Apoptosis of mouse hippocampal cells induced by Taenia crassiceps metacestode factor.

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.

A Taenia crassiceps factor induces apoptosis of spleen CD4+T cells and TFG-ß and Foxp3 gene expression in mice.

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)-ß 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.

A Taenia crassiceps metacestode factor enhances ovarian follicle atresia and oocyte degeneration in female mice.

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.

Disminución de las transfusiones alogénicas por la generalización del uso de recuperadores de sangre postoperatorios en cirugía de prótesis de rodilla / Decrease in allogenic transfusions due to the spread of use of postoperative retransfusion systems in knee replacement surgery

Objetivos. Los equipos medicoquirúrgicos disponen de múltiples herramientas para reducir la necesidad de transfusión alogénica perioperatoria. En nuestro centro se ha generalizado el uso de recuperadores de sangre filtrada en el postoperatorio de la cirugía de prótesis total de rodilla desde 2006. El objetivo de este estudio fue evaluar si el uso de recuperadores de sangre filtrada disminuyó la tasa de transfusiones en nuestra práctica clínica habitual. Material y métodos. Estudio de cohortes retrospectivo: grupo 2004, pacientes intervenidos de prótesis total de rodilla durante 2004, antes del uso de recuperadores de sangre filtrada; y grupo 2008, pacientes intervenidos de prótesis total de rodilla durante 2008, usando recuperadores. Se registraron sexo, niveles de hemoglobina preoperatorio y al cuarto día, masa total eritrocitaria perdida, reinfusión de sangre recuperada y transfusión de banco durante la estancia hospitalaria. Resultados. Ambos grupos fueron homogéneos en cuanto a sexo, hemoglobinas en el preoperatorio y el cuarto día, y masa total eritrocitaria perdida. La proporción de pacientes transfundidos fue significativamente menor en el grupo 2008 frente al grupo 2004 (20,18 vs. 42,19%), con un riesgo relativo de ser transfundido de 0,47 y NNT de 4,54; p = 0,0017. Conclusiones. En nuestro centro el uso de recuperadores disminuyó la proporción de pacientes transfundidos durante el ingreso para cirugía de prótesis total de rodilla, aunque este resultado no puede generalizarse debido a la ausencia de un criterio definido para indicar las transfusiones(AU)

Objectives. Surgical teams have several tools in order to reduce the need for postoperative allogenic transfusion. Postoperative autotransfusion of unwashed shed blood has become common practice for total knee replacement surgery since 2006 in our hospital. This study was designed to evaluate if this practice has reduced allogenic blood transfusions. Material and methods. A retrospective study comparing two cohorts, group 2004 with patients operated on for total knee replacement during the year 2004, before the use of the retransfusion system, and group 2008, patients operated on in the year 2008, with regular use of the retransfusion system. Gender, preoperative and postoperative haemoglobin levels, total amount of calculated erythrocytes lost, reinfusion of shed blood and allogenic blood transfusion during hospital stay were recorded. Results. Both groups were similar as regards gender, preoperative and postoperative hemoglobin levels, and total amount of erythrocytes lost. The proportion of transfused patients was significantly lower in group 2008 versus group 2004 (20.18% versus 42.19%), with a relative risk of being transfused of 0.47 and a NNT of 4.54. P = .0017. Conclusions. In our hospital the use of postoperative retransfusion systems has reduced the proportion of transfused patients during hospitalization for total knee replacement surgery, although this result cannot be generalized due to the lack of a fixed transfusion trigger(AU)

[Decrease in allogenic transfusions due to the spread of use of postoperative retransfusion systems in knee replacement surgery]. / Disminución de las transfusiones alogénicas por la generalización del uso de recuperadores de sangre postoperatorios en cirugía de prótesis de rodilla.

OBJECTIVES: Surgical teams have several tools in order to reduce the need for postoperative allogenic transfusion. Postoperative autotransfusion of unwashed shed blood has become common practice for total knee replacement surgery since 2006 in our hospital. This study was designed to evaluate if this practice has reduced allogenic blood transfusions. MATERIAL AND METHODS: A retrospective study comparing two cohorts, group 2004 with patients operated on for total knee replacement during the year 2004, before the use of the retransfusion system, and group 2008, patients operated on in the year 2008, with regular use of the retransfusion system. Gender, preoperative and postoperative haemoglobin levels, total amount of calculated erythrocytes lost, reinfusion of shed blood and allogenic blood transfusion during hospital stay were recorded. RESULTS: Both groups were similar as regards gender, preoperative and postoperative hemoglobin levels, and total amount of erythrocytes lost. The proportion of transfused patients was significantly lower in group 2008 versus group 2004 (20.18% versus 42.19%), with a relative risk of being transfused of 0.47 and a NNT of 4.54. P=.0017. CONCLUSIONS: In our hospital the use of postoperative retransfusion systems has reduced the proportion of transfused patients during hospitalization for total knee replacement surgery, although this result cannot be generalized due to the lack of a fixed transfusion trigger.

Definición de caso sospechoso de gripe A(H1N1) 2009: razones para la discordancia / No disponible

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Purification and characterization of a metacestode cysteine proteinase from Taenia solium involved in the breakdown of human IgG.

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.

Automatic analysis of immunocytochemically stained tissue samples.

An automatic colour image segmentation and cell counting software system has been developed for immunocytochemical analysis of stained tissue samples. The system was designed to count the total number of positive and negative cells in tissue samples treated with cytokine DNA probes from pigs naturally parasitised with Taenia solium metacestodes, using in situ hybridisation. A reaction index was calculated as the ratio of the number of cells with a positive reaction to the total number of cells (positives plus negatives) for each of five different probes. The objectives of automatic counting were to improve the reproducibility of the analysis and reduce the processing time of large image batches. A fast KNN classifier was used for colour segmentation. Watershed segmentation combined with edge detection was used to isolate individual cells that were then automatically labelled, using the results of the corresponding colour segmented image. Validation was performed on 122 non-training digital images with a total of 1069 positive cells and 1459 negative cells, with the following results: a mean true positive rate of 90.2% for positive cells and a mean true positive rate of 85.4% for negative cells. The corresponding mean false positive rates were 9.6% and 6.6%. The mean reaction index error of the automatic analysis was 5.35%. The processing of each digital image took 10 s on a Pentium IV PC.

Neurocysticercosis: relationship between the developmental stage of metacestode present and the titre of specific IgG in the cerebrospinal fluid.

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.

A cysteine protease from Taenia solium metacestodes induce apoptosis in human CD4+ T-cells.

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.

A factor isolated from Taenia solium metacestodes stimulates T lymphocytes to proliferate and produce gamma interferon.

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.

Taenia solium: a cysteine protease secreted by metacestodes depletes human CD4 lymphocytes in vitro.

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.

Impairment of the inflammatory reaction on implanted Taenia solium metacestodes in mice by a T. solium RNA-peptide: a scanning electron microscopy study.

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.

Depressed immunity to a Salmonella typhimurium vaccine in mice experimentally parasitized by Taenia crassiceps.

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.

A Taenia solium metacestode factor nonspecifically inhibits cytokine production.

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.

Field trial for reducing porcine Taenia solium cysticercosis in Mexico by systematic vaccination of pigs.

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.

Immunosuppression and inhibition of inflammation in mice induced by a small Taenia solium RNA-peptide to implanted T. solium metacestodes.

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.

[Diaphragmatic paralysis after the catheterization of the internal jugular vein]. / Parálisis diafragmática post cateterización de la vena yugular interna.

The complications derived from the puncture and catheterization of the veins of the neck, cause symptoms and signs on physical examination which facilitate their diagnosis. The phrenic paralysis which we present, is characterized by few accompanying symptoms, with the elevation of the hemidiaphragm being a radiological finding. The suspicion is confirmed by fluoroscopy. We reviewed the mechanisms, the cases described in the literature, and the possible risk factors.

Suppression of murine lymphocyte proliferation induced by a small RNA purified from the Taenia solium metacestode.

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.

Immune response impairment, genotoxicity and morphological transformation induced by Taenia solium metacestode.

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.