Immunohistochemical and Immunocytochemical Analyses in Patients with Vitreoretinal Lymphoma.

Purpose: The aim of this study was to analyze immunohistochemical and immunocytological findings by examining enucleated eyes and vitreous cell block (CB) in patients with vitreoretinal lymphoma (VRL).Methods: Histological specimens were obtained from two enucleated eyes with VRL associated with neovascular glaucoma. CB specimens were prepared in 18 patients from diluted waste fluids containing shredded vitreous. Histological and cytological specimens were submitted for hematoxylin-eosin staining and immunopathological analyses.Results: Both specimens demonstrated massive infiltration of large lymphoma cells. The lymphoma cells were positive for CD20 and MUM-1 in enucleated eyes. Membranous immunoreactivity for CD20 was observed in lymphoma cells in CB with VRL. Bcl-6 and MUM-1 were marked in five and eight out of nine cases examined, respectivelyConclusions: Cytological findings in CB specimens indicated similar histopathological characteristics of enucleated eyes. CB specimens obtained from vitreous waste diluted fluids may serve as effective materials for cytological diagnosis of VRL.

Association Study of ARMC9 Gene Variants with Vogt-Koyanagi-Harada Disease in Japanese Patients.

Purpose: To investigate whether variants in the ARMC9 gene encoding KU-MEL-1 are associated with Vogt-Koyanagi-Harada (VKH) disease in a Japanese population. Methods: We recruited 380 Japanese patients with VKH disease and 744 Japanese healthy controls to genotype seven single-nucleotide polymorphisms (SNPs) in ARMC9. We also performed imputation analysis of the ARMC9 region and 195 imputed SNPs were included in the statistical analysis. Results: We observed an increased frequency of the A allele of rs28690417 in patients compared with controls (P = 0.0097, odds ratio (OR) = 1.46). The A allele had a dominant effect on VKH disease risk (P = 0.011, OR = 1.51). However, these significant differences disappeared after Bonferroni correction (corrected P > 0.05). The remaining 201 SNPs did not show any significant association with disease risk. Conclusions: Our study suggests that ARMC9 variants do not play a critical role in the development of VKH disease.

Long-term efficacy of infliximab treatment and the predictors of treatment outcomes in patients with refractory uveitis associated with Behçet's disease.

OBJECTIVE: To assess the long-term efficacy and safety of infliximab (IFX) treatment for refractory uveitis associated with Behçet's disease (BD) and to identify predictors of long-term IFX therapy outcomes. METHODS: We retrospectively studied 44 consecutive BD patients with uveitis who were started on IFX therapy and analyzed the efficacy and safety of IFX and the treatment continuation rate. To determine predictors of IFX responsiveness, we analyzed the clinical characteristics of the patients who received regular maintenance therapy and those who required treatment intensification. The serum cytokine levels prior to IFX were measured through the Bio-Plex human cytokine assays. RESULTS: IFX significantly reduced the frequency of ocular attacks and improved the visual acuity of patients with BD-related uveitis. However, approximately half of the patients required dose escalations, necessitating a shortening of the intervals between IFX infusions due to loss of efficacy during the 5-year treatment. The frequency of ocular attacks was significantly higher in patients with complete BD than in patients with incomplete BD. A multiplex cytokine analysis revealed that patients with BD-related uveitis exhibited increased serum IL-2, IL-6, IL-8, and MCP-1 levels. Moreover, among BD patients, the serum IL-2 and IL-6 levels were particularly high in those who maintained remission and received regular IFX treatments. CONCLUSION: We confirmed the long-term efficacy and tolerability of IFX in patients with BD-related uveitis. Our results indicate that complete BD may be less responsive to IFX and that the pretreatment serum cytokine profiles may be useful for predicting the long-term IFX therapy outcomes.

Clinical Features of Systemic Metastatic Retinal Lymphoma in Japanese Patients.

PURPOSE: Systemic metastatic retinal lymphoma (SMRL) originates in systemic organs. It has been reported to exhibit clinical features similar to those of primary vitreoretinal lymphoma (PVRL). We report six cases of SMRL in a single-center survey in Japan. METHODS: The clinical and pathologic features in SMRL at the Kyushu University Hospital were retrospectively studied. RESULTS: The mean patient age at the onset of ocular involvement was 75.3 years. Four patients had brain involvement. The primary sites were: breast (2); chest (1); testis (1); intestinal tract (1); and nasal sinus (1). In all patients, the cytology of vitreous samples indicated diffuse large B-cell lymphoma (DLBCL). CONCLUSIONS: DLBCL is the most common subtype in our study. The prevalence of CNS involvement in patients with SMRL is similar to that with PVRL. The testis and breast may be common sites of origin for SMRL.

Choroidal thickening prior to anterior recurrence in patients with Vogt-Koyanagi-Harada disease.

AIM: To assess choroidal thickness changes associated with anterior segment recurrences in patients with Vogt-Koyanagi-Harada (VKH) disease using enhanced depth imaging optical coherence tomography (EDI-OCT). METHODS: EDI-OCT images were obtained periodically from 11 patients with VKH disease (22 eyes) who were followed-up due to anterior segment recurrences. Subfoveal choroidal thickness (SCT) values at the following stages were evaluated: (1) during the remission phase, (2) 1 month before detecting the anterior recurrence, (3) during the anterior recurrence and (4) after systemic prednisolone (PSL) treatment leading to remission. In comparison with SCT values in remission as baseline, the changing ratios of SCT were statistically analysed at subsequent three stages. RESULTS: The average of the SCT changing ratios compared with the remission phase significantly increased to 1.45±0.11 during anterior segment recurrences (p=0.00044) lacking any funduscopic signs of posterior involvement. Interestingly, the average SCT ratio 1 month before detecting the recurrence had already increased to 1.30±0.08 (p=0.002). After the PSL treatment, the ratio of SCT recovered to 0.95±0.03, which was equivalent to the remission level. However, in patients with their remission SCT values less than 240 µm, the SCT ratio did not increase significantly at any time points evaluated. CONCLUSIONS: The choroid in eyes with VKH disease thickened in association with the anterior segment recurrence, and this thickening was observed prior to the recurrence. EDI-OCT may be useful for detecting latent choroidal inflammation in VKH disease, whereas it may not for patients with the relatively thin choroid. TRIAL REGISTRATION NUMBER: The trial registration number of the internal review board of Hokkaido University Hospital is 014-0384.

Distinct Profiles of Soluble Cytokine Receptors Between B-Cell Vitreoretinal Lymphoma and Uveitis.

PURPOSE: To determine the profiles of soluble cytokine receptors and cytokines, including mostly their ligands, in the vitreous humor of patients with B-cell vitreoretinal lymphoma (VRL) and uveitis. METHODS: Vitreous samples were collected from immunocompetent patients with VRL (n = 21), uveitis (n = 20), and idiopathic epiretinal membrane (n = 21) as controls. Cytometric beads assay were used to determine the vitreous concentrations of soluble receptors and cytokines. RESULTS: Vitreous levels of soluble IL-2 receptor α (sIL-2Rα), sIL-6R, soluble tumor necrosis factor receptor (TNFR) 1, sTNFR2, soluble vascular endothelial growth factor receptor (sVEGFR) 1, sVEGFR2, and IL-10 were higher in patients with VRL than in those with uveitis and controls, whereas those of sIL-1R1, sIL-1R2, and sIL-4R were higher in patients with uveitis than those with VRL and controls. In analyses in patients with VRL, elevation of sVEGFR1 and sVEGFR2 levels was more prominent in patients with systemic metastatic retinal lymphoma (SMRL) than in those with primary VRL/primary central nervous system lymphoma (PVRL/PCNSL). Furthermore, sIL-2Rα levels were increased in patients with VRL who developed subretinal lesions compared with in those who mainly had vitreous cavity opacity, positively correlated with the density of CD3+ cells in the vitrectomy cell blocks. CONCLUSIONS: The profiles of soluble cytokine receptors and cytokines in patients with VRL were different from those with uveitis. In addition, sVEGFR1 and sVEGFR2 levels may be differential diagnostic markers between PVRL/PCNSL and SMRL, and sIL-2Rα levels can anticipate infiltration of VRL cells into the subretina and/or retina.

[A case report of malignant lymphoma receiving infliximab therapy with Behçet's disease].

BACKGROUND: To report a case of malignant lymphoma occurring in Behçet's disease (BD) with infliximab therapy. CASE: A 62-year-old man was diagnosed with BD in 1997. Despite treatment with colchicine, cyclosporine and prednisolone, he had frequent bilateral posterior ocular attacks. He was started on infliximab in August 2007 and for 6 months had no ocular attacks. Cyclosporine was therefore reduced. After 4 years of infliximab administration, he had neither ocular attacks nor general symptoms. However, he had general malaise and weight loss from the end of March 2012. Peripheral blood examination showed abnormal cells, so we terminated the infliximab. Bone marrow aspiration showed diffuse proliferation of medium to large lymphoid cells, and the histological diagnosis was diffuse large B-cell lymphoma. He was treated with 8 cycles of chemotherapy and 4 times intrathecal chemotherapy, and is now in remission. After termination of infliximab, he had no further ocular attacks. CONCLUSION: Although malignant lymphoma associated with BD is rare, attending ophthalmologists need to keep it in mind.

EBI3 is pivotal for the initiation of experimental autoimmune uveitis.

Murine experimental autoimmune uveitis (EAU) is a model for human autoimmune uveitis, whose pathogenesis is caused by both Th1 and Th17 cell responses. Epstein-Barr virus-induced gene 3 (EBI3) is a component of the heterodimeric cytokines: interleukin (IL)-27 and IL-35. Although IL-27 was shown to initiate Th1 cell development, it is also recognized as a negative regulator of fully activated CD4+ T cells, including Th17 cells. Recently, IL-35 also has also been reported to play immunosuppressive roles in autoimmunity. To investigate the roles of EBI3 in EAU, EBI3(-/-) mice were immunized with human interphotoreceptor retinoid binding protein peptide 1-20 (IRBP) to induce EAU. We observed that the clinical score in EBI3(-/-) mice was diminished compared with that in EBI3(+/+) mice up to day 22 after immunization, whereas the score in EBI3(-/-) mice reached the same levels as that of EBI3(+/+) mice after day 28. Histological analysis revealed a significant reduction of cellular infiltration into the retina in EBI3(-/-) mice on day 16. Although Th1 cell responses and IRBP-specific IL-10 production were reduced in EBI3(-/-) mice, the development of Th17 cell responses was unaffected on day 9. On day 21, Th1 cell responses and IRBP-specific IL-10 production was restored to the same levels as that in EBI3(+/+) mice, and Th17 cell responses significantly increased in EBI3(-/-) mice. Furthermore, Foxp3 expression in CD4+ T cells was comparable between EBI3(+/+) and EBI3(-/-) mice on days 9 and 21. Therefore, these results indicate that EBI3 may be important in EAU initiation by Th1 cell responses and may suppress EAU by inhibition of both Th1 and Th17 cell responses in the late/maintenance phase.

Behçet's disease ocular attack score 24: evaluation of ocular disease activity before and after initiation of infliximab.

PURPOSE: We developed a novel scoring system for uveitis due to Behçet's disease (BD), termed Behçet's disease ocular attack score 24 (BOS24), and examined its validity and usefulness by estimating changes in ocular disease activities both before and after initiation of infliximab therapy. METHODS: BOS24 consists of a total 24 points divided into 6 parameters of ocular inflammatory symptoms. To examine the validity of our scoring system, 5 uveitis specialists examined the severity of 50 ocular attacks in clinical charts using both our system and a physician's impression score (grade 1-10). In addition, ocular disease activities both before and after initiation of infliximab were retrospectively examined in 150 cases of ocular BD using BOS24. RESULTS: The average BOS24 for the 5 doctors was highly correlated with the average physician's impression score (p < 0.0001), whereas the coefficient of variance for BOS24 among doctors was much lower than that for the physician's impression score (p < 0.0001). Summation of BOS24 over a 6-month period (BOS24-6M) was significantly reduced after starting infliximab therapy (p < 0.0001). The average BOS24 for individual ocular attacks was also significantly decreased after starting infliximab, with scores for the posterior pole and fovea notably improved. CONCLUSIONS: BOS24 was highly related to severity noted by the physician's impression and had a low level of variability among the examined doctors. Using our novel scoring system, infliximab therapy was shown to reduce not only the frequency of ocular attacks, but also the severity of each attack. BOS24 is a promising tool for evaluating ocular BD activities.

Unilateral superior laryngeal nerve lesion in an animal model of dysphagia and its effect on sucking and swallowing.

We tested two hypotheses relating to the sensory deficit that follows a unilateral superior laryngeal nerve (SLN) lesion in an infant animal model. We hypothesized that it would result in (1) a higher incidence of aspiration and (2) temporal changes in sucking and swallowing. We ligated the right-side SLN in six 2-3-week-old female pigs. Using videofluoroscopy, we recorded swallows in the same pre- and post-lesion infant pigs. We analyzed the incidence of aspiration and the duration and latency of suck and swallow cycles. After unilateral SLN lesioning, the incidence of silent aspiration during swallowing increased from 0.7 to 41.5%. The durations of the suck containing the swallow, the suck immediately following the swallow, and the swallow itself were significantly longer in the post-lesion swallows, although the suck prior to the swallow was not different. The interval between the start of the suck containing a swallow and the subsequent epiglottal movement was longer in the post-lesion swallows. The number of sucks between swallows was significantly greater in post-lesion swallows compared to pre-lesion swallows. Unilateral SLN lesion increased the incidence of aspiration and changed the temporal relationships between sucking and swallowing. The longer transit time and the temporal coordinative dysfunction between suck and swallow cycles may contribute to aspiration. These results suggest that swallow dysfunction and silent aspiration are common and potentially overlooked sequelae of unilateral SLN injury. This validated animal model of aspiration has the potential for further dysphagia studies.

Differential involvement of two cortical masticatory areas in modulation of the swallowing reflex in rats.

To clarify the functional role of cortical descending inputs involved in the swallowing reflex, the effect of electrical stimulation of two cortical masticatory areas (CMAs: A- and P-area) on rhythmic jaw movements (RJMs) and superior laryngeal nerve (SLN)-evoked swallows were studied. RJMs and swallowing reflex were elicited by repetitive electrical stimulation of CMAs and the SLN, respectively. The electromyographic activities of jaw-closer (masseter), jaw-opener (digastric), and laryngeal-elevator (thyrohyoid) muscles were recorded to identify the RJMs and swallowing reflex. The number of evoked swallows was significantly lower, and swallowing interval was significantly longer during A-area stimulation compared with those without stimulation. Conversely, these parameters were not significantly altered during P-area stimulation. The inhibition of swallows by A-area stimulation was not affected by an increase in sensory input by wooden stick application between upper and lower teeth, or A-area stimulation preceding SLN stimulation. The present findings suggest that the swallowing reflex is inhibited by activation of the A-area, but not the P-area. Since no changes in swallows were seen after the increase in intraoral sensory input and prior activation of masticatory central pattern generator (CPG), swallowing inhibition may be mediated by direct inputs from the A-area or inputs via the masticatory CPG into the swallowing CPG.

The digastric muscle is less involved in pharyngeal swallowing in rabbits.

The swallowing reflex is centrally programmed by the lower brain stem, the so-called swallowing central pattern generator (CPG), and once the reflex is initiated, many muscles in the oral, pharyngeal, laryngeal, and esophageal regions are systematically activated. The mylohyoid (MH) muscle has been considered to be a "leading muscle" according to previous studies, but the functional role of the digastric (DIG) muscle in the swallowing reflex remains unclear. In the present study, therefore, the activities of single units of MH and DIG neurons were recorded extracellularly, and the functional involvement of these neurons in the swallowing reflex was investigated. The experiments were carried out on eight adult male Japanese white rabbits anesthetized with urethane. To identify DIG and MH neurons, the peripheral nerve (either DIG or MH) was stimulated to evoke action potentials of single motoneurons. Motoneurons were identified as such if they either (1) responded to antidromic nerve stimulation of DIG or MH in an all-or-none manner at threshold intensities and (2) followed stimulation frequencies of up to 0.5 kHz. As a result, all 11 MH neurons recorded were synchronously activated during the swallowing reflex, while there was no activity in any of the 7 DIG neurons recorded during the swallowing reflex. All neurons were anatomically localized ventromedially at the level of the caudal portion of the trigeminal motor nucleus, and there were no differences between the MH and DIG neuron sites. The present results strongly suggest that at least in the rabbit, DIG motoneurons are not tightly controlled by the swallowing CPG and, hence, the DIG muscle is less involved in the swallowing reflex.

Effects of electrical stimulation of the superior laryngeal nerve on the jaw-opening reflex.

The present study aimed to examine whether the jaw-opening reflex (JOR) is modulated during swallowing, and if so, to compare the modulation between the low- and high-threshold afferent-evoked reflex responses. Experiments were carried out on 11 anesthetized rabbits. The inferior alveolar nerve was stimulated to evoke the JOR in the digastric muscle. The stimulus intensity was either 1.5 (low threshold) or 4.0 (high threshold) times the threshold for eliciting the JOR. As a conditioning stimulation, the superior laryngeal nerve (SLN) was repetitively stimulated to evoke the swallowing reflex. The stimulus intensity ranged from 0.6 to 8.0 times the threshold to evoke the swallowing reflex during SLN stimulation over 20s. Electromyographic (EMG) activities of the digastric and mylohyoid muscles were recorded, and the peak-to-peak EMG amplitude of the digastric muscle was measured and compared with and without SLN stimulation, as well as with and without swallowing. Comparisons were also made between low- and high-threshold afferent-evoked JORs. The JOR was strongly suppressed during SLN stimulation. The degree of suppression increased and the latency for the JOR was delayed when the stimulus current applied to the SLN was increased. Such modulation was apparent when the low-threshold afferent-evoked JOR was recorded. Effects of motor outputs of swallowing events and those of single-pulse stimulation of SLN on the inhibition of the JOR were not noted. These results suggest that the JOR evoked by both the low- and high-threshold afferents was inhibited during laryngeal sensory input and following swallowing, probably to prevent opposing jaw movements evoked by oral sensory input during swallowing.