Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1ß, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 µM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Upward displacement of the odontoid process into the foramen magnum: a palaeopathological case.

An upward displacement of the odontoid process into the foramen magnum was observed in the skeletal remains of a young male unearthed from a 14th to 17th century cemetery in the north-eastern Italy. Examination of skull bone vestiges and computed tomography scan analysis of the axis exhibited a clear-cut contact zone between the odontoid process and the anterior border of the foramen magnum. In addition, the odontoid process appeared backward deviated. Findings suggest a possible diagnosis of basilar impression/invagination. This anomalous contact may cause compression of neural and vascular structures with a multifaceted series of clinical symptoms. We are unable to set our finding into a complete presumptive diagnostic outline because there is no chance to estimate either the magnitude of the whole craniovertebral junction defect but we believe that the present case contributes to the general knowledge of the craniovertebral region and to bone pathology in ancient times.

Capitate-trapezoid synostosis: analysis of an Early Bronze Age case and review of the literature.

BACKGROUND: Carpal synostoses are congenital defects characterised by complete or incomplete coalition of two or more carpal bones. Although most of these defects are discovered only incidentally, sometimes they become clinically manifest. Among the different types of carpal coalition, the synostosis between capitate and trapezoid bones is quite rare, with only sparse data available in the literature. The aim of this report was to describe a case of capitate-trapezoid synostosis (CTS) observed in an ancient human skeleton, as well as to scrutinise the pertinent literature in order to assess for the characteristics of this type of defect, including its potential relevance to clinical practice. MATERIALS AND METHODS: We studied the skeletal remains of an Early Bronze Age male warrior affected by incomplete CTS. Macroscopic and radiological examination of the defect was carried out. We also performed a comprehensive PubMed search in the Medline and other specialty literature databases to retrieve and analyse data relevant to the subject under consideration. RESULTS AND CONCLUSIONS: The present case is the most ancient CTS ever found. In those literature-reported cases accompanied by careful anatomical description, such as the present one, incomplete coalition invariably occurs between the dorsal surfaces of the two bones, this characteristic emerging as a distinctive morphological trait. Literature analysis further suggests that the true prevalence of CTS is likely to be higher than estimates based on data gathered from radiology series, and that this defect may be associated with pain and carpal bossing more frequently than generally thought.

Hepatopulmonary fusion in a newborn. An uncommon intraoperatory finding during right congenital diaphragmatic hernia surgery: case description and review of literature.

Hepatic pulmonary fusion is a rare malformation associated with right congenital diaphragmatic hernia (CDH), often only discovered during surgical repair of the defect. Fourteen previous cases have been reported in the literature. We describe a case of a full term male newborn with prenatal ultrasound diagnosis of right CDH who underwent a thoracoscopy converted to a thoracotomy, due to this rare aforementioned intraoperative incidental finding. We reviewed the previous reported literature, especially focusing on the chosen surgical approach, concluding that an early and appropriate preoperative imaging investigation may be crucial for the best management of these kinds of patients.

[Practical strategies for promoting breastfeeding in neonatal intensive care]. / Strategie pratiche di promozione del latte materno in TIN.

Strategies for promoting the use of human milk in NICU Human milk has several advantages in the nutrition of very-low-birthweight (VLBW) and high risk infants admitted to neonatal intensive care unit (NICU). Limited data available demonstrate that at discharge from NICU breastfeeding rate is relatively low. In a recent italian study (Davanzo R et aL; Pediatric and Perinatal Epidemiology 2009) only 30.5% of VLBW infants were exclusively breast fed and among the exclusively breastfed infants only 10% sucked directly at the breast. Strategies to promote breastfeeding and the use of human milk in NICUs are needed. They are well known, yet limitedly applied. Among them current literature lists: (1) access at anytime for both parents to NICU; (2) specific knowledge of the science of lactation and multidisciplinary breastfeeding training as provided by the Baby Friendly Hospital Training package; (3) peer support in hospital; (4) kangaroo mother care; (5) breastmilk expression using simultaneous pumping with an electric pump particularly in the first 2 weeks. On the contrary, pharmaceutical galactagogues and cup feeding have little benefit.

Mast cells and basophils: a potential link in promoting angiogenesis during allergic inflammation.

Mast cells and basophils are granulated metachromatic cells which possess complex and partially overlapping roles in acquired and innate immunity, including both effector and regulatory activities. Mast cells and basophils cooperate in exacerbating and/or modulating inflammation as well as in mediating subsequent tissue repair. Mast cells release a series of potent proangiogenic molecules during inflammation that stimulate vessel sprouting and new vessel formation. Recent data suggest that basophils may also play a role in inflammation-related angiogenesis, principally but not exclusively through the expression of several forms of vascular endothelial growth factors and their receptors. This review focuses on the potential cooperative link between mast cells and basophils in promoting angiogenesis during allergic inflammation. We discuss the multifaceted roles of mast cells and basophils in inflammatory mechanisms of allergic diseases and whether these cells can be both source and target of proangiogenic mediators.

Clinical and genetic characterization of Italian patients affected by CINCA syndrome.

OBJECTIVE: We report the experience of the Italian Registry of patients affected by chronic infantile neurological, cutaneous, articular (CINCA) syndrome. The clinical and genetic features of 12 unrelated Italian patients with CINCA syndrome are described, focusing on the possible influence of the presence of CIAS1/cryopyrin mutations on the phenotype of the disease and on its prognosis. METHODS: The clinical features of 12 Italian CINCA patients were evaluated. Genomic DNA of the patients was sequenced using specific primers for CIAS1 and ASC genes. RESULTS: Our patients shared typical CINCA characteristics and, sometimes, remarkable perinatal events, peculiar of CIAS1-mutated patients. Seven patients carried CIAS1 missense mutation, localized within the nucleotide binding domain of cryopyrin. Four previously described mutations and three new heterozygous CIAS1 missense mutations were identified. ASC gene, encoding for a direct interactor of cryopyrin, was not mutated in Italian CINCA patients. Finally, we reported the efficacy and safety of anti-IL1 therapy (Anakinra) in seven patients with a particularly severe CINCA phenotype. CONCLUSION: Despite some common signs-used as syndrome hallmarks-we observed a high variability in symptoms, genetic results and outcomes in Italian CINCA patients. In contrast with other authors, we cannot find out any correlation between mutations in CIAS1 and CINCA severity, but we underlined the concomitance of perinatal events and mental retardation only in CIAS1 mutated subjects. Finally, we confirmed the efficacy of Anakinra treatment, both in CIAS1-mutated and non-mutated patients.

Kidney length and scarring in children with urinary tract infection: importance of ultrasound scans.

BACKGROUND: Many studies have demonstrated that dimercaptosuccinic acid (DMSA) scintigraphy is the most sensitive diagnostic method in the identification of irreversible renal lesions (scars) in children with previous episodes of acute pyelonephritis (APN). This study assessed the reliability of ultrasound in identifying reflux nephropathy in children with acute pyelonephritis with or without vesicoureteric reflux (VUR). METHODS: Eighty children (45 female and 35 male, age range 5 months to 10 years, average age 2 years 1 month) with a positive history for at least one episode of APN participated in this study. All children underwent voiding cystourethrography, DMSA scintigraphy 4 to 8 months after the most recent episode of APN, and an ultrasound test evaluation less than 2 months after DMSA scintigraphy. RESULTS: Voiding cystourethrograms showed VUR in 52 children (68%); 13 of these were bilateral, for a total of 65 refluxing kidney units of the 154 (42%) evaluated; DMSA scintigram was normal for 108 of 154 kidneys (70%). Of the 65 kidneys with VUR, DMSA scintigram displayed normal findings in 29 cases (45%) and pathologic findings in 36 (55%). In the 79 nonrefluxing kidneys, DMSA scintigram was normal in 69 cases (87%). The relative risk of scarring in VUR kidneys is 2.6. The ultrasound study recorded a maximum longitudinal diameter between the 5th and 95th percentiles in 80 of 89 (81%) kidneys without VUR and in 21 of 65 (32%) with VUR. A significant correlation was found between maximum longitudinal diameters and DMSA scintigraphic findings in kidneys with VUR and those without VUR, respectively. CONCLUSION: This study establishes that ultrasound scans, by means of a simple and reproducible measurement technique, maximum longitudinal diameter, have a predictive value with regard to the presence of scars, with few exceptions. This finding, in our opinion, could lead to a decrease in the number of invasive procedures, in particular DMSA scan, in patients with APN.

A light and electron microscopic quantitative analysis of nerve-immune cell contacts in the gut-associated lymphoid tissue of the mouse colon.

Light microscopy (LM) histochemistry and transmission electron microscopy (EM) have been used to investigate the structural relationship between immunocompetent cells and enteric nerves in the gut-associated lymphoid tissue (GALT) of the mouse colon, i.e., a) the scattered immune cells in the lamina propria, b) the lymphoid follicles and c) the cryptopatches. Nerve-immune cell contacts have been quantified by LM, using the osmium-zinc iodide procedure for visualizing nerve fibers. Appositions of nerves to immune cells have been studied by EM when the distance between the immune cell plasma membrane and the neurilemma was 200 nm or less. In the diffuse GALT a), the LM incidence of nerve-lymph cell and nerve-plasma cell contacts has been calculated to be one and half and, respectively, three times greater than would be expected by chance alone (P < 0.0001 in both cases). EM showed close apposition of axonal varicosities, mostly containing 60 nm diameter dense-cored vesicles, to B lymphoblasts/immunoblasts or plasma cells. In isolated lymphoid follicles b), nerve-immune cell contacts were identified almost exclusively in the T-cell dependent parafollicular regions; the incidence of such contacts, calculated by LM, did not exceed expected theoretical values. By EM, apposition of nerve varicosities to small/middle-sized lymphocytes containing cytoplasmic lysosomal granules was seen sporadically. Examination of nerve-immune cell contacts in cryptopatches c), a recently identified extrathymic T-cell generating compartment, allowed recognition of a small proportion of nerve-lymph cell structural interactions, both at LM and EM. This study provides systematic quantitative data on the microanatomical relationship between enteric nerves and immune cells in the various GALT compartments. Findings suggest that such nerve-immune cell contacts might represent the structural foundation for communication between enteric nerves and the GALT.

Crying of a newborn child: alarm signal or protocommunication?

The purpose of the study was to explore whether the new-born cry is a simple alarm signal or differentiated cries with different meanings. 12 digital audio taped recordings of 6 full-term healthy babies were analysed. Cries of 6 newborns in this preliminary study were recorded in a pain condition after a prick for the hematic check-up the third day after delivery and then while crying spontaneously in the cradle. The sounds were sampled at 44100 Hz with a 16-bit resolution and converted to the .wav format. All the analyses were performed with a software written in the MAT-LAB environment. The most important result was that these new-born children modulated the supralaryngeal tract considerably more in cries following the painful stimulus than in "spontaneous" ones, as would be expected by the hypothesis of crying as "protolanguage."

Apposition of enteric nerve fibers to plasma cells and immunoblasts in the mouse small bowel.

Light (LM) and electron microscopy (EM) were used to investigate the structural relationship between enteric nerves and the population of immune cells in the mouse small bowel. By LM, the osmium-zinc iodide procedure was used for visualizing nerve fibers; the incidence of nerve-plasma cell contacts in the mucosa and submucosa was calculated to be approximately 4 times and, respectively, 3 times greater than would be expected by chance alone (P < 0.0001). EM revealed close, synaptic-like contacts between axonal varicosities and plasma cells or B immunoblasts. The results presented here provide systematic quantitative evidence that a structural foundation for communication between nerve fibers and B cells exists in the mouse small bowel.

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation.

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studies showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators.

Visualization of PGP 9.5 immunoreactive nerve terminals in the mouse snout epidermis.

Intraepidermal free nerve endings were investigated in the mouse snout skin by means of an immunohistochemical procedure using a rabbit antiserum against protein gene product 9.5 (PGP 9.5). Immunoperoxidase reactivity was detected in different subtypes of intraepidermal nerves and cells. The great majority of axons observed in the stratified epithelium were varicose; a small percentage was either smooth (non-varicose) or irregularly shaped. Intraepidermal nerves ended at different levels within the epidermis, often with a terminal knob-like swelling. Various patterns of intraepidermal innervation could be distinguished. Most fibres entering the epidermis originated from large bundles running a horizontal course below the dermo-epidermal junction. Such fibres ascended vertically through the stratified epithelium in a "candelabrum-like" fashion, without emitting collaterals. Other fibres branched profusely and ended in complex intraepidermal neural networks. Less frequently, intraepidermal fibres terminated with large irregularly shaped expansions of different morphologies. Some of these were the intraepidermal continuations of axons within Meissner's corpuscles. Some fibres appeared to come into contact with PGP 9.5-immunoreactive cells (which closely resembled Merkel cells) located in the stratum basale. Rare suprabasal dendritic cells (Langerhans cells?) also became visible.

Serotoninergic fibres form dense synaptic contacts with Purkinje cells in the mouse cerebellar cortex--an immunohistochemical study.

The distribution of serotonin immunoreactivity in the mouse cerebellar cortex was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger (1979) on epoxy embedded semithin sections. The great majority of serotonin-positive afferents distribute throughout the Purkinje cell layer and form dense synaptic contacts with the somata of the Purkinje neurons. Only a sparse immunostaining of serotoninergic fibres could be detected at the granular cell and molecular layers. The microanatomical organization of the serotoninergic projections to the mouse cerebellar cortex is quite different from that observed in other animal species. These findings suggest that in the mouse cerebellar cortex, the Purkinje cell population represents the main target for serotoninergic afferents. Our histochemical data provide morphological support for a series of electrophysiological observations which indicate serotonin as a potential modulatory neurotransmitter for Purkinje cell firing activity.

Suggestive evidence for a microanatomical relationship between mast cells and nerve fibres containing substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin in the rat mesentery.

A close microanatomical relationship between serotonin-positive mast cells and nerve fibres positive for substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin has been observed in whole-mount preparations of rat mesentery by an immunofluorescent double-staining procedure. Peptidergic fibres have been shown either to run in close proximity or come in direct contact with mast cells. This supports earlier morphological and immunohistochemical results suggesting an innervation of mast cells and provides a structural foundation for a series of pharmacological studies which outline the influence of various neuropeptides on mast cell secretory activity.