Activation of a novel p70 S6 kinase 1-dependent intracellular cascade in the basolateral nucleus of the amygdala is required for the acquisition of extinction memory.

Repeated presentations of a previously conditioned stimulus lead to a new form of learning known as extinction, which temporarily alters the response to the original stimulus. Previous studies have shown that the consolidation of extinction memory requires de novo protein synthesis. However, the role of specific nodes of translational control in extinction is unknown. Using auditory threat conditioning in mice, we investigated the role of mechanistic target of rapamycin complex 1 (mTORC1) and its effector p70 S6 kinase 1 (S6K1) in the extinction of auditory threat conditioning. We found that rapamycin attenuated the consolidation of extinction memory. In contrast, genetic deletion and pharmacological inhibition of S6K1, a downstream effector of mTORC1, blocked within-session extinction, indicating a role for S6K1 independent of protein synthesis. Indeed, the activation of S6K1 during extinction required extracellular signal-regulated kinase (ERK) activation in the basolateral nucleus of the amygdala (BLA) and was necessary for increased phosphorylation of the GluA1 (Thr840) subunit of the AMPA receptor following extinction training. Mice exposed to brief uncontrollable stress showed impaired within-session extinction as well as a downregulation of ERK and S6K1 signaling in the amygdala. Finally, using fiber photometry we were able to record calcium signals in vivo, and we found that inhibition of S6K1 reduces extinction-induced changes in neuronal activity of the BLA. These results implicate a novel ERK-S6K1-GluA1 signaling cascade critically involved in extinction.

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells.

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.

Dual role for transforming growth factor beta-dependent signaling in Trypanosoma cruzi infection of mammalian cells.

Expression of functional transforming growth factor beta (TGF-beta) receptors (TbetaR) is required for the invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi. However, the precise role of this host cell signaling complex in T. cruzi infection is unknown. To investigate the role of the TGF-beta signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TbetaR1 proteins that cannot induce growth arrest in response to TGF-beta but still transmit the signal for TGF-beta-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-beta signaling at invasion can be divorced from TGF-beta-induced growth arrest. TGF-beta pretreatment of mink lung cells expressing wild-type TbetaR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-beta to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-beta signaling, did not block infection by T. cruzi but did block the enhancement of infection by TGF-beta. Taken together, these results show that there is a dual role for TGF-beta signaling in T. cruzi infection. The initial invasion of the host cell is independent of both TGF-beta-dependent gene expression and growth arrest, but TGF-beta stimulation of infection requires a fully functional TGF-beta signaling pathway.

Cell-specific activation of nuclear factor-kappaB by the parasite Trypanosoma cruzi promotes resistance to intracellular infection.

The transcription factor nuclear factor-kappaB (NF-kappaB) is central to the innate and acquired immune response to microbial pathogens, coordinating cellular responses to the presence of infection. Here we demonstrate a direct role for NF-kappaB activation in controlling intracellular infection in nonimmune cells. Trypanosoma cruzi is an intracellular parasite of mammalian cells with a marked preference for infection of myocytes. The molecular basis for this tissue tropism is unknown. Trypomastigotes, the infectious stage of T. cruzi, activate nuclear translocation and DNA binding of NF-kappaB p65 subunit and NF-kappaB-dependent gene expression in epithelial cells, endothelial cells, and fibroblasts. Inactivation of epithelial cell NF-kappaB signaling by inducible expression of the inhibitory mutant IkappaBaM significantly enhances parasite invasion. T. cruzi do not activate NF-kappaB in cells derived from skeletal, smooth, or cardiac muscle, despite the ability of these cells to respond to tumor necrosis factor-alpha with NF-kappaB activation. The in vitro infection level in these muscle-derived cells is more than double that seen in the other cell types tested. Therefore, the ability of T. cruzi to activate NF-kappaB correlates inversely with susceptibility to infection, suggesting that NF-kappaB activation is a determinant of the intracellular survival and tissue tropism of T. cruzi.

Outcomes of annual tuberculosis screening by Mantoux test in children considered to be at high risk: results from one urban clinic.

BACKGROUND: In January 1994, the American Academy of Pediatrics recommended that annual screening with the purified protein derivative tuberculin skin test, Mantoux method, be used for tuberculosis screening in high-risk children. This test has a better sensitivity and specificity than the previously used multiple puncture test, and patients need to return for a reading done by palpation by a health care professional. OBJECTIVE: To estimate the prevalence of reactivity to purified protein derivative tuberculin in an urban primary care clinic whose patients meet high-risk criteria and to determine if annual screening is warranted, to determine the adherence to return to the clinic for reading of the skin test, and to describe the characteristics of patients who have tuberculosis infection and disease. RESEARCH DESIGN: Cross-sectional study. SETTING: Inner-city, hospital-based primary care pediatric clinic in Baltimore, MD. SUBJECTS: A total of 1433 consecutive children attending this clinic from March through September, 1994, who were at risk for tuberculosis because of frequent exposure to poor and medically indigent city dwellers. METHODS: The Mantoux test (5TU intradermal injection of purified protein derivative) was administered to children at annual health supervision visits. Patients were tracked to determine those who returned for a reading by a health care professional and find those with a positive Mantoux test. The charts of children with a positive test were reviewed. RESULTS: Five hundred seventy-three (40%) patients returned for a reading by a health care professional. Five patients had a positive Mantoux test, giving a prevalence rate of 0.8% of reactivity to purified protein derivative tuberculin. One child with a positive Mantoux test also had chest radiograph findings consistent with tuberculosis disease but was asymptomatic. CONCLUSIONS: In our city with a low prevalence of disease, children whose only risk factor for tuberculosis was exposure to poor and medically indigent city dwellers did not represent a high-risk group. Our results are supportive of the 1996 American Academy of Pediatrics screening statement that annual screening is not warranted. Sixty percent of children did not return for a reading of the Mantoux test by a health care professional. Alternative strategies that are more convenient for parents are needed to obtain accurate readings by health care professionals when skin testing is deemed necessary.

Modulation of protein kinase C activity in Plasmodium falciparum-infected erythrocytes.

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces many morphological and biochemical changes in the host cell. Host serine/threonine protein kinases could be involved in some of these processes. The aim of this study was to determine the effect of infection on red blood cell protein kinase C (PKC) and establish the importance of this enzyme in parasite growth and sexual stage differentiation. Phorbol myristate acetate (PMA)-induced translocation of erythrocyte PKC activity is impaired in erythrocytes enriched for mature asexual stage infected cells. Western blotting shows that this is due to a relative reduction in membrane PKC protein levels rather than inhibition of enzyme activity and analysis of PKC activity isolated from whole cell lysates by DE52 chromatography suggests that total activatable PKC levels are lower in infected erythrocytes. A reduction in PMA-induced activation is also observed in PKC assays performed in situ. Downregulation of erythrocyte PKC by overnight incubation with PMA before infection causes a significant decrease in the rate of the asexual growth, suggesting that the enzyme, although lost later in infection, may be important in the earlier development of the parasite. By contrast, the lack of PKC had no effect on the production of sexual stage parasites.

Plasmodium falciparum gametocyte adhesion to C32 cells via CD36 is inhibited by antibodies to modified band 3.

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.

CD36 and intercellular adhesion molecule 1 mediate adhesion of developing Plasmodium falciparum gametocytes.

Plasmodium falciparum trophozoite-infected erythrocytes adhere to the amelanotic melanoma C32 cell line in vitro. Here we demonstrate for the first time that immature gametocyte-infected erythrocytes also adhere to C32 cells, albeit at lower levels than trophozoites. However, anti-CD36 and anti-intercellular adhesion molecule 1 antibodies inhibit asexual and gametocyte adhesion by comparable percentages, suggesting a common dependency for binding to these cellular receptors.

Multiple tyrosine protein kinases structurally related to p56 lck are down-regulated following mitogenic stimulation of human T lymphocytes.

p56 lck is a well-characterized tyrosine protein kinase (TPK) which is thought to play a role in mitogenic signal transduction in T lymphocytes. Immunoblot analysis of human lymphocyte proteins using an antiserum cross-reactive with phosphotyrosine resulted in the detection of a 55-60 kDa protein band (presumably p56 lck) as well as several additional phosphotyrosyl proteins in lymphocyte extracts. All of these phosphotyrosyl proteins were down-regulated following mitotic stimulation. Autophosphorylation of lymphocyte microsomal fractions in the presence of [gamma-32P] ATP resulted in the labelling of p56 lck as well as other proteins of different molecular weights. Analysis of these labelled proteins by tryptic digestion resulted in strikingly similar peptide maps. The data suggest that lymphocytes may contain a family of TPKs structurally related to p56 lck. The down-regulation of the putative TPKs following mitogenic stimulation of lymphocytes with phytohaemagglutinin suggests that this family of TPKs may participate in mitotic signalling events, followed by their down-regulation.

An endogenous inhibitor of the protein tyrosine kinase activity of normal and malignant human lymphoid cells.

Tyrosine protein kinases (TPKs) have been implicated in mitotic signalling in a wide range of cells including lymphocytes. We describe here the partial characterization of a heat stable TPK inhibitor from both normal and malignant human lymphoid cells. Inhibitory activity was not attributable to contaminating ATPase, protease or phosphatase activities or to the Ca2+-binding protein S100. Preparations of the TPK inhibitor did not reduce the activity of cAMP-dependent protein kinase. While the inhibitor decreased the activity of TPKs towards an exogenous peptide substrate, it did not affect the autophosphorylation of microsomal TPKs. These results raise the possibility that the activity of TPKs in lymphoid cells may be regulated by an inhibitor protein.

Two major tyrosine protein kinases of resting human T lymphocytes are down-regulated following mitotic stimulation.

Human lymphocyte tyrosine protein kinases (TPKs) have been analyzed by gel-filtration chromatography. The major TPK species with activity towards an exogenous tyrosine-containing peptide had molecular masses of 70-100 kDa (TPK I) and 35-40 kDa (TPK II). TPKs I and II were distinct from the well-characterized autophosphorylating lymphoid cell TPK, pp56lck [(1983) J. Biol. Chem. 258, 10738-10742]. Both TPK I and TPK II were down-regulated following mitogenic stimulation of lymphocytes with phytohaemagglutinin. By contrast, pp56lck remained clearly detectable in stimulated lymphocytes. We suggest that TPKs I and II may play a role in the regulation of the lymphocyte cell cycle.