Renal thrombotic microangiopathy and nephrotic proteinuria induced by intravitreal injection of aflibercept for diabetic macular edema.

BACKGROUND: Vascular endothelial growth factor inhibitors (VEGFIs) are used to treat malignant neoplasms and ocular diseases by inhibiting angiogenesis. Systemic use of VEGFIs has various side effects, including hypertension, proteinuria, and thrombotic microangiopathy, but adverse events due to intravitreal injection of VEGFIs have not been fully clarified. Although age-related macular degeneration was initially the most common target of intravitreal injection of VEGFIs, it has also been applied sporadically for diabetic macular edema in recent years. Proteinuria following intravitreal injection of VEGFIs would be reversible. In patients with diabetes mellitus (DM), however, it would be difficult to determine whether kidney damage arises from the clinical course of DM or from intravitreal injection of VEGFIs for diabetic macular edema. CASE PRESENTATION: A 55-year-old woman with a 20-year history of type 2 DM began intravitreal injection of VEGFI (aflibercept, 2 mg every 4 weeks) for treatment of diabetic macular edema 2 years previously. She presented with leg edema, hypertension, and nephrotic-range proteinuria 14 months after the first injection. Histological examination of renal biopsy specimens revealed diabetic nephropathy with renal thrombotic microangiopathy probably associated with intravitreal injection of VEGFI. The patient's nephrotic syndrome completely improved at 6 months after simply discontinuing aflibercept. CONCLUSIONS: This is a precious report of pathologically investigated renal thrombotic microangiopathy leading to nephrotic syndrome due to intravitreal injection of aflibercept for diabetic macular edema in a patient with type 2 DM. Renal function and proteinuria should be monitored in diabetic patients who receive intravitreal injection of a VEGFI. If kidney damage develops independent of the clinical course of DM during intravitreal injection of a VEGFI, renal biopsy should be performed and intravitreal VEGFI injection discontinued.

Fluvastatin exerts an antitumor effect in vemurafenib-resistant melanoma cells.

Although vemurafenib has been shown to improve the overall survival of patients with metastatic melanoma harboring the BRAF V600E mutation, its efficacy is often hampered by drug resistance acquired within a relatively short period through several distinct mechanisms. In the present study, we investigated the effect of fluvastatin as a possible strategy to overcome such acquired resistance using a cultured cell line model. We established vemurafenib-resistant (VR) cells from three BRAF (V600E)-mutated melanoma lines (C32, HMY-1, and SK-MEL-28) and evaluated the mechanism of acquired resistance of VR cells by water-soluble tetrazolium salts assay, western blot, real-time quantitative PCR, and immunofluorescent microscopy. The efficacy of the combination of growth inhibitory effect of vemurafenib and fluvastatin on respective parental and VR cells were assessed by calculating combination index and western blot. IC50 values of three VR cells were ~5-100-fold higher than those for the respective parental cells. The VR cells derived from HMY-1 and SK-MEL-28 showed constitutive activation of AKT kinase, and the specific AKT inhibitor MK-2208 or the PI3K inhibitor wortmannin increased the cellular sensitivity to vemurafenib. Intriguingly, application of a statin-related drug, fluvastatin, also resulted in a synergistic increase of sensitivity to vemurafenib in the VR cells (combination index: 0.73-0.86) probably by alleviating constitutive AKT activation, whereas the same treatment did not notably alter the vemurafenib sensitivity of the parental cells. Our results suggest the possible usefulness of statin-related drugs for overcoming vemurafenib resistance acquired through constitutive activation of the PI3K-AKT axis.

[Adult-onset Henoch-Schönlein purpura mimicking infectious enteritis].

A 37-year-old male with chief complaints of vomiting, abdominal pain, and diarrhea presented to our hospital in June 2017. A blood test detected increased inflammatory response, and a computed tomography scan showed that wall thickening extended from the terminal ileum to the entire large intestine. Bacterial enteritis was suspected because his household members developed infectious enteritis; however, his symptoms did not improve after antibiotic treatment. High fever and peritoneal signs were observed on the 10th day of admission, and palpable purpura appeared on the lower extremities. The patient was administered methylprednisolone because Henoch-Schönlein purpura was also suspected. Subsequently, his symptoms improved, and the purpura disappeared.

Nucleus accumbens associated 1 is recruited within the promyelocytic leukemia nuclear body through SUMO modification.

Nucleus accumbens associated 1 (NACC1) is a cancer-associated BTB/POZ (pox virus and zinc finger/bric-a-brac tramtrack broad complex) gene, and is involved in several cellular functions in neurons, cancer and stem cells. Some of the BTB/POZ proteins associated with cancer biology are SUMOylated, which appears to play an important role in transcription regulation. We show that NACC1 is SUMOylated on a phylogenetically conserved lysine (K167) out of three consensus SUMOylation motif sites. Amino acid substitution in the SIM sequence (SIM/M) within the BTB/POZ domain partially reduced K167 SUMOylation activity of NACC1. Overexpression of GFP-NACC1 fusion protein leads to formation of discrete nuclear foci similar to promyelocytic leukemia nuclear bodies (PML-NB), which colocalized with SUMO paralogues (SUMO1/2/3). Both NACC1 nuclear body formation and colocalization with SUMO paralogues were completely suppressed in the GFP-NACC1-SIM/M mutant, whereas they were partially maintained in the NACC1 K167R mutant. Confocal immunofluorescence analysis showed that endogenous and exogenous NACC1 proteins colocalized with endogenous PML protein. A pull-down assay revealed that the consensus motifs of the SUMO acceptor site at K167 and the SIM within the BTB/POZ domain were both necessary for efficient binding to PML protein. Our study demonstrates that NACC1 can be modified by SUMO paralogues, and cooperates with PML protein.

Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro.

Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.

[Health and garden connections between Japan and New Zealand: the impact of Bella and Frederic Truby King's visit to Japan in 1904].

Through the case-study of the visit of a prominent New Zealand medical reformer and his wife to Japan in 1904, this article examines new aspects of the health and environmental connections between Japan and New Zealand in the early twentieth century. At one level, the article analyses the broader context of interest in Japanese plants in New Zealand and the model of Japanese health reforms constituted by these connections. At another, it argues that subjects previously considered separate--such as modem health reform, scientific agriculture and gardening, and Japanese and New Zealand intellectual influences--need to be considered together as contemporaries understood them. Doing so, it suggests, enables the more accurate consideration of the intellectual and scientific worlds of the early twentieth century and hints at the global dimensions of aspects of thought and state and societal reform associated with modernity.

Immunohistochemical study of HER2 and TUBB3 proteins in extramammary Paget disease.

Metastatic extramammary Paget disease (EMPD) is a potentially fatal malignancy for which effective chemotherapy and good biomarkers are desirable for management. We investigated the status of human epidermal growth factor receptor (HER2) and neuronal ß-tubulin isotype (class III ß-tubulin; TUBB3), whose overexpression is a factor involved in resistance of tumor cells to taxane derivatives) in 32 patients with EMPD. HER2 status was evaluated by immunohistochemistry followed by fluorescence in situ hybridization, and TUBB3 status was evaluated by immunohistochemistry. On the basis of the US Food and Drug Administration-approved criteria, 20 (63%) of the 32 EMPD tumors were found to overexpress HER2. Positive immunoreactivity for TUBB3 was observed in 7 (22%) of the 32 patients. Although some clinicopathologic variables (nodule formation, depth of tumor cells, presence of lymph node metastasis, and serum carcinoembryonic antigen level) were significantly associated with disease outcome (P < 0.05), HER2 gain or aberrant TUBB3 expression showed no significant correlation. However, the higher incidence of HER2 gain and the relatively lower incidence of aberrant TUBB3 expression suggested that HER2-targeted immunotherapy combined with taxane derivatives is warranted for metastatic EMPD, and that HER2 and TUBB3 status might be a good biomarker for determining the most appropriate therapeutic modality.

Detection of human papillomavirus in a urothelial carcinoma mimicking urethral caruncle.

Primary carcinoma of the female urethra is an uncommon diagnosis, accounting for less than 0.02% of all carcinomas in women. Urothelial carcinomas occupying the distal urethra in young females are considered to be extremely rare. Here we report what we believe to be the sixth case of primary urothelial carcinoma in the published English-language literature. The patient, a 26-year-old woman, presented with a distal urethral lesion that resembled a caruncle, but which was proved to be a urothelial carcinoma on histopathological examination of the resected specimen. Human papillomavirus type 51 DNA was detected in the tumor by polymerase chain reaction and in situ hybridization. These findings suggest that human papillomavirus might be involved in a subset of urothelial carcinomas of the urethra.

Sexual Difference in the Optimum Environmental Conditions for Growth and Maturation of the Brown Alga Undaria pinnatifida in the Gametophyte Stage.

Undaria pinnatifida is an annual brown kelp growing naturally in coastal areas as a major primary producer in temperate regions and is cultivated on an industrial scale. Kelps have a heteromorphic life cycle characterized by a macroscopic sporophyte and microscopic sexual gametophytes. The sex-dependent effects of different environmental factors on the growth and maturation characteristics of the gametophyte stage were investigated using response surface methodology. Gametophytes were taken from three sites in Japan: Iwate Prefecture, Tokushima Prefecture, and Kagoshima Prefecture in order to confirm the sexual differences in three independent lines. Optimum temperature and light intensity were higher for males (20.7-20.9 °C and 28.6-33.7 µmol m-2 s-1, respectively) than females (16.5-19.8 °C and 26.9-32.5 µmol m-2 s-1), and maturity progressed more quickly in males than females. Optimum wavelengths of light for growth and maturation of the gametophytes were observed for both blue (400-500 nm, λmax 453 nm) and green (500-600 nm; λmax 525 nm) lights and were sex-independent. These characteristics were consistent among the three regional lines. Slower growth optima and progress of maturation could be important for female gametophytes to restrict fertilization and sporophyte germination to the lower water temperatures of autumn and winter, and suggest that the female gametophyte may be more sensitive to temperature than the male. The sexual differences in sensitivity to environmental factors improved the synchronicity of sporeling production.

A case of bone marrow involvement in sarcoidosis with crescentic glomerular lesions.

Renal and bone marrow involvements in sarcoidosis are rare. We experienced the case of a 67-year-old man with systemic sarcoidosis, with bone marrow involvement, hepatic involvement and a unique constellation of renal lesion with cellular crescent formation. Immunosuppressive therapy was helpful for maintaining the stability of his pancytopenia, hepatic function and renal function. To the best of our knowledge, the association between sarcoidosis, bone marrow involvement and crescentic glomerulonephritis has been reported in only few cases in literature.

DNA hypomethylation at the CpG island is involved in aberrant expression of the L1 cell adhesion molecule gene in colorectal cancer.

The L1 cell adhesion molecule (L1CAM) has been identified as a target gene of beta-catenin-TCF signaling in colorectal cancer (CRC) and associated with aggressive tumor behavior such as invasion and metastasis. We investigated the methylation status at the L1CAM gene promoter and/or L1CAM mRNA/protein expression in 4 CRC cell lines and 71 primary CRCs. Aberrant L1CAM expression was immuno histochemically observed in 31 (43.7%) of 71 cases, and correlated with advanced stage and presence of lymph node and distant metastases (P<0.05). Treatment with a demethylating agent induced L1CAM mRNA/protein expression in two cell lines lacking L1CAM expression. Bisulfite-modified genome sequencing suggested that DNA methylation status at core promoter and putative TCF-binding sites within the L1CAM promoter was correlated with L1CAM mRNA/protein expression in 4 CRC cell lines. Using the crypt isolation followed by bisulfite-modified genome sequencing and methylation-specific PCR methods, we confirmed that the DNA hypomethylation at core promoter and putative TCF-binding sites was well correlated with the aberrant L1CAM protein expression in primary CRC samples. These results suggest that DNA hypomethylation at the L1CAM CpG islands might induce L1CAM aberrant expression and contribute to the acquisition of aggressive tumor behavior in CRC.

Expression of mucosal addressin cell adhesion molecule-1 on the reticular framework between white pulp and the marginal zone in the human spleen.

The antigenic heterogeneity of the reticular framework of the white pulp and marginal zone is well documented in the human adult spleen. Immunostaining of α-smooth muscle actin characterizes the heterogeneity of the reticular framework of the white pulp and marginal zone. In the human spleen, the blood cells flow in an open circulation. T and B lymphocytes flow out from the arterial terminal, and migrate in the reticular framework. Homing of lymphocytes to lymphoid tissues is regulated by selective interactions between cell surface homing receptors and tissue vascular addressins at sites of lymphocyte recruitment from the blood. In the present study, mucosal addressin cell adhesion molecule-1 was selectively expressed on α-smooth muscle actin-positive reticular framework. The reticular framework may function in lymphocyte homing and segregation into the periarteriolar lymphoid sheath, lymph follicle and marginal zone.

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines.

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem-loop-mediated reverse transcription real-time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR-138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR-138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR-138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = -0.392, P = 0.148). We demonstrated that overexpression of miR-138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR-138 and the hTERT 3'-untranslated region by luciferase reporter assay. These results suggest that loss of miR-138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma.

Epigenetic modification is involved in aberrant expression of class III beta-tubulin, TUBB3, in ovarian cancer cells.

Aberrant expression of class III beta-tubulin, TUBB3, has been reported to be one of the important mechanisms responsible for taxane resistance in diverse human malignancies. We investigated aberrant TUBB3 expression and its epigenetic modification in 66 primary tumors and 3 cell lines (OVCAR-3, JHOC-5 and JHOC-8) of ovarian cancers. Overexpression of TUBB3 protein was observed in 56 (85%) of the 66 ovarian cancers, and was significantly associated with aggressive tumor behavior (advanced stage, presence of ascites, suboptimal cytoreduction at surgery and presence of lymph node metastasis) (P<0.05). Responses to treatment with a demethylating agent (5-aza-2'-deoxycytidine, 5-Aza-CdR) and a histone deacetylase inhibitor (4-phenylbutyric acid, PBA) differed among the ovarian cancer cell lines. In 2 cell lines with weak expression of TUBB3 protein (OVCAR-3 and JHOC-8), TUBB3 induction was independently induced by treatment with 5-Aza-CdR (JHOC-8) or PBA (OVCAR-3), while neither agent markedly altered TUBB3 mRNA/protein expression in a strongly TUBB3-expressing cell line (JHOC-5). A CpG island within intron 1 was hypermethylated in 1 cell line (JHOC-8) that expressed TUBB3 weakly and required 5-Aza-CdR treatment for gene expression. A CpG island of another cell line showing faint expression of TUBB3 protein (OVCAR-3), in which a significant gain of TUBB3 expression was induced by treatment with PBA but not with 5-Aza-CdR, was hypomethylated, similarly to a cell line (JHOC-5) showing constitutive expression of TUBB3. We evaluated methylation status in this region in 14 primary tumors using methylation-specific PCR, but there was no significant relationship with TUBB3 immunoreactivity. These findings suggest that aberrant expression of TUBB3 protein might be associated with aggressive behavior of ovarian cancers, and that epigenetic modulation (DNA methylation and chromatin acetylation) might be partly involved in TUBB3 expression.

Angiotensin II and epidermal growth factor receptor cross-talk mediated by a disintegrin and metalloprotease accelerates tumor cell proliferation of hepatocellular carcinoma cell lines.

AIM: The cross-talk pathway between angiotensin II (AngII) and the epidermal growth factor receptor (EGFR) mediated by epidermal growth factor (EGF)-like ligands cleaved by a disintegrin and metalloprotease (ADAM) has been elucidated in several cell types. Even though the liver is a representative angiotensinogen-producing organ, such cross-talk has never been elucidated in hepatocellular carcinomas (HCCs). We investigated whether AngII exerted a mitogenic effect on HCC cell lines through the AngII-EGFR cross-talk pathway. METHODS: We determined the expression and/or phosphorylation status of AngII receptor type 1 (AGTR1), ADAM9, ADAM17, ERK1/2, STAT3, AKT and EGFR in five HCC cell lines using Western blotting. Proliferation and invasion activities were measured by ATP and Matrigel invasion assays, respectively. RESULTS: AGTR1 was expressed ubiquitously in HCC cell lines. EGFR expression in HepG2 was relatively weaker than that in the remaining HCC cell lines. The phosphorylation status of EGFR, ERK1/2, STAT3 and AKT was upregulated by AngII treatment in two EGFR-overexpressing cell lines (Huh7 and PLC/PRF/5), but not in HepG2 (showing weak EGFR expression). AngII stimulation significantly accelerated proliferation and invasion activities in Huh7 and PLC/PRF/5, and was inhibited by pretreatment with an ADAM inhibitor. A selective AGTR1 blocker significantly repressed proliferation activity in both cell lines, but did not significantly repress the invasion activity. Both chemical agents and neutralizing antibodies against ADAMs (ADAM9 and ADAM17) and EGF-like ligands suppressed EGFR transactivation and/or subsequent phosphorylation of ERK1/2, STAT3 and AKT. CONCLUSION: These results suggest that AngII-EGFR cross-talk signaling mediated by ADAMs is involved in the proliferation and invasion activities of several HCC cell lines.

Note: Design and resonant condition measurement of the mushroom-shaped Al test cavity for critical magnetic field evaluation of superconducting thin-film sample.

We designed a mushroom-shaped Al test cavity for measurement of the critical magnetic field at a radio frequency microwave with a frequency of 5.2 GHz. The characteristics of the Al test cavity are characterized toward the evaluation of the superconducting multilayer thin films under high-power radio frequency microwaves at cryogenic temperatures by the Nb-based cavity. We evaluated a target resonant frequency, the separations of neighboring modes, and the electromagnetic field distribution of the target mode. The calculated frequency change was in good agreement with that obtained experimentally.

Effect of Liposomes with Different Double Arms Polyethyleneglycol on Hepatic Metastasis Model Mice and Evaluation Using a Fluorescent Imaging Device.

BACKGROUND: We have previously reported the synthesis of a novel polyethyleneglycol (PEG) lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DDA-PEG). This study aimed to clarify the anti-metastatic effect and localization of DDA-PEG-modified liposomes on a murine hepatic metastasis model. METHODS: M5076 ovarian sarcoma cells were inoculated for hepatic metastasis model mice. The accumulation of liposomes in the tumor and metastatic sites was detected by fluorescent imaging device. In metastasis study, doxorubicin (DOX) loaded DDA-PEG-modified liposome (DDA-LDOX) was injected. Alexa Fluor 790 NHS Ester loaded DDA-PEG-modified liposomes were used to detect fluorescence intensity at metastatic sites when visualized topically using a fluorescence imaging device. RESULTS: DDA-PEG-modified liposomes accumulated at the sites of hepatic metastasis but not in the normal hepatocytes. Furthermore, the DDA-LDOX inhibited metastasis in this model. The survival time of M5076 ovarian sarcoma bearing mice in DDA-LDOX group was longer than those in control, DOX solution and the other PEG-modified liposomal DOX groups, and the survival ratio in DDALDOX group remained 66.7% until 60 days after treatment. CONCLUSION: It is expected that the DDA-PEG-modified liposomes will extensively contribute in clinical practice as a superior drug carrier because this liposomes proved to be effective against metastasis.

A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance.

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5'-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.