Impact of protein pulse feeding on lean mass in malnourished and at-risk hospitalized elderly patients: a randomized controlled trial.

BACKGROUND & AIMS: Aging is associated with a blunted anabolic response to dietary intake, possibly related to a decrease in systemically available amino acids (AAs), which in turn may stem from increased splanchnic AA metabolism. This splanchnic sequestration can be saturated by pulse feeding (80% of daily protein intake in a single meal), enabling increased protein synthesis. This study aimed to evaluate the efficacy of a new nutritional strategy, termed protein pulse feeding. METHODS: This prospective randomized study (ClinicalTrials.gov registration number NCT00135590) enrolled 66 elderly malnourished or at-risk patients in an inpatient rehabilitation unit. All were given a controlled diet for 6 weeks. In a spread diet (SD) group (n = 36), dietary protein was spread over the four daily meals. In a pulse diet (PD) group (n = 30), 72% of dietary protein (1.31 g/kg weight/d on average) was consumed in one meal at noon. The patients were evaluated at admission and at 6 weeks for body composition [lean mass (LM), appendicular skeletal muscle mass (ASMM), and body cell mass (BCM) indices, measured by X-ray absorptiometry combined with bioelectrical impedance analysis] (primary outcome), hand grip strength, and activities of daily living (ADL) score. RESULTS: Protein pulse feeding was significantly more efficacious than protein spread feeding in improving LM index (mean changes from baseline for PD group: +0.38 kg/m(2); 95% confidence interval (CI), [0; 0.60]; for SD group: -0.21 kg/m(2); 95% CI, [-0.61; 0.20]; p = 0.005 between the two groups), ASMM index (+0.21 kg/m(2); 95% CI, [0; 0.34] and -0.11 kg/m(2); 95% CI, [-0.20; 0.09]; p = 0.022), BCM index (+0.44 kg/m(2); 95% CI, [0.08; 0.52] and -0.04 kg/m(2); 95% CI, [-0.09; 0.10]; p = 0.004). There was no significant effect for hand-grip strength or ADL score. CONCLUSIONS: This study demonstrates for the first time that protein pulse feeding has a positive, clinically relevant effect on lean mass in malnourished and at-risk hospitalized elderly patients.

[Utility of soluble transferrin receptor measurement for early diagnostic of iron deficiency in elderly hospitalized patients]. / Intérêt du dosage du récepteur soluble de la transferrine dans le diagnostic biologique précoce du déficit martial chez les patients âgés hospitalisés.

Diagnosis of iron deficiency is not easily performed in inflammatory situation as usually encountered in elderly hospitalized patients (>70 years old). At first, we determined serum soluble receptor transferrin (RsTf), RsTf/serum ferritin ratio (RsTf-F index) and biochemical and haematological values used to evaluate iron status, in iron-depleted subjects (ferritinemia≤50 µg/L) (group 2) (n=22, 82±7 years) or not (group 1, reference group) (n=18, 82±6 years), without inflammatory diseases. Relevance of the biological parameters to diagnose iron deficiency was evaluated (ROC curve) and a cut-off value of RsTf-F (>1.85) was established. Then, we selected 60 patients (group 3) suspect of iron deficiency as previously validated with an inflammatory syndrome (CRP>12 mg/L). Almost all patients (95%) presented at least one risk factor of iron deficiency (anticoagulant drugs, nutritional or gastrointestinal diseases). In group 3, index RsTf-F values were increased (RsTf-F: 2.69±0.82 versus group 1: 1.25±0.34, p<0.05), in anemic patients (women Hb<120 g/L, men Hb<130 g/L) (n=42) and in non-anemic patients (n=18) (respectively RsTf-F: 2.84±0.87 versus 2.35±0.58, p<0.05). Thus, in elderly patients with inflammatory disorders, RsTf-F index could suspect iron deficiency before appearance of biological anemia.

Neurotensin protects pancreatic beta cells from apoptosis.

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

[Factors of therapeutic imbalance of antivitamins K and their haemorrhagic consequences in elderly]. / Les facteurs de déséquilibre thérapeutique des antivitamines K et leurs conséquences hémorragiques en pratique gériatrique.

Objective. To determinate, for older subjects, specific factors of imbalance of the oral anticoagulant treatments. Method. We conducted an epidemiological and analytical case-control study, during 18 months, in 2 geriatrics centers. Each patient with excessive anticoagulation (INR>4.5) was matched with 2 controls under anticoagulant, whom the INR (international normalized ratio) had stayed in the therapeutic range. Results. One-hundred fifty nine subjects (53 cases and 106 controls) were included. Haemorrhagic complications has been observed in 19.2% of cases, versus 3.9% of the controls. Some medicines frequently prescribed to the old subjects were correlated at a risk of excessive anticoagulation: amiodarone (9.4% versus 0, p<0.004), acetaminophen (18.9% versus 0.9%, p<0.001), tramadol (5.6% versus 0, p<0.04), ofloxacine (11.3% versus 1.9%, p<0.001), and lactulose (11.3% versus 0, p<0.001). Furthermore, several acute states increase the risk of excessive INR to the old subjects: fever (p<0.001), malnutrition (p<0.001), dehydration (p=0.006), and acute diarrhea (p<0.001). Conclusion. Some specific geriatric factors raised may destabilize treatments by anticoagulants.

Pain in the elderly: Prospective study of hyperbaric CO2 cryotherapy (neurocryostimulation).

OBJECTIVE: To evaluate the analgesic effects of hyperbaric CO(2) cryotherapy in elderly inpatients. METHODS: An open-label prospective study was conducted in two geriatrics departments in patients with a broad range of pain characteristics. Each patient underwent a physical evaluation followed by hyperbaric CO(2) cryotherapy sessions, whose spacing and number were at the discretion of the physiotherapist. Patients completed a 100-mm visual analog scale for pain severity before and after the sessions. RESULTS: We included 51 patients, who were treated between May 2 and June 30, 2005. Mean age was 83.7years, and the female-to-male ratio was 4/1. The patients had acute or chronic pain whose origin was usually musculoskeletal (80.3%) or neurological (18.6%). Pain scores decreased significantly after four sessions, from 52mm to 13mm (P<0.001) in patients with acute pain and from 45mm to 13mm (P<0.001) in those with chronic pain. CONCLUSION: Hyperbaric CO(2) cryotherapy is an innovative tool that should be incorporated within the non-pharmacological armamentarium for achieving pain relief in older patients.

Very low oral doses of vitamin B-12 increase serum concentrations in elderly subjects with food-bound vitamin B-12 malabsorption.

The BOSSANOVA study, a randomized double-blind trial, was designed to test the ability of very low oral doses of vitamin B-12 to increase the serum vitamin B-12 concentration in elderly subjects with food-bound vitamin B-12 malabsorption, and to determine whether there was a dose response. We also aimed to quantitatively assess the most efficient dose to be added to flour in addition to folic acid (flour cofortification with vitamin B-12 and folic acid). Sixty-seven patients were randomly assigned to 1 of 6 groups receiving various daily oral doses of vitamin B-12 (i.e., 2.5, 5, 10, 20, 40, or 80 microg/d) for 30 d. The dose-response was tested for different biological variables using a mixed model, taking into account the variable's initial value (between-subject effect), a linear log-dose effect, and a linear log (dosextime) interaction, where time was d 15 or d 30. We planned to determine the amount of oral vitamin B-12 that would increase the serum vitamin B-12 concentration by 37 pmol/L (50 ng/L). Significant between-subject effects were found for serum vitamin B-12, plasma homocysteine, and methylmalonic acid concentrations, but a log-dose effect was found only for vitamin B-12 (P<0.001). The slope of the line tended to be higher (P=0.07) at d 30 than at d 15. For a mean serum vitamin B-12 increase of 37 pmol/L, a dose of 5.9 (95% CI, 0.9-12.1) microg/d was needed. We concluded that very low oral doses of vitamin B-12 increased serum vitamin B-12 concentrations in elderly subjects with subclinical vitamin B-12 deficiency, following a log-dose pattern. Our results could be beneficial in the design of a public health program for safe flour cofortification with folic acid.

Internalization and recycling properties of neurotensin receptors.

The targeting, internalization and recycling of membrane receptors in response to extracellular ligands involve a series of molecular mechanisms which are beginning to be better understood. The receptor-dependent internalization of neurotensin has been widely investigated using endogenous or heterologous receptor expression systems. This review focuses on the general properties of neurotensin sequestration and on the characterization of the receptors involved in this process.

Functional roles of the NTS2 and NTS3 receptors.

Neurotensin exerts its actions in the central nervous system and the periphery through three identified receptors. Two of them, the NTS2 and NTS3, display unusual properties either because of their complex signal transduction mechanisms (NTS2) or because of their structural composition as a non-G-protein-coupled receptor (NTS3). Here, we review the transduction mechanisms, cellular trafficking, and potential physiological roles of these two unconventional receptors.

Morphine and pain-related stimuli enhance cell surface availability of somatic delta-opioid receptors in rat dorsal root ganglia.

The present study demonstrates that perikaryaldelta-opioid receptors (deltaORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal deltaORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog omega-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of mu-opioid receptor (muOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of deltaORs observed after sustained morphine is attributable to stimulation of muORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Adelta fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 microm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal deltaORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to muOR agonists.

Geriatric Nutritional Risk Index: a new index for evaluating at-risk elderly medical patients.

BACKGROUND: Patients at risk of malnutrition and related morbidity and mortality can be identified with the Nutritional Risk Index (NRI). However, this index remains limited for elderly patients because of difficulties in establishing their normal weight. OBJECTIVE: Therefore, we replaced the usual weight in this formula by ideal weight according to the Lorentz formula (WLo), creating a new index called the Geriatric Nutritional Risk Index (GNRI). DESIGN: First, a prospective study enrolled 181 hospitalized elderly patients. Nutritional status [albumin, prealbumin, and body mass index (BMI)] and GNRI were assessed. GNRI correlated with a severity score taking into account complications (bedsores or infections) and 6-mo mortality. Second, the GNRI was measured prospectively in 2474 patients admitted to a geriatric rehabilitation care unit over a 3-y period. RESULTS: The severity score correlated with albumin and GNRI but not with BMI or weight:WLo. Risk of mortality (odds ratio) and risk of complications were, respectively, 29 (95% CI: 5.2, 161.4) and 4.4 (95% CI: 1.3, 14.9) for major nutrition-related risk (GNRI: <82), 6.6 (95% CI: 1.3, 33.0), 4.9 (95% CI: 1.9, 12.5) for moderate nutrition-related risk (GNRI: 82 to <92), and 5.6 (95% CI: 1.2, 26.6) and 3.3 (95% CI: 1.4, 8.0) for a low nutrition-related risk (GNRI: 92 to < or =98). Accordingly, 12.2%, 31.4%, 29.4%, and 27.0% of the 2474 patients had major, moderate, low, and no nutrition-related risk, respectively. CONCLUSION: GNRI is a simple and accurate tool for predicting the risk of morbidity and mortality in hospitalized elderly patients and should be recorded systematically on admission.

Neurotensin and the neurotensin receptor-3 in microglial cells.

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. The type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the neurotensin receptor-3 is also the only neurotensin receptor expressed, and its activation by neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1beta and tumor necrosis factor-alpha. This induction is dependent on both protein kinases pathways. We observed that the effect of neurotensin on the cytokine/chemokine expression is also inhibited by the neurotensin receptor-3 propeptide. This is the demonstration that the neurotensin receptor-3 is functional and mediates both the migratory action of neurotensin and its induction of chemokines/cytokines expression.

[Impact of nasal colonization by methicillin-resistant Staphylococcus aureus among geriatric intermediate care facility patients]. / Impact de la colonisation nasale par le Staphylococcus aureus résistant à la méticilline dans une unité de soins de suite gériatrique.

OBJECTIVE: To evaluate the impact of nasal carriage of Methicillin Resistant Staphylococcus aureus (MRSA) on antibiotic cost, infection morbidity, mortality and length of stay in a geriatric population. METHODS: 341 consecutive elderly patients (mean age 83.4 +/- 8.7 years) admitted to an intermediate care facility were prospectively include between November 1998 and October 1999. Nasal swab cultures were taken on admission. RESULTS: In sixty patients (17.6%) no nasal swab was taken. Among the 281 patients screened, 52 were identified as MRSA carriers. The principle predictive factors were: diabetes (p=0,046), sores (p=0,03), malnutrition (p=0,02), polypathology (p=0,02) and prolongation of previous hospitalisation (p=0,09). CONCLUSION: Nasal carriage of MRSA on admission to the facility was not a deleterious prognostic factor regarding duration of stay, infectious morbidity and antibiotic cost, but was associated with higher mortality risk.

[Morning stiffness]. / Raideur matinale.

STIFFNESS AND UNSTRETCHING: Morning stiffness in the elderly is a painful entity often ignored, but frequent in institutions. Unstretching in the morning is part of the criteria in the diagnosis of inflammatory pathologies, but morning stiffness in old patients appears different from that described in rheumatological diseases and arthrosis. FROM AN AETIOLOGICAL POINT OF VIEW: Various affections can be accompanied by morning stiffness in an elderly patient: arthrosis, but also various consequential affections, static disorders, deformity. We felt it was important to define the precise criteria that individualise this pathology so as to improve its management. MANAGEMENT: Non-medicinal measures often reduce the intensity and duration of this symptom and implicate all those attending to the patient. Medicinal treatment has a more limited place in the fragile elderly patient, but its role remains to be assessed.

[Headaches in the elderly]. / Céphalées du sujet âgé

ACCORDING TO AGE: It is generally thought that the prevalence of headaches decreases with ageing. However recent studies, with stricter epidemiology and methodology, clearly indicate that this decreases is less obvious than that perceived. PRIMARY AND SECONDARY HEADACHES: In elderly patients, primary headaches and notably migraine (often with altered presentation) are less frequent, even though new authentic cases may appear. However the other types of headache are clearly present. In particular, the incidence and prevalence of secondary headaches slightly increases and they represent up to 30% of all the headaches observed, compared with less than 10% in young or adult patients. The causes of such symptomatic headaches are multiple and it is important to be able to identify them since an aetiological treatment is often possible. REGARDING TREATMENT: The symptomatic treatment of headaches in the elderly follows the same principles as that of younger patients. However, the side effects and drug interactions related to pharmacological treatments can be disastrous in fragilised patients. It is therefore important to emphasize the interest of physical methods of analgesia, particularly adapted to the elderly. Some of these methods have demonstrated their efficacy and are recommended by international consensuses.

Neurotensin receptor-3/sortilin mediates neurotensin-induced cytokine/chemokine expression in a murine microglial cell line.

We show that the type I neurotensin receptor-3 (also called sortilin) is the only known neurotensin receptor expressed in a murine microglial cell line and that its activation leads to phosphorylation of both extracellular signaling-regulated (Erk1/2) and Akt kinases. Using semiquantitative reverse-transcriptase (RT) PCR, we demonstrate that neurotensin induces gene expression of several cytokines/chemokines including macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. This induction is dependent on both phosphatidylinositol 3-kinase and mitogen-activated protein kinases pathways. We observe that the effect of neurotensin on cytokine/chemokine expression is inhibited by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. These results demonstrate that the neurotensin receptor-3 is functional in microglial cells where it mediates the induction of chemokines/cytokines expression by neurotensin.

Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells.

The neurotensin receptor-3, originally identified as sortilin, is unique among neuropeptide receptors in that it is a single trans-membrane domain, type I receptor. To gain insight into the functionality of neurotensin receptor-3, we examined the neurotensin-induced intracellular trafficking of this receptor in the human carcinoma cell line HT29, which expresses both neurotensin receptor-1 and -3 sub-types. At steady state, neurotensin receptor-3 was found by sub-cellular fractionation and electron microscopic techniques to be predominantly associated with intracellular elements. A small proportion (approximately 10%) was associated with the plasma membrane, but a significant amount (approximately 25%) was observed inside the nucleus. Following stimulation with neurotensin (NT), neurotensin/neurotensin receptor-3 complexes were internalized via the endosomal pathway. This internalization entailed no detectable loss of cell surface receptors, suggesting compensation through either recycling or intracellular receptor recruitment mechanisms. Internalized ligand and receptors were both sorted to the pericentriolar recycling endosome/Trans-Golgi Network (TGN), indicating that internalized neurotensin is sorted to this compartment via neurotensin receptor-3. Furthermore, within the Trans-Golgi Network, neurotensin was bound to a lower molecular form of the receptor than at the cell surface or in early endosomes, suggesting that signaling and transport functions of neurotensin receptor-3 may be mediated through different molecular forms of the protein. In conclusion, the present work suggests that the neurotensin receptor-3 exists in two distinct forms in HT29 cells: a high molecular weight, membrane-associated form responsible for neurotensin endocytosis from the cell surface and a lower molecular weight, intracellular form responsible for the sorting of internalized neurotensin to the Trans-Golgi Network.

Risk factors for deep vein thrombosis in inpatients aged 65 and older: a case-control multicenter study.

OBJECTIVES: To identify independent risk factors of symptomatic deep vein thrombosis (DVT) in geriatric inpatients and to define high-risk patients likely to benefit from preventive treatment. DESIGN: Hospital-based case-control multicenter study with prospective data collection. SETTINGS: Geriatric university hospitals with long-, intermediate-, and short-term care facilities. PARTICIPANTS: All patients aged 65 and older in 19 geriatric departments were submitted to clinical surveillance over a 16-month period. MEASUREMENTS: Twenty-three potential risk factors of phlebitis were screened for. Comparison using logistic regression of 310 consecutive patients with symptomatic DVT versus 310 randomly selected controls was performed. The risk for symptomatic DVT in geriatrics was then scored from the clinical risk factors identified using multivariate analysis. This score is defined by the sum of the odds ratio (OR) of each risk factor present. RESULTS: Six factors were identified as independently related to the development of DVT: restriction of mobility (from OR=1.73, limited mobility without immobilization, to OR=5.64, bedridden during <15 days), aged 75 and older (OR=1.5/10 years), history of DVT or pulmonary embolism (OR=3.38), acute heart failure (OR=2.52), chronic edema of the lower limbs (OR=2.51), and paresis or paralysis of a lower limb (OR=2.06). The defined score of 8 or higher corresponded to an 88.7% probability of having symptomatic DVT. CONCLUSION: Treatments to prevent symptomatic DVT in hospitalized elderly should be evaluated on patients with these factors.

Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.