Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles.

Over the last 30 years, atomic force microscopy (AFM) has made several significant contributions to the field of biology and medicine. In this review, we draw our attention to the recent applications and promise of AFM as a high-resolution imaging and force sensing technology for probing subcellular vesicles: exosomes and other extracellular vesicles. Exosomes are naturally occurring nanoparticles found in several body fluids such as blood, saliva, cerebrospinal fluid, amniotic fluid and urine. Exosomes mediate cell-cell communication, transport proteins and genetic content between distant cells, and are now known to play important roles in progression of diseases such as cancers, neurodegenerative disorders and infectious diseases. Because exosomes are smaller than 100 nm (about 30-120 nm), the structural and molecular characterization of these vesicles at the individual level has been challenging. AFM has revealed a new degree of complexity in these nanosized vesicles and generated growing interest as a nanoscale tool for characterizing the abundance, morphology, biomechanics, and biomolecular make-up of exosomes. With the recent interest in exosomes for diagnostic and therapeutic applications, AFM-based characterization promises to contribute towards improved understanding of these particles at the single vesicle and sub-vesicular levels. When coupled with complementary methods like optical super resolution STED and Raman, AFM could further unlock the potential of exosomes as disease biomarkers and as therapeutic agents.

Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp. lactis†CNCM I-3446.

Non-digestible milk oligosaccharides were proposed as receptor decoys for pathogens and as nutrients for beneficial gut commensals like bifidobacteria. Bovine milk contains oligosaccharides, some of which are structurally identical or similar to those found in human milk. In a controlled, randomized double-blinded clinical trial we tested the effect of feeding a formula supplemented with a mixture of bovine milk-derived oligosaccharides (BMOS) generated from whey permeate, containing galacto-oligosaccharides and 3'- and 6'-sialyllactose, and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446. Breastfed infants served as reference group. Compared with a non-supplemented control formula, the test formula showed a similar tolerability and supported a similar growth in healthy newborns followed for 12 weeks. The control, but not the test group, differed from the breast-fed reference group by a higher faecal pH and a significantly higher diversity of the faecal microbiota. In the test group the probiotic B. lactis increased by 100-fold in the stool and was detected in all supplemented infants. BMOS stimulated a marked shift to a bifidobacterium-dominated faecal microbiota via increases in endogenous bifidobacteria (B. longum, B. breve, B. bifidum, B. pseudocatenulatum).

Sub-Retinal Injection of Human Lipofuscin in the Mouse - A Model of "Dry" Age-Related Macular Degeneration?

Lipofuscin (LF) accumulates during lifetime in the retinal pigment epithelium (RPE) and is thought to play a crucial role in intermediate and late age-related macular degeneration (AMD). In an attemt to simulate aged retina and to study response of retinal microglia and RPE cells to LF, we injected a suspension of LF into the subretinal space of adult mice. LF suspension was obtained from human donor eyes. Subretinal injection of PBS or sham injection served as a control. Eyes were inspected by autofluorescence and optical coherence tomography, by electroretinography and on histological and ultrastructural levels. Levels of cytokine mRNA were determined by quantitative PCR separately in the RPE/choroid complex and in the retina. After injection of LF, microglial cells migrated quickly into the subretinal space to close proximity to RPE cells and phagocytosed LF particles. Retinal function was affected only slightly by LF within the first two weeks. After longer time, RPE cells showed clear signs of melanin loss and degradation. Levels of mRNA of inflammatory cytokines increased sharply after injection of both PBS and LF and were higher in the RPE/choroid complex than in the retina and were slightly higher after LF injection. In conclusion, subretinal injection of LF causes an activation of microglial cells and their migration into subretinal space, enhanced expression of inflammatory cytokines and a gradual degradation of RPE cells. These features are found also in an aging retina, and subretinal injection of LF could be a model for intermediate and late AMD.

Sub-Retinal Injection of Human Lipofuscin in the Mouse - A Model of "Dry" Age-Related Macular Degeneration?

Lipofuscin (LF) accumulates during lifetime in the retinal pigment epithelium (RPE) and is thought to play a crucial role in intermediate and late age-related macular degeneration (AMD). In an attemt to simulate aged retina and to study response of retinal microglia and RPE cells to LF, we injected a suspension of LF into the subretinal space of adult mice. LF suspension was obtained from human donor eyes. Subretinal injection of PBS or sham injection served as a control. Eyes were inspected by autofluorescence and optical coherence tomography, by electroretinography and on histological and ultrastructural levels. Levels of cytokine mRNA were determined by quantitative PCR separately in the RPE/choroid complex and in the retina. After injection of LF, microglial cells migrated quickly into the subretinal space to close proximity to RPE cells and phagocytosed LF particles. Retinal function was affected only slightly by LF within the first two weeks. After longer time, RPE cells showed clear signs of melanin loss and degradation. Levels of mRNA of inflammatory cytokines increased sharply after injection of both PBS and LF and were higher in the RPE/choroid complex than in the retina and were slightly higher after LF injection. In conclusion, subretinal injection of LF causes an activation of microglial cells and their migration into subretinal space, enhanced expression of inflammatory cytokines and a gradual degradation of RPE cells. These features are found also in an aging retina, and subretinal injection of LF could be a model for intermediate and late AMD.

Change in bile salt dependent lipase in human breast milk during extended lactation.

Two hundred eighty-one milk samples collected from Zaïrian nonprivileged, undernourished mothers, in series of nine groups from 1 month to 18 months after parturition, and 66 milk samples collected from French privileged mothers in series of four groups from 2 days to 16 months postpartum, were analyzed for their lactose, lipid and protein contents. In addition, the activity of bile salt-dependent lipase (esterase), which may play an important role in the newborn infant's lipids digestion, was measured. After the first month postpartum, independent of the nutritional state of the mother, sugar and protein concentrations were identical. Lipid content of French mothers' milk was lower in transitional milk, but appeared constant in mature milk with an average value of 29.1 +/- 5.8 mg/mL of milk. In Zaïrian mothers' milk, the lipid content of mature milk plateaued at around 50-55 mg/mL independent of the stage of lactation. Bile salt-dependent lipase showed constant esterase activity within the lactation stage in privileged mothers' milk, but decreased by almost 80-90% during the first four months of lactation in undernourished mothers. The data suggest that milk from nonprivileged mothers may lose some of its ability to hydrolyze milk lipid esters, which could also be of consequence to the infant's normal growth in view of its effect on the esters of the lipid-soluble vitamins A, E and D.

[Early continuous positive pressure in the labor room]. / Pression positive continue précoce en salle de travail.

BACKGROUND: Indication for intubation and mechanical ventilation in premature infants may be reduced by initiating continuous positive airway pressure (CPAP) in delivery room. POPULATION AND METHODS: Immediately after birth, respiratory support with CPAP was given to all infants with gestational age less than 32 weeks. In case of apnea or progressing symptoms with hypoxemia or carbonic acidosis, with PCO2 increasing to more than 60 mmHg, infants were treated with nasotracheal intubation and ventilation. RESULTS: One hundred and fifty one infants, with mean gestational age 29.6 +/- 1.9 weeks and mean birth weight 1,326 +/- 378 g were delivered in the obstetrical department of Marseille. In delivery room, 63% were treated with CPAP, and only 13% with nasotracheal intubation. The need for subsequent mechanical ventilation was reduced to 40% of the population. Surfactant therapy was used in 17% of this cohort. Two infants were given surfactant and extubated. Three of 14 deaths (9.2%) were caused by respiratory disease. CONCLUSIONS: Early CPAP reduces the indication of mechanical ventilation in premature infants. Incidence of pulmonary complications such as pneumothorax or bronchopulmonary dysplasia is low among those infants who require mechanical ventilation later. Early CPAP takes place in a general policy to decrease neonatal morbidity.

[Extreme prematurity: the limits of neonatal resuscitation]. / Extrême prématurité: les limites de la réanimation néonatale.

How far providing neonatal intensive care to extremely low birth weight infants is appropriate is still a highly controversial issue. Decision making when a poor prognosis has been established may be facilitated by consensus based recommendations and rigorous procedures. In the very majority of situations, the provision of intensive care is advocated at birth a priori. A decision of treatment withholding or withdrawal may eventually be made secondarily, in the case major neurological complications, likely to induce severe long term deficits, are evidenced. In any case, an ethical policy focused on each infant's best interest is justified, while the adoption of a systematic, gestational age or birth weight based restriction of access to intensive care may not be acceptable in most countries. Rigorous criteria must be fulfilled for end of life decision making and procedures. Continuous assistance to the patient and to the parents is key determinant.

[Idiopathic thrombopenic purpura and pregnancy]. / Purpura thrombopénique idiopathique et grossesse.

Idiopathic thrombocytopenic purpura occurring in pregnancy has a risk for the fetus of severe thrombocytopenia. The handling of these cases obstetrically is controversial. Our experience and that derived from reading the literature suggests that counting the number of maternal platelets does not reflect on the risk for the fetus and the neonate. On the other hand it does seem that estimating the levels of circulating platelet antibodies in the maternal plasma does give an indication of platelet destruction in the fetus. Corticosteroid treatment improves greatly the number of maternal platelets and is probably good treatment for the fetus in utero. Giving gamma-globulins intravenously to the pregnant woman or to the newborn is an interesting therapeutic measure seeing how it is free of danger. How useful this treatment is in pregnancy has yet to be determined.

[Glucose-6-phosphate dehydrogenase et neonatal jaundice]. / Déficit en glucose-6-phosphate déshydrogénase et ictère néonatal.

OBJECTIVE: Since 1986, quantification of G6PD activity has been a routine test for all babies born at the public maternity hospitals of Marseilles. The objective of our study was to determine the prevalence of G6PD deficiency in the population tested and to evaluate the relative risk of neonatal jaundice in newborns with G6PD deficiency. METHODS: Neonatal screening is performed on cord blood by spectrophotometric measurements of G6PD activity. A group of 7779 newborns was studied retrospectively. The occurrence of neonatal jaundice was evaluated in 85 children with G6PD deficiency and compared to 85 children with normal G6PD activity. RESULTS: The incidence of G6PD deficiency in male newborns was found to be 2.1%. The relative risk for neonatal jaundice in the G6PD deficient population compared to the non-deficient population is estimated to be 2.6. CONCLUSION: Neonatal jaundice with pathological hyperbilirubinemia develops more frequently in cases of G6PD deficiency. The early characterization of G6PD activity provides an etiological diagnosis for neonatal jaundice, as well as the opportunity to give the newborn's family information concerning hemolytic crisis prevention.

[Obstetric and neonatal consequences of urinary infection during pregnancy]. / Conséquences obstétricales et néonatales de l'infection urinaire gravidique.

In this article, the authors study the real obstetrical and neonatal effects of urinary infection in pregnant women. Basing themselves on 225 cases of gravidic urinary infection, the authors discuss asymptomatic bacteriuria, the need of early diagnosis in risk patients, and the use of regular post-treatment monitoring, in view of the fact that, even when well-treated, this complaint is apt to recur.

[Neonatal resuscitation and preventive continuous positive pressure ventilation]. / Réanimation néonatale et pression positive continue prophylactique.

One of the aspects of prematurity in neonates is the respiratory distress syndrome. Although treatment with mechanical ventilation reduced the mortality rate, bronchopulmonary dysplasia still develops in many neonates. We have attempted to reduce intubation and mechanical ventilation by using, in the delivery room, humidified and warmed gas with fractional inspired oxygen as low as possible to obtain SaO2 between 85 and 95%. The gas was administered with a face mask using continuous positive air pressure 3-5 cm H2O. Seventeen out of 66 premature neonates born before the 35th week of gestation were ventilated immediately (n = 11) or subsequently (n = 6). Seven out of 26 infants (27%) born between 30 and 32 weeks required mechanical ventilation. In contrast, ventilation was necessary for eight out of 16 premature neonates born before the 29th week of gestation. Mortality rate was 6% (4/66) in the latter group (< 29 weeks), and only one neonate developed bronchopulmonary dysplasia.

Isoproterenol infusion increases the maximal tubular capacity of phosphate reabsorption.

Isoproterenol, a beta-adrenoreceptor agonist, decreases urinary phosphate (Pi) excretion; however, plasma phosphate concentration also decreases. The purpose of the present study was to determine the effect of isoproterenol infusion on phosphate reabsorption with concomitant phosphate infusions and in the presence and absence of parathyroid hormone (PTH). Clearance experiments were performed on male Sprague-Dawley rats which were acutely thyroparathyroidectomized (TPTX) and successive infusions of phosphate (1, 2, and 3 mumol/min) were used to determine the maximal tubular capacity of phosphate reabsorption (TmPi) factored for the glomerular filtration rate (GFR) in four groups of rats. In the saline-infused control group the TmPi/GFR was 2.87 +/- 0.19 mumol/ml (n = 8). When isoproterenol was infused intravenously at a rate of 0.005 mg/kg/min, urinary cAMP excretion was significantly increased and the TmPi/GFR was 3.53 +/- 0.17 mumol/ml (n = 10, p less than 0.05). In the PTH-infused group (33 U/kg bolus followed by a sustaining infusion of 1 U/kg/min) TmPi/GFR was 1.69 +/- 0.15 mumol/ml (n = 9). Coadministration of isoproterenol and PTH significantly increased the TmPi/GFR to 3.25 +/- 0.64 mumol/ml (n = 9). Basal cAMP excretion was similar in both groups. These results demonstrate that the stimulation of renal beta-adrenoreceptors by isoproterenol infusion markedly increases phosphate reabsorption and reverses the decrease in the maximal tubular capacity of phosphate reabsorption induced by PTH infusion.

[Neonatal consequences of cesarean section on the presumably healthy infant]. / Conséquences néo-natales de la césarienne sur enfant présumé sain.

The authors studied the effects on the child of 196 caesarian sections performed in the absence of foetal distress. The following were noted for each child: the time before the first cry, the Apgar score, pH and the need or not for resuscitation techniques. Each feature was studied in terms of different factors: foetal, obstetric, anaesthetic and surgical. The conclusions were as follows: caesarian section, which remains the best means of preventing obstetric trauma, has its own direct complications which are linked to the conditions in which the operation takes place. These can be reduced to a minimum or even completely suppressed if the caesarian section is performed under ideal conditions: few or no depressant drugs before the operation (the use of diazepam for induction should be abandoned); inclined position of 15 degrees, even of the mother has never suffered from utero-caval syndrome and if possible on a heated mattress; extraction of the infant between the 5th and 15th minutes; finally, and above all, prior labour is desirable whenever obstetric conditions permit.

Effect of renal interstitial infusion of L-dopa on sodium and phosphate excretions.

It has been hypothesized that dopamine synthesized by the proximal tubule can act as a paracrine substance that regulates reabsorption by the proximal tubule. The present study was performed to study the effects of the stimulation of endogenous synthesis of dopamine by infusion of L-DOPA directly into the renal interstitium on sodium and phosphate excretions and to determine the roles of D1 and D2 receptors in the response. The infusion of L-DOPA (50 microg/kg/min) into the renal interstitium through an implanted matrix significantly increased the fractional excretion of sodium (FENa) from 1.0%+/-0.2% to 3.1%+/-0.6% and the fractional excretion of phosphate (FEPi) from 23%+/-3% to 36%+/-3%, P < .05, n = 10. The infusion of D1 receptor antagonist SCH23390 or SKF83566 (5 microg/kg/min) into the renal interstitium blocked the natriuretic (FENa 1.5%+/-0.2% to 1.9%+/-0.4%) and phosphaturic (FEPi 41%+/-3% to 41%+/-4%) effects of L-DOPA infusion. The infusion of the D2 receptor antagonist sulpiride at a rate of 4 microg/kg/min into the renal interstitium also attenuated the natriuretic (FENa 1.3%+/-0.3% to 1.6%+/-0.5%) and phosphaturic effects of L-DOPA infusion (FEPi 36%+/-5% to 39%+/-5%). We conclude that the renal interstitial infusion of L-DOPA increases sodium and phosphate excretions and that these responses are mediated by D1 and D2 receptors.

Fetal chest wall hamartoma: a case report.

A fetal chest wall hamartoma successfully treated by surgical resection after birth is described. Ultrasonography showing a heterogeneous partially calcified thoracic tumor allowed in utero diagnosis.

[Intrathoracic gas effusions of iatrogenic origin in neonatal reanimation]. / Epanchements gazeux intra-thoraciques iatrogènes en réanimation néo-natale

Intra-thoracic gaseous collections, pneumodiastinum and pneumothorax, have become more common since the use of positive expiratory pressure, either with spontaneous ventilation or in association with artificial ventilation. In our experience, the occurence rate of such collections has increased from 7 p. 100 in 1970 to 19 p. 100 in 1974, related to the use of continuous positive pressure of long duration in the treatment of idiopathic respiratory distress or hyaline membrane disease. These gaseous collections were initially associated with a high mortality (50 p. 100) which is now practically nil. Improved knowledge of the clinical and radiological signs and the insertion of thoracic drains in the presence of the slightest doubt represent the explanation of this reduced mortality.

[PAH clearance measurement without urine samples in the newborn infant with respiratory distress]. / Clearance du PAH sans prélèvements d'urines chez le nouveau-né présentant une détresse respiratoire

PAH clearance was carried out in 12 newborns, hospitalized in the infantile resuscitation unit for respiratory distress. 6 of these children weighed less than 2.5 kg, 4 had hyaline membrane disease, 6 had either amniotic abnormalities or transitory tachypnea, 2 were surgical patients: one right diaphragmatic hernia, one post-operative respiratory complication after intervention for neonatal occlusion. In 9 cases the newborn was under controled artificial ventilation associated with PEEP at 5 to 7 cm of water. In all of the cases, the hemodynamic, metabolic and blood gas conditions were normal. A control series of 11 newnorn was carried out in a pediatric unit, the clearance was done without urine samples, the rough value of the figures found varied from 5.5 ml per minute to 30 ml per minute in the respiratory distress series and 16 to 62 ml per minute in the control series. The analysis of these results in rendered difficult by the juxtaposition of several factors: Choice of a reference criterion: body surface area, PAH space, patient's weight theoretical weight of the kidneys. The factor of prematurity. The problem of the date of the investigation in comparison with the date of birth.

Fetomaternal hemorrhage: diagnostic problems. Three case reports.

This report describes 3 successive cases in which fetomaternal hemorrhage was suspected but confirmed in only 2. The manifestations of fetomaternal hemorrhage are often nonspecific and diagnosis can be difficult. We discuss diagnostic methods, especially the value of the Kleihauer-Betke test and maternal serum alpha-fetoprotein measurement, and approach.