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.

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.

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.

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.

Osteopontin as biomarker in early invasion by squamous cell carcinoma in tongue.

BACKGROUND: Osteopontin (OPN) expression in squamous cell carcinoma (SCC) of the tongue has not been clearly elucidated. METHODS: We selected 46 cases of tongue SCC and investigated the expression of OPN by immunohistochemical staining. The immunopositive reaction and score for each case were semiquantitatively evaluated. RESULTS: Scores were significantly higher in carcinoma nests than in neighboring normal epithelium or epithelial dysplasia. The OPN was expressed clearly in the cytoplasm of carcinoma cells. In cases of early invasive carcinoma, in particular, expression of OPN showed a remarkable increase at the invasion front compared with the non-invaded regions. However, there was no significant correlation between expression of OPN in the primary tumor nest and lymphatic metastasis, recurrence, or survival rate. CONCLUSION: This suggests that OPN is a useful biomarker of early invasion by SCC in tongue.

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.

Magnetic resonance imaging and computed tomography in the diagnoses of brain metastases of lung cancer.

We evaluated the usefulness of double-dose (0.2 mmol/kg of gadoteridol) contrast-enhanced magnetic resonance imaging (C-E MRI) in detecting brain metastases of lung cancer. We prospectively enrolled 134 patients with lung cancer who had no neurologic symptoms and who underwent a staging work-up. Patients were assigned to receive both contrast-enhanced computerized tomography (C-E CT) and double-dose C-E MRI. Double-dose C-E MRI detected brain metastases in 19 patients, while C-E CT detected brain metastasis in only 12 of the 19 (P = 0.02). The 3-month survival rate for patients in double-dose C-E MRI group was found to be 2.06 times that of patients in a C-E CT group (P = 0.029), although the survival rate fell to 1.45 (P = 0.387) at 6 months. The results imply that double-dose C-E MRI changed the clinical stage of lung cancer patients. We concluded that double-dose C-E MRI improves the rate of detection of brain metastases during the initial staging of lung cancer.

Hydrostatic brain edema: basic mechanisms and clinical aspect.

The term hydrostatic brain edema results from unfavorable hydrtostatic pressure gradiernts between blood vessels and brain tissue. Arterial hypertension combined with decompressive craniectomy produces extensive brain edema in the arterial boundary zone of the decompressive area. The increased hydrostatic pressure gradient enhances tissue damage and causes the biphasic opening of the blood-brain barrier (BBB). The hydrostatic pressure alone is capable of causing the initial BBB opening and this induced edema results in derangement of cerebral microcirculation and metabolism. With morphological opening and metabolic damage of the vascular wall, the second BBB opening is elicited by the amplified hydrostatic pressure gradient, which is similar to that in vasogenic edema. In the clinical aspect, the beneficial effect of decompressive craniectomy in the treatment of uncontrollable ICP and brain edema remains controversial. External decompression may have adverse effects on severe brain edema and swelling. In this regards, control of driving force for the formation of brain edema could be the treatment of choice as an initial step. In addition, recent reports provided by MR imaging indicated new information on the pathophysiological features of the patients with acute hypertension. In the patients with hypertension due to reversible posterior leucoencephalopathy (RPL) syndrome, MR images show reversible signal abnormalities in the bilateral occipital lobes, suggesting hydrostatic brain edema.

Extracellular superoxide dismutase following cerebral ischemia in mice.

We describe the changes in extracellular superoxide dismutase (EC-SOD) following cerebral ischemia in mice. Mice were subjected to transient forebrain ischemia and reperfusion. The measurements of EC-SOD using ELISA showed increased brain EC-SOD after 24 h of reperfusion. The immunohistochemical examination showed that EC-SOD immunoreactivity in cortical and striatal capillary wall was conspicuous after 3 h. EC-SOD immunoreactivity was also noted in cortical neurons after 24 h. Northern blot analysis showed an increased EC-SOD mRNA expression in the brain after 24 h. In situ hybridization study demonstrated no mRNA expression of EC-SOD following ischemia and reperfusion in the capillary wall. These findings suggest that serum EC-SOD might accumulate on brain endothelial cells, while cortical neurons produce EC-SOD themselves after cerebral ischemia with reperfusion.

A role of glial fibrillary acidic protein in hippocampal degeneration after cerebral trauma or kainate-induced seizure.

Astrocytes perform a variety of functions in the adult central nervous system (CNS). Recent evidence suggests the robust upregulation of glial fibrillary acidic protein (GFAP) after CNS insult. However, little is known about the role of GFAP in the hippocampal degeneration after brain injury. We herein compared the GFAP knockout (KO) and wild type (WT) mice on the histological and behavioral outcome in response to cerebral trauma or kainic acid (KA)-induced seizure. Although all KO mice showed hippocampal CA3 neuronal degeneration. WT mice did not show any neuronal degeneration in CA3 subfield at 72 hrs after trauma. Thereafter, KO mice showed a higher susceptibility to KA-induced seizures and an increased number of pyknotic CA3 neurons 72 hrs after KA administration. These results indicate that GFAP plays a crucial role in the hippocampal neurodegeneration after CNS insult.

Mitogen-activated protein kinases phosphorylation in posttraumatic selective vulnerability in rats.

Mitogenic stimulation of the Mitogen-activated protein kinase (MAPK) pathway modulates the activity of many transcriptional factors leading to biological responses. Of these, three MAPK cascades are well characterized as extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 pathways. The aim of this study was to investigate the topographic distribution and the role of activated MAPK pathways after fluid percussion injury (FPI) in rats. In the present results, FPI significantly induced ERK- and JNK-phosphorylation, but not p38-phosphorylation in the cortex and hippocampus at the injury site. The immunoreactivity for phospho-ERK was localized in the superficial neuronal layers, dentate hilar neurons, and the damaged CA3 neurons after 30 mins of FPI. Double immunostaining showed that phospho-ERK was prominent in astrocytes 6 hrs after TBI. The current results suggest that MAPK pathways are involved in signal transduction after FPI.

Focal brain edema and natriuretic peptides in patients with subarachnoid hemorrhage.

Central salt wasting syndrome may be caused by pathological increases in serum natriuretic peptides after subarachnoid hemorrhage (SAH). However, it is unclear as to why the serum concentration of atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) increases in the subacute phase of SAH. The present study was designed to assess the correlation between focal brain edema and serum concentration of ANP or BNP in patients with SAH. Focal brain edema was found in 8 SAH-patients and peaked between days 4 and 7 of SAH. The mean serum ANP and BNP levels in patients with focal brain edema were significantly higher than those in patients without focal brain edema between days 4 and 14 of SAH. These results suggest that focal brain edema might correlate with increased levels of ANP and BNP in the subacute phase of SAH.

Vascular endothelial growth factor expression in pituitary adenomas.

Vascular endothelial growth factor (VEGF) is known to be a mediator of angiogenesis and vascular permeability. A cystic component and hemorrhage are often found in pituitary adenomas. In the present study we assess the VEGF expression based on immunohistochemical examinations in 48 pituitary adenomas. All the adenomas showed some VEGF immunoreactivity mainly in the cytoplasm of tumor cells. Of the 48 adenoma-cases, 16 cases had a strong VEGF immunoreactivity, 26 cases had a moderate one, and 6 cases had a weak one. On the MR images, a cystic component was found in 16 cases (33.3%), and a hemorrhage was found in 18 cases (37.5%). The VEGF immunoreactivity had a significant relationship with the cystic component but neither the hemorrhage, size, recurrence, or HE classification. These findings suggest that VEGF might play a potential role in the pathogenesis of cystic formation in pituitary adenomas.