Masayuki Noritake

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.

Heart rate variability in patients with diabetes mellitus, ischemic heart disease, and congestive heart failure

The prognosis of patients with heart disease and prediction of sudden cardiac death can be assessed through heart rate variability, an indirect measure of abnormal autonomic control. The authors have evaluated the heart rate variability by 24-hour ambulatory electrocardiographic monitoring in 25 diabetic patients, 19 ischemic heart disease patients, 18 congestive heart failure patients, and 10 normal subjects. Thirteen diabetic patients had autonomic neuropathy and 12 patients did not. Heart rate variability index (mean SD) in patients with diabetes mellitus, ischemic heart disease, and congestive heart failure was significantly lower (34.5 +/- 12.6 ms, 43.7 +/- 15.4 ms, and 34.6 +/- 15.8 ms vs 65.6 +/- 16.7 ms, p less than 0.05) than that of normal subjects. Mean SD was significantly lower in patients with autonomic neuropathy as compared to patients without autonomic neuropathy (26.4 +/- 6.5 ms vs 44.2 +/- 11.0 ms, p less than 0.05) mean SD as compared to survivors: 49 +/- 7 ms in patients with mild ischemic heart disease, 48 +/- 15 ms in patients with severe ischemic heart disease, and 23 +/- 7 ms in patients who died. Similarly, the mean SD in 4 congestive heart failure patients who died was lower significantly (p less than 0.05) than in those who survived (19.0 +/- 5.6 ms vs 40.0 +/- 14.5 ms). Among congestive heart failure patients, clinical improvement by therapy was associated with a significant increase in mean SD. When the mean SD of 30 ms was used as the cutoff point for detection of autonomic dysfunction or patient death, specificity exceeded 90% and sensitivity was 75%.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes

Although the standard assays for reactive oxygen species have been based on the measurement of those released into the extracellular environment, the microbicidal capacity to the engulfed microorganisms is mainly dependent on those released into the intracellular environment, such as phagosomes. We studied intracellular oxidative activities of individual phagocytes by dichlorofluorescein (DCFH) oxidation assay to investigate the relationship between the reactive oxygen species released intracellularly and the impaired microbicidal capacity in diabetic patients. Time courses of intracellular production of hydrogen peroxide by polymorphonuclear leucocytes (PMNL) and monocyies were observed at the resting condition and after the stimulation with phorbol myristale acetate (PMA: 160 nm) by flow cytometry. Thirty‐four patients with non‐insulin‐dependent diabetes mellitus (NIDDM) and 23 age‐matched healthy volunteers were subjected to the studies. PMNL from patients with NIDDM showed a significantly decreased capacity to produce hydrogen peroxide after the stimulation (P < 0·05 at 15 min, P < 0·01 at 30 and 45 min). By contrast, inlracellular hydrogen peroxide production by monocytes at the resting condition and an early stimulatory phase (8 min after the stimulation) was significantly (P< 0·01) enhanced in patients with NIDDM compared with that in controls. Both the changes of inlracellular hydrogen peroxide production observed in PMNL and monocytes from patients with NIDDM were in association with an increased haemoglobin Alc level in crythrocytes. but did not relate to total cholesterol and triglyceride levels in the serum. The possible mechanisms of these dissociated changes in hydrogen peroxide producing capacity of phagocytes from patients with NIDDM are discussed.

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”.

Somatosensory Conduction Delay in Central and Peripheral Nervous System of Diabetic Patients

Fifty-four diabetic patients with or without clinical evidence of neuropathy and with no clinical evidence of CNS dysfunction were studied by somatosensory-evoked potentials after electrical stimulation of the median nerve at the wrist and recorded from the scalp electrode against a noncephalic reference. Peripheral conduction index, calculated as the distance from the wrist to the C7 spinous process divided by the P9 latency, was significantly decreased (P <0.01) in diabetic patients (69.81 ± 6.47 m/s) compared with 28 age-matched nondiabetic subjects (76.85 ± 5.65 m/s). The P11–P13 interpeak latency, representative of the transit time from the dorsal column at the level of the sensory input into the cervical cord to the brain stem along the somatosensory pathways (CCT1), and the P13-N19 interpeak latency, representative of the transit time from the brain stem to the somatosensory cortex (CCT2), were significantly increased in diabetic patients (CCT1, 2.51 ± 0.63 ms; CCT2, 5.76 ± 0.92 ms) compared with nondiabetic subjects (CCT1, 2.28 ± 0.36 ms, P <0.05; CCT2, 5.18 ± 0.51 ms, P <0.01). We conclude that, in diabetic patients, neurophysiological abnormalities may be present in two distinct parts of the CNS and the peripheral nervous system.

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.

Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp.

BACKGROUND To determine myo-inositol more accurately, we improved the enzymatic cycling method. METHODS We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol. We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration. RESULTS The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l. Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively. The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr). From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively. With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively. CONCLUSIONS The myo-inositol measurement method demonstrated high specificity and yielded accurate results. The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT.

Therapeutic effect of granulocyte colony‐stimulating factor and cephem antibiotics against experimental infections in neutropenic mice induced by cyclophosphamide

Effects of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) against severe infections in cyclophosphamide (CY)‐induced neutropenic mice were investigated by its single use or by its combination with cephem antibiotics. Treatment with rhG‐CSF increased the number of peripheral blood leucocytes and strikingly shortened the duration of the neutropenic state. Most of the regained population in the peripheral blood leucocytes were neutrophils. Functions of neutrophils, such as phagocytic activity, superoxide release, and expression of Mac‐1 molecules, were remarkably enhanced by administration of rhG‐CSF. When rhG‐CSF was administered to CY‐induced neulropenic mice before infection, protective effects against various kinds of bacteria were remarkable. On the other hand, such remarkable effects were not observed when rhG‐CSF was administered after infections. However, even in the case of post‐infectious treatment, a combination therapy of rhG‐CSF with cephem antibiotics, particularly with Cefodizime (CDZM), showed a significant improvement in the survival rate and a decrease in the number of viable bacteria in the liver. These results suggest that a combination therapy of rhG‐CSF with cephem antibiotics, especially with CDZM, is an efficient regime against severe infections in patients with ncutropenia induced by a heavy anti‐tumour chemotherapy.