 chickens died by the 13th day. Immunization with SG induced immunity in chickens of all genotypes. Indigenous naked neck and normally feathered chickens showed a higher survival rate when compared with B-380 chickens. Immunized Nana chickens showed the highest antibody titres (P<0.05) as well as the highest thymidine incorporation in peripheral blood lymphocytes stimulated with con A or SG antigens (P<0.05). The results show that Nana chickens are the most resistant to SG infection and the best responders to vaccination with SG antigens.

We have previously reported that a factor secreted by the metacestode of Taenia solium (MF) is able to transform Syrian hamster embryo cells. The aim of this study was to analyze the genotoxicity of MF in cultured human lymphocytes using the micronucleus assay. Results show a significantly high frequency of micronucleated cells in lymphocyte cultures treated with MF. Although further experiments are needed to determine whether this factor is also secreted by T. solium metacestodes in humans, analysis of the frequency of micronucleus induced in cultured human lymphocytes indicates that DNA instability induced by MF could represent a risk for malignant transformation., (Copyright 2003 Wiley-Liss, Inc.)

To detect IgG antibodies to Taenia solium, a controlled double-blind study was conducted using 91 coded cerebrospinal fluid samples from patients with neurocysticercosis (NCC) and other neurologic disorders. Samples were tested in an enzyme-linked immunosorbent assay (ELISA) using metacestode excretion/secretion antigens. The results were correlated with data from medical records on the diagnosis of NCC (based on computed tomography and magnetic resonance imaging criteria) and other neurologic disorders. The ELISA results were positive in 22 of the 24 cases with active NCC. In contrast, six cases with calcified cysts (inactive NCC), as well as one case in a transitional stage, were negative. One case with a calcified granuloma and another with a granuloma plus calcifications (classified as inactive NCC) had positive results. The remaining negative results corresponded to other neurologic disorders (58 cases). The results of the ELISA showed a significant difference between active and inactive NCC (P = 0.0034).

A metacestode factor (MF) isolated from live metacestodes of Taenia solium suppresses humoral and cellular responses, and inhibits the inflammatory reaction around metacestodes implanted subcutaneously in mice. When this MF is digested with RNase (dMF), it loses the suppressive capacity, but acquires T-cell stimulant ability. By filtering MF through a Bio-gel P6 column, two components were separated. The first (F1) was suppressive. while the second (F2) stimulated T cells to proliferate. In these experiments, F2 or dMF was used with mouse spleen cells in stimulation assays in vitro. Spleen cells from mice treated with F2 or dMF were also stimulated with concanavalin A (Con-A) ex vivo. Flow cytometry analyses were performed to estimate cell proliferation, intracellular cytokine production. and restoration of CD4 cells. Spleen lymphocytes from mice previously treated with F2 or dMF and then stimulated with Con-A ex vivo exhibited a significant increase in cell proliferation and gamma interferon production by CD4+ (P<0.05) and CD8+ cells. These effects were concentration-dependent and inversely correlated with the amount of dMF or F2. Similar results were observed in normal mouse spleen T cells incubated with F2 or dMF and Con-A in vitro. Finally, dMF induced a significant restoration of CD4-cells in mice depleted of these cells.

Excreted/secreted products from Taenia solium metacestodes cultured in vitro were analyzed for peptidase activity using peptide substrates Z-Phe-Arg-AFC, Arg-AFC, and Z-Gly-Gly-Arg-AFC and zymography studies. Specific inhibitor profiles revealed mainly cysteine and metalloprotease activities. Hydrolysis of substrate Z-Phe-Arg-AFC was augmented by the addition of L-cysteine and acid pH, consistent with cysteine protease activity. Cysteine protease activity was more prominent in supernatants from living metacestodes cultured in PBS than in either RPMI or RPMI plus fetal calf serum and was proportional to the number of metacestodes. Flow cytometry analysis showed depletion of human T lymphocytes cultured with living T. solium metacestodes. CD4(+) expression was significantly decreased when metacestode E/S products and L-cysteine were added to lymphocyte cultures (P = 0.027). This peptidase activity was inhibited by E-64 indicating that the depletion of CD4(+) cells was due to cysteine protease activity. Thus, T. solium metacestodes produce excretory/secretory proteases. These enzymes may cleave molecules critical for the host immune response allowing the parasites to survive in the host tissues., (Copyright 2000 Academic Press.)

To assess the immunological status of mice parasitized with Taenia crassiceps metacestodes, 6-month old female BALB/c mice experimentally parasitized with T. crassiceps and immunized with Salmonella typhimurium antigens were infected with S. typhimurium virulent bacilli (1.6 x LD50). Both T. crassiceps-parasitized and immunized and parasitized mice showed a very high susceptibility to infection (**P < 0.01) with higher bacteremia than control and immunized-control animals and produced a reduced IgG response to S. typhimurium, antigens (* P < 0.05). This indicates that T. crassiceps is able to preclude development of immunity to S. typhimurium, because appropriate antibody production to a heterologous antigenic stimulus did not take place, and the bacteremia results suggest the parasitosis altered the mononuclear phagocyte system. It has been demonstrated that Taenia solium metacestodes produce a small RNA molecule in culture which suppresses humoral and cellular responses against homologous antigens in mice. We propose that T. crassiceps may be actively synthesizing such a factor, apart from other simultaneously acting immunomodulatory mechanisms, to induce an immunosuppressed state favorable to its development in the host.

Inhibition of inflammation by a Taenia solium RNA-peptide (metacestode factor, MF) was studied by scanning electron microscopy (SEM). Viable (96%) T. solium metacestodes obtained from a naturally infected pig were dissected and implanted in treated and control mice, removed at 6 and 12 days postimplantation (p.i.), and studied by SEM. At day 6, metacestodes in control mice showed vigorous inflammation, whereas in mice treated with MF they were apparently intact with exiguous inflammation. Mice immunized with T. solium metacestode antigens showed a moderate inflammation; those treated with both MF and T. solium antigens presented scanty inflammation. At day 12, metacestodes presented copious inflammation and severe damage to the sucker tissues in mice immunized with T. solium; in mice treated with either MF or MF and T. solium antigens there was only discrete inflammation. These observations illustrate the central role of MF in the inhibition of the early events leading to the parasite's destruction by means of an inflammatory response.

Studies of the immune response in chronic helminth infections suggest that parasites modulate the host's immune response. Taenia solium metacestodes, in particular, produce molecules that down-regulate cell-mediated immunity. We have described a small RNA peptide termed metacestode factor (MF) that depresses the murine immune response to Salmonella typhimurium antigens. MF inhibits mitogen-induced proliferation, humoral and cellular responses to metacestode antigens, and inflammation surrounding metacestodes implanted subcutaneously in mice. To assess the effects of MF on cytokine production we stimulated murine spleen cells in vitro with concanavalin A and measured cytokine concentrations in the culture supernatants by enzyme-linked immunosorbent assay. When cultured with MF, the cells showed significantly decreased production of interleukin 2 (IL-2), interferon-gamma (IFN-gamma), and IL-4 as compared with mitogen alone. Exogenous rIL-2 and rIL-4 largely restored the proliferative response (85% and 71% of control cells, respectively). MF also decreased production of tumor necrosis factor-alpha (TNF-alpha) by macrophages stimulated with lipopolysaccharide and IFN-gamma. The TNF-alpha concentration was inversely correlated with the MF concentration. Experiments using spleen cells from mice treated with MF also showed a significant reduction in IL-4 concentration. These results suggest that MF inhibits cytokine production without regard to cell type or cytokine. This may explain the function of this molecule as an inhibitor of the host inflammatory and immune responses.

It has previously been demonstrated that immunization of pigs with a crude extract of Taenia solium metacestodes can confer a high level of protection against an egg challenge. Furthermore, vaccination of infected animals also induces an immune response against the larvae, which are either destroyed or rendered non-infectious. To assess the efficacy of immunization as a strategy for reducing the prevalence of porcine cysticercosis, a field trial of this vaccine was performed in an endemic area in the northern region of the Guerrero State, Mexico, Random samples of pigs belonging to 17 villages were examined for metacestodes by inspection of their tongues. Each animal was immunized with a dose of 150 micrograms of protein (antigenic extract from Taenia solium metacestodes) by the intramuscular route. A prevalence of 2.4% of porcine cysticercosis on average was found in these villages at the beginning of the trial (62 cysticercotic pigs out of 2650 inspected). Six of these villages were selected for the periodic vaccination of new random samples of pigs. A statistically significant decline in the prevalence of porcine cysticercosis was observed at the end of the trial, decreasing from 2.4% at the beginning of vaccination to 0.45% at the end of the trial. A reduction of 82% was observed in spite of the poor living conditions in these villages. These results are consistent with previous data and suggest that it may be possible to turn a susceptible pig population into a protected one by systematic vaccination.

Subcutaneous implantation of Taenia solium metacestodes in mice induces an inflammatory reaction made up mainly of neutrophils and eosinophils after 12 days. Administration of a small RNA-peptide (metacestode factor, MF) purified from T. solium metacestodes significantly reduces the inflammatory site in both size and composition, yielding a very low number of eosinophils. The metacestodes implanted in control mice were completely destroyed and their remnants were surrounded by an intense inflammation predominantly made up of neutrophils and eosinophils. In contrast, metacestodes implanted in mice treated with MF showed apparently intact suckers, rostellum, hooks, and tegument. Inhibition of inflammation around the parasites was also observed in mice immunized with T. solium metacestode antigens and inoculated simultaneously with MF. Mice immunized only with T. solium metacestode antigens produced a granulomatous process around metacestodes that destroyed most of the large metacestode structures: suckers, rostellum, hooks, and tegument-wall tissues. Furthermore, treatment of mice with MF or implanted metacestodes decreased the antibody (P < 0.05) and cellular responses (P < 0.05) to metacestode antigens. The antibody responses was even lower when both of these treatments were given simultaneously. These findings support the idea that MF plays a key role in the down-regulation of the host immune response, contributing to the parasite's survival.

A substance from Taenia solium metacestodes that decreases lymphocyte proliferation induced by concanavalin A was isolated. The molecular weight of this substance was estimated to be slightly more than 1,450 Da. Crude metacestode factor was fractionated through a Bio-gel P-6 column. Peak 1 showed suppressive activity. After incubation with RNase the substance lost its activity. Incubation of this material with trypsin or papain increased its suppressive activity. It was stable at boiling temperature for 10 min. The incubation of this substance with murine macrophages had no effect on [3H]-thymidine uptake by cocultured fresh splenic lymphocytes stimulated with concanavalin A. Conversely, cocultures of lymphocytes pretreated with the substance and fresh splenic lymphocytes showed a decreased incorporation of [3H]-thymidine. These results suggest that this substance is a RNA-peptide molecule whose RNA moiety accounts for its suppressive activity. The findings also suggest that in vivo the factor may be a modulator of the immune response.

In chronic helminthic infections such as cysticercosis, where the parasites live for years, profound modulation of the host immune response has been reported. To evaluate the genotoxicity of a drug used to treat cysticercosis, we observed the occurrence of genetic damage in cultured lymphocytes from cysticercotic swine and patients who had not been exposed to the drug. The human lymphocytes also showed a slower proliferation. These data suggested that the disease itself was promoting genetic damage in host lymphocytes which, in part, could explain the retardation of the lymphocyte proliferation observed in cysticercotic patients. Pigs infected with Taenia solium cysticerci showed an increased lymphocyte proliferation for 6-8 weeks post infection, followed by an impaired proliferation after this period. Significant induction of sister-chromatid exchanges was also observed in lymphocytes from infected pigs after the 6th week post infection. Additionally, it was found that a factor secreted by the cysticerci morphologically transformed primary fibroblasts in culture. The results strongly suggest that the parasite produces genetic instability in the host cells, which could result in immunosuppression and malignant transformation of target cells.

An antigenic extract from Taenia solium metacestodes was evaluated for immunogenicity in pig populations from a large area of endemic porcine cysticercosis in the State of Guerrero, Mexico. A total of 3,295 pigs from 18 villages were immunized with a single dose of 250 micrograms of protein administered intramuscularly. Systematic immunization was also performed on pigs (1,076 immunizations) from two of the villages with the highest percentages of cysticercosis. A year after immunization, porcine cysticercosis decreased from 4.8% and 5.4% to 0%. Immunity against the T. solium metacestode was estimated in vitro by measurements of 3H-thymidine uptake and inhibition of leukocyte migration. Peripheral blood lymphocytes from immunized cysticercotic (pigs that had cysticercosis prior to immunization), cysticercotic immunized (pigs that acquired cysticercosis after immunization), and normal control pigs incorporated 3H-thymidine better than lymphocytes from cysticercotic pigs when stimulated with concanavalin A. A significant inhibition in the leukocyte migration inhibition test was also found in leukocytes from immunized cysticercotic pigs (P < 0.01). Histopathologic studies revealed granuloma formation surrounding the metacestodes of the immunized cysticercotic and cysticercotic immunized pigs. These metacestodes exhibited several stages of destruction. Large numbers of eosinophils were frequently observed in a close association with the degeneration and destruction of parasites. Metacestodes in control cysticercotic pigs were intact and surrounded by a minor inflammatory reaction. Finally, the rate of in vitro evagination of scolices was high in metacestodes obtained from cysticercotic pigs and low or absent in those from immunized pigs (P < 0.01).

Neurocysticercosis, caused by Taenia solium, is arguably the most common parasitic disease of the central nervous system. In taeniid infections of nonhuman mammals, there is strong evidence of immunity in the intermediate host to the invasive larvae (oncospheres). This immunity, which is mediated by antibody and complement, has been exploited to develop vaccines that effectively prevent infection. To examine the immune response in humans, T. solium eggs were hatched and activated in vitro. Activated oncospheres were incubated with heat-inactivated sera from patients with neurocysticercosis with or without complement (guinea pig serum). Controls included oncospheres plus complement alone, normal human serum alone, normal serum with complement, or buffer alone. Serum from infected patients, especially with complement, markedly reduced oncosphere mobility and led to disappearance of secretory vesicles and loss of membrane integrity. Viability as assessed by staining with dimethyl-thiazolyl-diphenyl-tetrazolium was reduced from 92.5% in controls to 61.5% with immune serum and 38.8% with immune serum and complement (P < 0.01). Preliminary western blot analysis showed antigens at 22, 64, and 70 kDa recognized by all 3 sera, but not by control sera. These data suggest that sera from patients with cysticercosis can kill oncospheres in vitro and may be used to identify protective antigens.

Two cysticerci containing 2 scolices were found among several thousand Taenia solium metacestodes dissected from swine. Microscopic study of tissue sections revealed that both worms were equally well developed in 1 bladder worm, whereas 1 member of the other pair was incompletely formed.

Human neurocysticercosis results from infestation of the central nervous system with the metacestode form (tissue cyst) of Taenia solium. Cysticercosis is being increasingly recognized as a cause of neurologic symptoms in residents and emigrants from developing countries. Taeniid parasites have developed elaborate mechanisms to persist in the tissues of their intermediate hosts. The invasive larvae, termed oncospheres, are susceptible to antibody and complement. However, by the time that the host has generated an antibody response, the parasites have begun to transform to the more resistant metacestode form. The metacestodes also have means of evading complement-mediated destruction, including paramyosin, which inhibits C1q; taeniaestatin, which inhibits both classical and alternate pathways (likely by inhibiting factor D and C3 esterase); and sulfated polysaccharides, which activate complement away from the parasite. Similarly, antibody does not seem to be able to kill the mature metacestode. The parasites may even stimulate the host to produce antibody, which could be bound via Fc receptors, and used as a source of protein. Finally, taeniaestatin and other parasite molecules may interfere with lymphocyte proliferation and macrophage function, thus paralyzing the cellular immune response. Because the symptoms of neurocysticercosis are typically associated with a brisk inflammatory response, we hypothesize that disease is primarily the result of injured or dying parasites. This hypothesis raises important questions in assessing the role of chemotherapy in the management of neurocysticercosis as well as in evaluation of clinical trials, most of which have been uncontrolled.

The metacestode of Taenia solium persists for years in the human central nervous system. As proteolytic enzymes play an important role in the survival of tissues helminths, we examined extracts of T. solium metacestodes for proteolytic activity using 9 synthetic peptide substrates and 3 proteins (hemoglobin, albumin, and immunoglobulin G). The proteolytic enzymes were classified based on their inhibitor profiles. At neutral pH, aminopeptidase(arginine-7-amino-4-trifluoromethylcoumarin) and endopeptidase(benzyloxy-carbonyl-glycine-glycine-arginine-7-amino-4- trifluoromethylcoumarin) substrates were cleaved. Hydrolysis of both substrates was inhibited by chelating agents, which inhibit metalloproteases. Peak activity with both substrates eluted in gel filtration fractions corresponding to a molecular weight of about 104 kDa. Cysteine protease activity was identified, which cleaved benzyloxy-carbonyl-phenylalanine-arginine-7-amino- 4-trifluoromethylcoumarin (Z-Phe-Arg-AFC) and hemoglobin. Cleavage of Z-Phe-Arg-AFC was maximal at acid pH, was stimulated by thiols, and was inhibited by leupeptin and Ep459. Peak cysteine protease activity eluted in gel filtration fractions corresponding to a molecular weight of 32 kDa. Aspartic protease activity was identified by specific inhibition with pepstatin of acid digestion of hemoglobin and immunoglobulin G. Immunoglobulin digestion occurred at acid pH, with preferential degradation of the heavy chain. Upon gel filtration chromatography, the aspartic protease activity eluted as a broad peak with maximal activity at about 90 kDa. No serine protease activity was detected. None of the parasite enzymes digested albumin. Proteolytic enzymes of T. solium may be important for parasite survival in the intermediate host, by providing nutrients and digesting host immune molecules.

Cysticercosis caused by Taenia solium is associated with immunodepression of T and B lymphocytes. In order to ascertain if this parasite affects lymphocyte activity, a factor isolated from the parasite was tested on (3H) thymidine uptake by cultured human lymphocytes stimulated by phytohaemagglutinin. This dialysable factor had a molecular weight of less than 3500 Da, and was isolated from an extract of Cysticercus cellulosae. It decreased phytohaemagglutinin-stimulated uptake of (3H) thymidine. After the material was treated with RNase 'A', the suppressive activity was destroyed. It thus appears that the factor could correspond to an RNA fraction.

An immunogenic complex was obtained from Salmonella typhi by the bacterial acetone powder method. This complex induced in mice a high degree of protection against a challenge with the virulent Salmonella. This immunogenic complex was fractionated at least into 19 fractions when chromatographied on a DEAE-cellulose column. By SDS-polyacrylamide gel electrophoresis, 25 protein bands were observed. Eleven DEAE-cellulose fractions were tested in order to know their immunogenicity. Mice were inoculated with 10 micrograms of protein of each fraction. Seven days after, the mice received a booster. Thirty days after the first inoculation, the animals were challenged with S. typhi resuspended in chondroitin-sulphate at 13%, by the intraperitoneal route. Appropriate control mice were included; 30 min before the challenge, mice had been inoculated with 850 microgram of lead acetate by the intravenous route. The immunogenic complex protected 100% of mice; six of its fractions were good immunogens; one of them, the fraction 4, was shown to contain at least 3 proteins by electrophoresis assay. This fraction induced in mice a high degree of protection against the challenge by the virulent Salmonella. Finally, a ribonucleoprotein purified from this fraction was highly immunogenic to mice against the challenge by 10 LD50 of S. typhi (1 LD50 was equivalent to 2 X 10(6) CFU).

In hogs naturally infected with Taenia solium larvae (i.e., Cysticercus cellulosae), we studied the host response induced by antigens obtained from the larvae. Histopathological studies of cysticerci removed after 4 and 8 weeks of immunization showed an intense inflammatory reaction surrounding the larvae. The response was greater in the 8-week specimens. A dense layer of eosinophils was in close contact with the external membrane of the bladder wall and, in several cases, the eosinophils had infiltrated this tegument. Many eosinophils were seen in the spiral canal of larvae. This infiltration by eosinophils increased with time. Preparations from the 8-week samples showed many degenerated and disrupted eosinophils whose granules were found in close contact with the outer membrane of the larval tegument and, in some cases, had entered through the broken surface of this structure. More than 90% of the larvae were found in various stages of degeneration; the rest were completely destroyed and surrounded by a mass of eosinophils. After immunization, peripheral blood eosinophilia increased to 17%, whereas the eosinophilia of the control hog was 4% throughout the study. The larval worms removed from control hogs showed intact structures, with a low degree of infiltration by eosinophils and a discrete inflammatory reaction surrounding the bladder wall of the larvae.

Protection was induced in hogs against Taenia solium cysticercosis using an immunogenic complex obtained from its larval "bladder worm" form, Cysticercus cellulosae. Immunoelectrophoresis revealed that this complex contained at least eight antigens. In immunized hogs a total of 71 (mean 11.8) cysticerci were found, whereas in the control animals 397 (mean 74.9) were found. Histopathological studies showed that more than 40% of larvae obtained from immunized hogs were completely destroyed and the others were seen in various stages of degeneration. Eosinophils and mononuclear cells were observed infiltrating the internal structures of the larvae. Intense granulomatous reactions of eosinophils, lymphocytes, macrophages, epithelioid cells, plasma cells, and fibroblasts surrounded the larvae. Larvae from control hogs were intact and surrounded by a small inflammatory reaction. The cellular response was measured by the macrophage migration inhibition test, which was higher in immunized hogs when compared with control animals, either before the infection with T. solium eggs or before slaughter. No significant difference was found in the humoral response of immunized and control hogs.

A protein fraction, which consisted of at least 12 proteins, was obtained from the venom of Mexican scorpion Centruroides limpidus limpidus. The molecular weights of these proteins ranged between 9,800 and 163,000 daltons. This fraction was separated from the rest of the venom components, which were almost all neurotoxins, by chromatographying the venom obtained by electrical stimulation through a Sephadex G-50M column. This fraction was non-toxic for mice, even at dose of 200 micrograms/mouse. The most important is that it was able to induce immunity against C. l. limpidus venom, since 92.8% of the animals inoculated with three doses survived after the challenge with 39.2 micrograms of venom (2 DL50 for mice of 20 g); on the contrary, 88 min after the challenge, 100% of the control mice had already died. In another experiment, this immunogen was inoculated into mice three times at variable doses. Seven days after the last injection, each mouse was challenged with 19.6 micrograms of venom. In all controls the typical envenomation picture produced by scorpion venom was developed, and death was registered in 19% of the animals. In contrast, 87% of mice immunized with the highest dose failed to show signs of envenomation or died throughout the observation time. Only two immunized animals (13%) showed mild tachycardia and hyperpnea at 120 min post-challenge. Immunoelectrophoresis and immunodiffusion tests revealed that these proteins induced antibodies against components of the most toxic fraction.

An immunogenic complex was isolated from Salmonella typhi-murium and another one from Salmonella typhi Ty2. Both were prepared by the bacterial acetone powder method which eliminated the cell wall, the DNA almost completely and the membrane phospholipids. The complexes were denominated "New Vaccines". The S. typhi-murium new vaccine induced, even at doses of 0.5 microgram dry weight per mouse, a high degree of protection against the challenge of the virulent microorganism. By immunoelectrophoresis, 21 antigen-antibody systems could be detected, two of them corresponding to O antigens. The S. typhi Ty2 new vaccine induced better protection than the standard vaccine (heat-phenol inactivated typhoid vaccine) when both vaccines were compared in the relative potency test. Moreover, the new vaccine had very low toxicity when inoculated in humans at doses of 1 microgram dry weight, able to elicite a high antibody titre (1/1,790 mean of 10 sera) in 75% of the tested population, estimated by the complement fixation test. In contrast, the standard vaccine induced a low antibody titre (1/222, mean of 5 sera) in 50% of the humans inoculated with 1 X 10(8) bacterial cells. The new vaccine did not induce undesirable effects whereas the standard vaccine induced an important inflammatory process in 100% of the cases, with intense local pain in 67% after 24 h post-first inoculation as well as other less severe symptoms.

Ribosomal preparation obtained from Salmonella typhimurium, used as a vaccine, was able to induce protection of 100% on mice challenged with 5.5 X 10(9) CFU of S. typhimirium administered by oral route. Effective dose at 50% was of 0.1085 mug, expressed as ribonucleic acid (RNA). This vaccine induced an important humoral response as soon as in nine days. The ribosomal preparation used in this work was maintained at 4 degrees C at least for eighty two days. This fact shows an adequate stability of this biological product.