Genome-Wide B Cell, CD4+, and CD8+ T Cell Epitopes That Are Highly Conserved between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Targets for Preemptive Pan-Coronavirus Vaccines.

Over the last two decades, there have been three deadly human outbreaks of coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years caused by yet another spillover of a bat-derived SARS-like coronavirus (SL-CoV) into humans. Determining the Ag and the human B cells, CD4+ and CD8+ T cell epitope landscapes that are conserved among human and animal coronaviruses should inform in the development of future pan-coronavirus vaccines. In the current study, using several immunoinformatics and sequence alignment approaches, we identified several human B cell and CD4+ and CD8+ T cell epitopes that are highly conserved in 1) greater than 81,000 SARS-CoV-2 genome sequences identified in 190 countries on six continents; 2) six circulating CoVs that caused previous human outbreaks of the common cold; 3) nine SL-CoVs isolated from bats; 4) nine SL-CoV isolated from pangolins; 5) three SL-CoVs isolated from civet cats; and 6) four MERS strains isolated from camels. Furthermore, the identified epitopes: 1) recalled B cells and CD4+ and CD8+ T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2, and 2) induced strong B cell and T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice. The findings pave the way to develop a preemptive multiepitope pan-coronavirus vaccine to protect against past, current, and future outbreaks.

MYTHS AND TRUTHS OF THE ASSOCIATION OF RETINAL VASCULAR OCCLUSION WITH COVID-19.

PURPOSE: To critically review data published in the recent past to scrutinize a causal relationship between retinal vascular occlusion and COVID-19. METHOD: A comprehensive literature search was performed on Pubmed with the key words retinal vascular occlusion, retinal vein occlusion, retinal artery occlusion, and COVID-19. RESULTS: A total of 17 case reports were published during this period, and 10 were on retinal vein occlusion and 7 on retinal artery occlusion. Most of the published reports lacked convincing evidences in one or the other aspects, such as insufficient laboratory workup or presence of multiple confounding risk factors. CONCLUSION: In this index article, strength of the data is insufficient to establish a definitive cause-and-effect relationship of retinal vascular occlusive disorders with COVID-19. Hence, clinicians can continue to manage these cases according to the standard guidelines until there are more robust evidences to support this association to alter the diagnostic and treatment modalities.

PHASE 2 STUDY OF THE SAFETY AND EFFICACY OF BRIMONIDINE DRUG DELIVERY SYSTEM (BRIMO DDS) GENERATION 1 IN PATIENTS WITH GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To evaluate the safety and efficacy of Brimonidine Drug Delivery System (Brimo DDS), a biodegradable intravitreal implant, in the treatment of geographic atrophy (GA) secondary to age-related macular degeneration. METHODS: Phase 2, randomized, multicenter, double-masked, 24-month study. Study eyes were treated (Day 1; Month 6 retreatment) with Brimo DDS 132 µg (n = 49), Brimo DDS 264 µg (n = 41), or sham procedure (n = 23). The primary timepoint for efficacy analysis was Month 12. RESULTS: Mean GA area growth at Month 12 was 1.78 mm2, 1.59 mm2, and 2.19 mm2 in the Brimo DDS 132 µg, 264 µg, and sham groups, respectively. Geographic atrophy area growth was consistently smaller with Brimo DDS 132 and 264 µg than sham; between-group differences were significant (P ≤ 0.032) at Month 3. In patients with baseline lesion area ≥6 mm2 (two-thirds of patients), GA lesion area and effective radius growth was reduced with Brimo DDS 132 and 264 µg at Month 12 (P ≤ 0.050 vs. sham). Treatment-related adverse events were usually injection procedure-related. CONCLUSION: Brimo DDS demonstrated a favorable safety profile and reduced GA lesion area growth at Month 3. Lesion growth at Month 12 was reduced in patients with baseline GA lesion area ≥6 mm2. The results support Phase 3 development.

Current role of intravitreal injections in Irvine Gass syndrome-CRIIG study.

OBJECTIVE: To analyze the role of intravitreal anti-vascular endothelial growth factor (anti-VEGF) or steroid injection for the management of Irvine Gass syndrome. METHODS: It is an interventional, retrospective, multicenter study. One hundred and thirty-two injections were given in 79 eyes of 72 patients with Irvine Gass syndrome. Patients were treated with at least one intravitreal injection of either anti-VEGF or steroid. Outcomes were measured at 12 months (± 1 week). [Ranibizumab (Lucentis; Genentech, South San Francisco, CA) (Razumab; Intas Pharmaceutical Ltd, Ahmedabad, India) Bevacizumab (Avastin; Genentech, South San Francisco, CA) or Aflibercept (Eylea; Regeneron, Tarrytown, NY)] or steroids [Dexamethasone implant (Ozurdex, Allergan Inc, Irvine, CA) or intravitreal triamcinolone)]. RESULTS: Intravitreal injections were initiated in (67.6%) of eyes within 14 weeks of diagnosis. Intravitreal dexamethasone implant was used as the initial intravitreal therapy in (73.4%) of eyes. More than fifty percent (54.5%) of the patients were switched from anti-VEGF to Intravitreal dexamethasone implant. Reduction in the mean CMT was 336.7 ± 191.7 and 160.1 ± 153.1 microns in eyes treated within four weeks and more than 14 weeks from diagnosis (p = 0.005). Mean ETDRS letter gain was 16.7 ± 12.9 and 5.2 ± 9.2 in eyes treated within 4 weeks and more than 14 weeks from diagnosis (p = 0.004). Three eyes injected with intravitreal dexamethasone implant reported an intraocular pressure spike of > 25 mmHg which was controlled with topical medications. No other ocular or systemic adverse events were observed. CONCLUSION: Study results suggest that physicians tend to introduce intravitreal therapy within 14 weeks of diagnosis. The most common therapy at initiation and for the switch is intravitreal dexamethasone implant. Patients treated early (within 4 weeks) respond better in terms of structure and function.

Age-related macular degeneration (AMD) mitochondria modulate epigenetic mechanisms in retinal pigment epithelial cells.

Mitochondrial damage and epigenetic modifications have been implicated in the pathogenesis of Age-related Macular Degeneration (AMD). This study was designed to investigate the effects of AMD/normal mitochondria on epigenetic regulation in human transmitochondrial retinal pigment epithelial (RPE) cells in vitro. Human RPE cybrid cell lines were created by fusing mitochondria-deficient (Rho0) ARPE-19 cells with platelets obtained from either AMD patients (AMD cybrids) or normal subjects (normal cybrids). Therefore, all cybrids had identical nuclei (derived from ARPE-19 cells) but mitochondria derived from either AMD patients or age-matched normal subjects. AMD cybrids demonstrated increased RNA/protein levels for five methylation-related and four acetylation-related genes, along with lower levels of two methylation and three acetylation genes compared to normal cybrids. Demethylation using 5-Aza-2'-deoxycytidine (DAC) led to decreased expression of VEGF-A gene in AMD cells. Trichostatin A (TSA), an HDAC inhibitor, also influenced protein levels of VEGF-A, HIF1α, NFκB, and CFH in AMD cells. Our findings suggest that retrograde signaling leads to mitochondria-nucleus interactions that influence the epigenetic status of the RPE cells and this may help in the identification of future potential therapeutic targets for AMD.

Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes.

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

Corticosteroids and Anti-Complement Therapy in Retinal Diseases.

Corticosteroids are unique in that they are the one class of agents that acts upon most of the multiple processes in the pathophysiology of macular edema. Corticosteroids are capable of inhibiting prostaglandin and leukotriene synthesis as well as interfering with intercellular adhesion molecule-1 (ICAM-1), interleukin-6, VEGF-A, and stromal cell derived factor-1 pathways. Triamcinolone, dexamethasone, and fluocinolone have been extensively used in the treatment of retinal and choroidal vascular diseases. Sustained release implants of steroids have reduced the burden of repeated intravitreal injections necessary in most of the retinal diseases. Complement factors play an important role in the pathogenesis of age-related macular degeneration (AMD). Inhibitors of complement could provide a breakthrough in the treatment of dry AMD. Complement factor inhibitors, such as POT-4, lampalizumab, and eculizumab, have been tested in clinical trials for dry AMD with promising results. However, results of phase 3 trials are awaited.

Increased expression of ApoE and protection from amyloid-beta toxicity in transmitochondrial cybrids with haplogroup K mtDNA.

Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNP) patterns, represent populations of diverse geographic origins and have been associated with increased risk or protection of many diseases. The H haplogroup is the most common European haplogroup while the K haplogroup is highly associated with the Ashkenazi Jewish population. Transmitochondrial cybrids (cell lines with identical nuclei, but mtDNA from either H (n=8) or K (n=8) subjects) were analyzed by the Seahorse flux analyzer, quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC). Cybrids were treated with amyloid-ß peptides and cell viabilities were measured. Other cybrids were demethylated with 5-aza-2'-deoxycytidine (5-aza-dC) and expression levels for APOE and NFkB2 were measured. Results show K cybrids have (a) significantly lower mtDNA copy numbers, (b) higher expression levels for MT-DNA encoded genes critical for oxidative phosphorylation, (c) lower Spare Respiratory Capacity, (d) increased expression of inhibitors of the complement pathway and important inflammasome-related genes; and (e) significantly higher levels of APOE transcription that were independent of methylation status. After exposure to amyloid-ß1-42 peptides (active form), H haplogroup cybrids demonstrated decreased cell viability compared to those treated with amyloid-ß42-1 (inactive form) (p<0.0001), while this was not observed in the K cybrids (p=0.2). K cybrids had significantly higher total global methylation levels and differences in expression levels for two acetylation genes and four methylation genes. Demethylation with 5-aza-dC altered expression levels for NFkB2, while APOE transcription patterns were unchanged. Our findings support the hypothesis that mtDNA-nuclear retrograde signaling may mediate expression levels of APOE, a key factor in many age-related diseases. Future studies will focus on identification of the mitochondrial-nuclear retrograde signaling mechanism(s) contributing to these mtDNA-mediated differences.

Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial-nuclear interactions.

Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other 'modifiers' may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt-nuclear interactions.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Safety profile of ocriplasmin for the pharmacologic treatment of symptomatic vitreomacular adhesion/traction.

PURPOSE: To report the safety of intravitreal ocriplasmin injection based on 2 Phase 3 clinical trials in patients with symptomatic vitreomacular adhesion/vitreomacular traction, including when associated with full-thickness macular holes. METHODS: Safety analyses were based on 2 completed Phase 3 studies assessing intravitreal ocriplasmin injection. Adverse events (AEs), serious AEs, and suspected adverse drug reactions are reported. The authors also report AEs of special interest from 8 other completed Phase 2 studies and 2 ongoing studies. RESULTS: A total of 465 eyes were injected with ocriplasmin (125 µg), and 187 eyes were treated with placebo injection in Phase 3 studies. Overall AE rate was 69.0% in the placebo group and 76.6% for ocriplasmin-treated patients. Most AEs were in the study eye, mild or moderate in severity, and transient. All suspected adverse drug reactions were ocular; the majority was nonserious, of mild intensity, and transient. CONCLUSION: Intravitreal ocriplasmin injection provides a generally well-tolerated pharmacologic treatment option for patients with symptomatic vitreomacular adhesion/vitreomacular traction, including when associated with full-thickness macular holes ≤400 µm in diameter.

Dexamethasone intravitreal implant in previously treated patients with diabetic macular edema: subgroup analysis of the MEAD study.

BACKGROUND: Dexamethasone intravitreal implant 0.7 mg (DEX 0.7) was approved for treatment of diabetic macular edema (DME) after demonstration of its efficacy and safety in the MEAD registration trials. We performed subgroup analysis of MEAD study results to evaluate the efficacy and safety of DEX 0.7 treatment in patients with previously treated DME. METHODS: Three-year, randomized, sham-controlled phase 3 study in patients with DME, best-corrected visual acuity (BCVA) of 34-68 Early Treatment Diabetic Retinopathy Study letters (20/200-20/50 Snellen equivalent), and central retinal thickness (CRT) ≥ 300 µm measured by time-domain optical coherence tomography. Patients were randomized to 1 of 2 doses of DEX (0.7 mg or 0.35 mg), or to sham procedure, with retreatment no more than every 6 months. The primary endpoint was ≥ 15-letter gain in BCVA at study end. Average change in BCVA and CRT from baseline during the study (area-under-the-curve approach) and adverse events were also evaluated. The present subgroup analysis evaluated outcomes in patients randomized to DEX 0.7 (marketed dose) or sham based on prior treatment for DME at study entry. RESULTS: Baseline characteristics of previously treated DEX 0.7 (n = 247) and sham (n = 261) patients were similar. In the previously treated subgroup, mean number of treatments over 3 years was 4.1 for DEX 0.7 and 3.2 for sham, 21.5% of DEX 0.7 patients versus 11.1 % of sham had ≥ 15-letter BCVA gain from baseline at study end (P = 0.002), mean average BCVA change from baseline was +3.2 letters with DEX 0.7 versus +1.5 letters with sham (P = 0.024), and mean average CRT change from baseline was -126.1 µm with DEX 0.7 versus -39.0 µm with sham (P < .001). Cataract-related adverse events were reported in 70.3% of baseline phakic patients in the previously treated DEX 0.7 subgroup; vision gains were restored following cataract surgery. CONCLUSIONS: DEX 0.7 significantly improved visual and anatomic outcomes in patients with DME previously treated with laser, intravitreal anti-vascular endothelial growth factor, intravitreal triamcinolone acetonide, or a combination of these therapies. The safety profile of DEX 0.7 in previously treated patients was similar to its safety profile in the total study population. TRIAL REGISTRATION: ClinicalTrials.gov NCT00168337 and NCT00168389, registered 12 September 2005.

Dexamethasone Intravitreal Implant as Adjunctive Therapy to Ranibizumab in Neovascular Age-Related Macular Degeneration: A Multicenter Randomized Controlled Trial.

PURPOSE: To evaluate the efficacy and safety of dexamethasone intravitreal implant 0.7 mg (DEX) as adjunctive therapy to ranibizumab in neovascular age-related macular degeneration (nvAMD). PROCEDURES: This was a 6-month, single-masked, multicenter study. Patients were randomized to DEX implant (n = 123) or sham procedure (n = 120) and received 2 protocol-mandated intravitreal ranibizumab injections. The main outcome measure was injection-free interval to first as-needed ranibizumab injection. RESULTS: DEX increased the injection-free interval versus sham (50th percentile, 34 vs. 29 days; 75th percentile, 85 vs. 56 days; p = 0.016). 8.3% of DEX versus 2.5% of sham-treated patients did not require rescue ranibizumab (p = 0.048). Visual acuity and retinal thickness outcomes were similar in DEX and sham-treated patients. Only reports of conjunctival hemorrhage (18.2 vs. 8.5%) and intraocular pressure elevation (13.2 vs. 4.2%) were significantly different in the DEX versus the sham treatment groups. CONCLUSION: DEX reduced the need for adjunctive ranibizumab treatment and showed acceptable tolerability in nvAMD patients.

Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G.

BACKGROUND: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. METHOD: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. RESULT: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. CONCLUSION: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells.

Sustained delivery fluocinolone acetonide vitreous implants: long-term benefit in patients with chronic diabetic macular edema.

PURPOSE: To present the safety and efficacy of intravitreal implants releasing 0.2 µg/day fluocinolone acetonide (FAc) in patients with chronic versus nonchronic diabetic macular edema (DME). To assess ocular characteristics, anatomic changes, and re-treatment and ancillary therapies that may explain the differential treatment effect seen with intravitreal implants releasing FAc 0.2 µg/day in patients with chronic and nonchronic DME. An overall benefit-to-risk assessment for the FAc 0.2-µg/day and FAc 0.5-µg/day doses has been reported previously. DESIGN: Preplanned subgroup analysis of chronic (duration of diagnosis, ≥3 years) and nonchronic (duration of diagnosis, <3 years) DME in patients from 2 randomized, sham injection-controlled, double-masked, multicenter clinical trials. PARTICIPANTS: Patients with persistent DME despite 1 or more macular laser treatment were randomized 1:2:2 to sham injection (n = 185), FAc 0.2 µg/day (n = 375), or FAc 0.5 µg/day (n = 393). METHODS: Patients received study drug or sham injection and after 6 weeks were eligible for rescue laser. Based on re-treatment criteria, additional masked study drug could be given after 1 year. MAIN OUTCOME MEASURES: Percentage of patients with improvement of 15 letters or more from baseline. Secondary outcomes included other parameters of visual function and foveal thickness. RESULTS: At month 36, the difference between FAc 0.2 µg/day and sham control in the percentage of patients who gained 15 letters or more was significantly greater in chronic DME patients (FAc 0.2 µg/day, 34.0% vs. sham, 13.4%; P<0.001), compared with patients with nonchronic DME (FAc 0.2 µg/day, 22.3% vs. sham, 27.8%; P = 0.275). The greater response in patients with chronic DME was not associated with baseline ocular characteristics, changes in anatomic features, or differences in re-treatment or ancillary therapies. The ocular adverse event profile for FAc 0.2 µg/day was similar regardless of DME duration. CONCLUSIONS: This is the first published analysis correlating duration of diagnosis of DME with treatment effect. In patients with chronic DME, FAc 0.2 µg/day provides substantial visual benefit for up to 3 years and would provide an option for patients who do not respond to other therapy.

Onset and duration of visual acuity improvement after dexamethasone intravitreal implant in eyes with macular edema due to retinal vein occlusion.

PURPOSE: To evaluate the onset and duration of improvement in best-corrected visual acuity (BCVA) in eyes treated with dexamethasone intravitreal implant 0.7 mg (DEX implant) for macular edema after branch or central retinal vein occlusion. METHODS: Post hoc analysis of data from 2 previously reported multicenter, 6-month, randomized sham-controlled clinical trials. Patients received a single DEX implant (n = 427) or sham procedure (n = 426) in the study eye. The primary endpoint was the percentage of eyes with ≥ 15-letter improvement in BCVA from baseline at postimplant Day 7. RESULTS: The baseline mean BCVA was 20/80. At Day 7, 10.3% of DEX implant-treated eyes versus 4.0% of sham-treated eyes (P < 0.001) had ≥ 15-letter improvement in the BCVA, and 27.2% of DEX implant-treated eyes versus 10.6% of sham-treated eyes had ≥ 10-letter improvement (P < 0.001). The mean improvement at Day 7 was 5.3 letters (branch retinal vein occlusion, 5.1; and central retinal vein occlusion, 5.8) with DEX implant and 1.6 letters (branch retinal vein occlusion, 2.3; and central retinal vein occlusion, 0.1) with sham (P < 0.001). The mean time from initial observation of ≥ 15-letter BCVA gain to the last observation of ≥ 15-letter BCVA gain was 70 days. CONCLUSION: Dexamethasone intravitreal implant treatment led to improvement in BCVA compared with sham procedure as early as postimplant Day 7. The duration of ≥ 3-line improvement was typically 2 to 3 months.