Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Thrombin regulation in newborn infants

The coagulation system of newborn infants differs markedly from that of older children and adults. The activities of most coagulation factors and anticoagulants are low, leading to altered regulation in the formation of the key enzyme, thrombin. Timely and adequate generation of thrombin is essential, as thrombin activates platelets and many coagulation factors, cleaves fibrinogen into fibrin and activates the antithrombotic and anti-inflammatory protein C pathway. On the other hand, excess thrombin may promote thrombotic complications and exacerbate harmful inflammatory reactions. Despite the characteristic features, the newborn coagulation system can be considered physiological, since healthy newborns rarely show haemorrhagic or thrombotic complications. Sick newborns, however, often encounter clinical situations that challenge their coagulation system. The aim of this study was to clarify the behaviour of the neonatal coagulation system in selected clinical situations, with a special emphasis on the generation of thrombin. Thrombin was measured by in vivo thrombin generation markers and by thrombin generation potential in vitro. The patient groups included sick newborns undergoing intensive care and receiving fresh-frozen plasma (FFP), requiring exchange transfusions (ET) or presenting with a congenital heart defect requiring open heart surgery. Additionally, healthy newborns with inherited heterozygous factor V Leiden (FVL) mutation were studied. Thrombin generation potential was also analysed in cord plasma of healthy infants and in adults. Healthy as well as sick newborn infants showed lower total thrombin generation potential in vitro but faster initiation of thrombin generation than adults. These findings were qualitatively similar when plasma was supplemented with platelets. Platelets, however, significantly altered the effect of the major anticoagulant, activated protein C (APC), on thrombin generation potential. In accordance with previous studies, thrombin generation in healthy newborn platelet-poor plasma was resistant to the anticoagulant effects of APC, but when the plasma was supplemented with platelets APC attenuated thrombin generation significantly more in newborns than in adults. In vivo generation of thrombin was elevated in nearly all of the sick newborn infants. The low-volume FFP transfusion as opposed to the change from neonatal to adult blood in ET exerted markedly different effects on neonatal thrombin generation. FFP reduced the in vivo generation of thrombin in those newborns with the highest pretransfusional thrombin generation, thus acting as an anticoagulant agent. In those infants with lower pretransfusional thrombin generation, the effect of FFP on thrombin generation was fairly neutral. On the other hand, the combination of red blood cells and FFP, used to perform ET, significantly increased the in vivo thrombin formation and shifted the balance in the newborn coagulation system to the procoagulant direction. Cardiopulmonary bypass (CPB) also significantly increased the in vivo thrombin generation, but the thrombin generation profile during CPB differed from that previously observed in adults. Escalation of thrombin at early reperfusion was not observed in newborns; in adults, its occurrence is associated with postoperative myocardial damage. Finally, in healthy newborns with FVL heterozygosity, faster initiation of thrombin generation was observed compared with controls. Interestingly, FV level was lower in FVL-heterozygous infants, possibly to counteract the procoagulant effects induced by FVL. In conclusion, unique features regarding thrombin regulation in newborn infants were observed. These features included a novel platelet effect on the regulation of the protein C pathway. The clinical challenges mainly seemed to shift the balance in the coagulation system of newborns to the procoagulant direction. Blood component transfusions markedly affected coagulation in a manner specific to the product but that could also be altered by the clinical situation. Overall, the results highlight the need for understanding developmental haemostasis for both diagnostic and therapeutic purposes.

Effect of colostrum feeding method and presence of dam on the sleep, rest and sucking behaviour of newborn calves

"In rats, sucking milk reduces anxiety and promotes non-rapid eye movement (NREM) sleep, and in calves it induces resting but the effect on sleep is unknown. Here, we investigated how calves' sleep was affected by colostrum feeding methods. Forty-one calves were blocked by birth date and randomly allotted within blocks to the experimental treatments. Calves were housed for four days either with their dam (DAM) or individually with warm colostrum feeding (2 L four times a day) from either a teat bucket (TEAT) or an open bucket (BUCKET). DAM calves suckled their dam freely. Calves' sleeping and sucking behaviour was filmed continuously for 48 h at the ages of two and three days. Behavioural sleep (BS) was defined as calves resting at least 30 s with their head still and raised (non-rapid eye movement) or with their head against their body or the ground (rapid eye movement, REM). Latency from the end of colostrum feeding to the start of BS was recorded. We compared behaviour of TEAT calves with that of DAM and BUCKET calves using mixed models. Milk meal duration was significantly longer for TEAT calves than for BUCKET calves (mean +/- S.E.M.; 8.3 +/- 0.6 min vs. 5.2 +/- 0.6 min), but equal to that of DAM calves. We found no effect of feeding method on the duration of daily BS (12 h 59 min I h 38 min) but we found a tendency for the daily amount of NREM sleep; BUCKET calves had less NREM sleep per day than TEAT calves (6 h 18 min vs. 7 h 48 min, S.E.M. = 45 min) and also longer latencies from milk ingestion to BS (21.9 +/- 2.0 min vs. 16.2 +/- 2.0 min). DAM calves slept longer bouts than TEAT calves (10.8 +/- 1.0 min vs. 8.3 +/- 1.0 min) and less often (78 +/- 4 vs. 92 +/- 4). Sucking colostrum from a teat bucket compared with drinking from an open"

Snoring and sleep apnea in children

Snoring is a primary and major clinical symptom of upper airway obstruction during sleep. Sleep-disordered breathing ranges from primary snoring to significant partial upper airway obstruction, and obstructive sleep apnea. Adult snoring and obstructive sleep apnea have been extensively studied, whereas less is known about these disorders in children. Snoring and more severe obstructive sleep apnea have been shown to have a harmful effect on the neurobehavioral development of children, but the mechanisms of this effect remains unknown. Furthermore, the correlation of this effect to objective sleep study parameters remains poor. This study evaluated the prevalence of snoring in preschool-aged children in Finland. Host and environmental risk factors, and neurobehavioral and neurocognitive symptoms of children suffering from snoring or obstructive sleep apnea were also investigated. The feasibility of acoustic rhinometry in young children was assessed. The prevalence and risk factors of snoring (I) were evaluated by a questionnaire. The random sample included 2100 children aged 1-6 years living in Helsinki. All 3- to 6-year-old children whose parents reported their child to snore always, often, or sometimes were categorized as snorers, and invited to participate to the clinical study (II-IV). Non-snoring children whose parents were willing to participate in the clinical study were invited to serve as controls. Children underwent a clinical ear-nose-throat examination. Emotional, behavioral, and cognitive performances were evaluated by Child Behavioral Checklist (CBCL), Wechsler Preschool and Primary Scale of Intelligence (WPPSI-R) and NEPSY-A Developmental Neuropsychological Assessment (NEPSY). Nasal volume was measured by acoustic rhinometry, and nasal resistance by rhinomanometry. Lateral and posteroanterior cephalometry were performed. A standard overnight ambulatory polysomnography was performed in the home environment. Twenty-six healthy children were tested in order to assess the feasibility of acoustic rhinometry in young children (V). Snoring was common in children; 6.3% of children snored always or often, whereas 81.3% snored never or occasionally. No differences were apparent between snorers and non-snorers regarding age, or gender. Pediatric snoring was associated with recurrent upper respiratory infections, otitis media, and allergic rhinitis. Exposure to parental tobacco smoke, especially maternal smoking, was more common among snorers. Rhinitis was more common among children who exposured to tobacco smoke. Overnight polysomnography (PSG) was performed on 87 children; 74% showed no signs of significant upper airway obstruction during sleep. Three children had obstructive apnea/hypopnea index (OAHI) greater than 5/h. Age, gender, or a previous adenoidectomy or tonsillectomy did not correlate with OAHI, whereas tonsillar size did correlate with OAHI. Relative body weight and obesity correlated with none of the PSG parameters. In cephalometry, no clear differences or correlations were found in PSG parameters or between snorers and non-snorers. No correlations were observed between acoustic rhinometry, rhinomanometry, and PSG parameters. Psychiatric symptoms were more frequent in the snoring group than in the nonsnoring group. In particular, anxious and depressed symptoms were more prevalent in the snoring group. Snoring children frequently scored lower in language functions. However, PSG parameters correlated poorly with neurocognitive test results in these children. This study and previous studies indicate that snoring without episodes of obstructive apnea or SpO2 desaturations may cause impairment in behavioral and neurocognitive functions. The mechanism of action remains unknown. Exposure to parental tobacco smoke is more common among snorers than non-snorers, emphasizing the importance of a smoke-free environment. Children tolerated acoustic rhinometry measurements well.

Cortical processing of speech and non-speech sounds in adults and newborns

Comprehension of a complex acoustic signal - speech - is vital for human communication, with numerous brain processes required to convert the acoustics into an intelligible message. In four studies in the present thesis, cortical correlates for different stages of speech processing in a mature linguistic system of adults were investigated. In two further studies, developmental aspects of cortical specialisation and its plasticity in adults were examined. In the present studies, electroencephalographic (EEG) and magnetoencephalographic (MEG) recordings of the mismatch negativity (MMN) response elicited by changes in repetitive unattended auditory events and the phonological mismatch negativity (PMN) response elicited by unexpected speech sounds in attended speech inputs served as the main indicators of cortical processes. Changes in speech sounds elicited the MMNm, the magnetic equivalent of the electric MMN, that differed in generator loci and strength from those elicited by comparable changes in non-speech sounds, suggesting intra- and interhemispheric specialisation in the processing of speech and non-speech sounds at an early automatic processing level. This neuronal specialisation for the mother tongue was also reflected in the more efficient formation of stimulus representations in auditory sensory memory for typical native-language speech sounds compared with those formed for unfamiliar, non-prototype speech sounds and simple tones. Further, adding a speech or non-speech sound context to syllable changes was found to modulate the MMNm strength differently in the left and right hemispheres. Following the acoustic-phonetic processing of speech input, phonological effort related to the selection of possible lexical (word) candidates was linked with distinct left-hemisphere neuronal populations. In summary, the results suggest functional specialisation in the neuronal substrates underlying different levels of speech processing. Subsequently, plasticity of the brain's mature linguistic system was investigated in adults, in whom representations for an aurally-mediated communication system, Morse code, were found to develop within the same hemisphere where representations for the native-language speech sounds were already located. Finally, recording and localization of the MMNm response to changes in speech sounds was successfully accomplished in newborn infants, encouraging future MEG investigations on, for example, the state of neuronal specialisation at birth.

Pitch discrimination in optimal and suboptimal acoustic environments: electroencephalographic, magnetoencephalographic, and behavioral evidence

Pitch discrimination is a fundamental property of the human auditory system. Our understanding of pitch-discrimination mechanisms is important from both theoretical and clinical perspectives. The discrimination of spectrally complex sounds is crucial in the processing of music and speech. Current methods of cognitive neuroscience can track the brain processes underlying sound processing either with precise temporal (EEG and MEG) or spatial resolution (PET and fMRI). A combination of different techniques is therefore required in contemporary auditory research. One of the problems in comparing the EEG/MEG and fMRI methods, however, is the fMRI acoustic noise. In the present thesis, EEG and MEG in combination with behavioral techniques were used, first, to define the ERP correlates of automatic pitch discrimination across a wide frequency range in adults and neonates and, second, they were used to determine the effect of recorded acoustic fMRI noise on those adult ERP and ERF correlates during passive and active pitch discrimination. Pure tones and complex 3-harmonic sounds served as stimuli in the oddball and matching-to-sample paradigms. The results suggest that pitch discrimination in adults, as reflected by MMN latency, is most accurate in the 1000-2000 Hz frequency range, and that pitch discrimination is facilitated further by adding harmonics to the fundamental frequency. Newborn infants are able to discriminate a 20% frequency change in the 250-4000 Hz frequency range, whereas the discrimination of a 5% frequency change was unconfirmed. Furthermore, the effect of the fMRI gradient noise on the automatic processing of pitch change was more prominent for tones with frequencies exceeding 500 Hz, overlapping with the spectral maximum of the noise. When the fundamental frequency of the tones was lower than the spectral maximum of the noise, fMRI noise had no effect on MMN and P3a, whereas the noise delayed and suppressed N1 and exogenous N2. Noise also suppressed the N1 amplitude in a matching-to-sample working memory task. However, the task-related difference observed in the N1 component, suggesting a functional dissociation between the processing of spatial and non-spatial auditory information, was partially preserved in the noise condition. Noise hampered feature coding mechanisms more than it hampered the mechanisms of change detection, involuntary attention, and the segregation of the spatial and non-spatial domains of working-memory. The data presented in the thesis can be used to develop clinical ERP-based frequency-discrimination protocols and combined EEG and fMRI experimental paradigms.

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Cathepsin D Deficiency - Molecular and Cellular Mechanisms of Neurodegeneration

Cathepsin D (CTSD) is a lysosomal protease, the deficiency of which is fatal and associated with neurodegeneration. CTSD knock-out mice, which die at the age of four weeks, show intestinal necrosis, loss of lymphoid cells and moderate pathological changes in the brain. An active-site mutation in the CTSD gene underlies a neurodegenerative disease in newborn sheep, characterized by brain atrophy without any changes to visceral tissues. The CTSD deficiences belong to the group of neuronal ceroid-lipofuscinoses (NCLs), severe neurodegenerative lysosomal storage disorders. The aim of this thesis was to examine the molecular and cellular mechanisms behind neurodegeneration in CTSD deficiency. We found the developmental expression pattern of CTSD to resemble that of synaptophysin and the increasing expression of CTSD to coincide with the active period of myelination in the rat brain, suggesting a role for CTSD in early rat brain development. An active-site mutation underlying the congenital ovine NCL not only affected enzymatic activity, but also changed the stability, processing and transport of the mutant protein, possibly contributing to the disease pathogenesis. We also provide CTSD deficiency as a first molecular explanation for human congenital NCL, a lysosomal storage disorder, characterized by neuronal loss and demyelination in the central nervous system. Finally, we show the first evidence for synaptic abnormalities and thalamocortical changes in CTSD-deficient mice at the molecular and ultrastructural levels. Keywords: cathepsin D, congenital, cortex, lysosomal storage disorder, lysosome, mutation, neurodegeneration, neuronal ceroid-lipofuscinosis, overexpression, synapse, thalamus

Adult-type hypolactasia: Genotype-phenotype correlation

Adult-type hypolactasia (primary lactose malabsorption, lactase non-persistence) is the most common enzyme deficiency worldwide, and manifests with symptoms of lactose intolerance such as abdominal pain, gas formation and diarrhea. In humans with adult-type hypolactasia, lactase activity is high at birth, but declines during childhood to about one-tenth of the activity at birth. In 2002, a one base polymorphism C/T-13910, located 14 kilobases from the starting codon of the lactase-phlorizin hydrolase (LPH) gene was observed to be associated with the persistence of lactase activity. The T-13910 allele (C/T-13910 and T/T-13910 genotypes) associates with persistence of lactase activity throughout life, whereas the C/C-13910 genotype associates with adult-type hypolactasia. In this thesis work, the timing and mechanism of decline of lactase enzyme activity during development was studied using the C/T-13910 polymorphism as a molecular marker. We observed an excellent correlation between low lactase activity and the C/C-13910 genotype in all subjects > 12 years of age, irrespective their ethnicity. In children of African origin, the lactase activity declined somewhat earlier than among Finnish children. Furthermore, we observed an increasing imbalance in the relative lactase mRNA expression from the C-13910 and T-13910 alleles in Finnish children beginning from five years of age. The genetic test for adult-type hypolactasia showed a sensitivity of 93% and a specificity of 100% in the Finnish children and adolescents > 12 years of age. The relation of milk consumption and the milk-related abdominal complaints to the C/T-13910 genotypes associated with lactase persistence/non-persistence was studied by a questionnaire-based approach in > 2100 Finns. Both Finnish children and adults with the C/C-13910 genotype consumed significantly less dairy products compared to those with the C/T-13910 and T/T-13910 genotypes. Flatulence was the only of the abdominal symptoms of lactose intolerance that subjects with the C/C-13910 genotype reported significantly more often than those with the C/T-13910 and T/T-13910 genotypes. A minor proportion (<10%) of subjects with the C/C-13910 genotype, nevertheless, reported drinking milk without any symptoms afterwards. There was no association between cow's milk allergy starting as a newborn and adult-type hypolactasia. In an association study an increased risk of colorectal cancer was observed among those with molecular diagnosis of adult-type hypolactasia. It warrants further studies to clarify whether the increased risk observed in the Finnish population is associated with lactose or decreased intake of dairy products in these subjects.

Äidinmaito - terveysjuomaa ja normaalibakteereita

The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

IL-10 in Human Newborns : Ontogeny of IL-10 secretion and relation to the secretion of IFN-g, immunoglobulin M, G, and A, and mononuclear cell composition in newborns

The purpose of this work was to elucidate the ontogeny of interleukin-10 (IL-10) secretion from newborn mononuclear cells (MCs), and to examine its relation to the secretion of interferon-g (IFN-g) and immunoglobulins (Igs). The initial hypothesis was that the decreased immunoglobulin (Ig) synthesis of newborn babies was the result of immature cytokine synthesis regulation, which would lead to excessive IL-10 production, leading in turn to suppressed IFN-g secretion. Altogether 57 full-term newborns and 34 adult volunteers were enrolled. Additionally, surface marker compositions of 29 premature babies were included. Enzyme-linked immunoassays were used to determine the amount of secreted IL-10, IFN-g, and Igs, and the surface marker composition of MC were analyzed with a FACScan flow cytometer. The three most important findings were: 1. Cord blood MC, including CD5+ B cells, are able to secrete IL-10. However, when compared with adults, the secretion of IL-10 was decreased. This indicates that reasons other than excessive IL-10 secretion are responsible of reduced IFN-g secretion in newborns. 2. As illustrated by the IL-10 and IFN-g secretion pattern, newborn cytokine profile was skewed towards the Th2 type. However, approximately 25% of newborns had an adult like cytokine profile with both good IL10 and IFN-g secretion, demonstrating that fullterm newborns are not an immunologically homogenous group at the time of birth. 3. There were significant differences in the surface marker composition of MCs between individual neonates. While gestational age correlated with the proportion of some MC types, it is evident that there are many other maternal and fetal factors that influence the maturity and nature of lymphocyte subpopulations in individual neonates. In conclusion, the reduced ability of neonates to secrete Ig and IFN-g is not a consequence of high IL-10 secretion. However, individual newborns differ significantly in their ability to secrete cytokines as well as Igs.