Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

Pathogenesis of Salmonella-induced enteritis

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis.

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, ß-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by ß-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations.

Understanding the mechanisms of lung mechanical stress

Physical forces affect both the function and phenotype of cells in the lung. Bronchial, alveolar, and other parenchymal cells, as well as fibroblasts and macrophages, are normally subjected to a variety of passive and active mechanical forces associated with lung inflation and vascular perfusion as a result of the dynamic nature of lung function. These forces include changes in stress (force per unit area) or strain (any forced change in length in relation to the initial length) and shear stress (the stress component parallel to a given surface). The responses of cells to mechanical forces are the result of the cell's ability to sense and transduce these stimuli into intracellular signaling pathways able to communicate the information to its interior. This review will focus on the modulation of intracellular pathways by lung mechanical forces and the intercellular signaling. A better understanding of the mechanisms by which lung cells transduce physical forces into biochemical and biological signals is of key importance for identifying targets for the treatment and prevention of physical force-related disorders.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

Oxysterols are 27-carbon atom molecules resulting from autoxidation or enzymatic oxidation of cholesterol. They are present in numerous foodstuffs and have been demonstrated to be present at increased levels in the plasma of patients with cardiovascular diseases and in atherosclerotic lesions. Thus, their role in lipid disorders is widely suspected, and they might also be involved in important degenerative diseases such as Alzheimer's disease, osteoporosis, and age-related macular degeneration. Since atherosclerosis is associated with the presence of apoptotic cells and with oxidative and inflammatory processes, the ability of some oxysterols, especially 7-ketocholesterol and 7β-hydroxycholesterol, to trigger cell death, activate inflammation, and modulate lipid homeostasis is being extensively studied, especially in vitro. Thus, since there are a number of essential considerations regarding the physiological/pathophysiological functions and activities of the different oxysterols, it is important to determine their biological activities and identify their signaling pathways, when they are used either alone or as mixtures. Oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever. Moreover, a substantial accumulation of polar lipids in cytoplasmic multilamellar structures has been observed with cytotoxic oxysterols, suggesting that cytotoxic oxysterols are potent inducers of phospholipidosis. This basic knowledge about oxysterols contributes to a better understanding of the associated pathologies and may lead to new treatments and new drugs. Since oxysterols have a number of biological activities, and as oxysterol-induced cell death is assumed to take part in degenerative pathologies, the present review will focus on the cytotoxic activities of these compounds, the corresponding cell death signaling pathways, and associated events (oxidation, inflammation, and phospholipidosis).

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17% protein (normal-protein diet; NP) or 6% protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The β-cell mass was significantly reduced (≅65%) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30% reduction in insulin receptor substrate-1 and a 70% increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43% in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71% in LP islets compared to NP islets (P < 0.05). Therefore, the reduced β-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.

Glutamate-N-methyl-D-aspartate receptor modulation and minocycline for the treatment of patients with schizophrenia: an update

Growing consistent evidence indicates that hypofunction of N-methyl-D-aspartate (NMDA) transmission plays a pivotal role in the neuropathophysiology of schizophrenia. Hence, drugs which modulate NMDA neurotransmission are promising approaches to the treatment of schizophrenia. The aim of this article is to review clinical trials with novel compounds acting on the NMDA receptor (NMDA-R). This review also includes a discussion and translation of neuroscience into schizophrenia therapeutics. Although the precise mechanism of action of minocycline in the brain remains unclear, there is evidence that it blocks the neurotoxicity of NMDA antagonists and may exert a differential effect on NMDA signaling pathways. We, therefore, hypothesize that the effects of minocycline on the brain may be partially modulated by the NMDA-R or related mechanisms. Thus, we have included a review of minocycline neuroscience. The search was performed in the PubMed, Web of Science, SciELO, and Lilacs databases. The results of glycine and D-cycloserine trials were conflicting regarding effectiveness on the negative and cognitive symptoms of schizophrenia. D-serine and D-alanine showed a potential effect on negative symptoms and on cognitive deficits. Sarcosine data indicated a considerable improvement as adjunctive therapy. Finally, minocycline add-on treatment appears to be effective on a broad range of psychopathology in patients with schizophrenia. The differential modulation of NMDA-R neurosystems, in particular synaptic versus extrasynaptic NMDA-R activation and specific subtypes of NMDA-R, may be the key mediators of neurogenesis and neuroprotection. Thus, psychotropics modulating NMDA-R neurotransmission may represent future monotherapy or add-on treatment strategies in the treatment of schizophrenia.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Short-term exposure of mice to cigarette smoke and/or residual oil fly ash produces proximal airspace enlargements and airway epithelium remodeling

Chronic obstructive pulmonary disease (COPD) is associated with inflammatory cell reactions, tissue destruction and lung remodeling. Many signaling pathways for these phenomena are still to be identified. We developed a mouse model of COPD to evaluate some pathophysiological mechanisms acting during the initial stage of the disease. Forty-seven 6- to 8-week-old female C57/BL6 mice (approximately 22 g) were exposed for 2 months to cigarette smoke and/or residual oil fly ash (ROFA), a concentrate of air pollution. We measured lung mechanics, airspace enlargement, airway wall thickness, epithelial cell profile, elastic and collagen fiber deposition, and by immunohistochemistry transforming growth factor-β1 (TGF-β1), macrophage elastase (MMP12), neutrophils and macrophages. We observed regional airspace enlargements near terminal bronchioles associated with the exposure to smoke or ROFA. There were also increases in airway resistance and thickening of airway walls in animals exposed to smoke. In the epithelium, we noted a decrease in the ciliated cell area of animals exposed to smoke and an increase in the total cell area associated with exposure to both smoke and ROFA. There was also an increase in the expression of TGF-β1 both in the airways and parenchyma of animals exposed to smoke. However, we could not detect inflammatory cell recruitment, increases in MMP12 or elastic and collagen fiber deposition. After 2 months of exposure to cigarette smoke and/or ROFA, mice developed regional airspace enlargements and airway epithelium remodeling, although no inflammation or increases in fiber deposition were detected. Some of these phenomena may have been mediated by TGF-β1.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 μg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 μg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 μg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

Nitro-fatty acids: novel anti-inflammatory lipid mediators

Nitro-fatty acids are formed and detected in human plasma, cell membranes, and tissue, modulating metabolic as well as inflammatory signaling pathways. Here we discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, redirecting arachidonic acid metabolism and signaling. We also analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases.

The role of necroptosis in neurosurgical diseases

Programmed necrosis or necroptosis is an alternative form of cell death that is executed through a caspase-independent pathway. Necroptosis has been implicated in many pathological conditions. Genetic or pharmacological inhibition of necroptotic signaling has been shown to confer neuroprotection after traumatic and ischemic brain injury. Therefore, the necroptotic pathway represents a potential target for neurological diseases that are managed by neurosurgeons. In this review, we summarize recent advances in the understanding of necroptotic signaling pathways and explore the role of necroptotic cell death in craniocerebral trauma, brain tumors, and cerebrovascular diseases.

Restoration of sensory dysfunction following peripheral nerve injury by the polysaccharide from culinary and medicinal mushroom, Hericium erinaceus (Bull.: Fr.) Pers. through its neuroregenerative action

Abstract Peripheral nerves have the unique capability to regenerate after injury. Insights into regeneration of peripheral nerves after injury may have implications for neurodegenerative diseases of the nervous system. We investigated the ability of polysaccharide from Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats by daily oral administration. In sensory functional recovery test, the time taken for the rats to withdraw its hind limb from contact with the hot plate was measured. The test revealed acceleration of sensory recovery in the polysaccharide group compared to negative controls. Further, peripheral nerve injury leads to changes at the remotely located DRG containing cell bodies of sensory neurons. Immunofluorescence studies showed that Akt and p38 MAPK were expressed in DRG and strongly upregulated in polysaccharide group after peripheral nerve injury. The intensity of endothelial cells antigen-1 that recognized endothelial cells in the blood vessels of distal segments in crushed nerves was significantly higher in the treated groups than in the negative control group. Our findings suggest that H. erinaceus is capable of accelerating sensory functional recovery after peripheral nerve injury and the effect involves the activation of protein kinase signaling pathways and restoration of blood-nerve barrier.