Inhibition of RhoA reduces propofol-mediated growth cone collapse, axonal transport impairment, loss of synaptic connectivity, and behavioural deficits.

BACKGROUND: Exposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function. METHODS: RhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7. RESULTS: Propofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment. CONCLUSION: Inhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.

Two-year Experience of the "Organ Donation Week" of the Federal University of Health Sciences of Porto Alegre, Brazil.

BACKGROUND: Today, Brazil is the second country of the world in number of transplants. Nonetheless, waiting lists are getting longer. This lack of organs occurs mostly because of people's reduced knowledge about the donation process. With the aim of changing this scenario, in 2013 and 2014, "Organ Donation Week" events were held at the Federal University of Health Sciences of Porto Alegre. METHODS: During the 2 years, documentaries followed by a cycle of debates with experts in this area were exhibited. In 2013, a "flash-mob" took place, with the purpose of performing a "transplant waiting list" around the perimeter of Santa Casa's Hospital Complex. In 2014, a morning full of educational activities was planned for the pediatric patients from the Santo Antônio Children's Hospital and their relatives. RESULTS: It is estimated that approximately 1774 people were directly reached by the projects. Among these people, we can include medical students, healthcare professionals, university staff, transplanted patients, and their families. We believe that education and consciousness are central points in the donation and transplant process. Through this project, we could inform people about it, solving their doubts and myths and stimulating this kind of conversation among the family circle, making the moment when the family must make the decision much easier. CONCLUSIONS: Education and public awareness are essential for enhancing the number of organ donations. Therefore, events such as "Organ Donation Week" should be encouraged among medical schools.

Implementing Activities Developed by the Organ Transplantation Academic Society of the Hospital Dom Vicente Scherer: A Pilot Study.

BACKGROUND: The number of academic societies has been growing significantly in Brazilian universities, offering an extra opportunity for the development of educational activities and research. Because organ donation and transplantation is an area still insufficiently approached during the graduation of health professionals, we evaluated how academic societies might be a valuable tool. METHODS: Participants of the course promoted by the Organ Transplantation Academic Society of the Hospital Dom Vicente Scherer were evaluated through the use of a questionnaire and cognitive tests with 16 multiple-choice questions about topics approached during the course, before and after the lectures. Topics approached consisted of a general introduction about transplantation in Brazil, brain death, organ allocation and removal, post-transplant follow-up, and clinical cases. RESULTS: Of the 45 participants, 30 answered the tests at both times. The subjects were students of medicine, nursing, and phonoaudiology; 93.3% were organ donors, 84.6% said their families knew about this decision, and 65% had relatives who were organ donors. The mean score of correct answers was 7.63 of 16 before the activities and 12.54 after activities, demonstrating a 64.4% improvement. CONCLUSIONS: The improvement in performance suggests that academic societies are a useful resource for educational purposes and for students to get a deeper insight about organ donation and transplantation.

Andrographis paniculata decreases fatigue in patients with relapsing-remitting multiple sclerosis: a 12-month double-blind placebo-controlled pilot study.

BACKGROUND: Andrographis paniculata (A. paniculata), a medicinal plant, has shown anti-inflammatory, neuroprotective and antifibrotic effects in animal models as well as clinical efficacy in different studies, including an anti-fatigue effect in autoimmune diseases such as rheumatoid arthritis. In multiple sclerosis (MS), fatigue is rated as one of the most common and disabling symptoms. In the present trial, we investigated the effect of A. paniculata on relapse rate and fatigue in relapsing-remitting MS (RRMS) patients receiving interferon beta. METHODS: A randomised double-blind placebo-controlled trial assessed the effects of 170 mg of A. paniculata dried extract tablet b.i.d. p.o. on relapse rate and fatigue using the Fatigue Severity Scores (FSS) over 12 months in RRMS patients receiving interferon. The Expanded Disability Status Scale (EDSS) score, inflammatory parameters and radiological findings were also investigated. Twenty-five patients were enrolled, and twenty-two patients were ultimately analysed and randomised to the active or placebo group. RESULTS: Patients treated with A. paniculata showed a significant reduction in their FSS score as compared to the placebo, equivalent to a 44 % reduction at 12 months. No statistically significant differences were observed for relapse rate, EDSS or inflammatory parameters, with a trend in reducing new lesions among the A. paniculata group. One patient in the A. paniculata group presented with a mild and transient skin rash, which was alleviated with anti-histamine treatment for three weeks. CONCLUSION: A. paniculata was well tolerated in patients and no changes in clinical parameters were observed. A. paniculata significantly reduces fatigue in patients with RRMS receiving interferon beta in comparison to placebo and only interferon beta treatment. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02280876 ; Trial registration date: 20.10.2014.

cIAP1 regulates TNF-mediated cdc42 activation and filopodia formation.

Tumour necrosis factor-α (TNF) is a cytokine endowed with multiple functions, depending on the cellular and environmental context. TNF receptor engagement induces the formation of a multimolecular complex including the TNFR-associated factor TRAF2, the receptor-interaction protein kinase RIP1 and the cellular inhibitor of apoptosis cIAP1, the latter being essential for NF-κB activation. Here, we show that cIAP1 also regulates TNF-induced actin cytoskeleton reorganization through a cdc42-dependent, NF-κB-independent pathway. Deletion of cIAP1 prevents TNF-induced filopodia and cdc42 activation. The expression of cIAP1 or its E3-ubiquitin ligase-defective mutant restores the ability of cIAP1(-/-) MEFs to produce filopodia, whereas a cIAP1 mutant unable to bind TRAF2 does not. Accordingly, the silencing of TRAF2 inhibits TNF-mediated filopodia formation, whereas silencing of RIP1 does not. cIAP1 directly binds cdc42 and promotes its RhoGDIα-mediated stabilization. TNF decreases cIAP1-cdc42 interaction, suggesting that TNF-induced recruitment of cIAP1/TRAF2 to the receptor releases cdc42, which in turn triggers actin remodeling. cIAP1 also regulates cdc42 activation in response to EGF and HRas-V12 expression. A downregulation of cIAP1 altered the cell polarization, the cell adhesion to endothelial cells and cell intercalation, which are cdc42-dependent processes. Finally, we demonstrated that the deletion of cIAP1 regulated the HRas-V12-mediated transformation process, including anchorage-dependent cell growth, tumour growth in a xenograft model and the development of experimental metastasis in the lung.

Efficacy of an Andrographis paniculata composition for the relief of rheumatoid arthritis symptoms: a prospective randomized placebo-controlled trial.

Andrographis paniculata (Burm. f.) Wall ex Nees (Acanthaceae) possesses anti-inflammatory effects, attributed to the main constituent andrographolide proposed as alternative in the treatment of autoimmune disease. A prospective, randomized, double blind, and placebo-controlled study in patients with rheumatoid arthritis (RA) was performed. Tablets (Paractin) made of an extract of A. paniculata (30% total andrographolides) were administered three times a day for 14 weeks, after a 2-week washout period to 60 patients with active RA. The primary outcomes were pain intensity measured using a horizontal visual analog pain scale (VAPS). In addition, ACR, EULAR, and SF36 clinical parameters were recorded. The intensity of joint pain decreased in the active vs placebo group at the end of treatment, although these differences were not statistically significant. A significant diminishing for week in tender joint -0.13 95% confidence interval (CI; -0.22 to 0.06; p = 0.001), number of swollen joints -0.15 95%CI (-0.29 to -0.02; p = 0.02), total grade of swollen joint -0.27 95%CI (-0.48 to -0.07; p = 0.010), number of tender joints -0.25 95%CI (-0.48 to -0.02; p = 0.033), total grade of swollen joints -0.27 95%CI (-0.48 to -0.07; p = 0.01), total grade of tender joints -0.47 95%CI (-0.77 to -0.17; p = 0.002) and HAQ -0.52 95%CI (-0.82 to -0.21; p < 0.001) and SF36 0.02 95%CI (0.01 to 0.02; p < 0.001) health questionnaires was observed within the group with the active drug. Moreover, it was associated to a reduction of rheumatoid factor, IgA, and C4. These findings suggest that A. paniculata could be a useful "natural complement" in the treatment of AR; however, a larger trial and a more extended period of treatment is necessary in order to corroborate these results.

A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma.

A pair of isogenic colon carcinoma cells, SW480 and 620, was used to investigate the mechanisms of acquired tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance during tumour progression. Whereas primary tumour SW480 cells are sensitive to TRAIL-induced apoptosis, metastatic SW620 cells are resistant. The apoptotic signalling activated by TRAIL in SW480 cells is a type II pathway. We show that in SW620 cells, although caspase-8 is recruited and activated at the death-inducing-signalling complex and Bid is cleaved, this does not lead to caspase-9 activation. Comparison of Bcl-2, Bcl-xL and Mcl-1 levels in both cell lines showed no difference. In SW620 cells transfected with a tBid-GFP construct, tBid-GFP was correctly localized to the mitochondria. Thus, the resistance of SW620 cells is at the level of the mitochondria that can withstand large amounts of tBid. Although caspase-3 was directly cleaved by caspase-8 in SW620 cells to yield the p20 fragment, no further autocatalytic maturation into the p17 fragment was observed. We show that, in contrast to SW480 cells, the SW620 cell line expresses high amounts of X-linked inhibitor of apoptosis (XIAP). Downregulation of XIAP with bortezomib or small-interfering RNA was sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis in the absence of SMAC/Diablo or cytochrome c release from the mitochondria. Thus, SW620 cells have developed a dual resistance to TRAIL-induced apoptosis: a block at the level of the mitochondria and, after a conversion to a type I pathway, an increased expression of XIAP which inhibits this pathway.

Soluble interleukin-2 receptor and metalloproteinase-9 expression in head and neck cancer: prognostic value and analysis of their relationships.

In a series of 84 head and neck patients, a statistically significant correlation was observed between high serum soluble interleukin (IL)-2 receptor alpha (sIL-2Ralpha) (P = 0.034) and metalloproteinase-9 (MMP-9) concentrations (P = 0.036) at diagnosis and a shorter survival of these patients. As MMP-9 has been shown to mediate cleavage of IL-2Ralpha (CD25) by preactivated T cells, we looked for a relationship between MMP-9 expression and soluble IL-2Ralpha serum concentrations in these cancer patients. We did not find any correlation between intratumoral expression of MMP-9 or serum MMP-9 concentrations and serum sIL-2Ralpha levels. These results led us to reassess the role of MMP-9 in the release of sIL-2Ralpha. Treatment of Kit225 leukaemic cells with recombinant MMP-9 slightly decreased membrane CD25 expression and was associated with an increased concentration of sIL-2Ralpha in the supernatants. However, using a selective inhibitor of MMP-9 we did not succeed in specifically inhibiting the release of sIL-2Ralpha by the Kit225 cell line or by phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells. In addition, in a preclinical mouse model, basal serum sIL-2Ralpha concentrations and sIL-2Ralpha production by activated cells were not altered in MMP-9-deficient mice compared to wild-type mice. Interestingly, a broad spectrum metalloproteinase inhibitor inhibited the release of sIL-2Ralpha by PHA-activated peripheral blood mononuclear cells, suggesting that in contrast with current views concerning the major role of MMP-9 in the cleavage of membrane IL-2Ralpha, other proteases are involved in the shedding of sIL-2Ralpha. MMP-9 and sIL-2Ralpha appear therefore as independent prognostic markers in head and neck cancers.

Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways.

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ralpha and gammac and only binds IL-4, whereas, in the type II receptor, IL-4Ralpha dimerizes with IL-13Ralpha1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Ralpha2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Ralpha2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Ralpha2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Ralpha2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Ralpha2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Ralpha2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Ralpha2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins.

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.

Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase.

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.

A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners.

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing.

Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.

The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis.

In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.

Cdk2 associates with MAP kinase in vivo and its nuclear translocation is dependent on MAP kinase activation in IL-2-dependent Kit 225 T lymphocytes.

Cell proliferation is controlled by cdk2 which in association with cyclin E and A regulates G1/S transition and S phase progression. cdk2 activation is dependent on its localization in the nucleus where regulatory mediators are found. We report that activation of cdk2 is associated with the formation of cdk2/MAP Kinase complexes. cdk2 associates with both inactive and activated MAP Kinase. Prevention of MAP Kinase activation by the MEK inhibitor PD98059 inhibits both activation and nuclear localization of cdk2 and S phase entry. These findings indicate that the nuclear translocation of cdk2 is associated with the formation of molecular complexes containing active MAP Kinase and is dependent on MAP Kinase activation. Oncogene (2000) 19, 4184 - 4189

Il-4 and IFN-gamma mRNA induction in human peripheral lymphocytes specific for beta-lactam antibiotics in immediate or delayed hypersensitivity reactions.

Beta-lactam antibiotics elicit CD4+ and CD8+ T-cell-mediated immune responses that play a central role in allergic reactions. However, the involvement of a type 1- (Th1 or Tc1) or a type 2-like (Th2 or Tc2) differentiation in drug allergy remains unclear. We investigated the expression of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) mRNA by quantitative reverse transcription and polymerase chain reaction (RT-PCR) in patient-derived peripheral blood lymphocytes following specific in vitro stimulation. Samples were collected from a total of 19 patients who had developed immediate or delayed clinical manifestations of hypersensitivity to beta-lactam and from 11 control subjects. Peripheral blood mononuclear cells (PBMCs) were stimulated with either free antibiotics or antibiotic-human serum albumin (HSA) conjugates. Specific induction of IFN-gamma mRNA expression was observed in 11 of 11 allergic patients with immediate reactions, in 6 of 8 patients with delayed reactions, and in 4 of 11 control subjects. IL-4 mRNA expression was induced in 5 of 11 allergic individuals with immediate reactions but in none of the 8 patients with delayed responses and none of the 11 control subjects. IL-4 mRNA expression was only induced following activation with free drugs, while IFN-gamma mRNA expression was predominantly induced in CD4+ T cells following stimulation with HSA-conjugated drugs. Immediate-type hypersensitivity to beta-lactams was not associated with a pure type 2-like response when PBMCs were specifically stimulated in vitro: Some patients with well-documented history of beta-lactam-induced immediate allergic reaction showed a high IFN-gamma response. Contact dermatitis involved Tc1 and Th1 cells and other delayed hypersensitivity reactions to beta-lactams were restricted to Th1 responses.

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle.

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.

IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes.

The stress-activated pathways leading to activation of p38 MAP kinase (p38 MAPK) and c-jun N-terminal kinases (JNK) have been shown to be activated by pro-inflammatory cytokines, physical and chemical stresses as well as a variety of hematopoietic growth factors. One exception is interleukin (IL)-4, which does not activate this pathway in hematopoietic cell. We report here that in A431, a keratinocytic cell line, IL-4 activates Rac and Cdc42 and their downstream effector p21-activated kinase (PAK). Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of PAK and leading to activation of p38 MAPK, since IL-4 stimulates tyrosine phosphorylation of p38 MAPK and increases its catalytic activity. As A431 cells are able to produce IL-6 in response to IL-4 stimulation, we assessed the involvement of p38 MAPK in IL-6 gene expression. A pyrimidazole compound, SB203580, a specific inhibitor of p38 MAPK, inhibits production and gene expression of IL-6. SB203580 reduced significantly the stability of IL-6 mRNA. Here we provide evidence that p38 MAPK is activated in response to IL-4 and is involved in IL-6 synthesis by stabilizing IL-6 mRNA.