Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment.
Background: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3′,5-L-triiodothyronine (T3) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (–59/–52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (–101/–94). Methods: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (–242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T3. Results: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T3 while they increased by 1 mM 8Br-cAMP (± T3). Site-4 was protected from DNase I digestion with nuclear extracts from T3-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T3 + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Krüppel family. A TRβ2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T3. A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T3. Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T3. Discussion: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRβ2 is bound; pCREB and TR do not present mutual interference on their binding sites.
Intestinal infection with the protozoan parasite Entamoeba histolytica elicits a local immune response with rising of specific secretory IgA (sIgA) antibodies detectable in several compartments associated to mucosa. Anti-amoebic sIgA antibodies have been reported in faeces, saliva, bile and breast milk from dysenteric patients and research trying to elucidate their role in protection has recently intensified. IgA antibodies inhibit the in vitro adherence of E. histolytica trophozoites to epithelial cell monolayers by recognizing several membrane antigens, including the galactose-binding lectin (Gal-lectin), main surface molecule involved in adherence, and the serine and cystein-rich proteins, all of them potential vaccine candidates. In fact, the presence of sIgA anti-Gal lectin in faeces of patients recovered from amoebic liver abscess (ALA) was associated with immunity to E. dispar. Moreover, the combined nasal and intraperitoneal vaccination of C3H/HeJ mice with native and recombinant Gal-lectin protected mice against an intracecal challenge with virulent E. histolytica trophozoites, protection that seemed to be associated with the induction of specific intestinal sIgA antibodies. Therefore, the stimulation of intestinal secretory response by mucosal delivery of amoebic antigens has been positioned as a promising strategy for inducing protection against human amoebiasis.
Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline-stimulated TRH transcription.
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