Alterations in the innate and adaptive immune system in a real-world cohort of multiple sclerosis patients treated with ocrelizumab.

B cell depletion by the anti-CD20 antibody ocrelizumab is effective in relapsing-remitting (RR) and primary progressive (PP) multiple sclerosis (MS). We investigated immunological changes in peripheral blood of a real-world MS cohort after 6 and 12 months of ocrelizumab. All RRMS and most PPMS patients (15/20) showed treatment response. Ocrelizumab not only reduced CD20+ B cells, but also numbers of CD20+ T cells. Absolute numbers of monocytes, dendritic cells and CD8+ T cells were increased, while CD56hi natural killer cells were reduced after ocrelizumab. The residual B cell population shifted towards transitional and activated, IgA+ switched memory B cells, double negative B cells, and antibody-secreting cells. Delaying the treatment interval by 2-3 months increased mean B cell frequencies and enhanced naive B cell repopulation. Ocrelizumab reduced plasma levels of interleukin(IL)-12p70 and interferon(IFN)-α2. These findings will contribute to understanding ineffective treatment responses, dealing with life-threatening infections and further unravelling MS pathogenesis.

Extended interval dosing of ocrelizumab in patients with multiple sclerosis is not associated with meaningful differences in disease activity.

Risk concerns related to ocrelizumab treatment for multiple sclerosis (MS) during the COVID-19 pandemic caused infusion delays with extended interval dosing (EID). We reviewed medical records of patients on ocrelizumab to determine whether EID maintains its effectiveness compared to standard interval dosing (SID). Among 361 patients, 231 (64%) and 123 (34%) had at least one infusion with infusion intervals of ⩾8 months and ⩾12 months, respectively. There were no differences in demographics or clinical profiles between the SID and EID groups. No significant differences between rates of breakthrough activity among relapsing-remitting patients were observed between SID (three patients) and EID (seven patients).

Exploring the effects of extended interval dosing of natalizumab and drug concentrations on brain atrophy in multiple sclerosis.

BACKGROUND: Extended interval dosing (EID) of natalizumab treatment is increasingly used in multiple sclerosis. Besides the clear anti-inflammatory effect, natalizumab is considered to have neuroprotective properties as well. OBJECTIVES: This study aimed to study the longitudinal effects of EID compared to standard interval dosing (SID) and natalizumab drug concentrations on brain atrophy. METHODS: Patients receiving EID or SID of natalizumab with a minimum radiological follow-up of 2 years were included. Changes in brain atrophy measures over time were derived from clinical routine 3D-Fluid Attenuated Inversion Recovery (FLAIR)-weighted magnetic resonance imaging (MRI) scans using SynthSeg. RESULTS: We found no differences between EID (n = 32) and SID (n = 50) for whole brain (-0.21% vs -0.16%, p = 0.42), ventricular (1.84% vs 1.13%, p = 0.24), and thalamic (-0.32% vs -0.32%, p = 0.97) annualized volume change over a median follow-up of 3.2 years. No associations between natalizumab drug concentration and brain atrophy rate were found. CONCLUSION: We found no clear evidence that EID compared to SID or lower natalizumab drug concentrations have a negative impact on the development of brain atrophy over time.

Memory B cell-guided extended interval dosing of ocrelizumab in multiple sclerosis.

BACKGROUND: Ocrelizumab (OCR) is an anti-CD20 monoclonal antibody approved for the treatment of relapsing-remitting and primary-progressive multiple sclerosis (MS). We aimed to evaluate the effectiveness of an individualized OCR extended interval dosing (EID), after switching from standard interval dosing (SID). METHODS: This was a retrospective, observational, single-centre study including MS patients regularly followed at the Neurocenter of Southern Switzerland. After a cumulative OCR dose ⩾1200 mg, stable patients were switched to EID (OCR infusions following CD19+ 27+ memory B cell repopulation). RESULTS: A total of 128 patients were included in the study, and 113 (88.3%) were switched to EID with a median interval of 9.9 (8.8-11.8) months between infusions. No clinical relapses occurred; 2 (1.8%) patients experienced disability worsening. Three (2.7%) and 2 (1.8%) patients experienced new T2 brain and spinal lesions, respectively. There was a mild decrease in IgG and IgM concentrations during both SID and EID OCR regimens (ß = -0.23, p = 0.001 and ß = -0.07, p < 0.001, respectively). CONCLUSION: Switch to personalized dosing of OCR based on CD19+ 27+ memory B cell repopulation led to a great extension of the interval between infusions, with maintained clinical and radiological efficacy. Given the potential advantages in terms of safety and health costs, EID OCR regimens should be further investigated.

High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro.

INTRODUCTION: The humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing-remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown. OBJECTIVE: Here we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood-brain barrier (BBB) under physiological flow in vitro. RESULTS: Making use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of ß2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow. CONCLUSION: Taken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

Fingerprick blood samples to measure serum natalizumab concentrations.

BACKGROUND: Natalizumab via subcutaneous administration was recently approved for patients with multiple sclerosis. OBJECTIVE: In light of personalized extended dosing, in which treatment intervals are prolonged to a concentration cut-off, it would be preferable to measure natalizumab drug concentrations in capillary blood. METHODS: In this cross-sectional study in patients treated with intravenous (IV) natalizumab, capillary blood samples by fingerprick and venous blood samples were collected in 30 participants prior to IV administration of natalizumab. RESULTS: Natalizumab concentrations were similar with a mean bias of -0.36 µg/mL (95% CI: 1.3 to -2 µg/mL). CONCLUSIONS: This study shows that physicians can monitor natalizumab drug concentrations by a fingerprick, which could be used for personalized extended dosing.

Serum neurofilament light levels in natalizumab-treated patients with multiple sclerosis who switch to extended interval dosing from every-4-week dosing in real-world clinical practice.

BACKGROUND: Serum levels of neurofilament light chain (sNfL) are a potentially useful biomarker for assessing the efficacy of multiple sclerosis (MS) treatments. OBJECTIVE: To compare levels of sNfL in patients with MS who switched from natalizumab every 4 weeks (Q4W) to extended interval dosing (EID) and patients who remained on Q4W dosing in real-world clinical practice. METHODS: This was a retrospective analysis of samples from patients treated with natalizumab from 2010 to 2015 at a single center in the United States. Levels of sNfL were compared in patients who stayed on Q4W dosing or who switched to EID (parallel-arm analyses) and during Q4W and EID periods in patients who switched to EID (pre- and post-switch analyses). RESULTS: The analysis included 139 patients (Q4W: n = 79; EID: n = 60). After adjustment, levels of sNfL did not significantly differ between patients who remained on Q4W dosing and those who switched to EID in parallel-arm analyses (adjusted Q4W-EID difference = 0.51 pg/mL; p = 0.60) or pre- and post-switch analyses (adjusted difference = 0.96 pg/mL; p = 0.10). CONCLUSION: These sNfL biomarker results suggest that the effectiveness of natalizumab is maintained in patients who switch from Q4W dosing to EID.

Ocrelizumab extended-interval dosing in multiple sclerosis during SARS-CoV-2 pandemic: a real-world experience.

BACKGROUND AND PURPOSE: During the COVID-19 pandemic, ocrelizumab administration was frequently postponed because of a lack of safety information and to favour vaccination. The clinical implications of ocrelizumab administration delay in multiple sclerosis (MS) patients were assessed. METHODS: Relapsing (RMS) and primary progressive (PPMS) MS patients receiving ocrelizumab for at least 6 months at our centre were retrospectively classified, according to the possible occurrence of a delay (≥4 weeks) in treatment administration. Patients were categorized in the extended-interval dosing (EID) group in the presence of at least one delayed infusion; otherwise they were considered as part of the standard interval dosing (SID) cohort. MS history, magnetic resonance imaging examinations and B-cell counts were also retrospectively collected and analysed. RESULTS: A total of 213 RMS and 61 PPMS patients were enrolled; 115 RMS and 29 PPMS patients had been treated according to the SID regimen, whilst 98 RMS and 32 PPMS patients were included in the EID cohort. Average follow-up after delay was 1.28 ± 0.7 years in the EID cohort. In RMS, comparing SID and EID patients, no differences were found considering the occurrence of clinical relapses (9.6% vs. 16.3%, p = 0.338), magnetic resonance imaging activity (9.8% vs. 14.1%, p = 0.374) or disability progression (11.3% vs. 18.4%, p = 0.103). Similar findings were observed in PPMS patients. In the pooled EID group, treatment delay correlated with CD19-positive relative (r = 0.530, p < 0.001) and absolute (r = 0.491, p < 0.001) cell counts, without implications on disease activity. CONCLUSIONS: Sporadic ocrelizumab administration delay granted sustained treatment efficacy in our cohort. Prospective data should be obtained to confirm these observations and set up systematic extended-interval regimens.

Targeting CD20 in multiple sclerosis - review of current treatment strategies.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS) that mostly manifests as irreversible disability. The aetiopathogenesis of MS is still unclear, although it was initially thought to be primarily mediated by T-cells. Research into the immune concepts of MS pathophysiology in recent years has led to a shift in the understanding of its origin i.e. from a T-cell-mediated to a B-cell-mediated molecular background. Thus, the use of B-cell-selective therapies, such as anti- -CD20 antibody therapy, as expanded therapeutic options for MS is now strongly supported. This review provides an up-to-date discussion on the use of anti-CD20 targeted therapy in MS treatment. We present a rationale for its use and summarise the results of the main clinical trials showing the efficacy and safety of rituximab, ocrelizumab, ofatumumab, and ublituximab. Future directions that show selectivity to a broader population of lymphocytes, such as the use of anti-CD19 targeted antibodies, as well as the concept of extended interval dosing (EID) of anti-CD20 drugs, are also discussed in this review.

Natalizumab concentrations during pregnancy in three patients with multiple sclerosis.

In women with very active multiple sclerosis (MS), natalizumab can be continued during pregnancy to prevent rebound disease activity. Our aim was to evaluate changes in serum natalizumab trough concentrations during pregnancy. Blood samples of 3 patients were collected before, during, and after pregnancy. Natalizumab trough concentrations gradually decreased during pregnancy. The patient with the lowest trough concentrations during the third trimester was treated with extended interval dosing (EID). After delivery, natalizumab concentrations increased to similar levels as before pregnancy. All patients remained clinically and radiologically stable. MS neurologists should be aware of decreasing natalizumab concentrations during pregnancy, especially in patients with low initial trough concentrations and patients with EID.

Optimizing gentamicin conventional and extended interval dosing in neonates using Monte Carlo simulation - a retrospective study.

BACKGROUND: Although aminoglycosides are routinely used in neonates, controversy exists regarding empiric dosing regimens. The objectives were to determine gentamicin pharmacokinetics in neonates, and develop initial mg/kg dosing recommendations that optimized target peak and trough concentration attainment for conventional and extended-interval dosing (EID) regimens. METHODS: Patient demographics and steady-state gentamicin concentration data were retrospectively collected for 60 neonates with no renal impairment admitted to a level III neonatal intensive care unit. Mean pharmacokinetics were calculated and multiple linear regression was performed to determine significant covariates of clearance (L/h) and volume of distribution (L). Classification and regression tree (CART) analysis identified breakpoints for significant covariates. Monte Carlo Simulation (MCS) was used to determine optimal dosing recommendations for each CART-identified sub-group. RESULTS: Gentamicin clearance and volume of distribution were significantly associated with weight at gentamicin initiation. CART-identified breakpoints for weight at gentamicin initiation were: ≤ 850 g, 851-1200 g, and > 1200 g. MCS identified that a conventional dose of gentamicin 3.5 mg/kg given every 48 h or an EID of 8-9 mg/kg administered every 72 h in neonates weighing ≤ 850 g, and every 24 and 48 h, respectively, in neonates weighing 851-1200 g, provided the best probability of attaining conventional (peak: 5-10 mg/L and trough: ≤ 2 mg/L) and EID targets (peak:12-20 mg/L, trough:≤ 0.5 mg/L). Insufficient sample size in the > 1200 g neonatal group precluded further investigation of this weight category. CONCLUSIONS: This study provides initial gentamicin dosing recommendations that optimize target attainment for conventional and EID regimens in neonates weighing ≤ 1200 g. Prospective validation and empiric dose optimization for neonates > 1200 g is needed.

Treatment modifiers across different regimens of natalizumab treatment in MS: An Italian real-world experience.

Despite its widespread use in clinical practice, the effectiveness of natalizumab extended interval dosing (EID) adopted from treatment start across different treatment intervals and individual modifiers (body mass index - BMI) is still under-investigated. Here, seven-hundred and forty-five multiple sclerosis (MS) patients, exposed to natalizumab for 3.30 â€‹± â€‹1.34 years, were retrospectively enrolled in an observational multicenter study. After stratifying patients in EID or standard interval dosing (SID), we assessed differences in time to relapse, MRI activity and Expanded Disability Status Scale (EDSS) progression. The primary analysis was conducted on patients exposed to EID interval from 5 weeks and 1 day to 7 weeks, while a secondary analysis included also EID periods up to 8 weeks. An additional analysis explored the impact of BMI. No differences in time to first relapse, time to radiological activity, time to EDSS progression or time to EDA (evidence of disease activity) were detected between SID and EID group (EID interval from 5 weeks to 1 day to 7 weeks). When including EID periods from 7 weeks and 1 day to 8 weeks, the EID group showed a trend towards higher risk of experience clinical relapses than the SID group. A higher EDA risk was also identified in EID patients with BMI above median. In conclusion, a higher risk of relapses seems to occur for EID above 7 weeks. Independently from the EID scheme adopted, higher BMI increases the risk of EDA in these patients.

Influence of personalized extended interval dosing on the natalizumab wearing-off effect - a sub-study of the NEXT-MS trial.

BACKGROUND AND OBJECTIVES: Wearing-off symptoms during natalizumab treatment in multiple sclerosis are characterized by an increase of MS-related symptoms prior to natalizumab administration. The influence of extended interval dosing (EID) on wearing-off symptoms are important to consider, as this might cause hesitancy in initiating or continuing EID. METHODS: Participants of the NEXT-MS trial, in which treatment intervals are adjusted based on drug concentrations, were divided into two groups: an extended group containing participants with at least one week of additional interval extension, and a group with a fixed interval during the trial (range 4-7 weeks). Changes in the occurrence, frequency, onset, and severity of wearing-off symptoms were evaluated. RESULTS: 255 participants were included (extended group n = 171, fixed group n = 84). The odds on occurrence of wearing-off symptoms in the extended group did not increase after extending the treatment interval. Additional analyses for frequency, onset, and severity of wearing-off symptoms showed no changes over time. Mean decrease in natalizumab drug concentration did not influence the frequency of wearing-off symptoms. DISCUSSION: Wearing-off symptoms were not reinforced by further extending the natalizumab interval. Wearing-off symptoms might increase in a minority of patients after EID, although our data support the view that wearing-off symptoms appear to be unrelated to the decrease in natalizumab trough drug concentrations.

Natalizumab extended interval dosing: what about wearing-off effect?

BACKGROUND: Up to two thirds of patients with multiple sclerosis (MS) under natalizumab report a resurgence of symptoms at the end of the natalizumab cycle (wearing-off (WO) effect). At the outbreak of COVID-19, in line with the international recommendations for MS management, our centre switched all clinically stable patients on natalizumab therapy for more than one year from standard interval dosing (SID) to extended interval dosing (EID) with every six weeks infusions. This study aimed to evaluate the impact of EID in WO in MS patients under natalizumab. METHODS: An observational retrospective study in patients with MS under natalizumab on EID was conducted. A questionnaire regarding current (on EID) and past (on SID) experience of WO effect was applied. RESULTS: Seventy-six patients were included. No significant differences were found in the annual relapse rate after the switch to EID (p = 0.083). However, there was a significant increase in the proportion of patients complaining of WO from 38.2% to 56.6% (p = 0.001). Moreover, patients with WO on SID, referred a significant increase in severity (p = 0.019) and duration of WO symptoms (p = 0.029), due to an anticipation of the symptoms relative to the day of natalizumab infusion (p = 0.019), when switching to EID. Symptoms improved with treatment maintenance in 23.3% of patients; instead, a reduction in interval dosing was needed in 54.8% with symptom improvement. CONCLUSION: WO affects a significant proportion of MS patients under natalizumab. Its prevalence, severity, and duration increase on EID, therefore despite clinical effectiveness maintenance of this posology should be individualized.

COVID-19 outbreak in Italy: an opportunity to evaluate extended interval dosing of ocrelizumab in MS patients.

INTRODUCTION: During the COVID-19 pandemic, ocrelizumab (OCR) infusions for MS patients were often re-scheduled because of MS center's disruption and concerns regarding immunosuppression. The aim of the present study was to assess changes in OCR schedule during the first wave of pandemic in Italy and to evaluate the effect of delayed infusion on clinical/radiological endpoints. METHODS: Data were extracted from the Italian MS Register database. Standard interval dosing was defined as an infusion interval ≤ 30 weeks, while extended interval dosing was defined as an infusion interval > 30 weeks at the time of the observation period. Clinico-demographics variables were tested as potential predictors for treatment delay. Time to first relapse and time to first MRI event were evaluated. Cumulative hazard curves were reported along their 95% confidence intervals. A final sample of one-thousand two patients with MS from 65 centers was included in the analysis: 599 pwMS were selected to evaluate the modification of OCR infusion intervals, while 717 pwRMS were selected to analyze the effect of infusion delay on clinical/MRI activity. RESULTS: Mean interval between two OCR infusions was 28.1 weeks before pandemic compared to 30.8 weeks during the observation period, with a mean delay of 2.74 weeks (p < 0.001). No clinico-demographic factors emerged as predictors of infusion postponement, except for location of MS centers in the North of Italy. Clinical relapses (4 in SID, 0 in EID) and 17 MRI activity reports (4 in SID, 13 in EID) were recorded during follow-up period. DISCUSSION: Despite the significant extension of OCR infusion interval during the first wave of pandemic in Italy, a very small incidence of clinical/radiological events was observed, thus suggesting durable efficacy of OCR, as well as the absence of rebound after its short-term suspension.

Subcutaneous administration of natalizumab can lead to lower drug concentrations compared to intravenous administration.

BACKGROUND: Several studies reported lower drug concentrations with subcutaneous natalizumab compared to intravenous natalizumab. With the emergence of extended interval dosing, gaining more insight into lower concentrations after subcutaneous administration is essential. METHODS: We compared serum trough concentrations between subcutaneous and intravenous administration within a matched cohort (n = 50). RESULTS: Subcutaneous administration (n = 25) was associated with lower concentrations compared to intravenous administration (n = 25) (log-B=-0.28, p = 0.01). In an exploratory group of 11 patients receiving extended interval dosing of subcutaneous natalizumab, the median trough concentration was even lower. CONCLUSION: Subcutaneous natalizumab can lead to lower drug concentrations, potentially limiting extended interval dosing.

Patient Preference for Subcutaneous Versus Intravenous Administration with Every-6-Week Natalizumab (Tysabri®) Dosing: NOVA Phase IIIb Extension Study (Part 2).

INTRODUCTION: Following NOVA (part 1) and the approval of the subcutaneous (SC) route of administration of natalizumab by the European Medicines Agency, an extension phase of the NOVA phase IIIb study (part 2) was initiated to collect patient preference data for SC versus intravenous (IV) dosing in patients receiving every-6-week (Q6W) dosing of natalizumab. This study was performed to evaluate patient preference for SC versus IV natalizumab administration and explore the efficacy, safety, and pharmacology characteristics of both routes of administration. METHODS: In part 2, participants received natalizumab (Tysabri®) 300 mg via IV infusion Q6W for 36 weeks and then were randomized to 48 weeks of crossover treatment (24 weeks SC Q6W and 24 weeks IV Q6W, or vice versa). The primary endpoint was the proportion of participants who indicated a preference for natalizumab SC administration on the Patient Preference Questionnaire. RESULTS: A total of 153 participants were randomized in NOVA part 2. Of 123 with patient preference data, 108 (87.8%) preferred the SC route of administration for natalizumab over the IV route; 102 (82.9%) specified "requires less time in the clinic" as the reason for the SC preference. CONCLUSION: In NOVA (part 2), most participants on Q6W dosing of natalizumab preferred SC administration versus IV administration. CLINICALTRIALS: GOV: NCT03689972. INFOGRAPHIC.

Profiling of B cells and their subsets by whole blood gene expression analysis versus flow cytometry in multiple sclerosis.

We investigated if differentially expressed mRNA targets could be used as surrogate markers for circulating B cells and subsets. In paired blood samples from patients with untreated, anti-CD20-treated, fingolimod-treated, and natalizumab-treated multiple sclerosis, whole blood expression of CD19 correlated with B cell counts determined by flow cytometry, ROR1 with transitional B cells, TCL1A and ZNF727 with naïve B cells, NEXMIF with memory B cells and BCMA with plasmablasts. CD19 expression distinguished patients with B cell repletion and may be used as an alternative to flow cytometry, but NEXMIF was unsuitable for memory B cell monitoring in rituximab-treated patients.

Safety and efficacy of extended versus standard interval dosing of natalizumab in multiple sclerosis patients: a systematic review and meta-analysis.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory, immune-mediated disease affecting the central nervous system. Natalizumab, an FDA-approved monoclonal antibody for MS, has been explored for its off-label extended interval dosing (EID), suggesting a potential reduction in the risk of progressive multifocal leukoencephalopathy (PML) compared to standard interval dosing (SID). Our objective was to assess the efficacy and safety of EID in comparison to SID for natalizumab treatment in patients with MS. METHODS: We searched PubMed, Embase, WOS, Scopus, Ovid, Science Direct, Clinical trials.gov, and Cochrane Library. Our assessed outcomes were clinical relapses, MRI activity, change in expanded disability status scale [EDSS], and the risk of PML. The EID group was defined as 5 to 8 weeks [EID (Q5-8W)]. The analysis was conducted using RevMan ver. 5.4. The effect estimates were presented as a risk ratio [RR] or mean difference with 95% confidence intervals [CI] using SID group as the reference for comparisons. RESULTS: Fourteen studies met our inclusion criteria: 2 RCTs, 1 switched single-arm trial, and 12 observational studies. No significant differences were found in all efficacy outcomes of interest. Risk of clinical relapses [RR = 0.90, (95%CI 0.80, 1.02)], risk of new or newly enlarging T2 hyperintense MRI lesions [RR = 0.78, (95%CI 0.59, 1.04)], risk gadolinium enhancing lesions [RR = 1.30, (95%CI 0.98, 1.72)], change in EDSS [MD = 0.09 (95%CI - 0.57, 0.76)], risk of PML [RR = 1.09, 95%CI (0.24, 4.94)]. CONCLUSION: In summary, our meta-analysis indicates that natalizumab maintains its effectiveness under extended interval dosing [up to 8 weeks], presenting comparable risks for clinical relapses, MRI lesions, EDSS, and PML. Caution is advised given study limitations and heterogeneity. Robust conclusions necessitate well-designed high-quality prospective studies.