Ten-year follow-up of infliximab treatment for uveitis in Behçet disease patients: A multicenter retrospective study

PurposeTo evaluate 10-year outcome of infliximab (IFX) treatment for uveitis in Behçet disease (BD) patients using a standardized follow-up protocol.DesignRetrospective longitudinal cohort study.Participants140 BD uveitis patients treated with IFX enrolled in our previous study.MethodsMedical records were reviewed for demographic information, duration of IFX treatment, number of ocular attacks before IFX initiation, best corrected visual acuity (VA) at baseline and 1, 2, 3, 4, 5, and 10 years after IFX initiation, uveitis recurrence after IFX initiation and main anatomical site, concomitant therapies, and adverse events (AEs).Main outcome measures10-year IFX continuation rate and change in LogMAR VA.ResultsOf 140 BD patients, 106 (75.7%) continued IFX treatment for 10 years. LogMAR VA improved gradually after initiation of IFX, and the improvement reached statistical significance from 2 years of treatment. Thereafter, significant improvement compared with baseline was maintained until 10 years, despite a slight deterioration of logMAR VA from 5 years. However, eyes with worse baseline decimal VA < 0.1 showed no significant improvement from baseline to 10 years. Uveitis recurred after IFX initiation in 50 patients (recurrence group) and did not recur in 56 (non-recurrence group). Ocular attacks/year before IFX initiation was significantly higher in the recurrence group (2.82 ± 3.81) than in the non-recurrence group (1.84 ± 1.78). In the recurrence group, uveitis recurred within 1 year in 58% and within 2 years in 74%. Seventeen patients (34%) had recurrent anterior uveitis, 17 (34%) had posterior uveitis, and 16 (32%) had panuveitis, with no significant difference in VA outcome. In addition, logMAR VA at 10 years did not differ between the recurrence and non-recurrence groups. AEs occurred among 43 patients (30.7%), and 24 (17.1%) resulted in IFX discontinuation before 10 years.ConclusionsAmong BD patients with uveitis who initiated IFX, approximately 75% continued treatment for 10 years, and their VA improved significantly and was maintained for 10 years. Uveitis recurred in one-half of the patients, but visual acuity did not differ significantly from the patients without recurrence

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress

<div><p>Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking <i>Zip7</i> in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. <i>Zip7</i> deficiency led to the loss of <i>Olfm4</i>^<i>+</i> intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for <i>Zip7</i> in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.</p></div

ZIP7 is necessary for resolving ER stress responses.

<p>(A) ZIP7 subcellular localization. ZIP7 colocalizes with PDI in 293T cells. V5-tagged ZIP7 and PDI were stained with V5 (red) and PDI (green) antibodies. PDI was used as an ER marker. (B) (Left) Flow diagram for subcellular fractionation. NIH-3T3 cells were homogenized and centrifuged to obtain the supernatant (S1), which was centrifuged and subjected to discontinuous sucrose gradient fractionation. The supernatant (S3) and interface fraction were collected. The interface fraction was further centrifuged to obtain the ER-enriched pellet (P4). (Right) Immunoblots of subcellular fractions. ZIP7 was enriched in a fraction containing ER but not Golgi or mitochondria. Calnexin (ER), GM130 (Golgi), adenine nucleotide translocator (ANT, mitochondria), Lyn and GP130 (Plasma membrane), Tubulin (cytoskeleton), and Gapdh (cytosol) were used as markers for various organelles. (C) Quantitative PCR analysis of ER stress-related genes, <i>Derl3</i>, <i>Slc2a6</i>, <i>Creld2 and Herpud1</i> in control (<i>Zip7</i>^<i>+/-</i>, <i>Zip7</i>^flox/+/<i>Rosa26-CreERT2</i>) or <i>Zip7</i>^-/- (<i>Zip7</i>^flox/flox<i>/Rosa26-CreERT2</i>) MEF cells treated with 1 μM 4-OHT for 48 h. ER stress genes were upregulated in <i>Zip7</i>^<i>-/-</i> cells. (D) Overexpression of ZIP7 in MEF cells rescued ER stress gene (<i>Derl3</i>, <i>Creld2</i>) expression induced by <i>Zip7</i> deletion. <i>Zip7</i>^<i>+/-</i> or <i>Zip7</i>^<i>-/-</i> MEF cells were infected with mock or ZIP7-expressing retroviruses, treated with 1 μM 4-OHT for 48 h, and analyzed by quantitative PCR. (E) Thapsigargin or tunicamycin upregulated the transcription of <i>Zip7</i> and ER stress marker genes (CHOP, <i>Derl3</i>, and <i>Derl1</i>) in MEF cells. Cells were treated with 0.25 μM thapsigargin or 0.5 μg/mL tunicamycin for the indicated periods. Data are presented as mean ± SEM from at least three independent experiments.</p

ZIP7 is required for homeostasis of the intestinal epithelium.

<p>(A) Survival curves of <i>Zip7</i>^Cont (Cont: <i>Zip7</i>^Cont, <i>Zip7</i>^flox/+<i>/Vil-CreERT2</i>, blue line, n = 5) and <i>Zip7</i>^ΔIEC (ΔIEC: <i>Zip7</i>^ΔIEC, <i>Zip7</i>^flox/flox<i>/Vil-CreERT2</i>, red line, n = 7) mice. Tamoxifen (TM) was administered orally for five consecutive days (arrows) to induce <i>Zip7</i> deletion. (B) TM protocol used in Fig 2C. (C) H&E (Left), Ki67 staining (center), and TUNEL staining (right) of <i>Zip7</i>^Cont and <i>Zip7</i>^ΔIEC intestines. Deleting <i>Zip7</i> led to substantial damage to the intestine (left panels), loss of proliferative progenitors (center panels), and apoptosis (right panels). Scale bar: 100 μm. (D) Identification of <i>Olfm4</i>^<i>+</i> stem cells by <i>in situ</i> hybridization of <i>Olfm4</i> in the small intestines from <i>Zip7</i>^Cont and <i>Zip7</i>^ΔIEC mice. In the <i>Zip7</i>^ΔIEC intestine, <i>Olfm4</i>⁺ stem cells were lost after <i>Zip7</i> deletion. (E) Morphology of small intestinal organoids from <i>Zip7</i>^Cont or <i>Zip7</i>^ΔIEC mice. <i>Zip7</i>^Cont crypts (Cont) proliferated and formed budding structures during the first 6 days of culture. <i>Zip7</i>^ΔIEC crypts (ΔIEC) treated with 4-OHT progressively degenerated. Representative images from three independent experiments. Scale bar: 50 μm. (F) Quantification of the fate of organoids in E. The status of organoids was traced for 6 days. Organoids were scored as unaffected (white bars), affected (gray bars), or degenerated (black bars). (Cont Day 1: n = 83; Cont Day 2: n = 73; Cont Day 6: n = 63; ΔIEC Day 1: n = 96; ΔIEC Day 2: n = 120; ΔIEC Day 6: n = 42).</p

A schematic model of the ZIP7-dependent rapid epithelial proliferation in intestine.

<p>Intestinal stem cells located at the bottom of the crypt self-renew and generate proliferative cells. These cells proliferate vigorously, producing large numbers of differentiated cells. During this proliferation, ZIP7 is induced and resolves the upregulated ER stress, thereby ensuring proliferative responses in intestinal crypts.</p

Loss of <i>Zip7</i> leads to degeneration of Paneth cells.

<p>(A) Dissected Paneth cells and <i>Lgr5</i>⁺ intestinal stem cells were sorted by flow cytometry into CD24^hiSSC^hi (Paneth cells) and CD24^-EGFP⁺EpCAM^hi (stem cells) populations. (B) Quantitative PCR analysis of <i>Lgr5</i>, <i>Ang4</i> (Paneth cell marker), and <i>Zip7</i> genes in sorted stem cell (SC) and Paneth cell (PC) populations. (C) Identification of Paneth cells by <i>Defa1</i> (blue) and <i>Mmp7</i> (blue) <i>in situ</i> hybridization analysis in small intestine from <i>Zip7</i>^Cont and <i>Zip7</i>^ΔIEC mice. (D) Transmission electron microscopic analysis of the small intestine from <i>Zip7</i>^Cont and <i>Zip7</i>^ΔIEC mice, 3 days after tamoxifen treatment. Scale bar: 2 μm.</p

ZIP7 distribution in the mouse small intestine.

<p>(A) Quantitative PCR analysis of the relative expression of <i>Zip7</i>, <i>Lgr5</i>, and <i>Krt20</i> in villi and crypt. (B) ZIP7 protein levels in villi and crypt were analyzed by western blot of serial dilutions of villi and crypt protein lysates. (C) <i>In situ</i> hybridization of <i>Zip7</i> in small intestine tissues. Scale bar: 50 μm. (D) Simultaneous analysis of <i>in situ</i> hybridization of <i>Zip7</i> (magenta), EdU staining (green) and immunohistochemical E-cadherin staining (blue). Left: merged images of <i>Zip7</i> and E-cadherin. Center: merged images of EdU and E-cadherin. Right: merged images of <i>Zip7</i>, EdU and E-cadherin. Mice were injected with EdU 2 h before sacrifice. Cell borders were visualized by E-cadherin staining. Scale bar: 20 μm. (E) Simultaneous analysis of <i>in situ</i> hybridization of <i>Zip7</i> (magenta) and immunohistochemical E-cadherin staining (cyan). Left panel: merged images of E-cadherin and DIC. Center panel: merged images of <i>Zip7</i> (magenta), E-cadherin (Cyan) and DIC. Right panel: merged images of DIC, <i>Zip7</i>, E-cadherin and Hoechst33342. Arrows indicate Paneth cells. (F) Simultaneous analysis of <i>in situ</i> hybridization of <i>Zip7</i> (magenta), <i>Olfm4</i> (green) and immunohistochemical E-cadherin staining (blue). Top panel: merged images of <i>Olfm4</i> (green) and E-cadherin. Middle panel: merged images of <i>Zip7</i> (magenta) and E-cadherin. Bottom panel: merged images of <i>Zip7</i> (magenta), <i>Olfm4</i> (green) and E-cadherin (blue). Arrows indicate <i>Olfm4</i>-positive stem cells. Scale bar: 20 μm.</p