AAV gene therapy for Tay-Sachs disease.

Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months with an equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB administered intrathecally (i.t.), with 75% of the total dose (1 × 1014 vector genomes (vg)) in the cisterna magna and 25% at the thoracolumbar junction. Patient TSD-002 was treated at 7 months by combined bilateral thalamic (1.5 × 1012 vg per thalamus) and i.t. infusion (3.9 × 1013 vg). Both patients were immunosuppressed. Injection procedures were well tolerated, with no vector-related adverse events (AEs) to date. Cerebrospinal fluid (CSF) HexA activity increased from baseline and remained stable in both patients. TSD-002 showed disease stabilization by 3 months after injection with ongoing myelination, a temporary deviation from the natural history of infantile TSD, but disease progression was evident at 6 months after treatment. TSD-001 remains seizure-free at 5 years of age on the same anticonvulsant therapy as before therapy. TSD-002 developed anticonvulsant-responsive seizures at 2 years of age. This study provides early safety and proof-of-concept data in humans for treatment of patients with TSD by AAV gene therapy.

Ventriculogallbladder Shunt: Percutaneous Transhepatic Placement of the Distal Catheter.

BACKGROUND: Ventriculogallbladder (VGB) shunts are currently placed as a salvage procedure in treatment of hydrocephalus when all other options are exhausted. Although VGB shunts work well when successfully implanted, they are still rarely used because of the technical challenges with the traditional surgical technique. OBJECTIVE: To implant VGB shunts using a minimally invasive technique that is safer and less technically challenging. METHODS: We discussed our utilization of a percutaneous transhepatic approach to placement of the distal catheter into the gallbladder as a new technique that offers a solution to the established but surgically demanding current approach. We presented a complex patient with multiple failed shunts who underwent placement of a VGB shunt. RESULTS: The shunt was successfully placed and did not incur any complications or require any revisions in 5 yr. CONCLUSION: Given the safety and ease of this minimally invasive approach, we believe percutaneous transhepatic approach in VGB shunts should be considered as a reasonable option in patients with complex hydrocephalus.

Fatty Acid-binding Protein 4 Expression in Tumor Cells as a Potential Marker for Anaplastic Meningiomas.

Meningiomas are highly vascularized tumors originating from arachnoid cap cells of the leptomeninges. The majority of meningiomas are classified as World Health Organization (WHO) grade I and display a benign clinical course with a low risk of recurrence. In contrast, WHO grade III meningiomas carry a high risk of recurrence and poor prognosis. However, it is commonly recognized that histopathologic grading does not always reliably predict recurrence or progression of meningiomas. Fatty acid-binding protein 4 (FABP4) is a small molecular weight lipid chaperone that plays a proangiogenic role in vascular endothelial cells. FABP4 is not expressed in normal brain vasculature but is detected in some glioblastoma and arteriovenous malformations. The expression pattern of FABP4 in meningiomas have not been reported to date. We analyzed FABP4 expression in a cohort of paraffin-embedded meningioma specimens by immunohistochemistry and double immunofluorescence analyses. FABP4 expression was detected in a subset of endothelial cells in 47 of 48 meningioma samples analyzed. Interestingly, tumor cell-FABP4 expression was also detected in only 1 of 22 grade I, none of grade II, but 10 of 12 grade III meningiomas (P<0.0001). These results demonstrate that FABP4 is commonly expressed in meningioma vascular endothelial cells while tumor cell expression of FABP4 is primarily observed in anaplastic meningiomas. A combination of FABP4 immunostaining with histopathologic grading might provide a more accurate prediction of the biological behavior of meningiomas than histopathologic grading alone.

Volume and Infusion Rate Dynamics of Intraparenchymal Central Nervous System Infusion in a Large Animal Model.

Thalamic infusion of adeno-associated viral (AAV) vectors has been shown to have therapeutic effects in neuronopathic lysosomal storage diseases. Preclinical studies in sheep model of Tay-Sachs disease demonstrated that bilateral thalamic injections of AAV gene therapy are required for maximal benefit. Translation of thalamic injection to patients carries risks in that (1) it has never been done in humans, and (2) dosing scale-up based on brain weight from animals to humans requires injection of larger volumes. To increase the safety margin of this infusion, a flexible cannula was selected to enable simultaneous bilateral thalamic infusion in infants while monitoring by imaging and/or to enable awake infusions for injection of large volumes at low infusion rates. In this study, we tested various infusion volumes (200-800 µL) and rates (0.5-5 µL/min) to determine the maximum tolerated combination of injection parameters. Animals were followed for ∼1 month postinjection with magnetic resonance imaging (MRI) performed at 14 and 28 days. T1-weighted MRI was used to quantify thalamic damage followed by histopathological assessment of the brain. Trends in data show that infusion volumes of 800 µL (2 × the volume required in sheep based on thalamic size) resulted in larger lesions than lower volumes, where the long infusion times (between 13 and 26 h) could have contributed to the generation of larger lesions. The target volume (400 µL, projected to be sufficient to cover most of the sheep thalamus) created the smallest lesion size. Cannula placement alone did result in damage, but this is likely associated with an inherent limitation of its use in a small brain due to the length of the distal rigid portion and lack of stable fixation. An injection rate of 5 µL/min at a volume ∼1/3 of the thalamus (400-600 µL) appears to be well tolerated in sheep both clinically and histopathologically.

Case report: cranial angiosarcoma with multiple hemorrhagic brain metastasis in a child.

BACKGROUND AND IMPORTANCE: Angiosarcoma is a rare malignant tumor with an aggressive course and poor prognosis. It is typically seen in adults but very rarely seen in children. Angiosarcoma of the skull with brain metastasis is exceptionally rare. Due to the rare nature of these tumors, much is left unknown about clinical progression and treatment guidelines are not well established. CLINICAL PRESENTATION AND COURSE: A 14-year-old male patient presented with an enlarging mass on the parietal region of the head. Further investigations revealed a mass lesion involving scalp and skull tissue. Biopsy result showed angiosarcoma and the patient underwent multiple surgical interventions including scalp excision, craniectomy on tumor site, and excision of brain metastases. He also received chemotherapy and radiation therapy. Despite aggressive treatment, disease progression could not be controlled. CONCLUSION: Here we report a pediatric patient with intracranially invasive angiosarcoma of the scalp and skull, with recurrent hemorrhagic metastases to the brain. This is a very rare case in pediatric age group with very poor prognosis. Our patient had impressively longer survival than those reported in the literature despite multiple hemorrhagic brain metastasis and this is most likely related to our aggressive treatment strategy that includes multiple craniotomies for metastatic tumor resection in addition to neoadjuvant chemotherapy and radiation therapy. We believe optimal treatment of skull angiosarcoma in children should aim gross total resection of the skull tumor including involving scalp and dura as well as neoadjuvant chemotherapy and radiation therapy, and the patient should be followed closely with repeated brain MRI studies to pursue additional surgeries to remove brain metastasis if feasible.

A Safe and Reliable Technique for CNS Delivery of AAV Vectors in the Cisterna Magna.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

MRI Findings of Injury to the Longus Colli Muscle in Patients With Neck Trauma.

OBJECTIVE: The longus colli muscle (LCM) forms the bulk of the deep flexor muscles of the neck. To our knowledge, very little information on the effects of trauma on this muscle group has been published. We describe MRI findings related to injury of the LCM in patients with a history of neck trauma. MATERIALS AND METHODS: A radiology department database was searched to identify patient medical records from 2008 to 2013 that included the keywords "longus colli" and "deep flexors." Patients with fractures and ligament injuries were excluded. Patients with other obvious large soft-tissue injuries and nontraumatic conditions were also omitted. A total of 12 patients met the inclusion criterion of having an isolated or predominant injury to the LCM. Five patients had been involved in a motor vehicle accident, and seven patients had fallen. Eleven patients had undergone a CT examination before MRI was performed. RESULTS: No fractures were noted on CT. MRI examinations of the cervical spine were obtained for the following reasons: for increased prevertebral soft-tissue swelling noted on a CT scan plus neck pain (n = 6), for neck pain only (n = 4), or as part of a routine protocol for assessment of obtunded patients (n = 2). Eight of the 12 patients had isolated injury to the LCM. The remaining four patients also had minor injuries to the other neck muscles. The MR image showed swelling and T2 hyperintensity in the LCM and revealed free fluid in the prevertebral space. CONCLUSION: Isolated injury to the LCM may occur in neck injuries. The MRI findings indicating such injury include increased T2 signal, swelling of the muscle, and the presence of prevertebral fluid.

Fatty acid binding protein 4 expression in cerebral vascular malformations: implications for vascular remodelling.

AIM: Arteriovenous malformations (AVM) and cavernous malformations (CM) are the most commonly encountered cerebral vascular malformations, which are dynamic lesions with de novo growth potentials. Postnatal angiogenesis and vasculogenesis have been postulated to play a role in the pathogenesis of these malformations. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone, which is expressed in a subset of endothelial cells. FABP4 enhances the angiogenic responses of endothelial cells and is not expressed in normal cerebral vasculature. Herein, we investigated the hypothesis that FABP4 expression may be up-regulated in AVM and CM. METHODS: The abundance of FABP4 expression was analysed by immunohistochemistry on 35 paraffin-embedded AVM and CM sections. FABP4-expressing cells were further characterized by double immunofluorescence using antibodies against various cell-specific markers. RESULTS: Heterogenous FABP4 expression was detected in 100% AVM and 78% of CM samples. Endothelial cell FABP4 expression was present in 65% and 43% of AVM and CM, respectively. Interestingly, a population of FABP4-positive perivascular cells was detected in 100% of AVM and 86% of CM sections examined. These cells were negative for markers of macrophages and smooth muscle cells, but expressed vimentin, a marker of mesenchymal cells, including fibroblasts. CONCLUSION: FABP4 expression is detected in AVM and CM in a subset of endothelial cells and some perivascular fibroblast-like vimentin-positive cells.

Axial diffusivity of the corona radiata correlated with ventricular size in adult hydrocephalus.

OBJECTIVE: Hydrocephalus causes changes in the diffusion-tensor properties of periventricular white matter. Understanding the nature of these changes may aid in the diagnosis and treatment planning of this relatively common neurologic condition. Because ventricular size is a common measure of the severity of hydrocephalus, we hypothesized that a quantitative correlation could be made between the ventricular size and diffusion-tensor changes in the periventricular corona radiata. In this article, we investigated this relationship in adult patients with hydrocephalus and in healthy adult subjects. MATERIALS AND METHODS: Diffusion-tensor imaging metrics of the corona radiata were correlated with ventricular size in 14 adult patients with acute hydrocephalus, 16 patients with long-standing hydrocephalus, and 48 consecutive healthy adult subjects. Regression analysis was performed to investigate the relationship between ventricular size and the diffusion-tensor metrics of the corona radiata. Subject age was analyzed as a covariable. RESULTS: There is a linear correlation between fractional anisotropy of the corona radiata and ventricular size in acute hydrocephalus (r = 0.784, p < 0.001), with positive correlation with axial diffusivity (r = 0.636, p = 0.014) and negative correlation with radial diffusivity (r = 0.668, p = 0.009). In healthy subjects, axial diffusion in the periventricular corona radiata is more strongly correlated with ventricular size than with patient age (r = 0.466, p < 0.001, compared with r = 0.058, p = 0.269). CONCLUSION: Axial diffusivity of the corona radiata is linearly correlated with ventricular size in healthy adults and in patients with hydrocephalus. Radial diffusivity of the corona radiata decreases linearly with ventricular size in acute hydrocephalus but is not significantly correlated with ventricular size in healthy subjects or in patients with long-standing hydrocephalus.

Heart rate variability remains reduced and sympathetic tone elevated after temporal lobe epilepsy surgery.

PURPOSE: There is evidence of autonomic dysregulation in temporal lobe epilepsy. The structures removed during temporal lobectomy are important centers of central cardiovascular control; therefore surgery may conceivably alter the cardiovascular autonomic function. The effects of temporal lobectomy on autonomic cardiac control are controversial. We investigated the effects of temporal lobectomy on heart rate variability (HRV) in the early and late postoperative periods. METHODS: We used 1-h ECG recordings to assess heart rate variability by spectral analysis in 24 consecutive patients who underwent temporal lobectomy due to intractable temporal lobe epilepsy. ECG recordings were performed before and twice (early and late) after surgery. The results were compared with age and sex matched controls. RESULTS: When compared with controls, all the time and frequency domain indices (SDRR, RMSSD, TP, LF and HF) were significantly lower in the patient group before surgery. Findings were similar in the early and late post-operative periods except that the LF/HF ratio increased in the patient group after the late post-operative period. Within the patient group, compared to pre-operative results, normalized HF was increased in the early post-operative period; however in the late post-operative period, LF/HF ratio was increased. CONCLUSIONS: These findings show that in patients with intractable temporal lobe epilepsy, HRV is decreased globally in both sympathetic and parasympathetic domains. While the total HRV remains reduced throughout the postoperative periods, the LF/HF ratio, i.e., sympathovagal balance is altered, in favor of parasympathetic side early after surgery, but towards the sympathetic side after the first postoperative month.

Latex sensitization and allergy in children with spina bifida in Turkey.

PURPOSE: The latex allergy is known as a very frequent problem of children with spina bifida (SB). The aim of this prospective study is to determine the frequency of latex sensitization and allergy in Turkish children with SB and to evaluate the related risk factors. METHODS: We enrolled a total of 175 children and collected them in four groups of patients: children with SB, children who had neurosurgical disorders other than SB, atopic children with allergic diseases, and healthy children as control group. All cases were evaluated for latex sensitivity and allergy with skin prick tests and specific IgE. The diagnosis of latex allergy was confirmed with the latex provocation test. RESULTS: Ten children with SB, two children with other neurological disorders, and three atopic children with allergic disease were found "sensitized to latex". Among these sensitized children, only one child with SB had latex allergy. Multivariate logistic regression analysis of the children with SB and other neurologic disorders and the whole group of patients showed that skin test positivity, the presence of noncentral nervous system (CNS) surgery, and the number of the operations were significant risk factors for latex sensitization. CONCLUSIONS: Our study showed that rates of latex sensitivity and allergy in children with SB in Turkey are significantly lower than published rates in literature. We also found that atopy, the presence of non-CNS operations in children with SB and other neurologic disorder, and total number of operations in the whole group of patients are the most important risk factors for latex sensitization.

Diffuse villous hyperplasia of the choroid plexus and its surgical management.

Diffuse villous hyperplasia of the choroid plexus (DVHCP) is a very rare cause of hydrocephalus in children. Only 12 cases of DVHCP have been reported in the literature. In this report the authors describe a new case of a patient with DVHCP who was diagnosed prenatally with hydrocephalus. In a comprehensive literature review and discussion, the authors also discuss radiological and histological characteristics of the reported cases, treatment approaches, and surgical modalities that have been used in the treatment of these patients.

Dysembryoplastic neuroepithelial tumor located in pericallosal and intraventricular area in a child. Case report.

Dysembryoplastic neuroepithelial tumors (DNETs) are frequently seen in children and young adults with intractable epilepsy, and are typically located in the temporal cortex. Extracortical location of DNET is a very rare occurrence. The authors report on a child with a unique extracortical location of DNET with an extensive involvement to the supracallosal cistern and callosum, septum pellucidum, and lateral ventricle ependyma. The authors discuss the radiological and pathological characteristics of the lesion and reiterate the difficulty in differentiating the histological characteristics of central neurocytoma and DNET in extracortical locations and its significance for further management planning.

Epidermoid cyst of the brain stem symptomatic in childhood.

BACKGROUND: Epidermoid cysts may remain asymptomatic for a long period of time due to their slowly growing pattern corresponding with the normal human skin turnover time and due to soft and light cyst content. They do not cause compression of neural and vascular structures initially that is why almost all of the cases in the literature are diagnosed during adulthood. METHODS: We report here an epidermoid cyst in childhood, which was located in the medulla oblongata, unusually and atypically with liquefied cyst content. The liquefaction may occur due to an intrauterine or early childhood infection. The reported case also suffered previously a severe respiratory infection. Although the cyst is located in and around a highly eloquent neural area, plasticity of the brain stem prevented neurological deterioration due to this very slow growing extraaxial mass lesion. The ordinary cyst content found in the center of the cyst cavity during the operation suggested that the same ordinary material, which was previously at the periphery, ran to get liquefied in time. CONCLUSION: We suggest that the symptoms of this present case appeared very early due to liquefaction of the cyst content with compression and displacement of the brain stem caudally. The recent infection process may predispose the pathological condition.

Gelastic seizures due to right temporal cortical dysplasia.

Gelastic seizures are an uncommon seizure type. They are most frequently observed in patients with hypothalamic hamartoma. Their association with other types of cerebral lesions is rare. Depending on the location of the lesion, gelastic seizures may or may not be accompanied by a subjective feeling of mirth. The pathophysiological mechanisms of this type of seizure are still undefined, and little is known about which pathways promote laughter and its emotional content, mirth. We present a young man with drug-resistant, gelastic seizures due to focal cortical dysplasia of the right inferior temporal gyrus. The lesion was evident on cranial MRI. Interictal EEG displayed a right temporal focus, whereas ictal EEG was not informative. Ictal loss of consciousness precluded reporting of any possible emotional experience. The patient underwent surgical resection of the lesion and has been seizure-free with anti-epileptic medication for two years. Although various anatomical regions may elicit laughter, in view of the current literature it seems that the anterior cingulate region is involved in the motor aspects of laughter, while the basal temporal cortex is involved in the processing of mirth. The fact that the present case exhibited gelastic seizures stresses once more the importance of the baso-lateral temporal cortex in the genesis of this type of seizures.[Published with video sequences].

Surgical management of temporal lobe tumor-related epilepsy in children.

OBJECT: Slow-growing, low-grade temporal lobe tumors are one of the most common causes of epilepsy in children. Although there are numerous consistent features in this patient group, consensus about the management and surgical approach is lacking. In this study the authors review the clinical, pathological, and radiological features as well as outcome data obtained in 29 pediatric patients with temporal lobe tumor-related epilepsy and discuss the surgical treatment strategies. METHODS: In patients who presented with intractable seizures secondary to mass lesions and underwent comprehensive epilepsy workup, the tumor was resected and the diagnosis confirmed by pathological examination. A minimum follow-up period of 16 months was required. Medical records were reviewed for details of seizure type and duration, electrophysiological data, imaging studies, operative notes, pathological examination reports, and follow-up data. The surgical approach was as follows. The lesionectomy with/without cortical resection was performed in all cases of lateral temporal tumors. Lesionectomy was performed with/without cortical resection in cases of basal temporal tumors if the mesial structures were radiologically normal. Mesial temporal tumors were excised, as were the remaining mesial temporal structures in the nondominant hemisphere; however, if the tumor was in the dominant hemisphere, lesionectomy was performed only if the remaining mesial structures were radiologically normal. Twenty-nine patients between 2 and 18 years of age were identified. Most tumors were located in the mesial temporal lobe. All patients underwent resection of the tumor with or without mesial and cortical structures. The most common pathological entity was dysembryoplastic neuroepithelial tumor. Sixty-nine percent of the patients remained seizure free (Engel Class I) and 14% experienced significant improvement (Engel Class II) after surgery. Outcome was better in the patients who underwent gross-total tumor resection. CONCLUSIONS: Mesially located low-grade neoplasms were the most frequently observed mass lesions in children with temporal tumor-related epilepsy in this series. Resection of the tumor with or without amygdalohippocampectomy provides a high rate of seizure-free outcome. It is the author's opinion that temporal lobe tumors should be managed based on the subgroups defined by their anatomical locations. If the tumor is located in or in proximity to eloquent cortex, we recommend functional magnetic resonance imaging and invasive monitoring techniques to map the eloquent cortex and epileptogenic zone, thereby tailoring the resection.

A giant intramedullary spinal epidermoid cyst of the cervicothoracic region.

Intramedullary epidermoid cysts of the spinal cord are rare lesions. The authors report a case of giant intramedullary epidermoid cyst at the cervicothoracic region. A 6-year-old boy presented with motor and sensory loss related to a spinal intramedullary epidermoid cyst. Almost all cyst content was liquid and it was surgically removed totally in two successive operations. To the author's knowledge, this is the largest intramedullary epidermoid cyst published in the literature and no other case of intramedullary epidermoid cyst with a complete liquid content has been reported before. The authors discuss the clinical features, MR imaging characteristics and surgical findings of this rare tumor and review the associated literature.

Eosinophilic granuloma of the skull associated with epidural haematoma: a case report and review of the literature.

INTRODUCTION: Eosinophilic granuloma (EG), a benign bone lesion, represents a focal form of histiocytosis X. Here, the authors report a case of an extremely rare presentation of a solitary EG of the skull. CASE REPORT: A 9-year-old boy presented with headache and vomiting for 3 days. His examination was unremarkable except for a tender mass in his left occipital region. Radiological studies revealed a huge bilateral epidural haematoma under the occipital bone, extending supratentorially and infratentorially, and a nonenhanced, epidural, soft tissue mass overlying the left transverse sinus. Evacuation of the epidural haematoma was performed and it was noted that the left transverse sinus wall was eroded by the EG at one point with venous oozing. REVIEW OF THE LITERATURE: The acute presentation of a solitary EG of the skull with an epidural haematoma was described in only four cases in the literature. This is the first documented case in terms of the origin of an epidural haematoma.

Radiation-induced malignant triton tumor associated with severe spinal cord compression. Case report and review of the literature.

Malignant triton tumor (MTT) is a variant of malignant peripheral nerve sheath tumors. The authors report a case of radiation-induced MTT in a patient with severe cervicothoracic cord compression and review the related literature. This 36-year-old man presented with pain and weakness in his left arm. His medical history was significant for a biopsy procedure involving the sampling of an aneurysmal bone cyst located at T1-3 near the left lung apex; this was performed 6 years prior to presentation and was followed by radiotherapy. Neurological examination demonstrated radicular findings involving the left C-8 and T-1 nerve roots. Neuroimaging studies revealed a large mass lesion extending from C-6 to T-2 along the vertebral column, invading the upper thoracic cavity and the adjacent lung apex, and infiltrating the paravertebral muscles. A subtotal resection was performed, but the tumor regrew extensively within a short time. It invaded the spinal canal and caused significant cord compression. The patient underwent surgery two more times for tumor debulking and to relieve progressive airway and spinal canal compromise. He eventually became quadriplegic, however, and died 13 months after diagnosis of MTT. This is the seventh case of radiation-induced MTT and the fifth of MTT with spinal canal involvement to be reported in the literature.

Tectal gliomas in children: the implications for natural history and management strategy.

Tumors involving the tectal region constitute a distinct subgroup of brain stem gliomas with an indolent clinical course. Here, we present the clinical and neuroradiologic features of 9 children with tectal tumors. All patients presented with signs and symptoms of hydrocephalus and were managed with ventriculoperitoneal shunt insertion. MRI studies revealed focal hyperintense lesions on T2-weighted images without any contrast enhancement, and no evidence of progression was demonstrated in any patient. We also reviewed the published series of tectal gliomas in the literature to compare with our results. Based on these and other published series, it was concluded that intrinsic tectal gliomas of childhood with sizes less than 2 cm in diameter and without any tumor extension or contrast enhancement constitute a specific subgroup of tectal masses which rarely display invasive clinical behavior and should be managed conservatively. CSF diversion procedures and long-term yearly follow-up examinations with MRI scans are sufficient in these patients.