Rituximab treatment in non-malignant inflammatory sensory polyganglionopathy

Non-malignant inflammatory sensory polyganglionopathy (NISP) is the most common form of acquired ganglionopathies, a rare and distinct subgroup of peripheral nerve diseases characterized by a primary degeneration of sensory neurons in dorsal root ganglia. There is a rational for the use of immune modifying agents (IMA) in NISP since an underlying auto-immune process is demonstrated or suspected. However, commonly used IMA such as corticosteroids, azathioprine, methotrexate or IVIG do not prevent disease's progression in the majority of patients. The use of newer IMA, especially those directed against B-cells, may be a therapeutic alternative. An interesting candidate is rituximab, a mouse-human chimeric anti CD20 antibody that specifically eliminates B-cells and B-cells precursors.Objectives: This is a prospective open label pilot study to determine the efficacy of rituximab treatment in NISP patients, who did not respond to commonly used IMA.Methods: Five patients (40% male) with a mean age of 55 years (range 49-67), diagnosed with NISP after extensive work-up, were treated (schema used for one cure: 2 9 1gr within 15 days interval). Neurological scores (NIS, ISSS, ODSS) and B cell counts were assessed at baseline and during clinical follow-up at 2 and 6 monthsto assess treatment efficacy.Results: The treatment was generally well tolerated. Minor adverse events reported were transient arterial hypotension during the infusions in two patients and intermittent mild leucopenia in one patient. Clinical evolution during the follow-up period was characterized by a stability of the functional scores in four patients (80%) and the continuation of disability progression of one patient (20%). CD4 cells disappeared in all patients after the second infusion, an effect that lasted until the follow up at 6 months.Conclusion: The preliminary results of this pilot study indicate that rituximab is generally well tolerated and prevents progression of disability during the 6-month observation period in NISP patients. Inclusion of additional patients and extending of the follow-up period are intended to further investigate the efficacy of rituximab in NISP.

Assessment of early-stage optic nerve invasion in retinoblastoma using high-resolution 1.5 Tesla MRI with surface coils: a multicentre, prospective accuracy study with histopathological correlation.

OBJECTIVES: To assess the accuracy of high-resolution (HR) magnetic resonance imaging (MRI) in diagnosing early-stage optic nerve (ON) invasion in a retinoblastoma cohort. METHODS: This IRB-approved, prospective multicenter study included 95 patients (55 boys, 40 girls; mean age, 29 months). 1.5-T MRI was performed using surface coils before enucleation, including spin-echo unenhanced and contrast-enhanced (CE) T1-weighted sequences (slice thickness, 2 mm; pixel size <0.3 × 0.3 mm(2)). Images were read by five neuroradiologists blinded to histopathologic findings. ROC curves were constructed with AUC assessment using a bootstrap method. RESULTS: Histopathology identified 41 eyes without ON invasion and 25 with prelaminar, 18 with intralaminar and 12 with postlaminar invasion. All but one were postoperatively classified as stage I by the International Retinoblastoma Staging System. The accuracy of CE-T1 sequences in identifying ON invasion was limited (AUC = 0.64; 95 % CI, 0.55 - 0.72) and not confirmed for postlaminar invasion diagnosis (AUC = 0.64; 95 % CI, 0.47 - 0.82); high specificities (range, 0.64 - 1) and negative predictive values (range, 0.81 - 0.97) were confirmed. CONCLUSION: HR-MRI with surface coils is recommended to appropriately select retinoblastoma patients eligible for primary enucleation without the risk of IRSS stage II but cannot substitute for pathology in differentiating the first degrees of ON invasion. KEY POINTS: • HR-MRI excludes advanced optic nerve invasion with high negative predictive value. • HR-MRI accurately selects patients eligible for primary enucleation. • Diagnosis of early stages of optic nerve invasion still relies on pathology. • Several physiological MR patterns may mimic optic nerve invasion.

Parésie trochléaire transitoire comme signe neurologique inaugural d'une panartérite noueuse [Transient trochlear nerve palsy as the presenting neurological sign of panarteritis nodosa]

INTRODUCTION: Panarteritis nodosa (PAN) is a systemic vasculitis affecting small and medium-sized arteries. Neuro-ophthalmological complications of PAN are rare but numerous, and may affect the eye, the visual and the oculomotor pathways. Such complications occur mainly in patients previously diagnosed with PAN. OBSERVATION: A 51-year-old woman presented with an isolated right trochlear (IV) palsy, in the setting of headaches and fluctuating fever of unknown etiology. Erythrocyte sedimentation rate was 13 mm and full blood cell count was normal. Previous chest X-ray and blood studies were negative for an infection or inflammation. Orbital and cerebral CT scan was normal. Spontaneous recovery of diplopia ensued over four days. Two days later, paresthesia and sensory paresis of the dorsal portion of the left foot were present. Lumbar puncture revealed 14 leucocytes (76 percent lymphocytes) with elevated proteins, but blood studies and serologies were negative. A diagnosis of undetermined meningo-myelo-radiculoneuritis was made. Because of a possible tick bite six weeks previously the patient was empirically treated with 2 g intravenous ceftriaxone for 3 weeks. Fever rapidly dropped. Six weeks after the onset of diplopia, acute onset of blindness in her right eye, diffuse arthralgias and fever motivated a new hospitalization. There was a central retinal artery occlusion of the right eye. Blood studies now revealed signs of systemic inflammation (ESR 30 mm, CRP 12 mg/L, ANA 1/80, pANCA 1/40, leucocytosis 12.4 G/L, Hb 111 g/L, Ht 33 percent). Biopsy of the left sural nerve revealed arterial fibrinoid necrosis. A diagnosis of PAN was made. CONCLUSIONS: Transient diplopia can be the heralding symptom of a systemic vasculitis such as PAN, giant cell arteritis and Wegener granulomatosis. In this patient the presence of accompanying systemic symptoms raised a suspicion of systemic inflammation, but the absence of serologic and imaging abnormalities precluded a specific diagnosis initially. A few weeks later, the presence of a second ischemic event (retinal) and positive blood studies led to a further diagnostic procedure. Oculomotor and abducens palsies have rarely been reported in association with PAN. We report the first case of trochlear nerve paresis as the inaugural neurological sign of PAN. This case highlights the importance of considering inflammatory systemic disorders in patients with acute diplopia particularly when they are young, lack vascular risk factors or cause, and complain of associated systemic symptoms.

Spinal cord T-cell infiltration in the spared nerve injury model of neuropathic pain: a time course study

Background : Numerous studies have shown that immune cells infiltrate the spinal cord after peripheral nerve injury and that they play a major contribution to sensory hypersensitivity in rodents. In particular, the role of monocyte-derived cells and T lymphocytes seems to be prominent in this process. This exciting new perspective in research on neuropathic pain opens many different areas of work, including the understanding of the function of these cells and how they impact on neural function. However, no systematic description of the time course or cell types that characterize this infiltration has been published yet, although this seems to be the rational first step of an overall understanding of the phenomenon. Objective : Describe the time course and cell characteristics of T lymphocyte infiltration in the spinal cord in the Spared Nerve Injury (SNI) model of neuropathic pain in rats. Methods : Collect of lumbar spinal cords of rats at days 2, 7, 21 and 40 after SNI or sham operation (n=4). Immunofluorescence detecting different proteins of T cell subgroups (CD2+CD4+, CD2+CD8+, Th1 markers, Th2 markers, Th17 markers). Quantification of the infiltration rate of the different subgroups. Expected results : First, we expect to see an infiltration of T cells in the spinal cord ipsilateral to nerve injury, higher in SNI rats than in sham animals. Second, we anticipate that different subtypes of T cells penetrate at different time points. Finally, the number of T lymphocytes are expected to decrease at the latest time point, showing a resolution of the process underlying their infiltrating the spinal cord in the first place. Impact : A systematic description of the infiltration of T cells in the spinal cord after peripheral nerve injury is needed to have a better understanding of the role of immune cells in neuropathic pain. The time course that we want to establish will provide the scientific community with new perspectives. First, it will confirm that T cells do indeed infiltrate the spinal cord after SNI in rats. Second, the type of T cells infiltrating at different time points will give clues about their function, in particular their inflammatory or anti-inflammatory profile. From there on, other studies could be lead, investigating the functional side of the specific subtypes put to light by us. Ultimately, this could lead to the discovery of new drugs targeting T cells or their infiltration, in the hope of improving neuropathic pain.

Schwann cell strip for peripheral nerve repair.

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration.

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.

Retinal degeneration depends on Bmi1 function and reactivation of cell cycle proteins.

The epigenetic regulator Bmi1 controls proliferation in many organs. Reexpression of cell cycle proteins such as cyclin-dependent kinases (CDKs) is a hallmark of neuronal apoptosis in neurodegenerative diseases. Here we address the potential role of Bmi1 as a key regulator of cell cycle proteins during neuronal apoptosis. We show that several cell cycle proteins are expressed in different models of retinal degeneration and required in the Rd1 photoreceptor death process. Deleting E2f1, a downstream target of CDKs, provided temporary protection in Rd1 mice. Most importantly, genetic ablation of Bmi1 provided extensive photoreceptor survival and improvement of retinal function in Rd1 mice, mediated by a decrease in cell cycle markers and regulators independent of p16(Ink4a) and p19(Arf). These data reveal that Bmi1 controls the cell cycle-related death process, highlighting this pathway as a promising therapeutic target for neuroprotection in retinal dystrophies.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

Characterization Of Cdk Expressions During Retinal Degeneration In Rd1 Mice

Purpose: In Rd1 mice, a PDE6ß mutation is responsible for the rapid loss of photoreceptors. We observed re-expression of cell cycle proteins during early stages of retinal degeneration and the deletion of Bmi1 markedly delayed photoreceptor death in Rd1;Bmi1-/- mice. The present study characterizes the link between the expression of CDKs and the apoptotic process in Rd1 photoreceptors.Methods: CDK expression levels were evaluated by immunostaining of wild-type, Rd1 and Rd1;Bmi1-/- eye sections. The role of CDKs in retinal degeneration is currently being investigated by treating Rd1 retinal explants with CDK inhibitors, and by injecting roscovitine-containing micelles into the vitreous of P10 Rd1 mice.Results: We show that some Rd1 photoreceptors express CDK4 already at P9, and that the number of CDK4-positive cells increases more than 6-fold by P11. CDK2 and CDK6 are also expressed in the mutant outer nuclear layer (ONL), however to a lesser extent than CDK4. Concomitant with the expression of CDKs, the apoptotic process in Rd1 photoreceptors is detected by TUNEL staining. Co-localization analyses suggest that CDK expression precedes photoreceptor cell death since TUNEL-single-positive cells are rarely detected at P9, and double-positive as well as TUNEL- or CDK4-single-positive cells are all present in P11 Rd1 retinas. The wild-type ONL does not contain any TUNEL- or CDK4-positive cells. Interestingly, Bmi1 deletion downregulates CDK4 expression in P12 Rd1;Bmi1-/- retinas, and influences the accumulation of cGMP in Rd1 retinas. More cGMP is detected in the P11 Rd1;Bmi1-/- ONL than in the Rd1 ONL, while it is strongly reduced at P15. To better characterize the link between CDK expression and retinal degeneration, current experiments include the analysis of CDK inhibition in Rd1 retinal explants and in mouse eyes injected with roscovitine-containing micelles.Conclusions: The time-course of cell cycle protein expression may be related to early events of the apoptotic process in Rd1 photoreceptors. Moreover, the loss of Bmi1 seems to interfere with the first stages of retinal degeneration and to influence the expression of CDK4. Further experiments will determine whether the deletion of Bmi1 prevents cell death through a direct CDK inhibition.

Isolated IVth (trochlear) nerve palsy due to basilar artery dolichoectasia.

BACKGROUND: Dolichoectasia (elongation, dilatation and tortuosity) of the basilar artery can cause an isolated cranial neuropathy. The trigeminal nerve and facial nerve are most frequently affected. Dysfunction of one of the ocular motor cranial nerves due to basilar artery dolichoectasia is uncommon, and an isolated IVth (trochlear) nerve palsy has not been previously described in the literature. HISTORY AND SIGNS: Two men, ages 70 and 59 years, respectively, presented with vertical diplopia due to a IVth nerve palsy. In one patient, the onset of the IVth nerve palsy was painless and gradual and in the other patient, the onset was acute and associated with periorbital pain. Neuroimaging in both patients revealed pathological tortuosity of the basilar artery around the midbrain and displacement of the artery toward the side of the affected trochlear nerve. THERAPY AND OUTCOME: The patients were observed clinically. One patient had gradual worsening of his palsy for three and one-half years then suffered a stroke. The second patient whose IVth nerve palsy had an acute onset experienced spontaneous resolution of his palsy but later developed dysfunction of other cranial nerves. CONCLUSIONS: Basilar artery dolichoectasia should be considered in the differential diagnosis of an isolated IVth nerve palsy. The clinical course may be variable, and the prognosis is not always benign.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

Long-term prognosis in patients with vasculopathic sixth nerve palsy.

PURPOSE: To better define the long-term prognosis in patients with a vasculopathic sixth nerve palsy (6NP), specifically addressing the degree of recovery and incidence of recurrent similar episodes. DESIGN: Observational case series. METHODS: Retrospective chart review. SETTING: An outpatient neuroophthalmic practice. STUDY POPULATION: Patients with one or more vascular risk factors and an acute, isolated 6NP that spontaneously recovered. OBSERVATION PROCEDURE: Information regarding resolution of the 6NP, subsequent vascular events and recurrent ocular motor nerve palsy was obtained from chart review of follow-up clinic visits, mailed questionnaires and telephone interviews. The duration of follow-up ranged from 2 to 13 years. MAIN OUTCOME MEASURES: Resolution of 6NP (complete or incomplete) and incidence of recurrent ocular motor nerve palsy. RESULTS: Fifty-nine patients were identified with a mean age of 65.3 years +/- 11.6 (range 34-90 years). Fifty-one patients (86%) experienced complete resolution of their first episode of vasculopathic 6NP and eight patients (14%) had incomplete resolution. A subsequent episode of ocular motor mononeuropathy occurred in 18 of 59 (31%) patients. The number of recurrences ranged from one (in 14 patients) to four (in one patient). There was no association between any risk factor and recurrence of ocular motor nerve palsy. Similarly, incomplete resolution of the vasculopathic 6NP was not associated with any risk factor. CONCLUSIONS: Patients with a vasculopathic 6NP usually have complete resolution of their ophthalmoplegia, but nearly one third of patients in our study later experienced at least one episode of recurrent vasculopathic ocular motor nerve palsy.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Differential neuroglycan C expression during retinal degeneration in Rpe65-/- mice.

PURPOSE: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65-/- mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression. METHODS: Retinas from C57/Bl6 wild-type and Rpe65-/- mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry. RESULTS: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65-/- retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65-/- mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected. CONCLUSIONS: During retinal degeneration in Rpe65-/- mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.

Fibrinmatrix erhöht Adhärenz von regenerativen peripheren Nervenzellen [Fibrin matrix enhances adherence of peripheral nerve regenerative cells]

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.