Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis

Mami Tomita; Mizuki Tagami; Norihiko Misawa; Atsushi Sakai; Yusuke Haruna; Shigeru Honda

doi:10.1371/journal.pone.0297201

. 2024 Jan 17;19(1):e0297201. doi: 10.1371/journal.pone.0297201

Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis

Mami Tomita ¹, Mizuki Tagami ^1,^*, Norihiko Misawa ¹, Atsushi Sakai ¹, Yusuke Haruna ¹, Shigeru Honda ¹

Editor: Jiro Kogo²

PMCID: PMC10793905 PMID: 38232093

Abstract

Purpose

To investigate relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis.

Methods

This retrospective study examined vitreous inflammatory cells (hyperreflective particles) of posterior uveitis on widefield optical coherence tomography (WOCT). We examined the number of hyperreflective particles (possibility of vitreous inflammatory cells) observed on WOCT and the correlations with interleukin-6 (IL-6) levels. The relationship between vitreous IL-6 levels and image findings from WOCT from 37 eyes (34 patients) with posterior uveitis were analyzed. Mean patient age was 63.4±15.7 years. (Mean± standard deviation) IL-6 concentration in vitreous humor was 79.9±7380.9 pg/mL Uveitis was infectious in 9 cases and non-infectious in 28 cases with multiplex polymerase chain reaction system. We measured the number and size of vitreous cells in the posterior vitreous, defined as the space between the upper vitreous and the internal limiting membrane on WOCT at the macular, upper, and lower regions. Image analysis software was also used for cell counting.

Results

A strong correlation was seen between human and software counts. Pearson’s correlation coefficient (PCC) was performed to compare categorial variables (on macular +0.866; upper cavity +0.713; lower cavity +0.568; total vitreous cavity +0.834; P<0.001 each). IL-6 levels correlated with both vitreous cell counts and cell counts observed on macular WOCT (human-counted group +0.339, P = 0.04; software-counted group +0.349, P = 0.03). Infectious uveitis showed higher IL-6 levels (P = 0.016) and high cell counts compared with non-infectious uveitis (P = 0.04).

Conclusions

Vitreous number of hyperreflective particles (cells) findings on WOCTcorrelated well with human and software cell counts. Vitreous cells findings on WOCT also correlated with IL-6 concentrations on macular.

Introduction

Uveitis refers to inflammation of the uvea, which comprises the iris, ciliary body and choroid. Posterior uveitis often results in vitreous opacity, leading to a loss of visual acuity. Uveitis can be infectious or non-infectious, with each requiring different approaches to therapy. Non-infectious uveitis often has a relationship with autoimmune or infectious diseases [1]. The most frequent cause of uveitis in Japan is sarcoidosis, accounting for 10.6% of cases, followed by Vogt–Koyanagi–Harada disease (8.1%), herpetic iritis (6.5%), and acute anterior uveitis (5.5%) [2], Sonoda et al. reported on 5328 patients with new-onset uveitis in 2016, finding that unclassified intraocular inflammation uveitis represented over one-third of cases, thus diagnosis and treatment of uveitis can thus be difficult. The sorting of uveitis based on the grade of inflammation may prove useful in determining therapeutic approaches.

Optical coherence tomography (OCT) is an imaging modality that possibility of enables evaluation of the inflammatory degree [3,4]. In addition, Widefield OCT (WOCT) also allows imaging of the peripheral retina comparing with normal OCT, critical inflammatory diagnosis of uveitis using non-invasive methods [5,6].

Interleukin (IL-6) is one of the cytokines involved in inflammatory reactions and immune responses [7–10]. Many recent reports have examined the relationship between uveitis and IL-6 level and several functions of IL-6 have been identified, allowing the development of biomedicines against non-infectious uveitis [11–16]. Several reports have already shown that IL-6 levels are increased in the aqueous humor in patients with uveitis [17–19].

Here we report changes in vitreous opacity in posterior uveitis as observed using WOCT imaging, IL-6 levels and their relationship. In addition to these investigations, although the number of cases is small, flow cytometry results were described in our manuscript for understanding characters of vitreous cells.

This study investigated properties and intraocular distribution of hyperreflective particles (possibility of vitreous inflammatory cells) on WOCT and the correlations with IL-6 levels and cell characters in uveitis patients.

Materials and methods

Selection of cases and collation of clinicopathological data

This retrospective review enrolled patients treated in the Ophthalmology Department at Osaka Metropolitan University between July 2018 and November 2022.

Study inclusion criteria included [1] the presence of posterior vitreous inflammation [2], unknown cause of posterior uveitis [3], Cases in which preoperative WOCT and Wide field Optical Coherence Tomography angiography (WOCTA) of En-face could be obtained. Study exclusion criteria included [1] Patients with active systemic inflammation [2], Cases couldn’t be observed vitreous cavity due to vitreous opacity in preoperative WOCT [3], The cases couldn’t take En-face images of WOCTA or Optos (Optos® 200Tx, Optos®, Dunfermline, U.K.). We evaluated 37 eyes from 34 patients with posterior uveitis. These patients were investigated as cases of non-classified uveitis, with vitrectomy performed for diagnosis.

Data from patients were retrospectively collected. This study was performed in accordance with the tenets of the Declaration of Helsinki. All study protocols were approved by the institutional review board at Osaka Metropolitan University conducted with approval from the institutional review board (Osaka Metropolitan University-2023-043) prior to commencement. Written informed consent had been obtained from all patients enrolled in the study for the storage of patient information in the hospital database and its use in research.

All eyes underwent ophthalmologic examination, which included measurement of best-corrected visual acuity (BCVA) using a Landolt C acuity chart at 5 m, slit lamp bio-microscopy, OCT (spectral-domain OCT, Spectralis; Heidelberg Engineering, Heidelberg, Germany), and WOCT (Xephilio OCT-S1; Canon, Tokyo, Japan). BCVA was converted to logMAR BCVA.

All cases underwent 27-gauge diagnostic vitrectomy surgery and levels of IL-6 were measured.

IL-6 levels were measured in vitreous samples by enzyme-linked immunosorbent assay (ELISA) using kits for human IL-6 (LUMIPULSE G1200, FUJIREBIO, Tokyo, Japan). All vitreous samples underwent multiplex polymerase chain reaction (PCR) analysis for the diagnosis of bacterial or viral uveitis. The PCR test is a strip PCR test that can identify 24 different types of pathogenic microorganisms: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, human T-cell lymphotropic virus (HTLV)-1/2, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), Propionibacterium acnes (P. acnes), Aspergillus, Fusarium, bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, fungal 28S rRNA, and Acanthamoeba [20].

When bacteria or virus was detected in vitreous samples, infectious uveitis was diagnosed. If no findings or results of morbid significance were obtained, the case was classified as non-infectious uveitis.

We observed the vitreous area on WOCT before vitrectomy and defined hyperreflective particles as cells [3]. Also, En-face WOCTA was taken in all cases. Macular was defined horizontal B-scan through the foveal central subfield [3,21]. Furthermore, we examined the choroidal structure using WOCTA or Optos 200Tx (Optos PLC, Dunfermline, Scotland) [22,23]. We identified the ampullae of the vortex vein inflow by imaging in En-face choroidal mode of OCTA or En-face mode of Optos. The B-scan around equatorial region was defined as a straight line connecting temporal and nasal of the ampullae vortex vein inflow [24]. Upper vitreous cavity area on WOCT was defined as B-scan with horizontal lines drawn at the inflow point of the vortex vein volume on the superotemporal and superonasal sides. For the lower vitreous cavity area of WOCT, like the upper one, we selected a B-scan around the area connecting inferotemporal and inferonasal sides with a horizontal line. If the temporal and nasal vortex venous ampullaes could not be connected horizontally, a horizontal line passing through the nasal vortex vein ampulla was used.

Cells observed on WOCT were counted for B-scan images through the macular (Fig 1A), upper vitreous cavity area around the equatorial region (Fig 1B) and lower vitreous cavity area (Fig 1C). Vitreous cells were counted in the vitreous space using WOCT by humans. Furthermore, the utility of ImageJ software was examined. We manually selected the vitreous area (Fig 1D–1F), if areas obviously depicting vitreous were misidentified as cells, the vitreous membrane was manually selected to be avoided. (Fig 1E and 1F). Then counted the number of cells present in that area using ImageJ software application (Fig 1G–1I). Measurements were made with reference to previous reports [25].

WOCT images were taken horizontally at a maximum of 23 mm and at a maximum depth of 5.3 mm; the average measured vitreous cavity area in 37 cases was 22.5 x 3.0 mm through the macular, 16.8 x 4.4 mm around upper around equatorial area, 15.9 x 3.8 mm around lower.

We compared the number of cells observed on WOCT with IL-6 levels and examined differences between infectious or non-infectious uveitis. All data were collected in an Excel spreadsheet (Microsoft, Redmond, WA, USA).

Imaging evaluations

In the vitreous area, hyperreflective particles were counted one by one.

Images in the lower parts of three cases were unclear in the delineation and were excluded from analysis.

In accordance with previous reports, “posterior vitreous cavity area” was measured as the space between the upper edge of the vitreous body and the inner boundary membrane, after manual drawing using the polygon selection tool in ImageJ software [3,25]. In this study, the presence or absence of posterior vitreous detachment was not considered. Data obtained from an independent scorer who had been blinded to cases were used for analysis. All WOCT images were imported into ImageJ software version 1.5 (National Institutes of Health, Bethesda, MD, USA). All assessments were applied to each image; image contrast was not adjusted before or after exporting the image from the OCT system.

Our WOCT device that could obtain up to 23 mm of widefield B-scan images in a single acquisition. The device had a depth of 5.3 mm. The transverse resolution was 20 μm.

The system has a digital axial resolution of 1.6 μm, which can be combined with the ability to average multiple scans (up to 200) to further improve image quality.

Two methods of counting were used: counting by human (Group 1); and counting by software (Group 2). We used ImageJ software for counting. ImageJ is a Java-based image-processing program developed by the National Institutes of Health and the Optical Computational Metrology Institute. We manually selected the vitreous area using the selection tools in ImageJ. Binarization is a type of image processing that divides the intensity of an image into binary values, such as 0 or 1 [26]. On the WOCT images, cells were binarised and automatically counted by the ImageJ software [27]. The image area to be binarized needs to be manually selected. The correlation between cell counts and IL-6 levels in the images from each case was analyzed in each group.

Correlation with IL-6 value

We compared logMAR BCVA between pre-vitrectomy and 3 months postoperatively, and examined relationships between BCVA and the number of cells.

IL-6 levels were separated into below 1000 and above 1001 pg/mL, then we investigated whether cell counts observed from WOCT were high.

We also investigated logMAR BCVA preoperatively and at 3 months postoperatively. The group showing improved vision was defined as those achieving two or more levels of improvement in BCVA. The group with no changes in visual acuity was defined as the unchanged group. Those patients in whom visual acuity worsened by two or more levels between preoperatively and 3 months postoperatively were defined as the worsening group in each group.

Flow cytometry

In flow cytometry, gating is the process of selecting a subset of cells from a larger population for further analysis. Gating was performed by setting up a series of gates or regions on a plot of the data generated by the flow cytometer.

They separate cell fractions using forward scatter (FSC) and side scatter (SSC). High score of FSC means that cells in the samples is large. Large cells were identified as gate(A).

We analyzed using the CD45-SSC gating method. The CD45-SCC gating method uses the fact that immature, proliferative haematopoietic cells express low levels the CD45 antigen. This allows selective analysis of tumor cells by excluding mature normal cells [28].

CD45 gating were enable to analysis CD45 diminish area as Gate1.

The number of cells observed in Gate 1 was calculated and compared with the number of cells observed on WOCT. This analysis included subjects showing positivity for CD3, CD8, CD19, CD20 and CD56. The composition of lymphocytes are shown. CD19 and CD20 represent the B-cell family and were investigated together. Seven cases in enough vitreous fluid were analyzed by flow cytometry analysis for detecting vitreous cells characters.

Statistical analyses

Clinical and histopathological characteristics are summarized using descriptive statistics. Correlations between immunohistochemical, demographic, and clinicopathological factor data were assessed using the t-test and chi-squared test. Statistical analyses were performed using SPSS Statistics software (version 22; IBM Japan, Tokyo, Japan). Values of P<0.05 were considered significant. IL-6 levels and cell counts observed on OCT were analyzed using the Mann-Whitney U-test.

Results

In total, 37 eyes from 37 patients were included in the study “Table 1”.

Table 1. Baseline characteristics of patients.

Participant Baseline Characteristics	total	infectious	Non-infectious
Eye,n (%)	37	9 (24.3)	28 (75.7)
Patients,n (%)	34	9 (26.5)	25 (73.5)
Males,n (%)	18	8 (44.4)	10 (55.6)
Females,n (%)	19	1 (5)	18 (95)
Age,years (Mean±SD)	67.5±15.7	67±18.8	67.5±14.2
Baseline logMAR BCVA (Mean±SD)	0.22±0.6	0.15±0.8	0.35±0.5
IL-6 levels, pg/mL (Mean±SD)	79.9±7380.9	1040±12990.6	73.5±990.5

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Abbreviation: BCVA: Best-corrected visual acuity, SD: Standard deviation, IL-6: Interleukin-6.

Table 1 shows patient characteristics. PCR revealed the cause of infection as acute retinal necrosis in 3 cases, cytomegalovirus in 1 case, bacterial (16-strip PCR) in 1 case, and human T-cell leukemia virus type 1 in 1 case. Almost all cases of non-infectious uveitis were idiopathic (n = 22), with 5 cases caused by sarcoidosis and 1 case caused by macular pit syndrome.

On the WOCT images, the more cells there were in Group 1, the more cells were observed in Group 2 as well. (Table 2, Fig 2A–2D).

Table 2. Numbers of cells in Group 1 and Group 2.

	Group 1 (n)	Group 2 (n)	PCC	P-value
Macular	27.7±45.0	95.5±76.3	+0.866	<0.001^*
Upper	8.5±22.9	61.5±53.8	+0.713	<0.001^*
Lower (n = 34)	8.7±17.0	56.7±37.7	+0.568	<0.001^*
Total (n = 34)	44.0±73.0	209.1±133.4	+0.834	<0.001^*

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Statistical analysis were performed using the Pearson’s correlation coefficient.

Group 1: Manual counting, Group 2: Software-based counting.

PCC: Pearson’s correlation coefficient.

*P<0.05.

Fig 2 — Group 1: Manual counting, Group 2: Software-based counting. (A) In macular images, correlations between Group 1 and Group 2. (B) In upper vitreous around the equator images, correlations between Group 1 and Group 2. (C) In lower vitreous around the equator images, correlations between Group 1 and Group 2. (D) In total number of cells analyzed in the macula, upper and lower, correlations between Group 1 and Group 2. (E) In macular images, correlations between Group 1 and IL-6 levels. (F) In upper vitreous around the equator images, correlations between Group 1 and IL-6 levels. (G) In lower vitreous around the equator images, correlations between Group 1 and IL-6 levels (n = 34). (H) In total number of cells analyzed in the macula, upper and lower, correlations between Group 1 and IL-6 levels (n = 34). (I) In macular images, correlations between Group 2 and IL-6 levels. (J) In upper vitreous around the equator images, correlations between Group 2 and IL-6 levels. (K) In lower vitreous around the equator images, correlations between Group 2 and IL-6 level (n = 34). (L) In total number of cells analyzed in the macula, upper and lower, correlations between Group 1 and IL-6 levels (n = 34). PCC: Pearson’s correlation coefficient, IL-6: Interleukin-6. **P<0*.05.

Cell counts were consistently higher in Group 2 than in Group 1. Each image showed a high correlation coefficient between groups.

All area showed significant findings. As more cells were observed in Group 1, more cells were also found in Group 2. We investigated the correlation between IL-6 levels and cell counts on WOCT in Group 1 (Fig 2E–2H). We found a correlation between IL-6 level and cell count in Group 1 from macular images (PCC +0.339, P = 0.04; Fig 2E). We got correlations between IL-6 levels and cell counts from WOCT in Group2 using “ImageJ” software (Fig 2I–2L). Likewise, in Group 2, significant differences were found only in macular images (PCC+0.349,P = 0.03; Fig 2I).

The number of cells in macular images thus correlated with IL-6 levels in both groups (Table 3).

Table 3. Correlation between IL-6 levels and cell counts on WOCT images.

	Group 1 PCC	P-value	Group 2 PCC	P-value
Macular	+0.339	0.0402^*	+0.349	0.0343^*
Upper	-0.009	0.96	+0.048	0.777
Lower (n = 34)	-0.008	0.96	-0.020	0.914
Total (n = 34)	+0.196	0.266	+0.208	0.238

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Statistical analysis were performed using the Pearson’s correlation coefficient.

Group 1: Manual counting, Group2: Software-based counting.

PCC: Pearson’s correlation coefficient.

*P<0.05.

Only images at the macular showed a significant correlation (P = 0.0402; Group1, P = 0.0343; Group2).

We added a study of macular images based on this high correlation between IL-6 levels and cell count. As a high correlation was found between Groups 1 and 2, further analyses were added for only the mechanical analysis group.

In IL-6 levels, no significant relationship was seen between IL-6 levels and number of cells, but higher cell numbers tended to be observed in cases with higher IL-6 levels (P = 0.068; Fig 3A).

Fig 3 — Relationship between IL-6 levels and the number of cells when the IL-6 value is bimilitarised (A). The number of vitreous particles as hyperreflective particles in infectious and non-infectious uveitis (B, C). (A) We separated two groups according to IL-6 levels below 1000 pg/mL above 1001 pg/mL. IL-6 levels tended to be higher with greater cell counts on WOCT, but the relationship was not significant using the t-test. (B) IL-6 levels were higher in infectious uveitis compared with noninfectious uveitis using the Mann-Whitney test. (C) Patients with infectious uveitis showed significantly greater cell counts than those with non-infectious uveitis using the t-test. IL-6: Interleukin-6. *P<0.05.

The 37 patients were divided into groups with infectious and non-infectious uveitis. In each group, we compared IL-6 levels and cell counts from macular images. The infectious uveitis group showed higher IL-6 levels than the non-infectious group (P<0.001; Fig 3B). This result was similar to findings from a previous report [29]. Mean IL-6 levels was 8689.6±13,778.6 pg/mL in the infectious group and 387.8±1008.7 pg/mL in the non-infectious group. We also compared cell counts between the infectious and non-infectious uveitis groups. Among infectious cases, many cells were seen in WOCT images, as previously reported (P = -0.021; Fig 3C) [25]. Mean cell count was 161.3±113.2 units in the infectious group and 74.4±47.6 units in the non-infectious group.

Correlation with IL-6 levels and changing of visual acuity

IL-6 levels were separated into below 1000 and above 1001 pg/mL, then we investigated whether cell counts observed from WOCT were high.

We also investigated logMAR BCVA preoperatively and at 3 months postoperatively. (P = 0.018; Fig 4).

We also considered the relationship between number of cells on WOCT images and lymphocyte count from flow cytometry (Table 4).

Table 4. Baseline characteristics of the 7 patients investigated by flow cytometry.

ID	sex	Age(years)	classification	The number of cells in WOCT (macular)	CD3 (n)	CD8 (n)	CD19+20 (n)	CD56 (n)
1	F	56	PIOL	38	48.5	41.2	13.8	3.6
2	F	76	Sarcoidosis	15	103.9	1.5	2.0	4.2
3	F	47	Idiopathic	123	44.3	10.0	6.9	4.2
4	M	44	Idiopathic	46	29.9	42.3	4.2	1.3
5	F	69	Idiopathic	28	338.1	18.7	7.5	3.6
6	M	80	Idiopathic	53	31.3	42.7	228.7	7.8
7	M	57	Idiopathic	51	16.0	122.9	122.9	5.7

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Abbreviations: PIOL: Primary Intraocular lymphoma.

There was no statistically significant difference between the observed the number of cells on WOCT and each lymphocyte (Fig 5).

Discussion

We performed vitrectomy in 37 cases of unclassified posterior uveitis, and analyzed IL-6 levels from vitreous samples. We also observed reflective particles on B-scan widefield OCT performed pre-vitrectomy. This study identified significant differences in the number of vitreous hyperreflective particles on WOCT images of the macular according to IL-6 levels.

Diagnostic vitrectomy is a very general and important clinical tool for diagnosing uveitis. The safety and utility of 27-gauge vitrectomy have recently been reported [30].

Focusing on molecular uveitis, IL-6 is a cytokine showing elevated production in autoimmune diseases and infections [31,32]. We have previously described some associations with IL-6 levels in posterior uveitis [29,30,33]. In addition, other reports have found elevated IL-6 in macular edema associated with uveitis, and inhibition of IL-6 may be an effective method for treating non-infectious uveitis [34–36]. However, the mechanisms by which inflammatory cells migrate into the eye during uveitis are not yet fully understood.

IL-6 mainly produced by T cells, B cells and monocytes/macrophages in the eyes with uveitis.

Other reports have examined where IL-6 is produced, such as the retinal pigment epithelium (RPE), Muller cells or retinal endothelial cells [37–39]. IL-6 has been reported to produce vascular endothelial growth factor (VEGF) [40–43]. VEGF is a signaling protein that plays a crucial role in promoting the growth of new blood vessels.

Signal transducer and activator of transcription (STAT) proteins are a family of cytoplasmic transcription factors that play important roles in signal transduction and gene expression regulation. STAT is phosphorylated by janus kinase (JAK), STAT regulate the expression of related genes.

This pathway is known as the Janus kinase/transcriptional activation signaling pathway (JAK/STAT) signaling pathway.

VEGF is said to be associated with the JAK/STAT pathway, although the underlying mechanisms remain unclear.

IL-6 also leads to the production of intercellular adhesion molecule1 (ICAM-1) [44–46]. ICAM-1 is a cell surface glycoprotein, acting as an adhesion receptor that is well known for controlling the recruitment of leukocytes from the circulation to sites of inflammation [47].

Both VEGF and ICAM-1 increase vascular permeability, and VEGF promotes neovascularization. Increased vascular permeability may thus be involved in the findings of vitreous cell infiltration and strong inflammation.

OCT is an important tool in clinical practice in ophthalmology. The resolution achievable by OCT has been increasing recently, and rapid imaging has become possible. In the treatment of uveitis, this is very important [48,49]. Currently, OCT is often used to determine treatment strategies. WOCT is capable of imaging the retina at a wide angle, allowing evaluation of the peripheral retina. Articles reporting on the relationships between WOCT and clinical findings are increasing [5,6]. Recent studies have attempted to analyze OCT from various angles using image analysis software.

In this study, we evaluated WOCT findings using the binarization function of ImageJ. We were able to detect vitreous cells from images using binarization.

We have reported for the first time that vitreous cells in the vitreous area of WOCT images were binarized using ImageJ, and comparison with IL-6 levels yielded significant results. When human observers counted the number of highly reflective particles on macular WOCT, correlations with IL-6 levels were similar to those seen from software counting. In this study, Group 1 (manual counting) had less the number cells on WOCT than Group 2 (software counting). Also, there were large differences in the number of cells counted between Group 1 and 2. This suggests that software may be able to capture brightness that humans cannot see visually. In all WOCT images of macular, upper and lower, there was a positive correlation between the number of cells counted by humans and the number of cells analyzed by the software.

Here we reported that hyperreflective particles in the vitreous cavity at the macular correlated with vitreous IL-6 levels.

Patients with macular edema and some severe retinal diseases have been shown to have high IL-6 levels, so the present results suggest that cell counts on WOCT might be involved with the severity of pathology [32]. Although no significant results were identified in this study, WOCT may be used in the future to evaluate the prognosis of visual acuity.

This study examined only IL-6, but many other inflammatory cytokines may be related to uveitis, and we intend to focus on other cytokines and interleukins in the future.

Limitations

All patients in this study were Asian, including one Vietnamese patient. The remaining patients were Japanese. The generalizability of the study findings to other ethnic groups needs to be examined in the future.

The study design was limited by the retrospective design and the relatively small number of subjects. Given the small sample size and the inclusion of patients from a single ethnic background, this study cannot be considered representative or to comprehensively cover all types of uveitis. Another limitation was various issues with images. The first problem is that the posterior vitreous cavity was selected for image analysis. Strictly speaking, the posterior vitreous cavity area includes the posterior vitreous precortical pocket, which is considered distinct from the vitreous area. We think that it is an important issue that the “posterior vitreous area” that is measured is not fair depending on the presence or absence of posterior vitreous detachment. A distinction between vitreous pockets and posterior vitreous areas should be considered in the future. Second, this study was performed using manual procedures. Selecting the vitreous cavity manually may introduce bias. Third, WOCT has a de-noising function, and sometimes does not capture images as is. The problem with this case series is the WOCT poor images. Images obtained by WOCT were not corrected. This is because it was thought that image correction would become arbitrary. The poor image is one of the problems, however we couldn’t find good idea to solve it. Further, vitreous cells are not always captured in a single shot. As further cases are accumulated, future investigations will be able to determine whether hyperreflective particles in WOCT images correlate with the prognosis of visual function.

Conclusions

Vitreous number of hyperreflective particles (cells) findings on WOCT correlated well with human and software cell counts. Vitreous cells findings on WOCT also correlated with IL-6 concentrations on macular.

Supporting information

S1 File

(XLSX)

Click here for additional data file.^{(18.7KB, xlsx)}

Data Availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Funding Statement

Funding: This work was supported by JSPS KAKENHI Grant Numbers 23K09013, the Charitable Trust Fund for Ophthalmic Research in Commemoration of Santen Pharmaceutical’s Founder, and 2023 Osaka Community Foundation　(Mizuki.Tagami.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0297201.r001

Decision Letter 0

Jiro Kogo

6 Nov 2023

PONE-D-23-27793Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitisPLOS ONE

Dear Dr. Tagami,

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The authors demonstrated that relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis. The manuscript contains many grammatical errors. In addition, the purpose of the study is ambiguous. The methods for counting vitreous cells are not clear. Major revision must be necessary.

1. Line 25-26. Purpose of this study is ambiguous. Is the purpose may be to clarify the relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis?

2. Line 56. “and acute anterior uveitis (5.5%),” should be “and acute anterior uveitis (5.5%) [2].”

3. Line 58. “represented over half of case, thus Diagnosis and treatment of” should be “represented over one-third of cases, thus diagnosis and treatment of”.

4. Line 62. “Widefield OCT angiography (WOCTA)” should be “Widefield OCT (WOCT)”.

5. Line 66-67. “the relationship between uveitis and IL-6 level and several functions of IL-6 have been identified” should be “relationship between uveitis and IL-6 levels in aqueous humor and significant roles of IL-6 in non-infectious uveitis have been identified”.

6. Line 69. “increased in uveitis” should be “increased in the aqueous humor in patients with uveitis”.

7. Line99-100. Please describe the details of multiplex PCR. What kinds of pathogens did you examine? Did you examine fugus or toxoplasma DNA? It is necessary to include references related to multiplex PCR.

8. Line 104-115. I cannot understand the meaning of Figure 1A, 1B, 1C, 1D, 1E and 1F. Where is the area of “through the macula (Figure 1A)”, “upper vitreous cavity around the equatorial region (Figure 1B)” and “lower vitreous cavity (Figure 1C)”? How to determine the position of the equator in a WOCT image?

9. Line 131-133. What is the area for counting cells in vitreous cavity in a WOCT image? (for example, 13mm x 5mm)

10. Line 140-142. If you defined “posterior vitreous area” as described here, the area for counting the number of cells varies greatly depending on the height of the PVD, and this is not fair for evaluation of the severity of inflammation in the vitreous cavity.

11. Line 150-151. The methods for “counting by software” should be described. References for the methods for “counting by software” are necessary.

12. Line 167-168. In flow cytometry analyses, it is common to separate cell fractions (erythrocytes, lymphocytes, monocytes, and granulocytes) using forward scatter and side scatter at first. Which cell fraction did you gate on?

13. Line 194-202. Numbers of cells in Group 1 (manual counting) and Group 2 (software-based counting) were quite different and more numbers of cells was counted by manual counting. Why is there such a big difference between manual counting and software-based counting?

14. Line 268. “Correlation with IL-6 value” should be “Correlation with IL-6 levels and changing of visual acuity”.

15. Line 277. “Fig 4. We compared visual acuity between preoperatively and 3 months postoperatively“ should be “Fig 4. Comparisons of IL-6 levels among three groups depending on improvement or deterioration of visual acuity”.

16. Line 283-291. The finding that there is a correlation between the number of cells in WOCT and CD56-positive cells in FACS is clearly due to the fact that one case of ARN had an exceptionally high number of CD56-positive cells. ARN is infectious uveitis, and the other eight cases are non-infectious. If you want to claim that there is a correlation between the cell count in WOCT and CD56-positive cells in FACS, you should exclude one case of ARN from the analysis.

17. Line 311-313. IL-6 may be mainly produced by T cells, B cells and monocytes/macrophages in the eyes with uveitis.

18. Line 329. “OCT is an important tool in ophthalmology” should be “OCT is an important tool in clinical practice in ophthalmology”.

19. Line 332. “Papers” should be “Articles”.

20. Line 337. “We evaluated WOCT findings” should be “In this study, we evaluated WOCT findings”.

21. Line 337-340. The methods for binarization of WOCT image and the methods for counting vitreous cells should be described in Materials and Methods section.

22. Line 368. “WOCTA” should be “WOCT”.

Reviewer #2: 3.11.2023

In this manuscript, the authors described “Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis”. The study will be very useful for the literature. The report is an interesting study, but it needed some suggestions for publication.

Here are the concerns for the authors;

Generally;

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2. Grammar should be checked.

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5. The findings given in the table should not be repeated in the text in the results section.

6. The units of evaluated parameters should be given clearly. For example, pg/mL for IL-6 value.

7. The % of demographic characteristics should also be given in Table 1.

8. The spelling features of Tables 1 and 2 are not the same.

9. All p values should be written in italic.

10. The resolution of the figures is low and should be corrected.

11. Citations should be corrected according to the journal instructions.

12. The lot number of kits should be stated.

13. The brand and model numbers of the devices used in the study should be stated.

14. What does the word “patiene” in Table 1 mean?

15. Significance levels of p values should be stated below the tables.

**********

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Reviewer #1: Yes: Toshikatsu Kaburaki

Reviewer #2: No

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PLoS One. 2024 Jan 17;19(1):e0297201. doi: 10.1371/journal.pone.0297201.r002

Author response to Decision Letter 0

4 Dec 2023

Journal Requirements:

・ Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

Response

Thank you for your advice. We modified sentences.

・ Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

Response

Thank you for your advice. This time, we decided not to deposit data into participating repositories.

・ Thank you for stating the following financial disclosure:

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response

We added the following.

L605-606

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

・ Please upload a new copy of Figure 3 as the detail is not clear. Please follow the link for more information: https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/"

Response

Thank you for pointing out. Figure 3 was uploaded a new copy.

#Reviewer1

・ Line 25-26. Purpose of this study is ambiguous. Is the purpose may be to clarify the relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis?

Response

Thank you for your advice. We modified as the following.

L18-19

Purpose: To investigate relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis

・ Line 56. “and acute anterior uveitis (5.5%),” should be “and acute anterior uveitis (5.5%) [2].”

Response

Thank you for your advice. We corrected the position of the word.

・ Line 58. “represented over half of case, thus Diagnosis and treatment of” should be “represented over one-third of cases, thus diagnosis and treatment of”.

Response

Thank you for your advice. We modified the sentences.

・ Line 62. “Widefield OCT angiography (WOCTA)” should be “Widefield OCT (WOCT)”.

Response

Thank you for your comment. We corrected the word.

・ Line 66-67. “the relationship between uveitis and IL-6 level and several functions of IL-6 have been identified” should be “relationship between uveitis and IL-6 levels in aqueous humor and significant roles of IL-6 in non-infectious uveitis have been identified”

Response

Thank you for your advice. We modified the sentences.

・ Line 69. “increased in uveitis” should be “increased in the aqueous humor in patients with uveitis”.

Response

Thank you for your advice. We corrected words.

・ Line99-100. Please describe the details of multiplex PCR. What kinds of pathogens did you examine? Did you examine fugus or toxoplasma DNA? It is necessary to include references related to multiplex PCR.

Response

Thank you for your advice. We added the following.

L99-105

The PCR test is a strip PCR test that can identify 24 different types of pathogenic microorganisms: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, human T-cell lymphotropic virus (HTLV)-1/2, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), Propionibacterium acnes (P. acnes), Aspergillus, Fusarium, bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, fungal 28S rRNA, and Acanthamoeba[20].

・ Line 104-115. I cannot understand the meaning of Figure 1A, 1B, 1C, 1D, 1E and 1F. Where is the area of “through the macula (Figure 1A)”, “upper vitreous cavity around the equatorial region (Figure 1B)” and “lower vitreous cavity (Figure 1C)”? How to determine the position of the equator in a WOCT image?

Response

Thank you for pointing out. We modified any definition as following.

Also, the resolution of Fig1 was corrected and some schema were added to show which area the B-scan image cavers.

L110-120

Also, En-face WOCTA was taken in all cases. Macular was defined horizontal B-scan through the foveal central subfield [3, 21]. Furthermore, we examined the choroidal structure using WOCTA or Optos 200Tx (Optos PLC, Dunfermline, Scotland) [22, 23]. We identified the ampullae of the vortex vein inflow by imaging in En-face choroidal mode of OCTA or En-face mode of Optos. The B-scan around equatorial region was defined as a straight line connecting temporal and nasal of the ampullae vortex vein inflow[24]. Upper vitreous cavity area on WOCT was defined as B-scan with horizontal lines drawn at the inflow point of the vortex vein volume on the superotemporal and superonasal sides. For the lower vitreous cavity area of WOCT, like the upper one, we selected a B-scan around the area connecting inferotemporal and inferonasal sides with a horizontal line. If the temporal and nasal vortex venous ampullaes could not be connected horizontally, a horizontal line passing through the nasal vortex vein ampulla was used.

・ Line 131-133. What is the area for counting cells in vitreous cavity in a WOCT image? (for example, 13mm x 5mm)

Response

Thank you for pointing out. We added the following.

L150-152

・ Line 140-142. If you defined “posterior vitreous area” as described here, the area for counting the number of cells varies greatly depending on the height of the PVD, and this is not fair for evaluation of the severity of inflammation in the vitreous cavity.

Response

Thank you for pointing out. We agree your opinion.

In this study, we investigated patients with or without posterior vitreous detachment. This will be a subject for future consideration.

We added following to “Materials and Methods” and “Discussion” about your opinion.

L163-164

In this study, the presence or absence of posterior vitreous detachment was not considered.

L427-433

Another limitation was various issues with images. The first problem is that the posterior vitreous cavity was selected for image analysis. Strictly speaking, the posterior vitreous cavity area includes the posterior vitreous precortical pocket, which is considered distinct from the vitreous area. We think that it is an important issue that the “posterior vitreous area” that is measured is not fair depending on the presence or absence of posterior vitreous detachment. A distinction between vitreous pockets and posterior vitreous areas should be considered in the future.

・ Line 150-151. The methods for “counting by software” should be described. References for the methods for “counting by software” are necessary.

Response

Thank you for your advices. We added the following.

L176-179

Binarization is a type of image processing that divides the intensity of an image into binary values, such as 0 or 1 [26]. On the WOCT images, cells were binarised and automatically counted by the ImageJ software [27]. The image area to be binarized needs to be manually selected.

26. Nagasato D, Mitamura Y, Egawa M, Kameoka M, Nagasawa T, Tabuchi H, et al. Changes of choroidal structure and circulation after water drinking test in normal eyes. Graefes Arch Clin Exp Ophthalmol. 2019;257(11):2391-9. Epub 20190805. doi: 10.1007/s00417-019-04427-7. PubMed PMID: 31378831.

27. Grishagin IV. Automatic cell counting with ImageJ. Anal Biochem. 2015;473:63-5. Epub 20141224. doi: 10.1016/j.ab.2014.12.007. PubMed PMID: 25542972.

・ Line 167-168. In flow cytometry analyses, it is common to separate cell fractions (erythrocytes, lymphocytes, monocytes, and granulocytes) using forward scatter and side scatter at first. Which cell fraction did you gate on?

Response

Thank you for your advice. We added the following.

L197-202

They separate cell fractions using forward scatter (FSC) and side scatter (SSC). High score of FSC means that cells in the samples is large. Large cells were identified as gate(A).

CD45 gating were enable to analysis CD45 diminish area as Gate1.

・ Line 194-202. Numbers of cells in Group 1 (manual counting) and Group 2 (software-based counting) were quite different and more numbers of cells was counted by manual counting. Why is there such a big difference between manual counting and software-based counting?

Response

Thank you for pointing out. There were difference among Group 1 and Group 2.

Group 1 (manual counting) had the number cells on WOCT less than of Group2.

We've added a discussion about this below to the “Discussion”.

L405-410

In this study, Group 1 (manual counting) had less the number cells on WOCT than Group 2 (software counting). Also, there were large differences in the number of cells counted between Group 1 and 2. This suggests that software may be able to capture brightness that humans cannot see visually. In all WOCT images of macular, upper and lower, there was a positive correlation between the number of cells counted by humans and the number of cells analyzed by the software.

・ Line 268. “Correlation with IL-6 value” should be “Correlation with IL-6 levels and changing of visual acuity”.

Response

Thank you for your advice. We corrected words.

・ Line 277. “Fig 4. We compared visual acuity between preoperatively and 3 months postoperatively“ should be “Fig 4. Comparisons of IL-6 levels among three groups depending on improvement or deterioration of visual acuity”.

Response

Thank you for your advice. We corrected words.

・ Line 283-291. The finding that there is a correlation between the number of cells in WOCT and CD56-positive cells in FACS is clearly due to the fact that one case of ARN had an exceptionally high number of CD56-positive cells. ARN is infectious uveitis, and the other eight cases are non-infectious. If you want to claim that there is a correlation between the cell count in WOCT and CD56-positive cells in FACS, you should exclude one case of ARN from the analysis.

Response

Thank you for pointing out. We agree your opinion.

We excluded the case of ARN.

Upon reviewing the cases, we found that Case 2 did not have idiopathic uveitis but PIOL.

we fixed it. PIOL is one of non-infectious uveitis, we thought that it was no problem if PIOL included in FACS.

The following was deleted.

L361-362

Only high CD56 score was associated with greater cell counts on WOCT using Pearson's correlation coefficient.

Also, We added the following.

L351-352

There was no statistically significant difference between the observed the number of cells on WOCT and each lymphocyte.

・ Line 311-313. IL-6 may be mainly produced by T cells, B cells and monocytes/macrophages in the eyes with uveitis.

Response

Thank you for your advice. We added following sentences.

L374

IL-6 mainly produced by T cells, B cells and monocytes/macrophages in the eyes with uveitis.

・ Line 329. “OCT is an important tool in ophthalmology” should be “OCT is an important tool in clinical practice in ophthalmology”.

Response

Thank you for your advice. We corrected words.

・ Line 332. “Papers” should be “Articles”.

Response

Thank you for your advice. We corrected words.

・ Line 337. “We evaluated WOCT findings” should be “In this study, we evaluated WOCT findings”.

Response

Thank you for your advice. We corrected words.

・ Line 337-340. The methods for binarization of WOCT image and the methods for counting vitreous cells should be described in Materials and Methods section.

Response

We agree with the reviewer’s opinion. I added the following sentence to the Materials and Methods section.

L174-176

ImageJ is a Java-based image-processing program developed by the National Institutes of Health and the Optical Computational Metrology Institute.

・ Line 368. “WOCTA” should be “WOCT”.

Response

Thank you for your advice. We corrected words.

#Reviewer2

・ Keywords should be ordered alphabetical.

Response

Thank you for your advice. We corrected words.

・ Grammar should be checked.

Response

Thank you for your advice. We confirmed grammar and modified.

・ Abstract structure should be the same as described in the journal.

Response

Thank you for your pointing out. We corrected sentences.

・ The inclusion and exclusion criteria determined for the study should be clearly stated in the materials and methods.

Response

Thank you for pointing out. We added sentences the following to the materials and methods.

L75-80

Study inclusion criteria included [1] the presence of posterior vitreous inflammation, [2] unknown cause of posterior uveitis, [3] Cases in which preoperative WOCT and Wide field Optical Coherence Tomography angiography (WOCTA) of En-face could be obtained. Study exclusion criteria included [1] Patients with active systemic inflammation, [2] Cases couldn’t be observed vitreous cavity due to vitreous opacity in preoperative WOCT, [3] The cases couldn’t take En-face images of WOCTA or Optos (Optos® 200Tx, Optos®, Dunfermline, U.K.).

・ The findings given in the table should not be repeated in the text in the results section.

Response

Thank you for your advice. I have deleted the following text.

L211

The 37 patients comprised 18 men and 19 women, with a mean age of 67.5±15.7 years. Baseline logMAR BCVA was 0.22±0.60. Mean IL-6 level was 79.9±7380.9 pg/mL (Table 1).

L216

Uveitis was infectious in 9 cases and non-infectious in 28 cases.

L262-264

(on macular +0.866; upper cavity +0.713; lower cavity +0.568; total vitreous cavity +0.834; P0.001 each).

L266-269

In macular images, a correlation was identified (P=0.04; E).

Upper vitreous around the equator with reference to the vortex vena cava (P=0.96; F).

Lower vitreous around the equator with reference to the vortex vena cava (n=34) (P=0.96; G) and total (sum of macular, upper and lower) (P=0.27 H) (n=34)

L271-276

A correlation was seen for macular images alone (P=0.03; I).

Upper vitreous around the equator with reference to the vortex vena cava (P=0.777; J).

Lower vitreous around the equator with reference to the vortex vena cava (n=34) (P=0.91; K) and total (sum of macular, superior and inferior) (P=0238; L) (n=34).

＊P0.05

L281-284

No significant difference was seen between upper (PCC+0.09, P=0.96; Figure 2F) and lower images (PCC-0.008, P=0.96; Figure 2G). The number of cells seen in the images in the macular, upper, and lower was examined by totaling, but there was no significant difference (PCC+0.196, P=0.27; Figure2H).

L286-288

Similar to the Human Count Group, no significant difference was observed in studies other than the macular region (PCC+0.048 P=0.777; Figure2J, PCC-0.020 P=0.91; Figure2K, PCC:+0.208 P=0.238 Figure2L).

・ The units of evaluated parameters should be given clearly. For example, pg/mL for IL-6 value.

Response

Thank you for your advice. We added the units of evaluated parameters clearly.

・ The % of demographic characteristics should also be given in Table 1.

Response

Thank you for your advice. We added the % of demographic characteristics in Tabel1.

・ The spelling features of Tables 1 and 2 are not the same.

Response

Thank you for your advice. We corrected the spelling feature of Table 1 and Table2.

・ All p values should be written in italic.

Response

Thank you for your advice. We corrected words.

・ The resolution of the figures is low and should be corrected.

Response

Thank you for pointing out. We corrected the solution of the figures. Also, we added the following to the discussion.

L435-438

The problem with this case series is the WOCT poor images. Images obtained by WOCT were not corrected. This is because it was thought that image correction would become arbitrary. The poor image is one of the problems, however we couldn’t find good idea to solve it.

・ Citations should be corrected according to the journal instructions.

Response

Thank you for pointing out. We corrected according to the journal instructions.

・ The lot number of kits should be stated.

Response

Thank you for pointing out. We added following sentences.

We refrained from adding the kit lot number because it varies depending on the date the sample was collected.

L97

kits for human IL-6 (LUMIPULSE G1200, FUJIREBIO, Tokyo, Japan)

・ The brand and model numbers of the devices used in the study should be stated.

Response

Thank you for pointing out. We added following sentences.

L166

National Institutes of Health, Bethesda, MD, USA

・ What does the word “patiene” in Table 1 mean?

Response

Thank you for pointing out. We corrected the word “patients”.

Participant

Baseline

Characteristics total infectious Non-infectious

Eye,n (%) 37 9 (24.3) 28 (75.7)

Patients,n (%) 34 9 (26.5) 25 (73.5)

Males,n (%) 18 8 (44.4) 10 (55.6)

Females,n (%) 19 1 (5) 18 (95)

Age,years (Mean±SD) 67.5±15.7 67±18.8 67.5±14.2

Baseline logMAR BCVA (Mean±SD) 0.22±0.6 0.15±0.8 0.35±0.5

IL-6 levels, pg/mL (Mean±SD) 79.9±7380.9 1040±12990.6 73.5±990.5

・ Significance levels of p values should be stated below the tables.

Response

Thank you for pointing out. We added significance levels of p value below the tables.

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PLoS One. doi: 10.1371/journal.pone.0297201.r003

Decision Letter 1

Jiro Kogo

28 Dec 2023

PONE-D-23-27793R1Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitisPLOS ONE

Dear Dr. Tagami,

Please submit your revised manuscript by Feb 11 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #1: (No Response)

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Reviewer #1: Yes

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Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: The authors demonstrated that relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis. The manuscript was well revised according to the reviewers’ suggestions. However, a few mistakes were remained to be corrected.

1. Line 204, line 353 and Fig 5. CD3 was examined in line 356 and Fig 5, whereas CD9 was examined in line 204. CD3 may be correct.

2. Line 344. In Table 4, there were no cases with ARN, thus “ARN acute retinal necrosis” should be erased.

**********

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Reviewer #1: Yes: Toshikatsu Kaburaki

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PLoS One. 2024 Jan 17;19(1):e0297201. doi: 10.1371/journal.pone.0297201.r004

Author response to Decision Letter 1

28 Dec 2023

#Reviewer1

1. Line 204, line 353 and Fig 5. CD3 was examined in line 356 and Fig 5, whereas CD9 was examined in line 204. CD3 may be correct.

2. Line 344. In Table 4, there were no cases with ARN, thus “ARN acute retinal necrosis” should be erased.

Response

Thank you very much for your detailed corrections and advice. The above has been corrected.

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PLoS One. doi: 10.1371/journal.pone.0297201.r005

Decision Letter 2

Jiro Kogo

2 Jan 2024

Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis

PONE-D-23-27793R2

Dear Dr. Tagami

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0297201.r006

Acceptance letter

Jiro Kogo

8 Jan 2024

PONE-D-23-27793R2

PLOS ONE

Dear Dr. Tagami,

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Data Availability Statement

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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PERMALINK

Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis

Mami Tomita

Mizuki Tagami

Norihiko Misawa

Atsushi Sakai

Yusuke Haruna

Shigeru Honda

Roles

Abstract

Purpose

Methods

Results

Conclusions

Introduction

Materials and methods

Selection of cases and collation of clinicopathological data

Fig 1. Measurement of the number of hyperreflective particles on WOCT.

Imaging evaluations

Correlation with IL-6 value

Flow cytometry

Statistical analyses

Results

Table 1. Baseline characteristics of patients.

Table 2. Numbers of cells in Group 1 and Group 2.

Fig 2. Relationship between Group 1 and Group 2.

Table 3. Correlation between IL-6 levels and cell counts on WOCT images.

Fig 3.

Correlation with IL-6 levels and changing of visual acuity

Fig 4. Comparisons of IL-6 levels among three groups depending on improvement or deterioration of visual acuity.

Table 4. Baseline characteristics of the 7 patients investigated by flow cytometry.

Fig 5. The number of lymphocytes as observed by flow cytometry was compared with cell counts on WOCT.

Discussion

Limitations

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Jiro Kogo

Roles

Author response to Decision Letter 0

Decision Letter 1

Jiro Kogo

Roles

Author response to Decision Letter 1

Decision Letter 2

Jiro Kogo

Roles

Acceptance letter

Jiro Kogo

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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