Efficacy and Safety of Bleselumab in Preventing the Recurrence of Primary Focal Segmental Glomerulosclerosis in Kidney Transplant Recipients: A Phase 2a, Randomized, Multicenter Study.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage kidney disease and frequently recurs after kidney transplantation. Recurrent FSGS (rFSGS) is associated with poor allograft and patient outcomes. Bleselumab, a fully human immunoglobulin G4 anti-CD40 antagonistic monoclonal antibody, disrupts CD40-related processes in FSGS, potentially preventing rFSGS. METHODS: A phase 2a, randomized, multicenter, open-label study of adult recipients (aged ≥18 y) of a living or deceased donor kidney transplant with a history of biopsy-proven primary FSGS. The study assessed the efficacy of bleselumab combined with tacrolimus and corticosteroids as maintenance immunosuppression in the prevention of rFSGS >12 mo posttransplantation, versus standard of care (SOC) comprising tacrolimus, mycophenolate mofetil, and corticosteroids. All patients received basiliximab induction. The primary endpoint was rFSGS, defined as proteinuria (protein-creatinine ratio ≥3.0 g/g) with death, graft loss, or loss to follow-up imputed as rFSGS, through 3 mo posttransplant. RESULTS: Sixty-three patients were followed for 12 mo posttransplantation. Relative decrease in rFSGS occurrence through 3 mo with bleselumab versus SOC was 40.7% (95% confidence interval, -89.8 to 26.8; P = 0.37; absolute decrease 12.7% [95% confidence interval, -34.5 to 9.0]). Central-blinded biopsy review found relative (absolute) decreases in rFSGS of 10.9% (3.9%), 17.0% (6.2%), and 20.5% (7.5%) at 3, 6, and 12 mo posttransplant, respectively; these differences were not statistically significant. Adverse events were similar for both treatments. No deaths occurred during the study. CONCLUSIONS: In at-risk kidney transplant recipients, bleselumab numerically reduced proteinuria occurrence versus SOC, but no notable difference in occurrence of biopsy-proven rFSGS was observed.

Immunohistochemical detection of citrullinated histone H3-positive neutrophils is useful for identifying active glomerular and interstitial lesions in antineutrophil cytoplasmic antibody-associated vasculitis.

AIMS: Activated neutrophils release neutrophil extracellular traps (NETs), resulting in a form of cell death called NETosis. NET formation is reportedly involved in the onset of systemic lupus erythematosus and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Citrullination of histones is a key step in NET formation, and the presence of citrullinated histones in neutrophils may be associated with disease induction and activity. The aim of this study was to investigate the relationship between infiltrating citrullinated histone H3 (H3Cit)-positive neutrophils and disease specificity and activity in various glomerulonephritides. METHODS AND RESULTS: We selected 32 kidney biopsies with glomerulonephritides, including AAV, lupus nephritis (LN), Henoch-Schönlein purpura nephritis (HSPN), and poststreptococcal acute glomerulonephritis (PSAGN). We examined the presence of H3Cit in infiltrating neutrophils and their association with necrotising, crescentic lesions and tubulointerstitial lesions. In PSAGN and HSPN, we found many myeloperoxidase (MPO)+ neutrophils in glomeruli; however, only a few were H3Cit+. In LN, MPO+ neutrophils mainly existed in the margins of glomerular tufts forming wire-loop lesions, and some of these were noted to be H3Cit+ neutrophils. In contrast, we found a significantly higher frequency of H3Cit+ neutrophils, despite the small number of MPO+ neutrophils, in microscopic polyangiitis in AAV. In particular, H3Cit+ neutrophils were prominent in necrotising lesions along the glomerular capillaries. Moreover, we also found H3Cit+ neutrophils in the interstitium, with marked peritubular capillaritis in AAV. CONCLUSIONS: H3Cit immunostaining is a useful tool for identifying activated neutrophils. The frequency of H3Cit+ neutrophils is not only a disease-specific marker but also a potential marker for disease activity in AAV.

Update on Recurrent Focal Segmental Glomerulosclerosis in Kidney Transplantation.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a clinicopathological syndrome characterized by nephrotic-range proteinuria with high incidence of progression to end-stage renal disease (ESRD). In primary FSGS, 40-60% of patients develop ESRD within 10-20 years. SUMMARY: Recurrence of FSGS after kidney transplantation is frequent and is associated with poor allograft survival. The risk factors for recurrent FSGS include onset of FSGS during childhood, rapid progression of primary FSGS to ESRD, history of recurrent FSGS in previous allograft, and diffuse mesangial hypercellularity or collapsing variant of FSGS in the native kidney. The early histological findings of recurrent FSGS consist of unremarkable glomerular changes on light microscopy but significant podocyte effacement on electron microscopy; the loss of foot processes with eventual dropout of podocytes leads to the development of segmental lesions in the glomerulus. Experimental and clinical data suggest the existence of circulating permeability factors, such as soluble urokinase-type plasminogen activator receptor (suPAR), cardiotrophin-like cytokine factor-1 (CLCF-1), CD40 axis, and apolipoprotein A-Ib (ApoA-Ib), in the pathogenesis of recurrent FSGS. These biomarkers including circulating permeability factors may facilitate earlier diagnosis of FSGS posttransplant and may guide in the development of novel therapies that may be more effective and improve long-term outcomes in kidney transplantation. Key Messages: Several studies have suggested the possible circulating permeability factors, such as suPAR, CLCF-1, CD40 axis, and ApoA-Ib, in the pathogenesis and disease progression of FSGS and recurrent FSGS. Further studies should be performed to elucidate the true essential biomarker(s) associated with the onset and progression of FSGS as well as recurrent FSGS.

Assessment of mucin-related gene alterations following treatment with rebamipide ophthalmic suspension in Sjögren's syndrome-associated dry eyes.

Ocular surface mucins are thought to play vital roles in maintaining the homeostasis of the pre-ocular surface tear film. We performed ocular surface tests with impression cytology to assess the expression levels of mucin-related genes on the ocular surface in healthy eyes. In addition, we investigated alterations in mucin-related gene expression secondary to treatment with rebamipide ophthalmic suspension in patients with Sjögren's syndrome-associated dry eyes (SS-DE). Thirty-three healthy individuals (control group) and 13 patients from our hospital with SS-DE were enrolled. Impression cytology was performed using Schirmer's test paper for RNA sampling. The mRNA levels of SAM-pointed domain-containing ETS-like factor (SPDEF), mucin 5AC (MUC5AC), and mucin 16 (MUC16) were determined using a real-time reverse transcription-polymerase chain reaction. The ocular surface test was performed once for the control group, and at baseline as well as 2, 4, 8, and 12 weeks after treatment in the Sjögren's syndrome-associated dry eyes group. mRNA levels of SPDEF, MUC5AC, and MUC16 were not significantly different between the control and SS-DE groups before rebamipide ophthalmic suspension treatment. SPDEF mRNA levels in control subjects were significantly correlated with levels of MUC5AC. Among SS-DE patients, SPDEF mRNA levels were significantly increased at 2, 4, and 8 weeks after treatment compared with baseline levels. MUC16 mRNA levels were significantly decreased from baseline levels at 4 and 8 weeks post-treatment. Ocular surface test using impression cytology is a clinically useful tool for assessing mucous conditions on the ocular surface and can be used to determine the effects of instillation treatment with eye drops that affect mucin production at the ocular surface.

Microincision vitrectomy surgery: experimental visualization and quantification of vitreous contamination.

BACKGROUND: To visualize and quantify vitreous contamination following microincision vitrectomy surgery (MIVS) using an experimental vitreous contamination model (EVCM). METHODS: Enucleated porcine eyes with fluoresbrite carboxylate microspheres applied to the conjunctival surface were used as a type 1 EVCM. Twenty-five- or 27-gauge (G) trocar cannulas were inserted through the conjunctiva and sclera, followed by the placing and opening of an infusion cannula. These procedures were monitored by an intraocular fiber catheter. Secondly, condensed microspheres were applied to an excised sheet of porcine sclera to serve as type 2 EVCM. Twenty-five- or 27-G trocar cannulas were inserted perpendicularly through the top of the sclera where the condensed microspheres were applied, an infusion cannula was inserted, 0.1 mL of saline solution injected through the infusion cannula, and samples collected. The fluorescence strength of samples was then measured using fluorophotometry. RESULTS: We visually detected fluorescent microspheres in 10/10 eyes with 25-G and 10/10 with 27-G MIVS. In the experimental quantification study, each MIVS gauge value was significantly higher than the control (P < 0.01). However, there was no significant difference between 25-G and 27-G MIVS. CONCLUSIONS: MIVS carries the risk of introducing contamination directly into the eyes when the trocar cannula is inserted and infusion cannula is opened, even when a 27-G MIVS is used. Our study has shown it is essential that the surgeon be aware of the possibility of introducing contamination from the conjunctiva at all times during MIVS.

Molecular Insights in Transmission of Cancer From an Organ Donor to Four Transplant Recipients.

Organ donors are systematically screened for infection, whereas screening for malignancy is less rigorous. The true incidence of donor-transmitted malignancies is unknown due to a lack of universal tumor testing in the posttransplant setting. Donor-transmitted malignancy may occur even when not suspected based on donor or recipient factors, including age and time to cancer diagnosis. We describe the detection of a gastrointestinal adenocarcinoma transmitted from a young donor to 4 transplant recipients. Multidimensional histopathologic and genomic profiling showed a CDH1 mutation and MET amplification, consistent with gastric origin. At the time of writing, one patient in this series remains alive and without evidence of cancer after prompt organ explant after cancer was reported in other recipients. Because identification of a donor-derived malignancy changes management, our recommendation is to routinely perform short tandem repeat testing (or a comparable assay) immediately upon diagnosis of cancer in any organ transplant recipient. Routine testing for a donor-origin cancer and centralized reporting of outcomes are necessary to establish a robust evidence base for the future development of clinical practice guidelines.

Ocular allergy test and biomarkers on the ocular surface: Clinical test for evaluating the ocular surface condition in allergic conjunctival diseases.

Allergic conjunctival diseases (ACDs) are inflammatory diseases of the conjunctiva and cornea caused predominantly by the IgE-mediated immediate hypersensitivity response. Allergic conjunctival diseases include allergic conjunctivitis, vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), and giant papillary conjunctivitis. In clinical practice of ACDs, an ocular allergy test using biomarker measurement is a crucial examination technique for diagnosing, evaluating severity, and determining the efficacy of medical treatment. The ocular allergy test includes the tear test for evaluating the concentration of biomarkers in tears and an ocular surface test for assessing the expression levels of messenger ribonucleic acid (mRNA) biomarkers on the ocular surface. The clinical usefulness of several biomarkers has been demonstrated in patients with ACDs; specifically, eosinophil cationic protein and eotaxin-2 as eosinophilic inflammation biomarkers; interleukin-4 and thymus and activation regulated chemokine (CCL17/TARC) as Th2 inflammation biomarkers; eotaxin, tumor necrosis factor-alpha and soluble IL-6 receptor as giant papillae biomarkers; and osteopontin and periostin as allergic inflammation and remodeling biomarkers. Furthermore, the ocular allergy test, quantitative evaluation methods using biomarkers have allowed for better understanding of the immunological and pathophysiological mechanisms of ACDs. Therefore, the search for a biomarker is important to make an ocular allergy test useful. In previous ocular allergy tests, the biomarkers for allergic inflammation in patients with chronic ACDs including VKC and AKC were substantial. However, the selection of biomarkers associated with the early phase reaction of immediate hypersensitivity and innate immunity responses needs to be addressed in future investigations.

Further Evidence That the Soluble Urokinase Plasminogen Activator Receptor Does Not Directly Injure Mice or Human Podocytes.

BACKGROUND: The role of the soluble urokinase plasminogen activator receptor (suPAR) in focal segmental glomerulosclerosis (FSGS) as the circulating factor or as a predictor of recurrence after transplantation remains controversial. Previously published studies in mice and isolated podocytes produced conflicting results on the effect of suPAR on podocyte injury, effacement of foot processes, and proteinuria. These discordant results were in part due to diverse experimental designs and different strains of mice. The aim of our study was to determine the reasons for the inconsistencies of the previous studies results with suPAR by using uniform methods and studies in different strains of mice. METHODS: We utilized a primary culture of human podocytes and 2 mouse models, the wild type (WT) and the urokinase plasminogen activator receptor (uPAR) KO (uPAR), in an attempt to resolve the reported conflicting results. RESULTS: In both WT and uPAR mouse models, injection of recombinant uPAR, even at a high dose (100 µg), did not induce proteinuria, effacement of podocytes, or disruption of the cytoskeleton. Injection of suPAR resulted in its deposition exclusively in the glomerular endothelial cells and not in the podocytes of WT mice and was not detected at the uPAR KO mice. Kidneys from patients with recurrent FSGS had negative immunostaining for uPAR. We also evaluated the effect of recombinant uPAR on primary culture of human podocytes. uPAR did not result in podocytes damage. CONCLUSIONS: suPAR by itself is not the cause for direct podocyte injury, in vitro or in vivo. These findings suggest a more complex and still poorly understood role of suPAR in FSGS.

Evaluation of vitreous levels of advanced glycation end products and angiogenic factors as biomarkers for severity of diabetic retinopathy.

BACKGROUND/AIMS: Glyceraldehyde-derived advanced glycation end products (glycer-AGE; also called Toxic-AGE [TAGE]) play a crucial role in the pathogenesis of diabetic angiopathy. However, the relationships between vitreous glycer-AGE levels and diabetic retinopathy (DR) severity, and between glycer-AGE levels and the levels of other angiogenic factors remain unknown. We investigated the correlation between levels of vitreous biomarkers, including glycer-AGE and angiogenic factors (vascular endothelial growth factor [VEGF], interleukin [IL]-8, leptin, placental growth factor [PlGF], endoglin, and fibroblast growth factor [FGF]-2) in patients with DR, using three DR staging groups. METHODS: In this cross-sectional study, we examined 33 eyes from 33 patients with diabetes mellitus who underwent a vitrectomy (non-proliferative DR [NPDR, n = 8]; PDR with simple vitreous haemorrhage [VH, n = 17]; or PDR with a fibrovascular proliferative membrane [FVM, n = 8]). Vitreous levels of glycer-AGE and VEGF were evaluated using enzyme-linked immunosorbent assays. Vitreous levels of IL-8, leptin, PlGF, endoglin, and FGF-2 were evaluated using beaded assay methods. RESULTS: Vitreous levels of glycer-AGE in the FVM group were significantly higher than those in the NPDR and VH groups (all p < 0.05). Vitreous levels of VEGF (r = 0.85, p = 1.7 × 10-6) and leptin (r = 0.60, p = 5.0 × 10-3) were significantly correlated with levels of PlGF. CONCLUSION: The two systems (VEGF-PlGF-leptin and glycer-AGE) were represented in these measured biomarkers. High vitreous levels of both VEGF and glycer-AGE may be linked to more severe DR, suggesting that anti-VEGF and anti-TAGE therapy may be an important part of the therapeutic strategy for DR.

Relationships between vitreous levels of soluble receptor for advanced glycation end products (sRAGE) and renal function in patients with diabetic retinopathy.

PURPOSE: We investigated the relationship between vitreous levels of soluble receptor for advanced glycation end products (sRAGE) and vascular endothelial growth factor (VEGF) and renal function, and correlations between vitreous sRAGE levels and proliferative diabetic retinopathy (PDR) activity. METHODS: We examined 33 eyes from 33 patients with diabetes mellitus who underwent a vitrectomy (eight patients in the non-PDR [NPDR] group and 25 in the PDR group). Serum creatinine levels and estimated glomerular filtration rate (eGFR) were measured and classified according to the chronic kidney disease (CKD)-staging method. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify vitreous sRAGE and VEGF levels. RESULTS: Vitreous sRAGE levels were significantly higher in PDR group compared to NPDR group (p = 0.00003). Vitreous sRAGE levels were significantly higher in patients with CKD stage 5 (end-stage renal failure or hemodialysis) than in patients with CKD stage 1 or 2 (p < 0.01) and 3 or 4 (p < 0.05), and were significantly correlated with eGFR (r = - 0.490, p = 0.007) and creatinine levels (r = 0.484, p = 0.006). Within the PDR group, patients with low (<27 pg/mL) sRAGE levels required repeat vitreous surgeries for early postoperative vitreous hemorrhage significantly more frequently than those with high (≥27 pg/mL) sRAGE levels (p = 0.0067). CONCLUSIONS: Vitreous sRAGE levels were significantly correlated with renal function, and low vitreous sRAGE levels in patients with PDR were associated with postoperative vitreous hemorrhage. Vitreous sRAGE may be a useful biomarker for renal dysfunction associated with diabetic retinopathy.

EXPERIMENTAL VISUALIZATION AND QUANTIFICATION OF VITREOUS CONTAMINATION FOLLOWING INTRAVITREAL INJECTIONS.

PURPOSE: To detect and quantify vitreous contamination after intravitreal injection using an experimental vitreous contamination model. METHODS: Enucleated porcine eyes served as a Type 1 experimental vitreous contamination model with fluoresbrite carboxylate microspheres applied to the conjunctival surface. Saline solution (0.05 mL) was injected using a 27-, 30-, or 32-gauge (G) needle. Injection procedures were monitored using an intraocular fiber catheter. Condensed microspheres were applied to an excised sheet of porcine sclera (Type 2 experimental vitreous contamination model). Saline solution (0.05 mL) was injected from the top of an applied condensed microsphere through the sclera using a needle of one of the aforementioned gauges, and samples were then collected. The fluorescence strength of samples was measured using fluorophotometry. RESULTS: We visually detected fluorescent microspheres in 10/10, 9/10, and 9/10 eyes injected with 27-G, 30-G, and 32-G needles, respectively. In the experimental quantification study, values at all needle gauges were significantly higher than those of controls (P < 0.01). Fluorescence strength was significantly higher in the 27-G group than in the 30- (P < 0.01) and 32-G (P < 0.01) groups. CONCLUSION: Intravitreal injection carries the risk of introducing contamination directly into the eyes even when a 32-G needle is used. Furthermore, the 27-G needle carries the highest contamination risk.

Interleukin-6 Is a Risk Factor for Atrial Fibrillation in Chronic Kidney Disease: Findings from the CRIC Study.

Atrial fibrillation (AF) is the most common sustained arrhythmia in patients with chronic kidney disease (CKD). In this study, we examined the association between inflammation and AF in 3,762 adults with CKD, enrolled in the Chronic Renal Insufficiency Cohort (CRIC) study. AF was determined at baseline by self-report and electrocardiogram (ECG). Plasma concentrations of interleukin(IL)-1, IL-1 Receptor antagonist, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor-ß, high sensitivity C-Reactive protein, and fibrinogen, measured at baseline. At baseline, 642 subjects had history of AF, but only 44 had AF in ECG recording. During a mean follow-up of 3.7 years, 108 subjects developed new-onset AF. There was no significant association between inflammatory biomarkers and past history of AF. After adjustment for demographic characteristics, comorbid conditions, laboratory values, echocardiographic variables, and medication use, plasma IL-6 level was significantly associated with presence of AF at baseline (Odds ratio [OR], 1.61; 95% confidence interval [CI], 1.21 to 2.14; P = 0.001) and new-onset AF (OR, 1.25; 95% CI, 1.02 to 1.53; P = 0.03). To summarize, plasma IL-6 level is an independent and consistent predictor of AF in patients with CKD.

Glomerular endothelial cell injury and focal segmental glomerulosclerosis lesion in idiopathic membranous nephropathy.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) lesions have often been discussed as a negative predictor in idopathic membranous nephropathy (MN). The mechanism of the development of FSGS lesion in MN is still uncertain. METHODS: From 250 cases of MN, 26 cases contained FSGS lesion. We compared the clinicopathological characteristics between MN cases with FSGS lesion [MN-FSGS(+)] and MN without FSGS lesion [MN-FSGS(-)], matched for gender, age, stage of MN. RESULTS: The glomerular filtration rate (eGFR) was significantly lower in MN-FSGS(+) cases compared to MN-FSGS(-), although nephrotic syndrome, hematuria, and systolic blood pressure levels were not significantly different between the two groups. Pathologically, glomeruli in MN-FSGS(+) cases showed narrowing and loss of glomerular capillaries with separating from GBM or disappearance of CD34+ endothelial cells, and accumulation of extracellular matrix (ECM) in capillary walls, indicating the development of glomerular capillary injury. These findings of endothelial injury were seen even in MN-FSGS(-) cases, but they were more prominent in MN-FSGS(+) than MN-FSGS(-) by computer assessed morphometric analysis. In MN-FSGS(+) cases, 44 out of 534 glomeruli (8.2%) contained FSGS lesions (n = 31, NOS lesion; n = 13, perihilar lesion). Significant thickness of GBM with ECM accumulation was evident in MN-FSGS(+) cases. Podocyte injury with effacement of foot processes was also noted, but the expression of VEGF on podocytes was not different between the two groups, which suggests that the significant thickness of capillary walls may influence the function of VEGF from podocyte resulting in the glomerular capillary injury that contribute to the development of FSGS lesion in MN. CONCLUSION: Glomerular capillary injury was seen in all MN cases. Furthermore, the prominent injuries of glomerular capillaries may be associated with the deterioration of eGFR and the formation of FSGS lesions in MN.

CCL20/MIP-3 alpha mRNA expression in the conjunctival epithelium of normal individuals and patients with vernal keratoconjunctivitis.

BACKGROUND: CCL20, the single chemokine ligand for CCR6, contributes to recruiting CCR6-expressing memory B cells, memory T cells, Th17 cells and dendritic cells, and is involved in regulating immune responses, homeostasis, and inflammation in mucosal tissues. METHODS: CCL20 messenger RNA (mRNA) expression was analyzed in the conjunctival epithelium in an in vivo study of patients with vernal keratoconjunctivitis (VKC group) and healthy volunteers (control group) using impression cytology. In vitro analysis of CCL20 mRNA was performed using cultured conjunctival epithelial cells (CECs). Real-time polymerase chain reaction was used to assess IL-8 and eotaxin-2 mRNA expression for comparison with CCL20 mRNA expression. RESULTS: In the control group, CCL20 mRNA expression was present in all conjunctival locations. However, CCL20 mRNA expression was significantly higher in the upper palpebral conjunctiva in the severe VKC group than in the mild VKC and control groups (p < 0.05, Steel test). In vitro stimulation of CECs with lipopolysaccharide (LPS) significantly increased CCL20 expression in a concentration-dependent manner that was significantly correlated with expression of IL-8 (p < 0.001, Spearman's rank correlation coefficient), but not eotaxin-2. CONCLUSION: We conclude that CCL 20 mRNA expression in the conjunctival epithelium plays a crucial role in regulating homeostasis at the ocular surface and in exacerbation of VKC.

[Evaluation of dectin-1 and BAFF expression in conjunctival epithelial cells].

PURPOSE: To investigate the expression of dectin-1 protein in conjunctival epithelial cells and the expression of dectin-1 and B-cell activating factor belonging to the tumor necrosis factor family (BAFF) mRNA in in vivo conjunctival epithelial cells (CECs) and in vitro cultured CECs, and its difference in topographical change and etiology of disorders. SUBJECTS AND METHODS: 1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs. The subjects were 12 eyes of 12 healthy volunteers (control group), 6 eyes of 6 patients with Sjögren syndrome (Sjögren group) and 10 eyes of 10 patients with vernal keratoconjuctivitis (VKC group). CECs were sampled by impression cytology using nitrocellulose membrane. The expression of dectin-1 in CECs was detected by immunofluorescence and the quantitative determination of dectin-1 mRNA and BAFF mRNA expression was performed by real-time polymerase chain reaction(real-time PCR). 2. Investigation of dectin-1 and BAFF expression using cultured CECs. Cultured CECs which were divided into an OK-432 addition group (addition concentrations: 0.02, 0.1, 0.5KU/mL), a lipopolysaccharide (LPS) addition group (addition concentrations : 80, 160, 320 microg/mL) and an additive-free group were cultured. Quantitative determination of dectin-1 mRNA and BAFF mRNA expression in cultured CECs was performed by real-time PCR. RESULTS: 1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs. In the control group, there was no significant topographical difference in the expression of dectin-1 and the amount of dectin-1 mRNA among superior, inferior tarsal conjunctiva and temporal bulbar conjunctiva. The levels of dectin-1 mRNA expression were 1.5 (0.1-4.0) [median value (range)] for the control group, 2.6 (1.1-4.8) for the Sjögren group and 3.6 (1.7-16.6) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p < 0.01, Kruskal-Walles H-test). The levels of BAFF mRNA expression were 2.8 (0.2-13.8) [median value (range)] for the control group, 6.3 (2.1-15.1) for the Sjögren group and 11.2 (3.5-70.8) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p < 0.01, Kruskal-Walles H-test). Moreover, regarding the relationship between expression level of dectin-1 mRNA and that of BAFF mRNA in all the subjects, there was a significant correlation between them (r = 0.75, p < 0.001, Spearman's rank coefficient). The levels of dectin-1 mRNA expression in the moderate and severe VKC group 9.2 (2.6-16.6) [median value (range)] were significantly higher than those in mild VKC group 2.8 (1.7-3.8) (p < 0.05, Mann-Whitney U-test). The levels of BAFF mRNA expression in the severe and moderate VKC groups 17.4 (9.1-70.8) [median value (range)] were significantly higher than those in the mild VKC group 4.3 (3.5-11.2) (p < 0.05, Mann-Whitney U-test). 2. Investigation of dectin-1 and BAFF expression by cultured CECs. In the OK-432 addition group, the expression levels of dectin-1 mRNA were increased dose-dependently due to the OK-432 stimulation (p < 0.05, Kruskal-Wallis H-test). Moreover, regarding the relationship between the expression level of dectin-1 mRNA and that of BAFF mRNA in all the cultured conjunctival epithelial cells stimulated by OK-432, there was a significant correlation between them (r = 0.85, p < 0.005, Spearman's rank coefficient). CONCLUSIONS: We concluded that dectin-1 expression in CECs was demonstrated, and expression of both dectin-1 and BAFF in CECs is thought to be involved in pathologic aggravation of allergic inflammatory in patients with VKC.

The use of rituximab and bendamustine in treating chronic lymphocytic leukaemia (CLL) in end-stage renal disease (ESRD).

A patient with a history of type 2 diabetes mellitus and chronic lymphocytic leukaemia has renal failure with large kidneys. The patient refused kidney biopsy to determine the aetiology of her renal failure. She uses peritoneal dialysis to treat renal failure. She received rituximab and bendamustine to treat chronic lymphocytic leukaemia. Adenopathy resolves with treatment and she does not experience any electrolyte disturbances or decrease in urine output as a result of chemotherapy in the setting of renal failure. Renal function did not recover with chemotherapy.

Evaluation of eosinophilic inflammation in a novel murine atopic keratoconjunctivitis model induced by crude Dermatophagoides farinae antigen.

BACKGROUND: The purpose of this study is to conduct a histopathological research of the conjunctival findings and eosinophilic inflammation of novel atopic keratoconjunctivitis in a NC/Nga mouse model using crude Dermatophagoides farina. METHODS: NC/Nga mice were sensitized by repeated topical applications of an ointment containing Dermatophagoides farinae body (Dfb). They were then divided into 4 groups depending on the following topical ophthalmic treatment: DFb group undergoing topical ophthalmic ointment containing Dfb; DFco group undergoing topical instillation of allergen extracts of Dermatophagoides farinae; Ba group undergoing topical ointment with substrate alone and NT group without after-topical ophthalmic treatment. At 24 hours after the last ophthalmic treatment, histopathological examination was performed. The density of the subepithelial infiltration of the eosinophils was determined. Serum total IgE and house-dust-mite (HDM)-specific IgE antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In the DFb group, the conjunctiva showed similar findings to those of atopic keratoconjunctivitis, i.e. intraepithelial pseudotubular formation, Torus-form infiltration due to massive lymphocytes in the palpebral conjunctiva and gelatinous hyperplasia in the limbus with subepithelial granuloma composed of lymphocytes and eosinophils. Subepithelial infiltration of eosinophil density in the DFb group [878.4 ± 399.7cells/mm2 (mean ± SD)] was significantly higher than in the other 2 groups (DFco 85.6 ± 40.1 Ba 49.2 ± 32.3) (P < 0.001). Total serum IgE concentration and HDM-specific serum IgE antibody concentration in the DFb group and the DFco group were significantly higher compared with those in the NT group. CONCLUSIONS: Topical application of an ointment containing DFb to both the skin and eyes of NC/Nga mice can induce an atopic keratoconjunctivitis (AKC) model in these mice.

Regulation of soluble interleukin-6 (IL-6) receptor release from corneal epithelial cells and its role in the ocular surface.

PURPOSE: Interleukin (IL)-6 signaling through its soluble receptor (sIL-6R) (IL-6 trans-signaling) plays an important role in various inflammatory states. We investigated production of sIL-6R in the corneal epithelium and examined the role of IL-6 trans-signaling in the cornea. METHODS: In-vitro experiments were performed using SV40-transformed human corneal epithelial cells (HCEC) and primary human corneal fibroblasts (HCF, keratocytes). Ectodomain shedding in HCEC was stimulated by adding phorbol myristate acetate (PMA, 3 µM: ) both with and without ectodomain shedding inhibition using TNF-α processing inhibitor-1 (TAPI-1, 250 ng/mL), and the concentration of sIL-6R in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Expression of differential sIL-6R mRNA splicing (DS-sIL-6R) in HCEC was examined by using reverse transcription (RT)-PCR. The recombinant IL-6 or combination of recombinant IL-6/sIL-6R was added to HCF culture medium and phosphorylation of STAT3 was analyzed by Luminex assay. Tear fluid from patients with Sjögren syndrome was collected and analyzed by ELISA for sIL-6R concentration. RESULTS: In HCEC culture medium, sIL-6R release was increased significantly (P < 0.01) by adding PMA and this increased release of sIL-6R was inhibited significantly by adding TAPI-1, indicating the participation of ectodomain shedding in sIL-6R production. In RT-PCR, DS-sIL-6R expression was noted in HCEC. IL-6/sIL-6R-induced STAT3 phosphorylation was recognized in cultured HCF, suggesting IL-6 trans-signaling induced inflammatory cellular signaling in HCF. In the tear fluid of the patients with Sjögren syndrome, sIL-6R expression was up-regulated (Sjögren syndrome; 2.38 ± 0.98 ng/mL, normal control; 0.16 ± 0.34 ng/mL). CONCLUSIONS: Production of sIL-6R was induced by both ectodomain shedding and mRNA splicing in the corneal epithelium. IL-6 trans-signaling can induce an inflammatory response in corneal fibroblasts. The up-regulation of sIL-6R in inflamed ocular surfaces suggests a pivotal role of sIL-6R at the ocular surface.

[Allergy related factors in tears of patients with vernal keratoconjunctivitis undergoing topical 0.1% tacrolimus treatment].

PURPOSE: To investigate, using tear fluid analysis, the effects of topical 0.1% tacrolims therapy on the pathophysiology of vernal keratoconjunctivitis (VKC). SUBJECTS AND METHODS: Subjects were 6 eyes of 6 patients with VKC who underwent topical 0.1% tacrolims treatment twice a day and 5 eyes of 5 healthy volunteers as a control. Using the filter paper method, the tear fluid of the patients was sampled both before and 4 +/- 2 weeks after the treatment and once from the control subjects. Eosinophil cationic protein (ECP) in the tears was examined by the chemiimmunoluminescent enzyme immunoassay method and the chemokine profile of the tears was analyzed using an antibody-array. RESULTS: In terms of the chemokine profile, growth related oncogene (GRO) -alpha, eotaxin-2 and thymus and activation-regulated chemokine (TARC) in the VKC were elevated compared with those in the controls, but they decreased significantly after the treatment (p<0.05). ECP concentrations in the tears were 3092 +/- 1658 ng/ml (average +/- S. D.) for the pretreatment base-line and 464 +/- 775 for the posttreatment. ECP values for the pre-treatment time were statistically significantly higher than those for the post-treatment in 5 patients (p<0.05). CONCLUSION: Topical tacrolims treatment of VKC can suppress allergic inflammation associated chemokines such as eotaxin-2 and TARC.

Soluble IL-6 receptor in vitreous fluid of patients with proliferative diabetic retinopathy.

PURPOSE: To identify the biological reaction of soluble interleukin-6 receptor (sIL-6R) in the vitreous of patients with proliferative diabetic retinopathy (PDR). METHODS: The subjects were 45 patients (45 eyes) with vitreoretinal diseases. The patients were divided into three groups: the PDR group comprised 28 patients (28 eyes) with PDR; the pre-proliferative diabetic retinopathy (PPDR) group comprised seven patients (seven eyes) with PPDR combined with diabetic macular edema; and the nondiabetic group comprised ten patients (ten eyes) with idiopathic macular hole or idiopathic epiretinal membrane. Vitreous samples were obtained at vitrectomy. sIL-6R, vascular endothelial growth factor (VEGF), and protein concentration in vitreous samples were determined by enzyme-linked immunosorbent assay (ELISA). sIL-6R levels in serum were also determined by ELISA in nine of the 28 patients with PDR and in six healthy volunteers as controls. RESULTS: In vitreous fluid, the levels of sIL-6R in the PDR group, PPDR group, and nondiabetic group were 612.7 +/- 233.8 (mean +/- SD), 746.3 +/- 523.1, and 215.4 +/- 98.3 pg/ml, respectively. Vitreous levels of sIL-6R in the PDR and PPDR groups were significantly higher than those in the nondiabetic group (PDR group, P < 0.0001; PPDR group, P < 0.01). In serum, the levels of sIL-6R were 39.38 +/- 9.43 ng/ml in the PDR group and 22.84 +/- 5.32 ng/ml in the control group. sIL-6R levels in serum in the PDR group were significantly higher than those in the control group (P < 0.01). A partial correlation analysis showed a significant correlation between the levels of sL-6R and VEGF in the vitreous in the PDR group (r = 0.34, P < 0.05). CONCLUSIONS: We conclude that the level of sIL-6R in vitreous fluid can be considered as a biomarker of PDR.