A Novel Compound Heterozygous Mutation in the CYP4V2 Gene in a Japanese Patient with Bietti's Crystalline Corneoretinal Dystrophy

Purpose: To describe the clinical and genetic characteristics of a Japanese family in which one member exhibited Bietti's crystalline corneoretinal dystrophy (BCD). Methods: Using direct sequencing, mutation screening was performed in the CYP4V2 gene of both the patient with BCD and her daughter. Ophthalmic examinations were performed to determine the clinical features of both subjects. Results: The 64-year-old female patient had a bilateral visual acuity of 0.4. Slit lamp examination revealed bilateral crystalline-like deposits at the superior limbus of the cornea. Fundus examination revealed there was chorioretinal atrophy along with numerous glistening yellowish-white crystalline deposits that were scattered throughout the posterior pole and the mid-peripheral retina. Standard flash electroretinography showed an extinguished electroretinogram and Goldmann kinetic perimetry detected a relative scotoma. Genetic analysis revealed that the patient had a heterozygous mutation in the CYP4V2 gene (IVS6-8delTCATACAGGTCATCGCG/GC), which is the most commonly found mutation in Japanese patients with BCD. Furthermore, the patient was also shown to have a novel heterozygous point mutation in exon 9 of the CYP4V2 gene (c.1168C>T). In contrast, her daughter exhibited no clinical findings for BCD even though she carried the same heterozygous mutation in the CYP4V2 gene (c.1168C>T). Conclusion: A novel compound heterozygous mutation was found in the CYP4V2 gene of a patient with BCD. This previously unreported c.1168C>T mutation causes a missense mutation (p.R390C) in the CYP4V2 protein

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs)

Human Subperitoneal Fibroblast and Cancer Cell Interaction Creates Microenvironment That Enhances Tumor Progression and Metastasis

<div><p>Backgrounds</p><p>Peritoneal invasion in colon cancer is an important prognostic factor. Peritoneal invasion can be objectively identified as periotoneal elastic laminal invasion (ELI) by using elastica stain, and the cancer microenvironment formed by the peritoneal invasion (CMPI) can also be observed. Cases with ELI more frequently show distant metastasis and recurrence. Therefore, CMPI may represent a particular milieu that facilitates tumor progression. Pathological and biological investigations into CMPI may shed light on this possibly distinctive cancer microenvironment.</p><p>Methods</p><p>We analyzed area-specific tissue microarrays to determine the pathological features of CMPI, and propagated subperitoneal fibroblasts (SPFs) and submucosal fibroblasts (SMFs) from human colonic tissue. Biological characteristics and results of gene expression profile analyses were compared to better understand the peritoneal invasion of colon cancer and how this may form a special microenvironment through the interaction with SPFs. Mouse xenograft tumors, derived by co-injection of cancer cells with either SPFs or SMFs, were established to evaluate their active role on tumor progression and metastasis.</p><p>Results</p><p>We found that fibrosis with alpha smooth muscle actin (α-SMA) expression was a significant pathological feature of CMPI. The differences in proliferation and gene expression profile analyses suggested SPFs and SMFs were distinct populations, and that SPFs were characterized by a higher expressions of extracellular matrix (ECM)-associated genes. Furthermore, compared with SMFs, SPFs showed more variable alteration in gene expressions after cancer-cell-conditioned medium stimulation. Gene ontology analysis revealed that SPFs-specific upregulated genes were enriched by actin-binding or contractile-associated genes including α-SMA encoding ACTA2. Mouse xenograft tumors derived by co-injection of cancer cells with SPFs showed enhancement of tumor growth, metastasis, and capacity for tumor formation compared to those derived from co-injection with cancer cells and SMFs.</p><p>Conclusions</p><p>CMPI is a special microenvironment, and interaction of SPFs and cancer cells within CMPI promote tumor growth and metastasis.</p></div

Subperitoneal fibroblasts (SPFs) actively contribute to cancer progression.

<p>(A, left) Xenograft tumor growth in mice injected with DLD-1 human colorectal cancer cells alone (blue line, 840.7±112.6 mm³ in 7 weeks), co-injected with DLD-1 cells and submucosal fibroblasts (SMFs; red line, 1178.0±177.6 mm³ in 7 weeks), and co-injected with DLD-1 cells and subperitoneal fibroblasts (SPFs; green line, 1672.8±214.7 mm³ in 7 weeks). The differences of tumor volume between DLD-1 cells alone and DLD-1 cells with SPFs, and between DLD-1 cells alone and DLD-1 cells with SMFs were statistically significant (<i>P</i><0.05). (A, right) Xenograft tumor growth in mice injected with Caco-2 human colorectal cancer cells alone (blue line, 308.6±127.7 mm³ in 9 weeks), co-injected with Caco-2 cells and SMFs (red line, 1363.1±284.3 mm³ in 9 weeks), and co-injected with Caco-2 and SPFs (green line, 2595.1±349.5 mm³ in 9 weeks). The differences of tumor volume between Caco-2 cells alone and Caco-2 cells with SPFs (<i>P</i><0.01), and between Caco-2 cells with SMFs and Caco-2 cells with SPFs (<i>P</i><0.05) were statistically significant. Xenograft tumors derived from co-injection of cancer cells and SPFs grew faster than those derived from injection of cancer cells alone, or co-injection of cancer cells and SMFs. (B, left) Xenograft tumor weight in mice injected with DLD-1 cells alone was 0.71±0.11 g, co-injected with DLD-1 cells and SMFs was 1.27±0.19 g, and co-injected with DLD-1 cells and SPFs was 1.82±0.28 g in 8 weeks. The differences of tumor weight between DLD-1 cells alone and DLD-1 cells with SPFs (<i>P</i><0.01), and between DLD-1 cells alone and DLD-1 cells with SMFs (<i>P</i><0.05) were statistically significant. (B, right) Xenograft tumor weight in mice injected with Caco-2 cells alone was 0.53±0.24 g, co-injected with Caco-2 cells and SMFs was 1.91±0.34 g, and co-injected with Caco-2 cells and SPFs was 3.66±0.45 g in 10 weeks. The differences of tumor weight between DLD-1 cells alone and DLD-1 cells with SPFs (<i>P</i><0.01), between DLD-1 cells alone and DLD-1 cells with SMFs (<i>P</i><0.05), and between DLD-1 cells with SPFs and DLD-1 cells with SMFs (<i>P</i><0.05) were statistically significant. Weights of xenograft tumors derived from co-injection of cancer cells with SPFs were higher than those of tumors derived from injection of cancer cells alone, or co-injection of cancer cells and SMFs (left: DLD-1, right: Caco-2). (C) Although the value did not reach statistical significance, xenograft tumors derived from co-injection of DLD-1 cells and SPFs showed twice the frequency of lymph node metastasis (n = 4) compared to those deriving from co-injection of DLD-1 cells and SMFs (n = 2). (D) Co-injection of cancer cells and SPFs result in enhanced tumor formation capacity. Results are presented as the mean ± SE of 8 mice (*<i>P</i><0.05. **<i>P</i><0.01).</p

Pathological features of tumor microenvironment explored by using area-specific tissue microarray.

<p>(A) Schema of the cancer microenvironment formed by peritoneal invasion (CMPI) and the cancer microenvironment formed by submucosal invasion (CMSI) defined as (a) invasive front with peritoneal invasion and (b) submucosal invasive front, respectively. (B) The distribution of fibrosis in human colon cancer tissue. Dark gray bars show the number of the cases with fibrosis over 50% of the core from each tumor area, and light gray bars show the number of the cases without extensive fibrosis. Core samples with CMPI showed a higher frequency of marked fibrosis than did core samples with CMSI (<i>P</i><0.01). (C) Distribution of α-SMA expression in human colon cancer tissue. CMPI showed higher α-SMA expressions than those seen in CMSI (<i>P</i><0.01). (D) Distribution of CD3-positive cells in human colon cancer tissue. Numbers of CD3-positive cells were not significantly different between CMPI and CMSI. (E) Distribution of CD68-positive cells in human colon cancer tissue. Numbers of CD68-positive cells were not significantly different between CMPI and CMSI. (F) Distribution of CD31-positive vessels in human colon cancer tissue. Numbers of the CD31-positive vessels were not significantly different between CMPI and CMSI. Results in (B) are presented by case numbers, and those in (C–F) are presented as the mean ± SD of 149 cases (**<i>P</i><0.01).</p

Gene expression profiles in subperitoneal fibroblasts (SPFs) and submucosal fibroblasts (SMFs) with and without cancer-cell-conditioned medium (CCCM) stimulation.

<p>(A) Red is the microarray profile in SMFs with CCCM stimulation, blue is SMFs without CCCM stimulation, green is SPFs with CCCM stimulation, and silver is SPFs without CCCM stimulation. Three-dimensional representation of principal component analysis (PCA) component 1, 2, and 3. (B) Two dimensional representation of PCA components 1 and 2 (upper), and PCA components 1 and 3 (lower). Fibroblasts formed independent clusters, depending on histoanatomical site and the presence of CCCM stimulation. (C) Supervised cluster analysis in fibroblasts also revealed distinct clusters depending on histoanatomical site and the presence of CCCM stimulation. (D) Gene ontology analysis of upregulated genes in SPFs compared with SMFs. (E) Gene ontology analysis of genes upregulated in SPFs with CCCM stimulation, compared with SMFs with CCCM stimulation. Most of the genes with increased expressions in SPFs were retained after CCCM stimulation; however, there were some differences, and the order of annotation clusters were changed after CCCM stimulation.</p

Gene modification in subperitoneal fibroblasts (SPFs) after cancer-cell-conditioned medium (CCCM) stimulation.

<p>(A) Genes upregulated by CCCM stimulation. (B) Genes downregulated by CCCM stimulation. (C) Top 3 annotation clusters in gene ontology analysis of SPFs-specific genes upregulated by CCCM stimulation. (D) Top 20 genes upregulated specifically in SPFs after CCCM stimulation. (E) Immunocytochemical α-SMA expression in SMFs after CCCM stimulation. (F) Immunocytochemical α-SMA expression in SPFs after CCCM stimulation. α-SMA expression was upregulated specifically in SPFs after CCCM stimulation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088018#pone.0088018.s002" target="_blank">Figure S2</a>).</p

Histological features of fibrosis in the cancer microenvironment formed by peritoneal invasion (CMPI).

<p>(A) Histological features of stromal component of CMPI. Both in CMPI and the cancer microenvironment formed by submucosal invasion (CMSI), plump spindle-shaped fibroblasts were major sources of the stroma. (B) Marked α-SMA expression was found in fibroblasts. (C) Higher magnification more clearly revealed plump spindle-shaped fibroblasts. (D) Using morphometric software, we successfully detected and analyzed α-SMA expression.</p