Non-invasive transcriptomic analysis using mRNAs in skin surface lipids obtained from children with mild-to-moderate atopic dermatitis.

BACKGROUND: Specimens for analysing the molecular pathology of skin disease are generally obtained through invasive methods, such as biopsy. However, less burdensome methods are desirable for paediatric patients. We recently established a method that comprehensively analyses RNA present in sebum (skin surface lipid-RNAs: SSL-RNAs) using a next-generation sequencer. Using this method, biological information can be obtained from the skin in a completely non-invasive manner. OBJECTIVES: To verify the applicability of the SSL-RNA method for analysis of paediatric skin and analyse the molecular pathology of mild-to-moderate atopic dermatitis (AD) in children. METHODS: We collected sebum specimens from the whole faces of 23 healthy children and 16 children with mild-to-moderate AD (eczema area and severity index (EASI) score: 5.9 ± 2.6) ranging in age from 6 months to 5 years, using an oil-blotting film. We then extracted SSL-RNAs from the samples and performed an AmpliSeq transcriptomic analysis. RESULTS: The expressions of genes related to keratinization (LCE, PSORS1C2, IVL and KRT17), triglyceride synthesis and storage (PLIN2, DGAT2 and CIDEA), wax synthesis (FAR2), ceramide synthesis (GBA2, SMPD3 and SPTLC3), antimicrobial peptides (DEFB1) and intercellular adhesion (CDSN), all of which are related to the skin barrier, are lower in children with AD than in healthy children. The children with AD also have higher expression of CCL17, a Th2-cytokine and an increased Th2-immune response as demonstrated by a gene set variation analysis. Moreover, KRT17 and CCL17 expression levels are significantly correlated with the EASI score. CONCLUSIONS: Molecular changes associated with abnormal immune responses and the epidermal barrier in children with mild-to-moderate AD can be determined using the SSL-RNA method. This non-invasive method could therefore be a useful means for understanding the molecular pathology of paediatric AD.

A teenage girl with green hair.

An adolescent girl with blond hair, her family, and classmates noted that her hair was progressively turning green. Initially the green color was thought to be secondary to chlorine from the local swimming pool. This was not the real cause. The chlorotrichosis was actually caused by an excessive amount of dissolved copper from copper pipes in the home plumbing system. Copper had leached from the plumbing and accumulated in the pipes because the rented house had been vacant for many months. Risk factors for chlorotrichosis include light-colored hair, copper plumbing, long periods when the water was not thoroughly flushed out of the copper pipes, and frequent shampooing.

Impact of screening with thyroid ultrasonography in myasthenia gravis patients.

OBJECTIVES: This study was conducted to screen thyroid abnormality evaluated with ultrasonography (US) in patients with myasthenia gravis (MG) and investigate further when malignancy is suspected. METHODS: Thyroid screening using US was conducted in 162 patients with MG. In cases where malignancy was suspected, further investigations were performed. RESULTS: Abnormal US findings were detected in 125 of 162 patients with MG (72 patients with nodules, 74 patients with cysts, 27 patients with diffuse findings such as enlargement, atrophy, a hypoechoic pattern or a heterogenous echoic pattern, and 28 patients with calcification). From among these 125 subjects, 30 patients underwent further examinations such as needle aspiration cytology. As a result, six patients (3.7% of 162 cases) were positive for papillary carcinoma. The size of the carcinoma in three patients was <10 mm, yet the stage of thyroid carcinomas was high (stage III or IVa) in all six cases. CONCLUSIONS: Our data suggest that the prevalence of thyroid carcinoma in cases of MG may be higher than that of the general population. Furthermore, in patients with MG, there is a possibility that the stage of the carcinoma is higher even when the carcinoma is of a very small size. Patients with MG, when diagnosed, should be advised to undergo US screening of the thyroid because most cases of thyroid carcinoma are highly curable.

Prognostic significance of AMP-activated protein kinase expression and modifying effect of MAPK3/1 in colorectal cancer.

BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.

Clock gene expression in peripheral leucocytes of patients with type 2 diabetes.

AIM/HYPOTHESIS: Recent studies have demonstrated relationships between circadian clock function and the development of metabolic diseases such as type 2 diabetes. We investigated whether the peripheral circadian clock is impaired in patients with type 2 diabetes. METHODS: Peripheral leucocytes were obtained from eight patients with diabetes and six comparatively young non-diabetic volunteers at 09:00, 15:00, 21:00 and 03:00 hours (study 1) and from 12 male patients with diabetes and 14 age-matched men at 09:00 hours (study 2). Transcript levels of clock genes (CLOCK, BMAL1 [also known as ARNTL], PER1, PER2, PER3 and CRY1) were determined by real-time quantitative PCR. RESULTS: In study 1, mRNA expression patterns of BMAL1, PER1, PER2 and PER3 exhibited 24 h rhythmicity in the leucocytes of all 14 individuals. The expression levels of these mRNAs were significantly (p < 0.05) lower in patients with diabetes than in non-diabetic individuals at one or more time points. Moreover, the amplitudes of mRNA expression rhythms of PER1 and PER3 genes tended to diminish in patients with diabetes. In study 2, leucocytes obtained from patients with diabetes expressed significantly (p < 0.05) lower transcript levels of BMAL1, PER1 and PER3 compared with leucocytes from control individuals, and transcript expression was inversely correlated with HbA(1c) levels (rho = -0.47 to -0.55, p < 0.05). CONCLUSIONS/INTERPRETATION: These results suggest that rhythmic mRNA expression of clock genes is dampened in peripheral leucocytes of patients with type 2 diabetes. The impairment of the circadian clock appears to be closely associated with the pathophysiology of type 2 diabetes in humans.

Role for cingulate motor area cells in voluntary movement selection based on reward.

Most natural actions are chosen voluntarily from many possible choices. An action is often chosen based on the reward that it is expected to produce. What kind of cellular activity in which area of the cerebral cortex is involved in selecting an action according to the expected reward value? Results of an analysis in monkeys of cellular activity during the performance of reward-based motor selection and the effects of chemical inactivation are presented. We suggest that cells in the rostral cingulate motor area, one of the higher order motor areas in the cortex, play a part in processing the reward information for motor selection.

Pathophysiology and diagnosis of spontaneous intracranial hypotension.

BACKGROUND: Spontaneous intracranial hypotension (SIH) has become a well-recognized syndrome. However, diagnosis of SIH is still challenging. The problem with SIH is that the precise mechanism of cerebrospinal fluid (CSF) leakage remains largely unknown and there is no definite diagnostic criterion in the imaging. METHODS: The clinical findings of our ten cases and 301 literature reports on SIH were investigated in a retrospective analysis to clarify the pathophysiology of CSF leakage, correlate the findings of imaging studies and determine the most adequate diagnostic criteria. RESULTS: The events precede symptoms of SIH were categorized as traumatic, secondary and strictly spontaneous (62%). The location of the spinal CSF leak remains undetectable in approximately 50% of cases reported. In 93% of patients, the CSF leakage sites were detected at the cervical or thoracic level of the spine. On recent MRI studies, 88% of patients showed spinal epidural fluid collections that most likely represent CSF leakage. MR myelography using heavily T2-weighted fast-spin-echo sequence can clearly demonstrate the site of CSF leakage. Although numerous treatment options are available, none of the treatments have been evaluated by randomized clinical trials. In 48% of papers, autologous epidural blood patch (EBP) was the treatment of choice in patients who have failed initial conservative management. Forty-nine percent of patients showed relief of symptoms after up to three repeated EBPs. CONCLUSION: We propose new diagnostic criteria of SIH to avoid misdiagnosis.

Optimization of DARTEL Settings for the Detection of Alzheimer Disease.

BACKGROUND AND PURPOSE: Although Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) has been introduced as an alternative to conventional voxel-based morphometry, there are scant data available regarding the optimal image-processing settings. The aim of this study was to optimize image-processing and ROI settings for the diagnosis of Alzheimer disease using DARTEL. MATERIALS AND METHODS: Between May 2002 and August 2014, we selected 158 patients with Alzheimer disease and 198 age-matched healthy subjects; 158 healthy subjects served as the control group against the patients with Alzheimer disease, and the remaining 40 served as the healthy data base. Structural MR images were obtained in all the participants and were processed using DARTEL-based voxel-based morphometry with a variety of settings. These included modulated or nonmodulated, nonsmoothed or smoothed settings with a 4-, 8-, 12-, 16-, or 20-mm kernel size. A z score was calculated for each ROI, and univariate and multivariate logistic regression analyses were performed to determine the optimal ROI settings for each dataset. The optimal settings were defined as those demonstrating the highest χ2 test statistics in the multivariate logistic regression analyses. Finally, using the optimal settings, we obtained receiver operating characteristic curves. The models were verified using 10-fold cross-validation. RESULTS: The optimal settings were obtained using the hippocampus and precuneus as ROIs without modulation and smoothing. The average area under the curve was 0.845 (95% confidence interval, 0.788-0.902). CONCLUSIONS: We recommend using the precuneus and hippocampus as ROIs without modulation and smoothing for DARTEL-based voxel-based morphometry as a tool for diagnosing Alzheimer disease.

Alteration of gap junction proteins (connexins) following lateral fluid percussion injury in rats.

Gap junctions are intercellular channels that mediate the cytoplasmic exchange of small hydrophilic molecules and are formed by a family of integral membrane proteins called connexins (Cxs). Cx43 is expressed predominantly in astrocytes, while Cx36 is expressed in neurons. In this study, we show alteration of Cx43 and Cx36 in the hippocampus after traumatic brain injury in rats. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion injury of moderate severity. Brain coronal sections were used for immunohistochemistry with Cx43 and Cx36 antibodies. Cx43 immunoreactivity was increased in reactive astrocytes in the damaged hippocampus 24 hours after injury, and persisted for 72 hours. On the other hand, Cx36 immunoreactivity increased in CA3 neurons 1 hour after injury, and decreased later. These results indicate that gap junctions might participate in the pathophysiological process after traumatic brain injury.

Correlation between reduction of syndecan-1 expression and clinico-pathological parameters in squamous cell carcinoma of tongue.

We examined expression of syndecan-1 in squamous cell carcinoma (SCC) of tongue using immunohistochemistry. Forty-three cases of SCC arising in lateral border of tongue were investigated. From the immunohistochemical staining pattern, the cases were divided into two groups based on expression of syndecan-1 at the supra-peripheral cells of the tumor nest: Group A, completely or mainly positive; Group B, sporadically positive or negative. Most poorly differentiated SCC cases were classified into Group B (81.8%). The number of Group B cases in T1-2 was different from that in T3-4. The number of cases where syndecan-1 expression was reduced was much greater in T3-4, and represented the majority of Group B (86.7%). More than 80% of Grade 4D cases were in Group B (83.3%) based on the Yamamoto-Kohama criteria. These results indicate that reduction of syndecan-1 correlates to histological grade, tumor size and mode of invasion in tongue SCC.

Establishment and characterization of a new human cultured cell line from a prolactin-secreting pituitary adenoma.

A new human cell line was established from a prolactin (PRL) secreting pituitary adenoma. This cell line, designated as HPA, initially produced and secreted PRL, but the ability was decreased with increasing passage number. After about 30 passages in vitro, these cells had a short doubling time (14 h) and a low plating efficiency (9%). When a minimum of 10(5) cells was injected per mouse, virtually all athymic nude mice developed a slow growing, nonmetastasizing tumor at the injection site about 30 days after injection. PRL production by the HPA cells after Day 150 was demonstrated by immunocytochemistry as well as radioimmunoassay. In addition, cimetidine (10(-4) M) had a significant stimulatory effect on PRL secretion by 4-day-cultured HPA cells. When the HPA cells were incubated in the presence of 5.0 and 10.0 nM bromocriptine, the proliferation rate was inhibited to 53.4 and 25.1% of untreated controls, respectively. On the other hand, the same concentrations of bromocriptine did not affect the proliferation rate of YK cells derived from human immature teratoma of the ovary. In addition, bromocriptine inhibited significantly the growth rate of xenotransplanted HPA but not YK cells. These results suggest that bromocriptine inhibits specifically the proliferation of HPA cells.

Insulin and glucagon secretion after pancreatectomies. Correlation of secretion and hormonal contents of remaining pancreas.

We studied whether insulin and glucagon secretory capacities change in relation to the corresponding hormonal contents in the pancreas remnant after pancreas resection. The following groups of dogs were used: sham operated (S-O), left pancreatectomized (L-Px), right pancreatectomized (R-Px), subtotal pancreatectomized (St-Px), and total pancreatectomized (T-Px). Endocrine functions were assessed by intravenous glucose tolerance test (IVGTT) and insulin tolerance test (ITT) in each dog under anesthesia before surgery and 1 wk after. In these five groups, the insulin secretory capacities, assessed as the integrated incremental secretion of immunoreactive insulin (sigma delta IRI) from the IVGTT, decreased to 95 +/- 11, 78 +/- 9, 48 +/- 8, 12 +/- 8, and -4 +/- 4% of the respective preoperative values, and these values were proportional to the percentage residual weight (100, 64 +/- 2, 35 +/- 2, 13 +/- 2, 0%) and IRI content (100, 59 +/- 4, 45 +/- 3, 10 +/- 2, 0%) of the pancreas remnant. After surgery, glucagon secretory capacity, the integrated incremental secretion of immunoreactive glucagon (sigma delta IRG) during the ITT, decreased significantly in the L-Px, St-Px, and T-Px groups but not in the R-Px group. The sigma delta IRG values as percentages of the preoperative values were 109 +/- 25, 46 +/- 11, 89 +/- 13, 19 +/- 11, and 3 +/- 3%, respectively, for the five groups. These values were proportional to the percentage residual IRG contents of the pancreas remnants (100, 48 +/- 6, 65 +/- 8, 12 +/- 2, 0%) rather than to the percentage residual pancreatic weights.(ABSTRACT TRUNCATED AT 250 WORDS)

Leptin receptor of Zucker fatty rat performs reduced signal transduction.

Zucker fatty (fa/fa) rats exhibit overt obesity, hypercholesterolemia, hyperlipidemia, and hyperglycemia as recessive traits. The fa mutation has been determined to be a missense mutation in the extracellular domain of the leptin receptor. We report herein the construction of CHO cells that stably express the fa-type leptin receptor and the characterization of this receptor using mRNA expression levels of the immediate early genes, c-fos, c-jun, and jun-B, which are induced by leptin as a criterion of signal transduction. The fa-type receptor not only exhibits a slightly reduced leptin-binding affinity, but also performs reduced signal transduction.

Poor capacity for proliferation of pancreatic beta-cells in Otsuka-Long-Evans-Tokushima Fatty rat: a model of spontaneous NIDDM.

Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat, a genetic model of spontaneous development of NIDDM, exhibits hyperglycemic obesity with hyperinsulinemia and insulin resistance similar to that in humans. It is still unclear whether a defect in the beta-cell proliferation per se is the primary pathogenetic event in OLETF rat. To determine whether it is, we used partially pancreatectomized rats as a model, with administration of phlorizin to control blood glucose level, to examine whether the capacity for proliferation of beta-cells during hyperglycemia or normoglycemia differs between OLETF and their diabetes-resistant counterparts, Long-Evans-Tokushima-Otsuka (LETO) rats. We also examined whether such a defect, if present, could be improved by nicotinamide. Male rats, 6 weeks of age, were allocated at random to two groups: 70% pancreatectomy (Px) and sham-pancreatectomy (sham). Each group was divided into four subgroups by date of killing after surgery: 3-day, 7-day, 28-day (treated with phlorizin, nicotinamide, or saline), and 91-day. A sustained hyperglycemia was evident in the Px OLETF rats after surgery, which was associated with insufficient proliferation of beta-cells, characterized by decrease in beta-cell labeling index in proportion to decrease in beta-cell mass and reduction in insulin content in the remnant pancreas. This defect was unaffected by restoration of normoglycemia induced by phlorizin injection. Administration of nicotinamide, however, ameliorated the sustained hyperglycemia by increasing beta-cell proliferation. These findings suggest that OLETF rats have poor capacity for proliferation of pancreatic beta-cells and that this change may be the critical pathogenetic event before the onset of overt diabetes.

Mechanism of hypoglycemia observed in a patient with insulin autoimmune syndrome.

A 21-year-old female patient complaining of frequent hypoglycemic attacks in the presence of a large amount of circulating insulin-binding antibodies without previous known immunization is described. In order to clarify the possible mechanism of the hypoglycemic attacks occurring in this new syndrome, changes in plasma glucose, plasma total and free immunoreactive insulin (IRI), and C peptide immunoreactivity (CPR) levels were investigated in the patient before, during, and after a three-hour glucose infusion. The character of her antibodies were also examined. An abrupt discontinuation of the glucose infusion caused a sharp decline in the plasma glucose level, reaching a nadir of 30 mg./100 nk, at 270 minutes; then she became unconscious. A huge amount of total IRI of 2,834 micron U./ml. was registered at 180 minutes, while the peak value of free IRI of 208 micronU./ml. was observed 45 minutes after the cessation of the glucose infusion. Plasma CPR was increased from high basal level, 19.6 ng./ml., to the maximum level of 29.2 ng./ml. The maximum insulin-binding capacity of IgG in the patient's serum was 6.25 mU./ml. The antibody-combining site was homogeneous, showing one high-affinity site (K: 1.1 X 10(9)M-1). Neither the prolonged fasting nor the administration of tolbutamide induced the hypoglycemic attack in the patient. The hypoglycemia may be explained by an unduly excessive amount of insulin liberated from a large pool of bound insulin irrespective of blood sugar level. The cause of the antibody production is also discussed.

Correlation between minimal secretory capacity of pancreatic beta-cells and stability of diabetic control.

The significance of the minimal secretory capacity of pancreatic beta-cells for the stability of the plasma glucose level was studied in 20 patients with insulin-dependent diabetes mellitus. Changes in plasma concentrations of major counterregulatory hormones in response to hypoglycemia were also investigated in these patients to clarify their contribution to diabetic brittleness. beta-Cell function was evaluated on the basis of elevation of plasma C-peptide immunoreactivity (CPR) during the intravenous glucagon test with a highly sensitive assay for plasma CPR that could detect as little as 0.03 ng/ml. After stimulation with glucagon, a significant increase in plasma CPR was observed in 10 of the patients whose beta-cell function had been evaluated as completely depleted by a conventional assay for plasma CPR. A clear inverse correlation was found between the secretory capacity of pancreatic beta-cells measured in this way and the degree of glycemic instability (r = -.74, P less than .01). Infusion of insulin at a rate of 0.15 U.kg-1.h-1 for 60 min caused a continuous decrease in the plasma glucose level, resulting in neuroglycopenia in 7 of the 10 CPR nonresponders but only 2 of the CPR responders. During insulin-induced hypoglycemia, plasma glucagon immunoreactivity did not increase in the CPR nonresponders but increased significantly in the CPR responders. A positive correlation was found between the minimal residual beta-cell capacity and the responsiveness of alpha-cells to hypoglycemia (r = .65, P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of exercise training and food restriction on endothelium-dependent relaxation in the Otsuka Long-Evans Tokushima Fatty rat, a model of spontaneous NIDDM.

We investigated whether endothelial function may be impaired in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of spontaneous NIDDM. The effect of exercise training and food restriction on endothelial function was also studied. OLETF rats were divided into three groups at age 16 weeks: sedentary, exercise trained, and food restricted (70% of the food intake of sedentary rats). Otsuka Long-Evans Tokushima rats were used as the age-matched nondiabetic controls. Endothelium-dependent relaxation of the thoracic aorta induced by histamine was significantly attenuated in the sedentary or food-restricted rats, and exercise training improved endothelial function. Relaxation induced by sodium nitroprusside, a donor of nitric oxide, did not differ significantly among groups. Both exercise training and food restriction significantly suppressed plasma levels of glucose and insulin and serum levels of triacylglycerol and cholesterol and reduced the accumulation of abdominal fat. Insulin sensitivity, as measured by the hyperinsulinemic-euglycemic clamp technique, was significantly decreased in sedentary rats but was enhanced in exercise-trained and food-restricted rats. The urinary excretion of nitrite was significantly decreased in sedentary and food-restricted rats compared with nondiabetic rats and was significantly increased in exercise-trained rats. These results indicate that exercise training, but not food restriction, prevents endothelial dysfunction in NIDDM rats, presumably due to the exercise-induced increase in the production of nitric oxide.

Impaired beta-cell function and deposition of fat droplets in the pancreas as a consequence of hypertriglyceridemia in OLETF rat, a model of spontaneous NIDDM.

Hypertriglyceridemia is known to be a feature of obesity-related NIDDM, but the patho-etiological significance of this association is obscure. The effects of triglycerides (TGs) on beta-cell function and morphological changes in pancreas were examined using in vivo and in vitro approaches in male OLETF rats at ages 6, 12, and 30 weeks, with their diabetes-resistant counterpart, LETO rats, as normal controls. The results showed that, in the fasting state, plasma TGs in OLETF rats were increased 2.5-fold at age 6 weeks, 3.3-fold at age 12 weeks, and 6.2-fold at age 30 weeks, compared with age-matched LETO rats. The TG content in islets from 12-week-old OLETF rats was significantly increased when compared with those from their age-matched counterparts, but this was not the case with the 6-week-old OLETF rats. Therefore, the islets from 6-week-old rats were cultured with either free fatty acids (FFAs; 1.0 mmol/l sodium oleate) or TG (5.0 mmol/l Intralipide) for 72 h. Several abnormalities in OLETF rats were evident, in contrast to the results from control LETO rats: 1) glucose-induced insulin secretion was more inhibited by either FFAs or TGs in the presence of 27.7 mmol/l glucose, a result associated, at least in part, with reduced glucokinase activity in the islets; 2) a marked elevation in TG content was found in the islets; and 3) the deposition of fat droplets in the enlarged islets, even in the beta-cells, was found by Oil Red O-insulin double staining at age 30 weeks. In conclusion, hypertriglyceridemia resulted in significant TG stores in the islets, which subsequently inhibited glucose-induced insulin secretion, at least in part, via reduced glucokinase activity in the islets. Fat droplets in islets, therefore, may play an important role in hastening the development of NIDDM in this rat model.