Imaging and lipidomics methods for lipid analysis in metabolic and cardiovascular disease.

Cardiometabolic diseases exhibit changes in lipid biology, which is important as lipids have critical roles in membrane architecture, signalling, hormone synthesis, homoeostasis and metabolism. However, Developmental Origins of Health and Disease studies of cardiometabolic disease rarely include analysis of lipids. This short review highlights some examples of lipid pathology and then explores the technology available for analysing lipids, focussing on the need to develop imaging modalities for intracellular lipids. Analytical methods for studying interactions between the complex endocrine and intracellular signalling pathways that regulate lipid metabolism have been critical in expanding our understanding of how cardiometabolic diseases develop in association with obesity and dietary factors. Biochemical methods can be used to generate detailed lipid profiles to establish links between lifestyle factors and metabolic signalling pathways and determine how changes in specific lipid subtypes in plasma and homogenized tissue are associated with disease progression. New imaging modalities enable the specific visualization of intracellular lipid traffic and distribution in situ. These techniques provide a dynamic picture of the interactions between lipid storage, mobilization and signalling, which operate during normal cell function and are altered in many important diseases. The development of methods for imaging intracellular lipids can provide a dynamic real-time picture of how lipids are involved in complex signalling and other cell biology pathways; and how they ultimately regulate metabolic function/homoeostasis during early development. Some imaging modalities have the potential to be adapted for in vivo applications, and may enable the direct visualization of progression of pathogenesis of cardiometabolic disease after poor growth in early life.

Intracellular distribution and stability of a luminescent rhenium(i) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging.

Optical epifluorescence microscopy was used in conjunction with X-ray fluorescence imaging to monitor the stability and intracellular distribution of the luminescent rhenium(i) complex fac-[Re(CO)3(phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence imaging techniques. X-ray fluorescence also showed that the rhenium complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.

Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes.

Fourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes.

Atg9 is required for intraluminal vesicles in amphisomes and autolysosomes.

Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and Lamp1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to amphisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/Lamp-1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within amphisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development.

Recurrent High-Flow Arterio-Venous Malformation of the Thyroid Gland.

BACKGROUND: Vascular malformations and hemangiomas of the thyroid gland are rare disorders. The first case of a patient with recurrent high-flow arterio-venous malformation of the right thyroid gland involving the right endolarynx is presented. PATIENT FINDINGS: In June 2013, a 42-year-old female patient presented to the surgical department with recurrent hoarseness and a soft, vibrating mass on the right side of her neck. In 1993, she underwent right subtotal hemithyroidectomy with embolization on the day before surgery for a high-flow arterio-venous malformation of the thyroid gland. Diagnostic work-up in 2013 demonstrated a complex recurrent high-flow arterio-venous malformation on the right side of her neck involving the endolarynx. Full function of the right vocal fold could not be ascertained. The lesion was embolized again and excised the following day. Intraoperative gross bleeding and scar tissue prevented visualization and monitoring of the recurrent laryngeal nerve. Gross bleeding was also noted on hemithyroidectomy after embolization in 1993. No therapy was needed for the endolaryngeal part of the lesion. Histology showed large arterio-venous malformations with thyroid tissue. She remains well without signs of recurrence 18 month later but with a definitive voice handicap. SUMMARY: This is the first report of a recurrent high-flow arterio-venous malformation originally developing from the right thyroid gland involving the right endolarynx. Counseling, diagnostic, and therapeutic work-up of the patient was possible only with an interdisciplinary team. The endolaryngeal part of the hemangioma dried out after embolization and completion hemithyroidectomy. Her hoarseness has greatly improved but a definitive voice handicap remains. CONCLUSION: High-flow arterio-venous malformations of the thyroid gland are a rare disease, and recurrent lesions have not been reported. Interdisciplinary management of these patients is mandatory due to the complex nature of the underlying pathology. Recurrence might develop after long free intervals.

[Psychogenic aphonia. A challenging diagnosis?]. / Psychogene Aphonie. Eine schwierige Diagnose?

BACKGROUND: Based on clinical experience in the treatment of psychogenic aphonic patients, the study aimed to analyse the time interval between symptom onset and diagnosis using concrete data and to identify the cause of delayed diagnosis. PATIENTS AND METHODS: The medical records of 14 patients (all female) with psychogenic aphonia first diagnosed at the ENT department of the Saarland University Clinic were evaluated in a retrospective study. RESULTS: The time interval between symptom onset and the diagnosis of psychogenic aphonia ranged from 1 to 32 weeks, with a mean time interval of 9 weeks. In addition to all patients consulting an ENT specialist, additional diagnostic procedures (primarily imaging) were performed in 6 of 14 patients before the correct diagnosis was made. In all, 13 patients had previously undergone some form of therapy, 11 of these receiving antibiotic treatment. CONCLUSIONS: The study confirms that unnecessary instrument-based"over-diagnosis", as well as ineffective treatment attempts lead to significant delays in the diagnosis of psychogenic aphonia and hence delays in the initiation of causal treatment.

[Subjective and objective hearing test results for children with orofacial clefts]. / Audiometrische Daten subjektiver und objektiver Hörprüfungen bei Kindern mit orofazialen Spaltbildungen--eine prospektive Längsschnittuntersuchung.

OBJECTIVE: The hearing status of children with orofacial clefts was analysed using objective hearing tests over an average of 38.7 months following soft palate closure. We investigated the influence of: timing of closure, type of cleft, importance of grommets insertion and concomitant craniofacial syndromes such as Pierre Robin (PRS). MATERIAL AND METHODS: 70 children were selected for prospective audiometric testing twice yearly. RESULTS: The average age at surgical closure was 8.3 months. More than half had PRS, but their hearing levels were not significantly different from those who did not. Neither did cleft type or timing of closure seem to have any influence. The portion of children with a hearing level better than 30 dB increased after repair from 38.6% to 81.4%--significant at 0.001 and remained stable more than 24 months later. In the group of children with concomitant grommets insertion, it was also found that also more than 80% achieved longstanding normal hearing. CONCLUSIONS: There is no long lasting hearing loss which has any relevance to speech. The only intervention of any consequence is insertion of pressure equalising/tympanostomy tubes (grommets) in cases of primary palatal clefts when hearing is substantially impaired at the time of soft palate closure.

Eggs of the Australian saltmarsh mosquito, Aedes camptorhynchus, survive for long periods and hatch in instalments: implications for biosecurity in New Zealand.

The Australian saltmarsh mosquito, Aedes camptorhynchus (Diptera: Culicidae), is a significant biting pest and disease vector and is the subject of an eradication programme in New Zealand (NZ), where it has been resident for more than 10 years. To better understand the ecology of this common and widespread pest, we studied egg longevity and hatching patterns in the laboratory. By regularly testing for the presence of viable embryos, we found that eggs may last more than 15 months when stored dry (13% viable at this time). Eggs display instalment hatching, with no more than 56% of a batch hatching upon first inundation. Further hatching may occur for at least six inundations and some unhatched eggs may remain viable even after this. Variation in hatching rates can be observed using different water types, with weaker hatching media stimulating lower hatching rates spread over more inundations. By applying average hatching rates to a non-linear model of natural egg attrition, we showed that egg batches exposed to three inundations should be exhausted (zero live eggs present) in approximately 11 months at the conditions tested here. These findings have implications for the current eradication programme for Ae. camptorhynchus in NZ and for our understanding of the ecology of a widespread and common disease vector in Australia.

[Dysphagia in children with cerebral palsy--fiberoptic-endoscopic findings]. / Dysphagie bei Kindern mit Infantiler Zerebralparese--Fiberoptisch-endoskopische Befunde.

OBJECTIVE: Cerebral Palsy (CP) is associated frequently with swallowing disorders. By means of fiberoptic endoscopy, pathological findings in dysphagic patients with CP have been intended to be described, to be quantified, and to be related to selected anamnestic aspects. PATIENTS AND METHODS: Swallowing competency has been analysed endoscopically in 54 patients with spastic tetraparesis (range of age: 3 months to 15 years and 11 months). RESULTS: Over 60% of all patients examined endoscopically showed deficits in terms of penetration (24%) or aspiration (39%) of saliva. Two-thirds of the patients who have been tested with food had a penetration or aspiration of food, more than half of them without airway protection by cough reflex. Patients who have been fed orally before examination reported aspirations of food for 40%. Interestingly, about 40% of the children without history of pneumonia had aspirations of saliva and food. Drooling phenomena where associated with aspirations of saliva in 54% and with aspiration of food in 53% of our patients. CONCLUSIONS: Fiberoptic endoscopy is useful to detect a potentially dangerous penetration or aspiration of saliva, secretions and food. Thereby it enables the physician to change airway and feeding management. This is of special importance as children who formerly have been fed orally without suspicion of risks and pneumonia anamnesis turned out to have severe swallowing deficits. A close connection between appearance of drooling phenomena and aspiration of saliva and food has also to be considered in children with CP.

[Dysphagia in children and young persons. The value of fiberoptic endoscopic evaluation of swallowing]. / Dysphagien im Kindes- und Jugendlichenalter. Zum Stellenwert der fiberoptisch-endoskopischen Schluckdiagnostik.

BACKGROUND: The aim of this retrospective study was to evaluate fiberoptic endoscopic examination of swallowing in the pediatric patient by focusing on its feasibility and typical complications. PATIENTS AND METHODS: From 1997 to 2005, 164 children and young persons with swallowing disorders, who had not previously undergone endoscopic examination, presented for fiberoptic endoscopic examination of swallowing. Of these patients, 47% were age 6 years or younger. The most frequent reasons for the swallowing disorders were neurogenous (87%). RESULTS: In 129 of the 164 patients (78.6%), relevant informations about swallowing pathology was obtained. Because of anatomical restrictions or lack of cooperation, endoscopic examination was impossible in eight cases (4.9%). In 27 patients (16.5%) the procedure had to be terminated early because of inadequate cooperation. Severe complications concerning the endoscopic procedure did not occur. CONCLUSIONS: Fiberoptic endoscopic examination of swallowing turned out to be a feasible tool for the diagnostics of pediatric swallowing disorders. Its complications are rare if basic precautions are respected.

Development of aerodynamic aspects in children's voice.

To determine the changes with increasing age in children this study measured the air flow and the acoustic signal in children aged 4-15 years. The subglottal pressure was estimated from the intraoral pressure during the phonation of the word 'ipipi'. From these parameters the glottal resistance and the glottal efficiency were calculated. Since these parameters depend on the sound pressure level, a linear regression of the parameters versus the sound pressure level was done, and the values for 75 dB SPL were taken for statistical analysis. The study found a subglottal pressure in younger children between 6 and 10 cm water column, which seemed to diminish with age. The phonatory flow rate was between 50 and 150 ml/s in younger children, which seemed to increase with age. The glottal resistance did not depend on age, values of 20-250 Ns/m5 at a sound pressure level of 75 dB exceed those for adults given in the literature. The measurements for the glottal efficiency showed a broad variability between 20 ppm and 400 ppm.

Methodological aspects of spontaneous crystalluria studies in calcium stone formers.

Despite nearly a half-century of study, the clinical value of spontaneous crystalluria (Cx) examinations in calcium stone formers (CaSF) is still uncertain. The analytical complexity of urine particle study is largely responsible for this situation. As a result, there is no consensus regarding technical methods in Cx with several techniques for urine sampling and three different instruments currently used for particle study, namely, particle counting (PC), light microscopy (LM) and petrographic microscopy (PM). In this work, we first examined urine sampling and instrument methods regarding their appropriateness for Cx studies. Then we performed a comparative analysis of Cx studies in CaSF. Despite many technical and clinical discrepancies, several studies agree that the frequency of "all particles" and of the weddellite and whewellite calcium oxalate (CaOx) crystalline phases are increased in CaSF as compared to normal subjects (NS). Particle sizes and aggregation ratio are also often increased. Altogether, these results reinforce the need for an efficient method for Cx studies in these patients. Examining each technique leads us to conclude that most particle parameters can be studied by "direct LM" observation of freshly voided urine samples, i.e., urine samples without any separation steps. For clinical applications, several examinations should be performed, first to define the specific Cx characteristics in a patient, then for the study of treatment efficiency on Cx control, and finally, during the patient follow-up. Due to Cx variability in each patient, the frequency of Cx examinations during each phase needs to be determined in longterm comparative prospective studies of CaSF.

Hyperhomocysteinaemia: a significant risk factor for cardiovascular disease in renal transplant recipients.

Moderate hyperhomocysteinaemia has been shown to constitute an independent risk factor for cardiovascular disease (CVD), a frequent cause of morbidity and mortality in renal transplant recipients (RTR). In these patients few data regarding both total homocysteine levels and their influence on cardiovascular risk have been reported. We therefore studied serum homocysteine levels in deep-frozen sera from 42 kidney transplant recipients with a follow-up of 11 +/- 4.5 years (mean +/- SD) after transplantation. Eighteen patients had one or more ischaemic events (CVD (+)) and 24 patients had none (CVD (-)). Serum samples had been drawn 1-6 months prior to the first vascular event in CVD (+) patients and serum storage time was comparable in both CVD (-) and CVD (+) patients. Serum homocysteine levels were measured using a radioenzymatic method. Mean homocysteine level was significantly higher in 42 RTR males and females (15.5 +/- 6.3, 13.5 +/- 5.5 microM respectively) compared with 35 control subjects matched for age and sex (8.7 +/- 1.9, 7.5 +/- 1.9 microM, P < 0.001). The difference in serum homocysteine levels between CVD (+) and CVD (-) RTR nearly reached statistical significance in male patients (18.6 +/- 7.8 versus 13.1 +/- 3.4 microM, P < 0.06) but not in female patients (P = NS). In the CVD (+) group 11/18 patients had homocysteine levels > 14 microM (the upper limit in healthy controls) versus 7/24 in the CVD (-) group (P = 0.04). In these patients we simultaneously measured in the same serum samples, serum triglycerides, and total and HDL cholesterol, and calculated LDL cholesterol. By stepwise discriminant analysis and by logistic regression analysis in this relatively small patient population, only serum triglycerides and homocysteine were selected as risk factors associated with CVD. We conclude that significant hyperhomocysteinaemia is present in renal transplant recipients and represents a potential risk factor for cardiovascular disease in these patients.

Urinary calculi: review of classification methods and correlations with etiology.

Current physical and chemical methods available for urinary stones analysis are critically reviewed. No one method is sufficient to provide all the clinically useful information on the structure and composition of the stones. We show that a combination of refined morphological and structural examination of stone with optical microscopy, complemented by compositional analysis using infrared spectroscopy of the core, cross-section and surface of calculi, provides a precise and reliable method for identifying the structure and crystalline composition, and permits quantification of stone components while being highly cost effective. Using such morphoconstitutional studies leads to a classification of urinary stones in seven distinctive types and twenty-one subtypes among monohydrate (whewellite) and dihydrate (weddellite) calcium oxalates, phosphates, uric acid, urates, protein, and cystine calculi. Furthermore, all of the recognized sub-types exhibit correlations with specific pathophysiologic conditions. We conclude that such morphoconstitutional refined analysis and classification of urinary calculi is of interest to properly identify the type of stone disease and provides clues to etiopathogeny.

Urolithiasis in patients with end stage renal failure.

Renal stones from 30 chronic hemodialysis patients were subjected to morphological study by means of microscopic examination and to constitutional analysis with infrared spectrophotometry. In 29 patients calculi could be classified into 3 main types: 1) protein stones made of pure proteins or with a protein core and less than 30% calcium oxalate (9 cases, or 30%)--they were observed predominantly in patients with primary glomerular disease, 2) oxalo-protein stones with a core of calcium oxalate and a total stone content of more than 30% calcium oxalate (15 cases, or 50%)--they appeared to be related to metabolic factors, such as high urinary oxalate and low urinary citrate concentration, and to iatrogenic factors, namely vitamin D3 and calcium salt supplementation, and 3) aluminum-magnesium urate stones, probably induced by aluminum overload (6 cases, or 20%). Thus, our study indicates that a significant proportion (70%) of stones formed by hemodialysis patients may be due to metabolic and iatrogenic factors. Our data suggest that accurate analysis of such stones provides useful information on pathogenetic factors and consequently may give clues to their prophylaxis.

[Distant metastases of epitheliomas of the upper respiratory and digestive tracts]. / Les métastases à distance des épithéliomas des V.A.D.S.

Distant metastases of ENT tumors are rare; when analysing 1.666 patients, we found them in 6.5%. They started especially from tumors of the hypopharynx (19.7%), oral cavity (7.6%), oropharynx (5.6%) and larynx (3.2%). 9 metastases were diagnosed, before therapy started, all the others appeared late, even later than 5 years after therapy in some cases of carcinoma of the larynx. The metastases were situated in the lung (71 cases), the skeleton (45 cases), the pleura (11 cases) and the liver (8 cases), but in rare instances in almost every organon. The main examinations for staging are therefore: the radiograph of the chest, the sonography of the abdomen and the scintigraphy of the skeleton. These examinations may be completed, if necessary, by the CT-scan. A patient with distant metastases never could be healed.

Vitamin D3 and glucose-6-phosphate dehydrogenase in rat duodenal epithelial cells.

A microdensitometric method was employed to determine enzyme activities in situ in undisrupted tissue rat duodenum. The effect of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on glucose-6-phosphate dehydrogenase (G6PD) activity and on the two utilization pathways of synthesized NADPH, H1 (mixed function oxidation) and H2 (biosynthesis), was studied. In normal animals, a crypt-to-villus gradient of G6PD activity and of both NADPH utilization pathways was observed. A high level of NADPH utilization occurred predominantly via the H2 pathway. In vitamin D-deficient rat animals, G6PD activity in the middle part of the villus was approximately 60% lower than in normal animals [10.05 +/- 0.35 vs. 3.95 +/- 0.26 (means +/- SE) A585.min-1.micron-3 X 10(5), P less than 0.001] with reduced NADPH utilization via the H2 pathway (8.39 +/- 0.49 vs. 2.73 +/- 0.43 A585.min-1.micron-3 X 10(5), P less than 0.001) but not the H1 pathway (1.65 +/- 0.17 vs. 1.22 +/- 0.19 A585.min-1.micron-3 X 10(5), P = NS). Intraperitoneal administration of 1,25(OH)2D3 (500 pmol) to vitamin D-deficient animals resulted in increased G6PD activity within 30 min (4.09 +/- 0.38 vs. 5.51 +/- 0.39 A585.min-1.micron-3 X 10(5), P less than 0.05), attaining normal levels within 2 h. The H2 but not the H1 pathway of NADPH utilization increased significantly in response to 1,25(OH)2D3. This increase is essentially located in the basal and middle parts of the villus. Thus 1,25(OH)2D3 may influence biosynthesis in the duodenum via stimulation of G6PD activity and the H2 pathway of NADPH utilization.

Comparative effects of 17 beta-estradiol, progestin R5020, tamoxifen and RU38486 on lactate dehydrogenase activity in MCF-7 human breast cancer cells.

The effects of 17 beta-estradiol (estradiol), synthetic progestin R5020 and their antagonists, tamoxifen (Tam) and synthetic RU38486 on lactate dehydrogenase (LDH) activity in MCF-7 human breast cancer cells during the growth period were studied. A specially developed quantitative cytochemical assay was used; LDH activity is expressed per cell, and is thus independent of the positive and negative growth effects of the hormones and antagonists. Estradiol and R5020 stimulated LDH activity after similar exposures (6-48 h) and the stimuli were concentration dependent over the range 10(-7) M to 10(-10) M. As for the antagonists, RU38486 stimulated LDH activity in much the same way as estradiol and R5020; Tam alone, on the other hand, does not stimulate LDH, but when added to estradiol, Tam inhibits estradiol mediated LDH activation. When present at half-stimulant concentration, estradiol + R5020 and estradiol + RU38486 exhibit additive effects on LDH activity. Thus LDH appears to be an interesting tool for the study of hormone and antagonist effects in MCF-7 breast cancer cells.