Yoshiya Katsura

Hepatocyte Growth Factor in Vitreous Fluid of Patients With Proliferative Diabetic Retinopathy and Other Retinal Disorders

OBJECTIVE To determine whether hepatocyte growth factor (HGF) is elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR). RESEARCH DESIGN AND METHODS Vitreous fluid samples were obtained at the time of vitreoretinal surgery from 73 eyes of PDR patients and from 17 eyes of nondiabetic patients (control subjects) who had macular hole, rhegmatogenous retinal detachment, or epiretinal membrane (9, 4, and 4 eyes, respectively) but no associated proliferative vitreoretinopathy. Stages of PDR were classified as active or quiescent. Concentrations of HGF and vascular endothelial growth factor (VEGF) in vitreous fluid were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS Intravitreous concentrations of HGF (median [range]) were significantly higher in diabetic patients with PDR (6.00 ng/ml [0.75−22.2]) than in control patients (2.86 ng/ml [0.75−5.80]). Intravitreous concentrations of VEGF were also higher in diabetic patients with PDR (1.62 ng/ml [0.15−7.9]) than in control patients (0.16 ng/ml [0.16−0.29]). Both VEGF and HGF concentrations were significantly higher in patients with active retinopathy than in those with quiescent retinopathy. However, vitreous concentrations of HGF were unrelated to those of VEGF CONCLUSIONS We found that levels of HGF in vitreous fluid of PDR patients are significantly higher than in nondiabetic patients and that the levels of HGF are elevated in the active PDR stage. This suggests that HGF stimulates or perpetuates neovascularization in PDR.

Effect of acarbose on postprandial lipid metabolism in type 2 diabetes mellitus

The effect of acarbose, an alpha-glucosidase inhibitor, on postprandial glucose and lipid metabolism was investigated in patients with type 2 diabetes mellitus. Twenty patients (10 men and 10 women) with type 2 diabetes mellitus were studied. A test meal was taken with or without 100 mg of acarbose. The levels of plasma glucose, and serum immunoreactive insulin, lipids, apolipoproteins, and remnant-like particle cholesterol were investigated. Acarbose inhibited the postprandial increase of both plasma glucose and serum immunoreactive insulin. Acarbose also significantly suppressed the increase of serum triglycerides at 60, 90, and 120 min (P < 0.05 to P < 0.01), and the increase of serum remnant-like particle cholesterol at 60 and 120 min (P < 0.05). Acarbose inhibited the postprandial decline of apolipoprotein C-II, and decreased the postprandial serum apolipoprotein C-III level. These results suggest that acarbose may improve postprandial hyperlipidemia as well as postprandial hyperglycemia in patients with type 2 diabetes mellitus.

The insulin ball

In August, 2005, a 62-year-old Japanese man, with a 20-year history of type 1 diabetes, was admitted to our hospital, for investigation of apparent insulin resistance. Since January, 2004, he had been taking insulin in a longacting (glargine) and a shortacting (lispro) form, at a total of 66 units a day. Despite the dose being gradually increased to 94 units a day, his blood glucose concen trations had subsequently been high, and poorly con trolled. Examination showed nothing of note. Blood tests gave a value for glycosylated haemoglobin (HbA1c) of 8∙6% (target value ≤6·5%). The patient’s body-mass index was 21∙1 kg/m2 (weight 57∙5 kg). Although we kept the patient on a strict diet, and increased the total daily dose of insulin to 116 units (lispro 94; glargine 22), his blood glucose concentration, before meals, remained 7∙8–21∙1 mmol/L. Blood tests showed normal concentrations of corticotropin, cortisol, thyroid hormones, glucagon, growth hormone, and carcino-embryonic antigen, and undetectable quantities of antibodies to insulin; urinary concen trations of meta nephrines were normal. Ultrasonography of the abdomen showed nothing of note. However, re-examination of the lower abdomen revealed two lumps, one on each side, under the skin; each contained a hard, irregular nodule, 3–4 cm in diameter. The patient had noticed the lumps several months previously; he routinely injected insulin into the nodules, since they were easily grasped, and injection was less painful than elsewhere. We admin istered insulin via a diff erent part of the abdomen; hypoglycaemia ensued. We rapidly decreased insulin doses. Good blood glucose concentrations were obtained with a total daily dose of 24 units (lispro 18; glargine 6). Abdominal MRI showed the nodules to lie in the fat under the skin; unlike fat, they had low signal intensity on T1-weighted (fi gure) and T2-weighted images. We took a biopsy sample of a nodule; histopathological analysis showed it to consist largely of amorphous, acellular, eosinophilic material. When the material was stained with Congo red, and seen with polarised light, we saw green birefringence, diagnostic of amyloid. On immunohistochemical testing, the amyloid deposit was positively stained by monoclonal antibodies to human insulin. When last seen, in November, 2008, the patient needed 66 units a day of insulin—injected outside the lumps, which were smaller than before. Common causes of insulin resistance include obesity; sever physical stress; prescribed drugs, such as glucocorticoids; and endocrine disorders, such as Cushing’s syndrome, phaeochromocytoma, or acromegaly. Rarer causes include mutations of the insulin receptor gene; antibodies to insulin, or to the insulin receptor; and lipodystrophy syndromes. Doctors should always consider if, and how, the patient is injecting insulin. Amyloid deposits consist of proteins arranged in a cross-β conformation: pathological amyloid deposits can be formed of various proteins, including α-synuclein in Parkinson’s disease, and prions in Creutzfeldt-Jakob disease. For amyloid to consist of insulin is rare. In all reported cases of insulin-induced amyloidosis, of which we are aware, the patient took non-human insulins: we speculate that aminoacid diff erences between endogenous and injected insulin may have contributed to the development of amyloidosis. Partly because use of nonhuman insulin is increasing, and partly because we have seen four cases recently, we speculate that insulininduced amyloidosis may be under diagnosed: for instance, by being mistaken for lipo hypertrophy. Injection into fatty lumps can reduce the eff ectiveness of insulin, though less markedly than does injection into amyloid. Amyloid lumps are typically harder, and more discrete, than fatty lumps of lipo hyper trophy; MRI can assist with diagnosis. We refer to amyloid lumps of insulin as “insulin balls”.

Basic Fibroblast Growth Factor Modulates the Surface Expression of Effector Cell Molecules and Primes Respiratory Burst Activity in Human Neutrophils

Basic fibroblast growth factor (b-FGF) mediates a variety of biological responses such as angiogenesis and hematopoiesis. We examined the effect of b-FGF on human neutrophil functions in vitro. The surface expression of effector cell molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. b-FGF increased the expression of CD11b leukocyte integrin and complement receptor type 1 on neutrophils and decreased the expression of L-selectin on neutrophils in a dose- and time-dependent manner. We also examined the effect of b-FGF on the respiratory burst activity in neutrophils. Although b-FGF alone did not induce intracellular oxidative product formation by neutrophils, it enhanced H2O2 production in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These findings suggest that b-FGF may participate in the inflammatory process via modulating the surface expression of effector cell molecules and enhancing respiratory burst activity in neutrophils.

Adsorption and preparation of human viruses using hydroxyapatite column.

The adsorption and chromatographic properties of hydroxyapatite sorbents for application to different viruses have been investigated. The strong adsorption of viruses was observed on macroporous hydroxyapatite with hydrophilic properties of the sorbent surface. The viruses were purified on this sorbent without loss of biological activity. The column can be used for virus vaccine production.

Insulin-derived Amyloidosis and Poor Glycemic Control: A Case Series

OBJECTIVES Insulin-derived amyloidosis is a rare skin-related complication of insulin therapy. The purpose of this study was to show the effects of insulin-derived amyloidosis on blood glucose levels, insulin dose requirements, and insulin absorption. METHODS Seven patients were found to have insulin-derived amyloidosis at the Tokyo Medical University Ibaraki Medical Center. The clinical characteristics and insulin therapy of the 7 patients were investigated. Insulin absorption was studied by comparing the serum insulin levels after insulin injections into insulin-derived amyloidosis sites versus injections into normal sites in 4 patients. RESULTS When the insulin-derived amyloidosis was discovered, the mean hemoglobin A1c level was 9.3%, and the mean daily insulin dose was 57 units. After changing the injection sites to avoid the insulin-derived amyloidosis, the blood glucose concentrations improved, and the mean daily insulin dose could be reduced to 27 units (P = .035; 53% reduction). The insulin absorption at insulin-derived amyloidosis sites was 34% of that at normal sites (P = .030). CONCLUSIONS Insulin-derived amyloidosis caused poor glycemic control and increased insulin dose requirements because of impairments in insulin absorption.

Increased mitochondrial uptake of rhodamine 123 by CDDP treatment.

Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5-fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells.

Effects of Combined Therapies with Protein-Bound Polysaccharide (PSK, Krestin®) and Fluorinated Pyrimidine Derivatives on Experimental Liver Metastases and on the Immunologic Capacities of the Hosts

An experimental model is introduced for the study of liver metastases using intrasplenically injected EL-4 and Lewis lung tumor cells. Fluorinated pyrimidine derivatives, 1-(2-tetrahydrofuryl)-5-fluorouracil and 5-fluorouracil, showed inhibitory effects on the frequencies of liver metastases. Immunosuppressive effects of these drugs were compared at the doses capable of showing 50% inhibition of the development of metastatic nodules. These derivatives strongly suppressed the phagocytic activity and the number of Kupffer cells of the liver and then the humoral response against sheep red blood cells, the delayed hypersensitivity against picryl chloride. On the contrary, combined administration of protein-bound polysaccharide (PSK) and 1-(2-tetrahydrofuryl)-5-fluorouracil showed no inhibitory effect on these activities.

Protein Kinase C-Mediated Regulation of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase Production in a Human Retinal Müller Cells

Purpose: Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix (ECM) proteins in the retina. Breakdown of ECM proteins results in neovascularization and tractional retinal detachment, which eventually lead to the symptoms of proliferative diabetic retinopathy. Müller cells are reported to be one of the MMP-producing cells in the retina. However, the molecular mechanism of MMP production derived from Müller cells remains to be fully elucidated. Materials and Methods: The human retinal Müller cell line (MIO-M1) was continuously-subcultured in high glucose (25 mM) condition. After the cells reached confluence, they were treated for 24 h with phorbol ester and/or a protein kinase C (PKC) inhibitor, GF109203X in high (25 mM) or low (5 mM) glucose condition. Gelatinase activities in conditioned medium were assessed using gelatin zymography. RT-PCR was performed to analyze the mRNA expression level of MMP-9. Western blot analysis used to detect the protein expression of tissue inhibitors of metalloproteinases (TIMPs). Electrophoresis mobility shift assay was conducted to examine transcription factors involved in MMP-9 transcription. Results: We demonstrated the protein kinase C (PKC)-mediated regulation of proMMP-9 transcription and protein expression through the action of phorbol ester, an activator of PKC. Moreover, we demonstrated the expression of TIMPs, known as natural inhibitors of MMPs at the protein level in a human retinal Müller cell line for the first time, and report that production of these proteins was also regulated in a PKC-dependent manner. Conclusion: Our results suggest that imbalance between MMP and TIMP proteins may promote neovascularization by PKC activation in retinal Müller cells. In addition, the development of novel compounds with regulatory action on MMP and TIMP production through inhibiting PKC activity in retinal Müller cells may lead to new therapeutic approaches for the treatment and prevention of diabetic retinopathy.

AIDS-like disproportion of minor T-cell subsets in Japanese patients with Wegener's granulomatosis

Fifteen Japanese patients with Wegeners granulomatosis (WG) were evaluated according to their lymphocyte subset abnormalities. Two colour immunofluorescent flow cytometry was used to distinguish the lymphocyte subset alterations. WG group showed a decrease in the percentage of CD4+ cells and the increase of CD8+ cells. Within the NK cell family, the functionally unidentified CD8+57+ cells were markedly elevated. The disproportion of the lymphocyte subsets (CD4+ decreases CD8+57+ increases) were similar to those of Acquired Immunodeficiency Syndrome (AIDS) and AIDS relating complexes (ARC). To assess silent infection of Human immunodeficiency virus (HIV), the polymerase chain reaction (PCR) was done for all patients to selectively amplify specific HIV proviral DNA sequences in peripheral blood mononuclear cells, HIV-1 proviral DNA was not found in any patients but these changes of WG might suggest a possible adaptive response to unknown viral infection.