Neurocysticercosis is still prevalent in Mexico.

In this work, we report the published cases of human and porcine cysticercosis, as well as Taenia solium taeniasis diagnosed in Mexico during the last 10 years. Numerical data allow us to state that this disease remains as a public health problem in our country. Whereas efficient tools have been developed for the diagnosis and prevention of cysticercosis, we strongly recommend further measures allowing the control and eventual eradication of this parasite in Mexico.

Quantitative and phenotypic analyses of lymphocyte-monocyte heterokaryons induced by the HIV envelope proteins: Significant loss of lymphoid markers.

Cells infected with the human immunodeficiency virus (HIV) can fuse with CD4(+) cells leading to the formation of multinucleated cells. The presence of multinucleated cells infected with HIV in tissues of patients has been documented, although their cellular composition and role in AIDS pathogenesis is still under study. Here, we present evidence of in vitro heterotypic lymphocyte-monocyte fusion in cocultures of lymphocytic Jurkat T cells expressing the HIV-1 gp120/gp41 glycoproteins (Env) and CD4(+) monocytic THP-1 cells. Using a previously characterized method that involves differential labeling of fusion partners with fluorescent probes and flow cytometry analysis after coculture, up to 20% of double fluorescent cells were detected in 48h. This double fluorescent cell population was produced by heterotypic lymphocyte-monocyte fusion as it was not observed when Jurkat T cells expressing a mutant non-fusogenic Env protein were used. Heterokaryon formation was inhibited by an anti-CD4 monoclonal antibody and the HIV-fusion inhibitor peptide T-20. About 68% of heterokaryons remained alive and non-apoptotic after 2days of coculture. In heterokaryons, CD4 was barely detectable and the expression of the CD3 and CD28 lymphoid markers was greatly reduced, whereas the expression of CD32 and the intracellular antigen CD68, both markers of monocytic cells, remained unchanged. In contrast with unfused T cells, heterokaryons only expressed very low levels of the lymphoid activation marker CD25 following treatment with PMA plus ionomycin. These studies point to the possible generation of lymphocyte-monocyte heterokaryons with a myeloid phenotype during HIV infection, with unknown consequences for AIDS pathogenesis.

Budding of Taenia crassiceps cysticerci in vitro is promoted by crowding in addition to hormonal, stress, and energy-related signals.

Taenia crassiceps cysticerci (cysts) reproduce by budding. The cysts' production of buds was measured in vitro to explore parasite and environmental-related factors involved in the extreme individual variation in parasite loads of inbred mice. Cysts were placed in in vitro culture for 10 days at initial parasite densities of 1, 5, 10 cysts/well in 1 ml of RPMI Medium 1640 without serum. Results showed that there is considerable intrinsic initial variation among inoculated cysts in their production of buds and that increasing parasite density (crowding) stimulates the overall production of buds and recruit into budding most of the cysts. Identical cultures were then subjected to various treatments such as heating and exposure to peroxide to induce stress, or to 17beta-estradiol, insulin, glucose, or insulin+glucose to supplement putatively limiting hormonal and energy resources. All treatments increased budding but the parasites' strong budding response to crowding alone overshadows the other treatments.

Decreased CD4 and wide-ranging expression of other immune receptors after HIV-envelope-mediated formation of syncytia in vitro.

In human HIV infection, multinucleated cells (syncytia) are formed by fusion of HIV-infected cells with CD4+ cells. In order to examine possible functional implications of syncytia formation for the immune response, the expression of important surface molecules by T-cell syncytia and surrounding cells that remain unfused (bystander cells) was analyzed in cocultures of HIV-Env- and CD4-expressing E6 Jurkat T cells. Fusion partners were differentially labeled with lipophilic probes, and syncytia and bystander cells were identified by flow cytometry. The cellular phenotype and response to activation stimulus after fusion were analyzed with antibodies coupled to third-party fluorochromes. Cocultured unfused E6 cells showed a marked decrease in CD4 expression, suggesting the selective recruitment of cells strongly expressing CD4 into syncytia. However, the incorporated CD4 was not detected in the syncytia, whereas the range of expression of CD28, ICAM-1, CXCR4 and CD3 was wider than that of unfused cells. Limited expression of CD4 in the bystander unfused population, as well as in the newly formed syncytia, would result in limitation of further viral entry and a failure to identify these cells, and it could partially contribute to functional impairment and a decrease in the number of CD4+ T cells in AIDS. Most of the syncytia were viable and expressed CD25 and IL-2 in response to activation by phorbol myristate acetate (PMA) and ionomicyn. Thus, syncytia populations harboring widely heterogeneous levels of receptors would constitute a potential source of anomalous immune function.

HIV-envelope-dependent cell-cell fusion: quantitative studies.

Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17-23; López-Balderas et al., 2007, Virus Res. 123, 138-146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env-mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env-mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

[Linking Nef with the immunological and virological determinants of AIDS]. / Vinculación de Nef con los determinantes inmunológicos y virológicos del SIDA.

Nef is an accessory protein from HIV-1 involved in viral replication, depletion of CD4 + T cells and progression to AIDS. The participation of Nef in altering several of the cellular and subcellular components has been studied in detail. The finding that infected patients over 15 years with HIV-1 variants that expressed a defective nef gene had not progressed to AIDS, suggested that Nef played a decisive role in the outcome of AIDS, through the regulation of different virological and immunological components. In addition, the mere expression of Nef is able to induce AIDS in transgenic mice and alter the kinetics of replication of the virus in infected monkeys. This review examines the relationship between the expression of Nef and the ultimate consequences of HIV-1 infection, taking into account the most relevant clinical parameters: viral load, counting of CD4 + T cells and progression to AIDS. Under this approach, it is emphasized the role of Nef as immunomodulator and also as independent mediator of damage to the immune system.

Treatment with dehydroepiandrosterone in vivo and in vitro inhibits reproduction, growth and viability of Taenia crassiceps metacestodes.

The aim of this work was to explore the effect of dehydroepiandrosterone (DHEA) on the establishment, growth and reproduction of the metacestode stage of the tapeworm Taenia crassiceps, both in vivo and in vitro. Administration of DHEA prior to infection in mice of both sexes reduced the parasite load by 50% compared with untreated mice. This protective effect was not associated with the immune response, since there was no effect of DHEA treatment on mRNA levels of IL-2, IFN-gamma, IL-4 or IL-10. DHEA treatment of infected mice increased androgen receptor expression in splenocytes of both sexes. Moreover, in vitro treatment of T. crassiceps with DHEA reduced reproduction, motility and viability in a dose- and time-dependent fashion. Results indicate that DHEA has strong negative direct modulatory effects on murine cysticercosis. We suggest the use of hormonal-analogues for protective purposes as a therapeutic approach to prevent murine cysticercosis.

Modified progesterone receptor expression in the hypothalamus of cysticercotic male mice.

Progesterone participates in numerous developmental and behavioral processes in the mammalian brain. The intracellular (is this really intracellular? nuclear membrane?) progesterone receptor is expressed as two isoforms: a full-length form (PR-B) and the N-terminally truncated one (PR-A). Experimental intraperitoneal infections with Taenia crassiceps in mice exhibit the tendency of the parasites to develop more rapidly in females. Male mice undergo a feminization process characterized by their oestrogenisation, deandrogenisation and loss of sexual and aggressive patterns of behavior. Hence, we suspected that changes in PR expression in the brain could be involved in the feminization of the infected male mice and in the loss of the sexual and aggressive behaviors. We have studied the expression of PR isoforms in the normal and infected male mouse brain. Transcripts of both receptor isoforms (PR-A and -B) were readily detectable in normal and infected mice, but differentially regulated during infection depending on the area of the brain studied. Although the precise role of progesterone in mediating the behavioral changes noted during infection is not fully understood, our data implicate a role for PR signaling in the feminization process. CNS activity is potentially involved in the network that regulates the oestrogenisation and deandrogenisation observed in chronically infected male mice, as well as in the behavioral peculiarities observed in this parasitic infection.

Tamoxifen treatment induces protection in murine cysticercosis.

Administration of tamoxifen (an antiestrogen) produced an 80% parasite load reduction in female mice, and a weaker effect of 50% in male mice. This protective effect was associated in both sexes, with an increase in the mRNA levels of interleukin (IL)-2 (a cytokine associated with protection against cysticerci) and IL-4 (no effect on infection). tamoxifen treatment modified 17-beta estradiol production in females, whereas serum testosterone was not affected. However, the expression of the 2 types of estrogen receptor (ER), i.e., ER-alpha and ER-beta, in the spleen of infected mice of both sexes, was decreased by tamoxifen treatment. In vitro, treatment of Taenia crassiceps with tamoxifen reduced reproduction and loss of motility. These results indicate that tamoxifen treatment is a new therapeutic possibility to treat cysticercosis, because it can act at both ends of the host-parasite relationship, i.e., by increasing the cellular immune response protective against the parasite and by directly affecting the parasite's reproduction and survival.

Renewed hope for a vaccine against the intestinal adult Taenia solium.

Review of experimental and observational evidence about various cestode infections of mammalian hosts revives hope for the development of an effective vaccine against adult intestinal tapeworms, the central protagonists in their transmission dynamics. As for Taenia solium, there are abundant immunological data regarding cysticercosis in humans and pigs, but information about human taeniasis is scarce. A single publication reporting protection against T. solium taeniasis by experimental primo infection and by vaccination of an experimental foster host, the immunocompetent female hamster, kindles the hope of a vaccine against the tapeworm to be used in humans, its only natural definitive host.

Effect of Transforming Growth Factor-ß upon Taenia solium and Taenia crassiceps Cysticerci.

Taeniids exhibit a great adaptive plasticity, which facilitates their establishment, growth, and reproduction in a hostile inflammatory microenvironment. Transforming Growth Factor-ß (TGFß), a highly pleiotropic cytokine, plays a critical role in vertebrate morphogenesis, cell differentiation, reproduction, and immune suppression. TGFß is secreted by host cells in sites lodging parasites. The role of TGFß in the outcome of T. solium and T. crassiceps cysticercosis is herein explored. Homologues of the TGFß family receptors (TsRI and TsRII) and several members of the TGFß downstream signal transduction pathway were found in T. solium genome, and the expression of Type-I and -II TGFß receptors was confirmed by RT-PCR. Antibodies against TGFß family receptors recognized cysticercal proteins of the expected molecular weight as determined by Western blot, and different structures in the parasite external tegument. In vitro, TGFß promoted the growth and reproduction of T. crassiceps cysticerci and the survival of T. solium cysticerci. High TGFß levels were found in cerebrospinal fluid from untreated neurocysticercotic patients who eventually failed to respond to the treatment (P = 0.03) pointing to the involvement of TGFß in parasite survival. These results indicate the relevance of TGFß in the infection outcome by promoting cysticercus growth and treatment resistance.

Neurocysticercosis: clinical, radiologic, and inflammatory differences between children and adults.

BACKGROUND: Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. NC is clinically heterogeneous, ranging from asymptomatic infection to severely incapacitating and even fatal presentations. Although NC affects adults and children, age-related factors have not been thoroughly studied. METHODS: We describe and compare the clinical, radiologic, and inflammatory features of pediatric and adult Mexican NC cases. Two hundred six NC cases (92 pediatric and 114 adult) diagnosed by computed tomography or magnetic resonance imaging were included. RESULTS: Seizures were more frequent in children (80.4% versus 56.1%), and intracranial hypertension and headaches were more frequent in adults (27.2% versus 15.2% and 35.1% versus 21.7%, respectively). Different causes underlie the different distribution of seizures and intracranial hypertension in the 2 patient groups. In pediatric NC patients, single colloidal parenchymal cysts were the most common radiologic findings compared with adults in whom multiple viable parasites in the basal subarachnoidal cisterns or in the ventricles were seen. Cerebrospinal fluid inflammation was greater in adults than in children (P = 0.02). CONCLUSIONS: This study documents significant age-related radiologic, clinical, and inflammatory differences in Mexican NC patients. Possible causes and relevance of these age-associated findings are discussed.

Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

The genome project of Taenia solium.

We have constituted a consortium of key laboratories at the National Autonomous University of Mexico to carry out a genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis, and, in conjunction with the Echinococcus granulosus and Echinococcus multilocularis genome project of expressed sequence tags (ESTs), will mark the advent of genomics for cestode parasites. Our project is planned in two consecutive stages. The first stage is being carried out to determine some basic parameters of the T. solium genome. Afterwards, we will evaluate the best strategy for the second stage, a full blown genome project. We have estimated the T. solium genome size by two different approaches: cytofluorometry on isolated cyton nuclei, as well as a probabilistic calculation based on approximately 2000 sequenced genomic clones, approximately 3000 ESTs, resulting in size estimates of 270 and 251 Mb, respectively. In terms of sequencing, our goal for the first stage is to characterize several thousand EST's (from adult worm and cysticerci cDNA libraries) and genomic clones. Results obtained so far from about 16,000 sequenced ESTs from the adult stage, show that only about 40% of the T. solium coding sequences have a previously sequenced homologue. Many of the best hits are found with mammalian genes, especially with humans. However, 1.5% of the hits lack homologues in humans, making these genes immediate candidates for investigation on pharmaco-therapy, diagnostics and vaccination. Most T. solium ESTs are related to gene regulation, and signal transduction. Other important functions are housekeeping, metabolism, cell division, cytoskeleton, proteases, vacuolar transport, hormone response, and extracellular matrix activities. Preliminary results also suggest that the genome of T. solium is not highly repetitive.

Regulation of the immune response to cestode infection by progesterone is due to its metabolism to estradiol.

The aim of this work was to investigate the role of progesterone during Taenia crassiceps cysticercosis, and the immunological mechanisms involved in its effects, by relating progesterone treatment to whole parasite counts, to host humoral and cellular immune response, to the presence or absence of nuclear receptors to sex steroids in splenocytes, and to serum sex steroid levels in infected mice of both genders. Progesterone treatment increased parasite loads two-fold in females and three-fold in males compared with control mice. The expression of the Th2 cytokine profile (IL-4, IL-6 and IL-10) was markedly increased in infected mice of both genders, while progesterone treatment returned this expression to basal levels. However, the Th1 cytokine profile (IFN-gamma and TNF-alpha) was not affected by infection, whilst progesterone treatment increased the expression of both cytokines two-fold compared to uninfected, infected and placebo-treated mice. Testosterone serum levels decreased in infected male mice by 95%, and treatment with progesterone did not affect them. In females, no change in testosterone levels was observed. Progesterone levels increased three-fold only in progesterone-treated infected mice of both sexes, while estradiol levels in female and male progesterone-treated infected mice increased two-fold compared to infected control mice. The infection markedly induced the expression of progesterone receptor (PR) isoforms A and B in splenocytes of infected mice of both genders (five-fold). Metabolism of progesterone to estradiol was demonstrated by the use of the anti-estrogen tamoxifen, which reduced parasite loads 100% in infected mice of both sexes treated with progesterone. These results suggest that progesterone, possibly through its metabolism to estradiol, affects establishment, growth and reproduction of the helminth parasite T. crassiceps.

Mortality and antibody responses of mice to three successive episodes of experimental scorpion (Centruroides limpidus limpidus) envenomation and immunological rescue.

Mortality rates of mice and their levels of anti-venom and anti-F(ab')2 antibodies were assessed after three episodes of subcutaneous envenomations with or without treatment with horse F(ab')2. Soluble venom from the Mexican scorpion Centruroides limpidus limpidus was used for these experiments. Repetition of episodes did not induce different mortality rates in untreated mice. F(ab')2 rescued about 85% of the mice in the first two episodes and 66% in the third, without distinction of gender or ostensible side-effects: a suggestion of selection of the most resistant mice. Surviving mice produced in vitro neutralizing antibodies to the scorpion venom and also antibodies to F(ab')2, when injected alone but more so if combined: a possible immunological adjuvant or alarm effect of the venom or of the cascading physiopathology of envenomation. In the few surviving mice, both anti-venom and anti-F(ab')2 antibodies increased significantly after the first envenomation but not thereafter, showing no correlation with mortality rates: a suggestion of their clinical irrelevance, the few hard-to kill mice appeared to resist envenomation by mechanisms other than antibody response. Injection of F(ab')2 alone induced production of detectable anti-venom antibodies in a few mice and injection of venom alone induced that of anti-F(ab')2 antibodies, perhaps due to trace amounts of venom in the high affinity fraction of F(ab')2 and to anti-idiotypic antibodies or polyclonal activity in the envenomation episode, respectively.

The network of antigen-antibody reactions in adult women with breast cancer or benign breast pathology or without breast pathology.

The Immunoglobulin G (IgG) antibody response to different protein antigens of the mammary ductal carcinoma by adult women affected by Breast Cancer (BC) distinguishes at least 103 proteins that differ in their molecular weights (MW). The IgG producing cell clones (nodes) coexist with each other in each individual organism and share energy resources among themselves, as well as factors that control the level of expression and Specificity of their IgG antibodies. So, it can be proposed that among them there is a Network of interconnections (links) unveiled by the antigens, which specifically react with the IgG antibodies produced by the clones. This Network possibly regulates IgG antibodies' activity and effectiveness. We describe the Network of nodes and links that exists between the different antigens and their respective IgG producing cell clones against the extracted protein antigens from the cells of the T47D Cell-Line, in 50 women with BC, 50 women with Benign Breast Pathology (BBP) and 50 women without breast pathology (H). We have found that women with BBP have the highest number of Links, followed by the H group and, lastly, the women with BC, a finding which suggests that cancer interferes with the Connectivity between the IgG producing cell clones and blocks the expression of 322 links in women with BBP and 32 links in women with H. It is also plausible that the largest number of links in the women with BBP indicates the Network's state of arousal that provides protection against BC. On the other hand, there were many missing links in the BC group of women; the clone which lost more links in the BC group was the hub 24, which point to some of the antigens of T47D as potentially useful as vaccines, as the immune system of women with BBP is well aware of them.

TH2 profile in asymptomatic Taenia solium human neurocysticercosis.

Neurocysticercosis (NC), a parasitic disease caused by Taenia solium, may be either asymptomatic or have mild to severe symptoms due to several factors. In this study, the immunological factors that underlie NC pleomorphism were studied. Ten of the 132 inhabitants of a rural community in Mexico (Tepez) had a computerized tomography (CT) scan compatible with calcified NC, and all were asymptomatic. Their immunological profiles were compared with those of 122 CT scan negative (non-NC) subjects from the same village. NC was associated with a TH2 response (IgG4, IL-4, IL-5, IL-13). Subjects from Tepez had higher levels of specific antibodies (IgG1, IgG2, IgG4, IgE) and specific cell proliferation than subjects from an area with low exposure (Ensenada). This suggests that non-NC subjects from Tepez had been exposed to T. solium and resisted infection in the brain. Distinct immunological profiles in equally exposed individuals differing in outcome of infection support the hypothesis of host-related factors in resistance to and pathogenesis of NC. This is the first study reporting the immunological profile associated with the asymptomatic form of NC.

Population genetic structure of Taenia solium from Madagascar and Mexico: implications for clinical profile diversity and immunological technology.

Taenia solium is a cestode parasitic of humans and pigs that strongly impacts on public health in developing countries. Its larvae (cysticercus) lodge in the brain, causing neurocysticercosis, and in other tissues, like skeletal muscle and subcutaneous space, causing extraneuronal cysticercosis. Prevalences of these two clinical manifestations vary greatly among continents. Also, neurocysticercosis may be clinically heterogeneous, ranging from asymptomatic forms to severely incapacitating and even fatal presentation. Further, vaccine design and diagnosis technology have met with difficulties in sensitivity, specificity and reproducibility. Parasite diversity underlying clinical heterogeneity and technological difficulties is little explored. Here, T. solium genetic population structure and diversity was studied by way of random amplified polymorphic DNA in individual cysticerci collected from pigs in Madagascar and two regions in Mexico. The amplification profiles of T. solium were also compared with those of the murine cysticercus Taenia crassiceps (ORF strain). We show significant genetic differentiation between Madagascar and Mexico and between regions in Mexico, but less so between cysticerci from different localities in Mexico and none between cysticerci from different tissues from the same pig. We also found restricted genetic variability within populations and gene flow was estimated to be low between populations. Thus, genetic differentiation of T. solium suggests that different evolutionary paths have been taken and provides support for its involvement in the differential tissue distribution of cysticerci and varying degrees of severity of the disease. It may also explain difficulties in the development of vaccines and tools for immunodiagnosis.