A sensor fusion approach for inertial sensors based 3D kinematics and pathological gait assessments: toward an adaptive control of stimulation in post-stroke subjects.

Pathological gait assessment and assistive control based on functional electrical stimulation (FES) in post-stroke individuals, brings out a common need to robustly quantify kinematics facing multiple constraints. This study proposes a novel approach using inertial sensors to compute dorsiflexion angles and spatio-temporal parameters, in order to be later used as inputs for online close-loop control of FES. 26 post-stroke subjects were asked to walk on a pressure mat equipped with inertial measurement units (IMU) and passive reflective markers. A total of 930 strides were individually analyzed and results between IMU-based algorithms and reference systems compared. Mean absolute (MA) errors of dorsiflexion angles were found to be less than 4°, while stride lengths were robustly segmented and estimated with a MA error less than 10 cm. These results open new doors to rehabilitation using adaptive FES closed-loop control strategies in "foot drop" syndrome correction.

Kinect-based assessment of proximal arm non-use after a stroke.

BACKGROUND: After a stroke, during seated reaching with their paretic upper limb, many patients spontaneously replace the use of their arm by trunk compensation movements, even though they are able to use their arm when forced to do so. We previously quantified this proximal arm non-use (PANU) with a motion capture system (Zebris, CMS20s). The aim of this study was to validate a low-cost Microsoft Kinect-based system against the CMS20s reference system to diagnose PANU. METHODS: In 19 hemiparetic stroke individuals, the PANU score, reach length, trunk length, and proximal arm use (PAU) were measured during seated reaching simultaneously by the Kinect (v2) and the CMS20s over two testing sessions separated by two hours. RESULTS: Intraclass correlation coefficients (ICC) and linear regression analysis showed that the PANU score (ICC = 0.96, r2 = 0.92), reach length (ICC = 0.81, r2 = 0.68), trunk length (ICC = 0.97, r2 = 0.94) and PAU (ICC = 0.97, r2 = 0.94) measured using the Kinect were strongly related to those measured using the CMS20s. The PANU scores showed good test-retest reliability for both the Kinect (ICC = 0.76) and CMS20s (ICC = 0.72). Bland and Altman plots showed slightly reduced PANU scores in the re-test session for both systems (Kinect: - 4.25 ± 6.76; CMS20s: - 4.71 ± 7.88), which suggests a practice effect. CONCLUSION: We showed that the Kinect could accurately and reliably assess PANU, reach length, trunk length and PAU during seated reaching in post stroke individuals. We conclude that the Kinect can offer a low-cost and widely available solution to clinically assess PANU for individualised rehabilitation and to monitor the progress of paretic arm recovery. TRIAL REGISTRATION: The study was approved by The Ethics Committee of Montpellier, France (N°ID-RCB: 2014-A00395-42) and registered in Clinical Trial (N° NCT02326688, Registered on 15 December 2014, https://clinicaltrials.gov/ct2/show/results/NCT02326688 ).

Proximal arm non-use when reaching after a stroke.

After a stroke, many people "cannot and do not" use their paretic upper limb. With recovery, some people "can but do not" use their paretic upper limb and this non-use should be counteracted with specific rehabilitation. The aim of the study was to quantify one aspect of the non-use: proximal arm non-use when reaching within one's arm length in 45 post-stroke and 45 age matched controls. Arm use refers to the contribution of the shoulder and elbow motion to the hand movement towards the target. Proximal arm non-use is calculated as the ratio of the difference between spontaneous arm use and maximal arm use. We found that proximal arm non-use has very good test-retest reliability, does not depend on time since stroke, increases with impairment (Fugl-Meyer) and loss of function (Box & Block), and most importantly, that 61% of patients with lower impairment (Fugl-Meyer >28/42) exhibit proximal arm non-use. We conclude that quantifying proximal arm non-use in post-stroke individuals provides novel information that complements routine clinical measures. It is likely that proximal arm non-use quantifies one aspect of the motor reserve that therapists can target in patient specific rehabilitation programs.

Kinematics in the brain: unmasking motor control strategies?

In rhythmical movement performance, our brain has to sustain movement while correcting for biological noise-induced variability. Here, we explored the functional anatomy of brain networks during voluntary rhythmical elbow flexion/extension using kinematic movement regressors in fMRI analysis to verify the interest of method to address motor control in a neurological population. We found the expected systematic activation of the primary sensorimotor network that is suggested to generate the rhythmical movement. By adding the kinematic regressors to the model, we demonstrated the potential involvement of cerebellar-frontal circuits as a function of the irregularity of the variability of the movement and the primary sensory cortex in relation to the trajectory length during task execution. We suggested that different functional brain networks were related to two different aspects of rhythmical performance: rhythmicity and error control. Concerning the latter, the partitioning between more automatic control involving cerebellar-frontal circuits versus less automatic control involving the sensory cortex seemed thereby crucial for optimal performance. Our results highlight the potential of using co-registered fine-grained kinematics and fMRI measures to interpret functional MRI activations and to potentially unmask the organisation of neural correlates during motor control.

Innovative technologies applied to sensorimotor rehabilitation after stroke.

Innovative technologies for sensorimotor rehabilitation after stroke have dramatically increased these past 20 years. Based on a review of the literature on "Medline" and "Web of Science" between 1990 and 2013, we offer an overview of available tools and their current level of validation. Neuromuscular electric stimulation and/or functional electric stimulation are widely used and highly suspected of being effective in upper or lower limb stroke rehabilitation. Robotic rehabilitation has yielded various results in the literature. It seems to have some effect on functional capacities when used for the upper limb. Its effectiveness in gait training is more controversial. Virtual reality is widely used in the rehabilitation of cognitive and motor impairments, as well as posture, with admitted benefits. Non-invasive brain stimulation (rTMS and TDCS) are promising in this indication but clinical evidence of their effectiveness is still lacking. In the same manner, these past five years, neurofeedback techniques based on brain signal recordings have emerged with a special focus on their therapeutic relevance in rehabilitation. Technological devices applied to rehabilitation are revolutionizing our clinical practices. Most of them are based on advances in neurosciences allowing us to better understand the phenomenon of brain plasticity, which underlies the effectiveness of rehabilitation. The acceptation and "real use" of those devices is still an issue since most of them are not easily available in current practice.

Therapeutic patient education for stroke survivors: Non-pharmacological management. A literature review.

OBJECTIVES: Focus on the different therapeutic patient education (TPE) programs for stroke survivors found in the literature. Verify their content and efficacy. METHOD: A literature review was conducted by searching for entries from 1966 to 2011 in the Medline and Cochrane Library databases. The references for the accepted articles were taken into consideration and the articles corresponding to the criteria inclusion but not present within the initial search were selected. The keywords used were "self care", "self management", "patient education" and "stroke". Given the multiplicity of symptoms that may be addressed in TPE programs, and following expert advice, the symptoms were grouped after expanding the bibliographic search using the following, additional keywords: "dysphagia"; "swallowing disorder"; "urinary incontinence"; "caregiver"; "fall prevention"; "falling"; "injury"; "shoulder pain"; "physical activity"; "exercise"; "aphasia" and "cognitive impairment". RESULTS: We found 30 article abstracts. In the end, we only accepted seven articles on general TPE programs that were well structured and detailed enough. The TPE programs found in the literature were often of questionable methodological quality. The multiplicity of symptoms led to very general TPE programs that covered all possible stroke after-effects. The purpose of these programs was to reduce stress and anxiety, to improve quality of life and to alleviate psychosocial after-effects. A change in caregiver and patient behavior was observed at times. We expanded the bibliographic search to include scientific arguments that could help implement TPE programs for more specific targets. CONCLUSION: TPE programs for stroke survivors could be improved by standardizing and assessing programs that focus on a specific problem caused by the various possible after-effects of strokes. In order to promote education for stroke survivors, specific training for health care professionals and appropriate funding are necessary.

Rehabilitation of arm function after stroke. Literature review.

INTRODUCTION: In the recent literature we can find many articles dealing with upper extremity rehabilitation in stroke patients. New techniques, still under evaluation, are becoming the practical applications for the concept of post-stroke brain plasticity. METHODS: This literature review focuses on controlled randomized studies, reviews and meta-analyses published in the English language from 2004 to 2008. The research was conducted in MEDLINE with the following keywords: "upper limb", "stroke", "rehabilitation". RESULTS: We reviewed 66 studies. The main therapeutic strategies are: activation of the ipsilesional motor cortex, inhibition of the contralesional motor cortex and modulation of the sensory afferents. Keeping a cortical representation of the upper limb distal extremity could prevent the learned non-use phenomenon. The modulation of sensory afferents is then proposed: distal cutaneous electrostimulation, anesthesia of the healthy limb, mirror therapy, virtual reality. Intensifying the rehabilitation care means increasing the total hours of rehabilitation dedicated to the paretic limb (proprioceptive stimulation and repetitive movements). This specific rehabilitation is facilitated by robot-aided therapy in the active-assisted mode, neuromuscular electrostimulation and bilateral task training. Intensifying the rehabilitation training program significantly improves the arm function outcome when performed during subacute stroke rehabilitation (< six months). Ipsilesional neurostimulation as well as mental practice optimize the effect of repetitive gestures for slight motor impairments. Contralesional neurostimulation or anesthesia of the healthy hand both improve the paretic hand's dexterity via a decrease of the transcallosal inhibition. This pathophysiological mechanism could also explain the positive impact of constraint-induced movement therapy (CI therapy) in an environmental setting for chronic stroke patients. CONCLUSION: To ensure a positive functional outcome, stroke rehabilitation programs are based on task-oriented repetitive training. This literature review shows that exercising the hemiparetic hand and wrist is essential in all stages of a stroke rehabilitation program. New data stemming from neurosciences suggest that ipsilesional corticospinal excitability should be a priority.

[Bickerstaff's syndrome presenting with coma, tetraplegia and blindness]. / Coma, tétraplégie flasque et cécité révélant un syndrome de Bickerstaff.

INTRODUCTION: Bickerstaff brainstem encephalitis is characterized by the occurrence of ataxia, ophthalmoplegia, motor weakness with areflexia and central nervous system symptoms, with drowsiness, pyramidal syndrome and sensorial symptoms. Diagnosis is based on MR findings and GQ1b antibodies. Treatment is not well known. OBSERVATION: We report a patient aged 39 years native of Laos who presented weakness, loss of reflexes, and drowsiness. Brain MR showed hyperintense signals in the brain stem. GQ1b antibodies were positive. The course was characterized by decrease of the weakness, normalization of MR and negativity of GQ1b antibodies. DISCUSSION: This observation underlines common features of Bickerstaff brainstem encephalitis, Miller Fisher syndrome and Guillain Barre syndrome. A favorable course and GQ1b antibodies are shared by these syndromes.

[Influence of acute aggravations on the development of long-term handicap in relapsing remitting multiple sclerosis: a clinical study in 99 patients]. / Influence des poussées sur la survenue d'un handicap au long cours dans la sclérose en plaques de forme rémittente: étude observationnelle de 99 patients.

INTRODUCTION: Rate of relapse occurring during the first 5 years of MS-RR is a prognosis factor of occurrence of disability or secondary progressive (SP) phase. Progressive phase, related to chronic axonal loss, is mainly considered as the principal factor of disability progression. Influence of acute relapses during the relapsing-remitting phase on disability development is not known as a prognosis factor. OBJECTIVES: To determine the influence of the exacerbations among patients with RR-MS after the second clinical event on the disability occurrence. METHODS: Diagnosis of multiple sclerosis was established according to Poser's classification. Disability measurement was made with the use of the Expanded Disability Status Scale (EDSS). The patients included in the study were classified as clinically definite RR-MS, with an EDSS score500 m. The study began at the time of the second clinical event and ended when an EDSS score of 4.0 was reached or when a SP phase was beginning or at the last follow-up visit date if these two stages were not reached. The primary outcome measure was the comparison of the risk and the average time to reach an EDSS>or=4.0 or a SP form according to the annual exacerbation rate (AER) using Kaplan-Meier survival curve. RESULTS: Among the 238 ms patients of the database, 136 patients were classified as having a definite RR-MS. Among these 136 patients, 99 patients could be included in the study according to the inclusion criteria. The median follow up of the patients since the first clinical event was 9.8 years (range 4 to 44). The average EDSS score was 0.7 at the beginning of the study and 2.3 at the end. 20.2p.cent of patients (n=20) reached an EDSS score of 4.0 or a SP-MS. The median AER was 0.4 and the average 0.62 (range 0 to 6.1). The time to reach the primary end point for 25p.cent of the population was 17.8 years in group with an AER<0.4 (group A) and 6.9 years in group with an AER>0.4 (group B) (logrank; p<0.0001). The relative risk for patients of the group B compared to group A to reach an EDSS of 4.0 or a SP form was 8.01 (IC-95p.cent: 2.74-23.46; p=0.0001). CONCLUSIONS: In spite of a limited number of patients, this study gives evidence that a high rate of acute exacerbations in RR-MS patients after the second clinical event may be an independent predictive factor of long-term residual disability progression. High relapse rate leads to a more frequent and faster SP or EDSS>4.0 occurrence.

[The assessment of autonomy in elderly people]. / Evaluation de l'autonomie de la personne âgée.

The assessment of autonomy in elderly people relies on various instruments that aim to evaluate and follow up patients, to measure the burden of care for the medical staff, or to properly distribute health budgets. In this article, we describe 3 clinical scales traditionally employed by gerontologists and specialists in geriatric rehabilitation. We intentionally left out generic scales such as the Barthel index and the Functional Independence Measure, which are well known by physiatrists. The Katz index is a scale of Activities of Daily Living, and the Lawton test is a scale of Instrumental Activities of Daily Living. We paid special attention to the AGGIR classification, which is the actual legal instrument for evaluating dependency in elderly in France, and whose first application is health resources management.

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

[Cerebral blood flow disturbances after anterior choroidal artery infarcts. Anatomical and functional correlates]. / Le débit sanguin cérébral après infarctus choroïdien antérieur. Correlations anatomique et foncionelles.

We have investigated the cortical and subcortical regional cerebral blood flow (rCBF) disorders resulting from infarcts of the anterior choroidal artery (AChA), and correlations with the severity of lesions, the physical and cognitive deficits, and the functional impairment. Eighteen patients presenting with recent anterior choroidal artery infarct without any other brain injury were examined at the secondary phase post-stroke using the single photon emission computed tomography technique and 133 Xenon inhalation. The rCBF and asymmetry indexes (AI) were calculated for 12 symmetrical hemispheric areas, and the cerebellum. The AI values were compared with those of 24 control subjects. The severity of the lesions was evaluated from CT scans or MRI. The neurological status (Orgogozo scale, walking disorders, MMSE, attention impairment, aphasia) and disability (functional independance measure: FIM) were assessed for each patient at the same time period. The relationships between rCBF disorders and brain lesions, and between the results of clinical investigations and rCBF disorders and brain lesions were assessed by linear regression analyses (stepwise variable selections, p=0.05). The AI values were significantly increased in the cerebral hemispheres, and this was most severe in the internal capsule (direct effect of the lesion) and the dorsolateral hemispheric cortex (diaschisis). Individual evaluations showed that AI were significantly increased in 13 patients in at least one ROI of the cerebral hemispheres, and in 3 patients in the internal capsule. Stepwise variable selections revealed that AI were best explained by the severity of the lesions in the internal capsule and the internal temporal area. The AI of the external temporal area and the internal capsule also helped explain the clinical (physical and cognitive) deficits. Thus, AChA infarcts may have relatively large effects on the central part of the lateral and dorsal cortex of the ipsilateral hemisphere. Subcortical and cortical consequences both contribute to explain the motor and cognitive deficits and disability.

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.

[Neuropsychological correlates of cerebral blood flow anomalies and MRI lesions after rupture of anterior communicating artery aneurysms]. / Corrélations neuropsychologiques des anomalies du débit sanguin cérébral et des lésions IRM après rupture des anévrysmes de l'artère communicante antérieure.

The aim of this study was to investigate relations between neuropsychological disorders resulting from rupture of aneurysms of the anterior communicating artery, regional cerebral blood flow anomalies and brain lesions revealed on MRI. Blood flow was analyzed in 22 consecutive patients at least 3 weeks after surgery using single photon emission computed tomography. Flow values were calculated in 10 regions of interest on each side of the brain. Attention, motor control, executive functions, short-term and long-term learning (verbal, visuo-spatial), categorical evocation, general intellectual performances were investigated. Flow drop was observed over frontal areas, which predominated on the right side. The correlation analyses showed that

CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

Hepatocyte-derived cell lines express a functional receptor for cardiotrophin-1.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study, we show that CT-1 binds to hepatocyte-derived cell lines of rat and human origin with high (Kd = 600-800 pM) and low (Kd approximately 3-6 nM) binding affinities. Treatment of HepG2 cells with CT-1 resulted in the induction of tyrosine phosphorylation of both transducing receptor subunits, gp130 and LIF receptor, and this phosphorylation was completely inhibited by a neutralizing anti-gp130 mAb. Addition of CT-1 to HepG2 or H35 cell cultures induced a dose-dependent production of several acute phase proteins (haptoglobin, fibrinogen, alpha1-acid glycoprotein, alpha2-macroglobulin). Moreover, the use of a neutralizing mAb to gp130 in cultures of HepG2 cells grown in the presence of CT-1, inhibited the induction of acute phase protein secretion, indicating an absolute requirement of gp130 in the formation of a functional CT-1 receptor. Altogether, these results suggest that CT-1 could play an important role in the regulation of hepatocyte metabolism in inflammatory responses.

Regulation of interleukin 6 expression by cardiotrophin 1.

Cardiotrophin 1 (CT-1) is a recently described cytokine sharing many biological properties with those reported previously for leukaemia inhibitory factor (LIF). In the present study we show that CT-1 binds to the KB epidermoid cancer cell surface through a tripartite receptor complex which includes the gp130 signal transducing protein, LIF receptor beta (LIFR beta) and a third component displaying a molecular weight of 80 kDa. CT-1 activates gp130 and LIFR beta transducing components, as attested by analysing their tyrosine phosphorylation level. The activation process is relayed to the nucleus by the recruitment of the STAT3 transcription factor. Analysis of KB cell line culture supernatants after CT-1 treatment indicates that CT-1 stimulates the production of interleukin 6 (IL-6) in a time- and dose-dependent manner. This stimulation of IL-6 production by CT-1 is associated with an increase in intracellular levels of IL-6 mRNA. This study suggests that at least in some pathological situations CT-1 might represent an immunomodulator regulating cytokine-induced gene products.

Alanine substitution for Thr268 and Asp269 of soluble ciliary neurotrophic factor (CNTF) receptor alpha component defines a specific antagonist for the CNTF response.

Ciliary neurotrophic factor (CNTF) associates with an alpha subunit (CNTFRalpha) of the receptor complex to initiate signal transduction by facilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) beta. CNTFRalpha is anchored to the membrane by a glycosylphosphatidylinositol linkage; however, a soluble form of the alpha subunit can still bind CNTF to recruit the signal transducing components of the receptor complex. In the present study we show that alanine substitution for residues Thr268 and Asp269 of the CNTFRalpha subunit results in a mutated receptor subunit (R3), which can bind CNTF with an affinity similar to that of the wild type CNTFRalpha but, when expressed as a soluble receptor subunit, lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3alpha inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFRalpha but not by IL-6 or oncostatin M. Similarly, CNTFR3alpha specifically antagonizes the induction of gp130 and LIFRbeta tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFRalpha in the HepG2 hepatoma cell line, as well as the subsequent events leading to haptoglobin synthesis. Positions 268 and 269 of CNTFRalpha appear to be critical for its interaction with gp130 and LIFRbeta, whereby alanine substitution of the residues at these positions results in antagonism of the CNTF-induced response.

Nerve growth factor is involved in the supportive effect by bone marrow--derived stromal cells of the factor-dependent human cell line UT-7.

We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte-macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS-5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble muNGF duplicate MS-5 effect and survival of CD34+/CD38- clonogenic progenitor cells promoted by MS-5 was unaffected by an anti-NGF and was not induced by soluble NGF alone or combined with SCF. In contrast, NGF in synergy with SCF supported the short-term maintenance of high numbers of CD34+/CD38+ mature erythroid progenitors probably through an indirect mechanism implying macrophages. These results suggest that NGF, in which the primary target cells are outside the hematopoietic system, is present in the marrow environment and might act at some steps of hematopoietic stem cell development. These results also underline that the response of cell lines and normal stem cells to stromal cells is mediated by different pathways.