Impairment of TNF-alpha production and action by imidazo[1,2- alpha] quinoxalines, a derivative family which displays potential anti-inflammatory properties.

In a previous study, we analysed the synthesis and properties of a series of imidazo[1,2-alpha]quinoxalines designed in our laboratory as possible imiquimod analogues. We found that these imidazo[1,2-alpha]quinoxalines were in fact potent inhibitors of phosphodiesterase 4 enzymes (PDE4). PDE4 inhibition normally results in an increase in intracellular cAMP which, in PBMC, induces the suppression of TNF-alpha mRNA transcription and thus cytokine synthesis. Such an effect is antagonistic to that of imiquimod. Furthermore, some TNF-alpha-induced activity, such as cell apoptosis which is dependent on the intracellular cAMP levels might also be affected. Therefore, by counteracting the properties of TNF-alpha and/or its production, the imidazo[1,2-alpha]quinoxalines could be considered as potential anti-inflammatory drugs. The present study was performed to confirm or refute this hypothesis. For this, we characterized the effects of imidazo[1,2-alpha]quinoxalines both on TNF-alpha activity and synthesis in regard to their ability to act as inhibitors of PDE4 (IPDE4). We found that the imidazo[1,2-alpha]quinoxalines dose-dependently prevented the TNF-alpha-triggered death of L929 cells, with the 8-series (-NHCH3 in R4) being the most potent. Moreover, when the effect of the 8-series on TNF-alpha production was investigated using gamma9delta2 T cells, it was observed that these compounds impaired the TCR:CD3-triggered TNF-alpha production. Structure-activity analysis revealed that these properties of the drugs did not coincide with their IPDE4 properties. This prompted further exploration into other signalling mechanisms possibly involved in TNF-alpha action and production, notably the p38 MAPK and the PI3K pathway. We demonstrate here that the imidazo[1,2-alpha]quinoxalines targeted these pathways in a different way: they activated the p38 MAPK pathway whilst inhibiting the PI3K pathway. Such effects on cell signalling could account for the imidazo[1,2-alpha]quinoxalines effects on 1) action and 2) production of TNF-alpha, which define these drugs as potential anti-inflammatory agents.

Relearning of Activities of Daily Living: A Comparison of the Effectiveness of Three Learning Methods in Patients with Dementia of the Alzheimer Type.

This study examined the effectiveness of three different learning methods: trial and error learning (TE), errorless learning (EL) and learning by modeling with spaced retrieval (MR) on the relearning process of IADL in mild-to-moderately severe Alzheimer's Dementia (AD) patients (n=52), using a 6-weeks randomized controlled trial design. The participants had to relearn three IADLs. Repeated-measure analyses during pre-intervention, post-intervention and 1-month delayed sessions were performed. All three learning methods were found to have similar efficiency. However, the intervention produced greater improvements in the actual performance of the IADL tasks than on their explicit knowledge. This study confirms that the relearning of IADL is possible with AD patients through individualized interventions, and that the improvements can be maintained even after the intervention.

The lectin jacalin triggers CD4-mediated lymphocyte signaling by binding CD4 through a protein-protein interaction.

The lectin jacalin specifically stimulates lymphocytes expressing the CD4 antigen. Recent studies have also demonstrated that this lectin interacts with CD4, inhibits in vitro HIV infection, and triggers cell signaling directly via CD4. Jacalin as a lectin was suggested to trigger CD4-mediated cell signaling by interacting with the oligosaccharide side chains of CD4 located on Asn271 and Asn3OO. Such a hypothesis was of importance because it implied that the glycosylated chains could represent a functional domain directly involved in CD4-related cell activation. We analyzed this possibility by studying the effect of hapten sugars on jacalin-induced CD4 cell signaling and jacalin/CD4 interaction, and by studying the binding capacities of the lectin toward glycosylated, deglycosylated, and unglycosylated CD4. The results presented in this study provide evidence that jacalin does not recognize the CD4 oligosaccharide chains and actually binds CD4 through a specific protein-protein interaction; as a consequence these results rule out the involvement of the CD4 saccharide moieties in CD4-mediated cell signaling triggered by the lectin.

Jacalin, a lectin that inhibits in vitro HIV-1 infection, induces intracellular calcium increase via CD4 in cells lacking the CD3/TcR complex.

The lectin jacalin interacts with the CD4 cell surface antigen; this lectin inhibits in vitro infection by human immunodeficiency virus type 1 without preventing virus binding on the host cell. The infection process is known to involve cellular events triggered by the binding of the viral external glycoprotein gp120 to CD4. Herein we demonstrate that jacalin induces cell signaling directly through the CD4 antigen and that independently of the CD3/TcR complex. The capacity of jacalin to trigger cell signals through the CD4 molecule is discussed in relation to its ability to inhibit HIV infection.

The T cell antigen receptor activates phosphatidylinositol 3-kinase-regulated serine kinases protein kinase B and ribosomal S6 kinase 1.

The present study has explored T cell antigen receptor-regulated serine kinases in human T cells. The results identify two phosphatidylinositol 3-kinase (PI3K)-controlled serine kinases operating downstream of the T cell receptor (TCR) in primary T cells: (i) protein kinase B whose activation regulates the phosphorylation of glycogen synthase kinase 3 and (ii) ribosomal S6 kinase 1, a kinase with a critical role in the regulation of protein synthesis and cell growth. T cells express two isoforms of S6k1: a 70 kDa cytoplasmic kinase and an 85 kDa isoform that has a classic nuclear localisation. TCR ligation triggers a parallel engagement of both the 70 and 85 kDa isoforms of S6k1 in a response that requires PI3K function.

Effector pathways regulating T cell activation.

Activation of T lymphocytes is a key event for an efficient response of the immune system. It requires the involvement of the T cell receptor antigen as well as costimulatory molecules such as CD28. Engagement of these receptors through the interaction with a foreign antigen associated with major histocompatibility complex molecules and CD28 counter-receptors B7.1/B7.2, respectively, results in a series of signaling cascades acting in synergy and which culminate in activation of interleukin-2 gene transcription and eventually cell proliferation. Many studies aimed at characterizing these specific effector pathways have been published; however, the actual signaling molecules that transduce activation signals from the cell membrane to the nucleus and that directly regulate interleukin-2 gene transcription are not yet completely defined and remain a matter of debate. In this commentary, we have attempted to analyze the results, which are sometimes diverging if not totally contradictory, characterizing effector pathways that possibly are triggered during T cell activation.

The Raf-1/mitogen-activated protein kinase kinase-1/extracellular signal-regulated-2 signaling pathway as prerequisite for interleukin-2 gene transcription in lectin-stimulated human primary T lymphocytes.

It has been shown that stimulation of lymphoid cells causes the activation of the extracellular signal-regulated-2 (ERK-2) which activates nuclear factor of activated T cells (NF-AT), a transcription factor involved in the regulation of interleukin-2 (1L2) gene transcription. ERK-2 is activated via a kinase cascade initiated by activation of the G protein p21Ras followed by phosphorylation and activation of Raf-1 and mitogen-activated protein kinase kinase-1 (MEK-1). Activation of this pathway has been described primarily in human T cell lines; however, using primary T lymphocytes from transgenic mice, a recent study has shown that a blockade of this cascade did not perturb lymphocyte stimulation and proliferation. In the present paper, we studied in human primary T cells the possible involvement of the Raf-1/MEK-1/ERK-2 pathway upon stimulation by jacalin, a mitogenic lectin which specifically stimulates CD4+ lymphocytes. We show here that the mitogen-activated protein (MAP) kinase pathway was stimulated in human purified lymphocytes upon activation with jacalin. Moreover, activation of this pathway appeared to be essential, since its blockade by a specific inhibitor of the MEK-1 kinase abolished IL2 gene transcription; in contrast, in T cells stimulated with phytohemagglutinin M(PHA), another potent T cell mitogenic lectin, blockade of MEK-1 reduced but did not totally inhibit either ERK-2 phosphorylation or IL2 mRNA expression. This shows, as already suggested, that another pathway in addition to the Raf-1/MEK-1/ERK-2 kinase cascade could be triggered in T cell activation. Jacalin stimulation therefore appeared to be a good model for the specific activation of the MAP kinase pathway in human primary T lymphocytes, which would allow the characterisation of drugs specifically targeted to this particular pathway.

Initiation and supervisory processes in schizophrenia and depression.

Clinical studies show that schizophrenic and depressive subjects have problems with daily life activities, and neuropsychological studies tend to explain these problems in terms of a dysexecutive syndrome. Verbal fluency and sentence arrangement are tasks considered to focus on two aspects of the dysexecutive syndrome known as initiation and supervision processes, respectively. In this study, we assessed performance in these two tasks in schizophrenia and depression. Twenty-six schizophrenic subjects (chronic schizophrenia, DSM IV definition) were compared with 26 control subjects balanced for sex, age and educational level, and 16 depressive subjects (major depression episode, DSM IV) were compared with 11 similarly balanced control subjects. Switching and clustering scores were evaluated during a semantic fluency task as two components underlying the initiation and organization processes. Capture errors specific to failure of the supervisory system and differences between the number of correct responses in two conditions (valid/invalid) were evaluated as indexes of the supervision process in a sentence arrangement task. In the semantic fluency task, switching scores were significantly lower in the schizophrenic and depressive subjects than in their respective controls. In the sentence arrangement task, only the schizophrenic subjects made significantly more capture errors than their controls and had significantly fewer correct sequences in invalid conditions than in valid conditions. This study shows a dissociation between supervision and initiation processes in two different psychiatric populations. Initiation is impaired, but supervision is preserved in depression, whereas both initiation and supervision are impaired in schizophrenia.

Inhibition of HIV Infection by Lectin Binding to CD4.

The progression of AIDS all over the world has led research laboratories to focus their energy on the identification of molecules that can impair the infection process, Every step of the viral rephcative cycle can be considered as a potential target to block infection. One of the first events in the infection of T-cells by the human immunodeficiency virus (HIV) is the binding of the viral envelope glycoprotein gp120 to the differentiation antigen CD4 expressed on the host cell (1-3). Among the therapeutic strategies aimed at controllmg HIV disease progression, one was to inhibit or perturb this interaction.

Efficacy of tianeptine vs placebo in the long-term treatment (16.5 months) of unipolar major recurrent depression*

To compare the efficacy and acceptability of tianeptine vs placebo in the long-term treatment of unipolar major recurrent depression, 268 hospitalized and ambulatory patients meeting DSM III-R criteria for major depression with a 21-item Hamilton depression rating scale (HDRS) score >/= 17 and at least one episode in the previous 5 years received tianeptine in a 6-week multicenter open study. At D42, 185 responders (intention-to-treat population) were randomized for ethical reasons into two unbalanced groups to receive tianeptine 37.5 mg/day (n= 111) or placebo (n= 74) for 16.5 months; 173 of the 185 responders were defined as strict responders (per-protocol population) by an HDRS score which was both < 15 and at least halved vs D1, combined with clinical confirmation by the investigator. The groups were similar at baseline except in the severity of the depressive episode (greater in the tianeptine group: 33 vs 18%, p= 0.018). Relapse (before 6 months) and recurrence (after 6 months) were defined by an HDRS score >/= 15 and/or clinical global impression score >/= 4, and clinical confirmation by the investigator. Visits were at D63 and M3, M6, M9, M12, M15, and M18. Efficacy was measured by the number of relapses and recurrences and their time of onset (Kaplan-Meier survival curve analysis). Between D42 and M18 (intention-to-treat population), relapse and recurrence were less frequent on tianeptine vs placebo (16 vs 36%, p= 0.002). Comparison over time also showed a higher proportion of patients without relapse or recurrence on tianeptine (p< 0.001); the intergroup difference increased with follow-up duration. Secondary analysis of relapse in the intention-to-treat group showed a higher proportion on placebo (p= 0.002); secondary analysis of recurrence over time showed that the difference in the percentages of recurrence-free patients was nonsignificant in the intention-to-treat population (p= 0.067) but significant in the per-protocol population (p= 0.036) in favor of tianeptine. Acceptability did not differ between the groups. Treatment-induced adverse events were rare and mild in both groups. These data support the use of tianeptine in the long-term treatment of unipolar major recurrent depression. Relapse and recurrence were decreased two- to threefold on tianeptine vs placebo with no difference in acceptability between the two groups. Copyright 1997 Doin Editeurs

Evaluation of the supervisory system in elderly subjects with and without disinhibition.

UNLABELLED: Disinhibition and irritability, defined as loss of behavioral and emotional control, are frequent in the elderly. The working hypothesis for this study was that these disorders are associated with a cognitive alteration of control processes that manifests as non-routine behavior because of the dysfunction of a general executive component known as the supervisory attentional system (SAS). METHODS: A total of 28 elderly subjects with mild cognitive impairment were recruited and divided into two groups using the Neuropsychiatric Inventory. Fourteen subjects were allocated to the disinhibited group and 14 subjects matched for age, sex and educational level formed a disinhibition-free control group. The neuropsychological battery included the following tests: Mini Mental Score Evaluation, Boston Naming test, Token test, Trail Making and Verbal Fluency. Two tasks were specifically designed to stress the SAS: 1) A specific verbal sentence arrangement task in which subjects had to use sequential reasoning with verbal material. Each test sequence consisted of a series of words shown in jumbled order. The construction of some sequences had to be done by using familiar routine associations (valid conditions). In contrast, other sequences required the overriding selection of familiar routine associations, which were inappropriate within the general context of the task (invalid conditions). 2) Using the Continuous Performance Test, four aspects were evaluated: sustained, selective, preparation and suppressive attention. RESULTS: The only group differences in neuropsychological test results were the following: 1) the sentence arrangement task. In comparison with the control group, the disinhibited group was impaired in invalid conditions and the calculated difference between the number of correct responses in invalid conditions minus that in valid conditions was significantly higher; and 2) the CPT. Disinhibited subjects had a significantly lower number of hits, exclusively in the 'suppressive attention' paradigm. These results suggest that subjects with disinhibition have impaired supervisory system function.

[Diagnostic criteria for fronto-temporal lobe degeneration]. / Critères diagnostiques des dégénérescences lobaires fronto-temporales.

Circumscribed atrophy of the frontal and temporal lobes (frontotemporal lobar degeneration) accounts for about one fifth of cases of primary degenerative dementia occurring before the age of 65. It produces three prototypical clinical syndromes. The most common is frontotemporal dementia, characterized by personality change and profound alteration in social conduct and associated with bilateral atrophy of the frontal and anterior temporal lobes. Progressive non-fluent aphasia is characterized by difficulty in verbal expression, anomia and phonemic errors in the presence of relative preservation of comprehension and associated with atrophy predominantly of the left hemisphere. In semantic dementia there is fluent speech with semantic errors and severely impaired comprehension and naming, together with a visual associative agnosia, resulting from bilateral atrophy of the inferior and middle temporal gyri. The clinical syndromes occur with either of two main histological types: prominent microvacuolar change, without specific histological features (frontal lobe degeneration-type), severe astrocytic gliosis with or without ballooned cells and inclusion bodies (Pick-type). To improve clinical recognition and advance understanding of this relatively common form of cerebral degeneration, members of an international workshop on Frontotemporal Lobar Degeneration developed consensus criteria, building upon earlier published clinical diagnostic guidelines for frontotemporal dementia. The consensus criteria reported here specify core and supportive features for each of the prototypical clinical syndromes: frontotemporal dementia, progressive aphasia and semantic dementia, as well as providing broad inclusion and exclusion criteria for the generic entity of frontotemporal lobar degeneration.

Inpatient mother-and-child postpartum psychiatric care: factors associated with improvement in maternal mental health.

PURPOSE: This study assessed the underexplored factors associated with significant improvement in mothers' mental health during postpartum inpatient psychiatric care. METHODS: This study analyzed clinical improvement in a prospective cohort of 869 women jointly admitted with their infant to 13 psychiatric Mother-Baby Units (MBUs) in France between 2001 and 2007. Predictive variables tested were: maternal mental illness (ICD-10), sociodemographic characteristics, mental illness and childhood abuse history, acute or chronic disorder, pregnancy and birth data, characteristics and mental health of the mother's partner, and MBU characteristics. RESULTS: Two thirds of the women improved significantly by discharge. Admission for 25% was for a first acute episode very early after childbirth. Independent factors associated with marked improvement at discharge were bipolar or depressive disorder, a first acute episode or relapse of such an episode. Schizophrenia, a personality disorder, and poor social integration (as measured by occupational status) were all related to poor clinical outcomes. DISCUSSION: Most women improved significantly while under care in MBUs. Our results emphasize the importance of the type of disease but also its chronicity and the social integration when providing postpartum psychiatric care.

Antigen receptor signal transduction: activating and inhibitory antigen receptors regulate STAT1 serine phosphorylation.

Antigen receptors are crucial regulators of the mammalian immune response. Immediate antigen receptor proximal signal transduction pathways mediated by tyrosine (Tyr) kinases are well defined. In contrast, much less is known about the network of serine (Ser) kinases and Ser kinase substrates that are linked to antigen receptor function. Here we describe a new signaling module for antigen receptors in lymphocytes; a Ser kinase pathway that phosphorylates Ser 727 in STAT1alpha, a member of the signal transducer and activator of transcription gene family. In the present study we have explored the regulation of STAT1 Ser 727 phosphorylation in human T and B lymphocytes and show that it is controlled by both positive and negative antigen receptor signaling cascades. Ligation of antigen receptors in both B and T cells induce a delayed but then sustained phosphorylation of STAT1 on Ser 727. STAT1 Ser phosphorylation is induced by the TCR in the absence of STAT1 Tyr phosphorylation, indicating that in T cells STAT1 Ser and Tyr phosphorylation are independent events. Antigen receptor regulation of STAT Ser phosphorylation is dependent on phosphatidylinositol 3-kinase-mediated signals. Furthermore, the negative regulatory receptor FcgammaRIIb, which mediates vital feedback control of B cell responses, prevents antigen receptor-induced phosphorylation of STAT1 Ser 727. The ability of antigen receptors to both positively and negatively regulate STAT1 Ser 727 phosphorylation reveals a Ser kinase network that operated during sustained responses to antigen receptor engagement.

Production of TNF-alpha by human V gamma 9V delta 2 T cells via engagement of Fc gamma RIIIA, the low affinity type 3 receptor for the Fc portion of IgG, expressed upon TCR activation by nonpeptidic antigen.

Human lymphocytes expressing the gammadelta TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vgamma9Vdelta2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vgamma9Vdelta2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vgamma9Vdelta2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vgamma9Vdelta2 T cells express FcgammaRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vgamma9Vdelta2 T cells. Indeed, ligation of CD16 on Vgamma9Vdelta2 T cells leads to TNF-alpha production. This TNF-alpha production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vgamma9Vdelta2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcgammaRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vgamma9Vdelta2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.

Evidence for a p21(ras)/Raf-1/MEK-1/ERK-2-independent pathway in stimulation of IL-2 gene transcription in human primary T lymphocytes.

T cell stimulation leads to triggering of signals transmitted from the cell membrane to the nucleus through TCR/CD3 proteins. Characterization of these signals largely results from the use of cell lines stimulated with anti-CD3 monoclonal antibodies. These studies have established that activation caused a rapid increase in the formation of GTP-bound Ras, which stimulates the mitogen-activated protein kinase pathway involving the extracellular-regulated kinase-2 (ERK-2) and activates the nuclear factor of activated T cells (NF-AT) that regulates interleukin-2 (IL-2) gene transcription. In the present study, we used human primary T cells, and we investigated the intracellular signals triggered by two different anti-CD3 monoclonal antibodies (UCHT1 and X-35), which both strongly induce cell proliferation. We found that, in contrast to the commonly used UCHT1, X-35 activated IL-2 gene transcription without stimulation of the Raf-1/mitogen-activated ERK kinase-1 (MEK-1)/ERK-2 phosphorylation cascade; we also showed that X-35 stimulation, which triggers an ERK-2-independent pathway, does not involve activation of p21(ras). In addition to demonstrating that activation of p21(ras) and of its Raf-1/MEK-1/ERK-2 effector pathway is not an event obligatorily triggered upon TCR/CD3 ligation, these results provide the first evidence of the existence of a p21(ras)/ERK-2-independent pathway for IL-2 gene transcription in human primary T lymphocytes.

Covariance analysis of protein families: the case of the variable domains of antibodies.

A nonrestrictive method for identifying covariance in protein families is described and applied to human and mouse germline Vkappa and VH sequence alignments. Amino acids that occur at each position in a sequence alignment are divided into two sets, called a word, by generating all possible combinations of alternative amino acids. Each word is associated with a pattern of changes. Words with identical patterns identify covariant positions. In antibody variable domains, the number of words generated ranged between 1103 and 2195 depending on the alignment, of which 4 to 12 % occurred in covariant pairs. Despite the nonrestrictive character of pattern generation, covariant residues did not reflect a random selection with respect to the nature of amino acid changes and/or their spatial proximity in a reference crystallographic structure. This approach allowed the identification of a covariance signal for positions with high variability, mostly located in the outer part of the common structural framework of antibody variable domains. Covariance in these regions may reflect the existence of alternative and mutually exclusive atomic arrangements that are compatible with antibody function. The method may be of general applicability to rationalize residue variability in protein families.

Isopentenyl pyrophosphate, a mycobacterial non-peptidic antigen, triggers delayed and highly sustained signaling in human gamma delta T lymphocytes without inducing eown-modulation of T cell antigen receptor.

The Vgamma9Vdelta2 T cell subset, which represents up to 90% of the circulating gammadelta T cells in humans, was shown to be activated, via the T cell receptor (TcR), by non-peptidic phosphorylated small organic molecules. These phosphoantigens, which are not presented by professional antigen-presenting cells, induce production of high amounts of interferon-gamma and tumor necrosis factor (TNF-alpha). To date, the specific signals triggered by these antigens have not been characterized. Here we analyze proximal and later intracellular signals triggered by isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vgamma9Vdelta2 T cells, and compare these to signals induced by the non-physiological model using an anti-CD3 antibody. During antigenic stimulation we noticed that, except for the proximal p56(lck) signal, which is triggered early, the signals appear to be delayed and highly sustained. This delay, which likely accounts for the delay observed in TNF-alpha production, is discussed in terms of the ability of the antigen to cross-link and recruit transducing molecules mostly anchored to lipid rafts. Moreover, we demonstrate that, in contrast to anti-CD3 antibody, IPP does not induce down-modulation of the TcR.CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-alpha.

Evidence for a CD4-associated calcium influx independent of the phosphoinositide transduction pathway in human T cells.

We recently showed, using human Jurkat T cell variants lacking the T cell receptor (TCR)/CD3 complex, that the lectin jacalin is able to trigger intracellular calcium increase provided that CD4 is expressed on the cell surface. Involvement of the CD4 molecule in jacalin-induced biological effects was furthermore demonstrated in differentiated U937 myelomonocytic cells expressing or not expressing CD4, and is confirmed here in human CD4-transfected mouse thymoma cells. In the present paper, we analyze the CD4-associated calcium response triggered by jacalin independently of the TCR/CD3 complex. We show that the observed calcium rise results from a direct long-lasting calcium influx from the outside without release of calcium from intracellular stores. We demonstrate that it is independent of the phosphoinositide phospholipase C transduction pathway. Moreover, we show that this peculiar calcium response can be blocked by protein tyrosine kinase inhibitors (herbimycin and genistein) giving evidence of the involvement of a protein tyrosine kinase, the best candidate of which is the CD4-associated p56lck. Altogether, our results suggest that, independently of the TCR/CD3 complex, CD4 may be involved in the triggering of a calcium signal dependent on a protein tyrosine kinase and independent of the phosphoinositide transduction pathway.