Male patients affected by mosaic PCDH19 mutations: five new cases.

Pathogenic variants in the PCDH19 gene are associated with epilepsy, intellectual disability (ID) and behavioural disturbances. Only heterozygous females and mosaic males are affected, likely due to a disease mechanism named cellular interference. Until now, only four affected mosaic male patients have been described in literature. Here, we report five additional male patients, of which four are older than the oldest patient reported so far. All reported patients were selected for genetic testing because of developmental delay and/or epilepsy. Custom-targeted next generation sequencing gene panels for epilepsy genes were used. Clinical data were collected from medical records. All patients were mosaic in blood for likely pathogenic variants in the PCDH19 gene. In most, clinical features were very similar to the female phenotype, with normal development before seizure onset, which occurred between 5 and 10 months of age, clustering of seizures and sensitivity to fever. Four out of five patients had mild to severe ID and behavioural problems. We reaffirm the similarity between male and female PCDH19-related phenotypes, now also in a later phase of the disorder (ages 10-14 years).

Hyperactive behavior in a family with autosomal dominant lateral temporal lobe epilepsy caused by a mutation in the LGI1/epitempin gene.

Autosomal dominant lateral temporal lobe epilepsy (ADLTE) is characterized by focal seizures with auditory features or aphasia. Mutations in the leucine-rich glioma-inactivated 1 (LGI1) gene have been reported in up to 50% of families with ADLTE. Attention-deficit/hyperactivity disorder (ADHD) symptoms have not yet been reported in these families. Clinical data were collected from a family with five affected members. Leucine-rich glioma-inactivated 1 exons and boundaries were sequenced by standard methods. Attention-deficit/hyperactivity disorder symptoms were scored based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria. Affected members had seizures with auditory features and psychic auras, and some experienced nightmares. A heterozygous c.431+1G>A substitution in LGI1 was detected in all members. Significantly more hyperactivity symptoms were found in family members carrying the LGI1 mutation. This study expands the phenotypic spectrum associated with ADLTE due to LGI1 mutation and underlines the need for more systematic evaluation of ADHD and related symptoms.

Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders.

Epilepsy and mental retardation limited to females (EFMR), caused by PCDH19 mutations, has a variable clinical expression that needs further exploration. Onset of epilepsy may be provoked by fever and can resemble Dravet syndrome. Furthermore, transmitting males have no seizures, but are reported to have rigid personalities suggesting possible autism spectrum disorders (ASD). Therefore, this study aimed to determine the phenotypic spectrum associated with PCDH19 mutations in Dravet-like and EFMR female patients and in males with ASD. We screened 120 females suffering from Dravet-like epilepsy, 136 females with EFMR features and 20 males with ASD. Phenotypes and genotypes of the PCDH19 mutation carriers were compared with those of 125 females with EFMR reported in the literature. We report 15 additional patients with a PCDH19 mutation. Review of clinical data of all reported patients showed that the clinical picture of EFMR is heterogeneous, but epilepsy onset in infancy, fever sensitivity and occurrence of seizures in clusters are key features. Seizures remit in the majority of patients during teenage years. Intellectual disability and behavioural disturbances are common. Fifty percent of all mutations are missense mutations, located in the extracellular domains only. Truncating mutations have been identified in all protein domains. One ASD proband carried one missense mutation predicted to have a deleterious effect, suggesting that ASD in males can be associated with PCDH19 mutations.

Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.

BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

The Eocene Arctic Azolla bloom: environmental conditions, productivity and carbon drawdown.

Enormous quantities of the free-floating freshwater fern Azolla grew and reproduced in situ in the Arctic Ocean during the middle Eocene, as was demonstrated by microscopic analysis of microlaminated sediments recovered from the Lomonosov Ridge during Integrated Ocean Drilling Program (IODP) Expedition 302. The timing of the Azolla phase (approximately 48.5 Ma) coincides with the earliest signs of onset of the transition from a greenhouse towards the modern icehouse Earth. The sustained growth of Azolla, currently ranking among the fastest growing plants on Earth, in a major anoxic oceanic basin may have contributed to decreasing atmospheric pCO2 levels via burial of Azolla-derived organic matter. The consequences of these enormous Azolla blooms for regional and global nutrient and carbon cycles are still largely unknown. Cultivation experiments have been set up to investigate the influence of elevated pCO2 on Azolla growth, showing a marked increase in Azolla productivity under elevated (760 and 1910 ppm) pCO2 conditions. The combined results of organic carbon, sulphur, nitrogen content and 15N and 13C measurements of sediments from the Azolla interval illustrate the potential contribution of nitrogen fixation in a euxinic stratified Eocene Arctic. Flux calculations were used to quantitatively reconstruct the potential storage of carbon (0.9-3.5 10(18) gC) in the Arctic during the Azolla interval. It is estimated that storing 0.9 10(18) to 3.5 10(18) g carbon would result in a 55 to 470 ppm drawdown of pCO2 under Eocene conditions, indicating that the Arctic Azolla blooms may have had a significant effect on global atmospheric pCO2 levels through enhanced burial of organic matter.

Limited costs of wrong root placement in Rumex palustris in heterogeneous soils.

Nutrient hot spots in the soil have a limited life span, but the costs and benefits for root foraging are still underexposed. We assessed short-term costs that may arise when a nutrient-rich patch induces root proliferation, but then rapidly disappears. Rumex palustris plants were grown with a homogeneous or a heterogeneous nutrient application. After root proliferation in a nutrient-rich patch, nutrient supply was switched from homogeneous to heterogeneous, and vice versa, or the patch location was changed. R. palustris proliferated its roots in the rich patch. After switching, the relative growth rates of the roots were adjusted to the novel pattern of nutrient availability. However, the changes in local root biomass lagged behind the rapid shift in nutrient supply, because the root mass realized in specific sectors could not be rapidly relocated. Despite this, R. palustris did not exhibit costs of switching in terms of biomass or nitrogen uptake. Our data suggest that rapid shifts in uptake rate and redistribution of nitrogen within the plant may have lowered the costs of incorrect root placement.

Management of otitis media with effusion in children.

Otitis media with effusion in children: B-ENT Guidelines. OME is highly prevalent among young children, with peak prevalences at around two and five years of age. Although serious complications are rare, the burden of OM is large with impaired quality of life and high direct and indirect socio-economic costs. To date, medical treatment of OME is not recommended because of the limited scientific evidence that this treatment is effective in the long term. Surgical candidacy for OME depends largely on hearing status, associated symptoms, the child's developmental risk and the anticipated chance of spontaneous resolution of the effusion. Ultimately, the recommendation for surgery must be individualized.

Elevated levels of myeloperoxidase, pro-inflammatory cytokines and chemokines in naturally acquired upper respiratory tract infections.

Upper respiratory tract infections (URTIs) are characterised by a neutrophilic mucosal infiltration. The purpose of this study was to investigate the time course of release of the cytokines/chemokines interleukins (IL) IL-1beta, IL-1ra, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, interferon-gamma (IFN-gamma) and monocyte chemotactic protein (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1), myeloperoxidase (MPO) and bradykinin in nasal secretions of patients with a naturally acquired URTI. A total of 117 healthy adult volunteers were recruited for baseline nasal lavages, 39 of whom developed URTI symptoms within 6 months and returned to our centre within 48 h. Lavages were performed daily during the symptomatic period and 3 weeks thereafter, with symptoms no longer present. Compared to baseline, significantly elevated concentrations of total protein, bradykinin, IL-1beta, TNF-alpha, IL-6, IL-8, MCP-1, IFN-gamma, MPO and sICAM-1 were detected in nasal lavage fluids of symptomatic patients, whereas IL-1ra remained unaltered. All studied variables reached baseline 3 weeks after the URTI. Naturally acquired URTI represent a limited, neutrophilic inflammatory reaction, orchestrated by the release of pro-inflammatory cytokines and chemokines.

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.

OBJECTIVE: The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS: The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS: Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Gap junctions in the rabbit sinoatrial node.

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.

Prophylactic cranial irradiation in limited disease small-cell lung cancer in complete remission: a retrospective analysis.

Recently a meta-analysis showed an improved survival probability of prophylactic cranial irradiation (PCI) in limited disease small-cell lung cancer (LD SCLC) in complete remission after chemotherapy. We evaluated treatment results of PCI+ and PCI- in these patients. Whether PCI (n = 65) or no PCI (n = 37) was administered did not depend either on patients or on tumour characteristics. After 2 years the incidence of brain metastases was 11% in PCI+ patients and 51% in PCI- patients. Both disease-free survival and overall survival were significantly longer after PCI. PCI reduces the incidence of brain metastases, prolongs brain metastases-free period, and overall survival in LD SCLC patients in complete remission after chemotherapy.

Turnover of Haemophilus influenzae isolates in otitis-prone children.

Arbitrarily primed PCR with primer ERIC2 and RAPD Ready-to-Go beads was applied to study the epidemiology of non-typable Haemophilus influenzae (NTHI) isolated from the nasopharynx of children with recurrent otitis media (otitis-prone children). Thirty-five otitis-prone children (OP-children) were included. Three pairs of siblings were identified in the study population. This study is part of a large prospective multicentric trial investigating the efficacy of a new conjugated pneumococcal vaccine in OP-children (OMAVAX trial). During a 2-year study period, NTHI strains were isolated from nasopharyngeal swabs and, when possible, middle ear aspirates were collected in OP-children between 1 and 6 years of age. In 20 out of the 35 children, 48 H. influenzae isolates were obtained simultaneously from different sites (left and/or right ear and/or nasopharynx) of the same child as well as from siblings or during initial and follow-up visits of the same child. Subsequent genotyping indicated substantial genetic diversity among the H. influenzae isolates studied, since for a total of 48 isolates in 20 OP-children, 29 different genotypes were observed. Simultaneous isolation for different sampling sites (ear or nasopharynx) as well as for siblings resulted mostly in identical fingerprints. Longitudinal follow-up of H. influenzae isolates in the nasopharynx almost always resulted in different genotypes. We can therefore conclude that both cross colonization (between sampling sites within the same patient and between siblings) and turnover of H. influenzae isolates are high in OP-children.

Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein connexin40.

BACKGROUND: Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts. METHODS AND RESULTS: Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. CONCLUSIONS: Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Viral rhinitis and asthma.

Upper respiratory tract infections are among the most common infectious diseases. Approximately 80% of the common colds are caused by rhinoviruses. Recently, rhinovirus colds have been linked with lower airway illnesses such as asthma exacerbations resulting in a considerable interest in the pathogenesis of lower respiratory tract pathology. The important role that allergic airway disease plays in virally induced changes in airway function has been experimentally shown in several studies. Unfortunately, the precise mechanisms by which viruses could induce lower airway symptoms have not yet been determined.

The common cold at the turn of the millennium.

Upper respiratory tract infections are one of the most common infectious diseases in man and are characterized by transient, relatively mild symptoms. Human rhinoviruses are known to be the major causative agent in adult common colds and their relative importance has further increased with the use of the sensitive RT-PCR technique. Characteristic for a common cold is the selective neutrophil recruitment and time-limited increase in mediator, cytokine, and chemokine concentrations that orchestrate chemotaxis, transmigration, and activation of inflammatory and immunocompetent cells. Common cold symptoms are found to correlate to rhinovirus-induced IL-8 elaboration and neutrophil activation. Treatment of rhinoviral upper respiratory tract infections consists of an inhibition of viral infection by antiviral agents and/or a reduction of symptoms by damping the host inflammatory response.

The common cold.

Upper respiratory tract infections are one of the most common infectious diseases in man and are mainly caused by rhinoviruses. A rhinoviral cold is characterized by a neutrophilic inflammatory reaction with relatively mild symptoms that rather result from the host inflammatory response to the virus than from a direct viral cytotoxic effect. As regulators of chemotaxis, transmigration and activators of inflammatory and immunocompetent cells, cytokines and mediators were shown to play a crucial role in the pathogenesis of a rhinovirus infection.

The immune response in adenoids and tonsils.

The adenoid and tonsils are lymphoid tissues located in the pharynx that play an important role in host defense against invading antigens of the upper respiratory tract. Histologically, these structures consist of four well-defined microcompartments which all participate in the immune response: the cryptepithelium, the follicular germinal center with the mantle zone and interfollicular area. With the uptake of antigen by M-cells present in the cryptepithelium a process is initiated which ultimately results in the generation and dissemination of antigen-specific memory and mainly dimeric IgA-producing effector B-lymphocytes. This process requires successful cognate interactions between antigen-presenting cells and lymphocytes and mutually between lymphocytes, which depend not only on antigen-specific signals but also on the expression of various complementary adhesion and costimulatory molecules.

An update on the pathophysiology of rhinovirus upper respiratory tract infections.

Upper respiratory tract infections are one of the most common infectious diseases in man and are characterized by relatively mild symptoms. However, complications of bacterial super-infection or asthma exacerbations are not seldomly seen. Most upper respiratory tract infections are caused by rhinoviruses. The rhinovirus is a non-enveloped 30 nm RNA-virus with over 100 serotypes that belongs to the Picornaviridae family and only replicates in primates. It is characterized by a single positive stranded genome acting not only as a template for RNA synthesis, but also encoding for a single polypeptide necessary for viral replication. The viral capsid has an icosahedral symmetry and demonstrates deep canyons, with a receptor-binding domain. Rhinoviruses are transmitted mainly via direct- or indirect contact with infected secretions and invade their host by binding to the ICAM-1 receptor on the nasal epithelium. Typical for rhinovirus upper respiratory tract infections are isolated scattered foci of infected epithelium, not showing any striking damage or cytopathic alterations, between large areas of normal epithelium. Today there is still little detailed knowledge on the pathophysiology of common cold, especially on the aspect of cellular migration and defense. A better understanding in mechanisms underlying this cellular response would not only have therapeutical consequences, but may also explain the relationship between viral infectious rhinitis and asthma or atopy. During a rhinovirus infection, a selective neutrophil and monocyte recruitment is observed. In vitro and in vivo data have demonstrated a time-limited, rhinovirus-induced increase in bradykinin, cytokine, chemokine and sICAM-1 concentrations. Epithelial derived proinflammatory cytokines initiate an adhesion cascade and activate T lymphocytes that create a TH1-type cytokine environment within the infected tissue, necessary to eradicate the virus infection. The selective recruitment of neutrophils seems linked to increased concentrations of the chemokine IL-8 and common cold symptoms. It is doubtful that the cytokine-regulated-production of specific neutralising immunoglobulins is necessary for recovery from viral illnesses and presumably only contributes to a late and temporary protection against rhinovirus reinfection. These observations confirm the crucial role that cytokines and mediators play in the pathogenesis of a rhinovirus infection by mediating chemotaxis, transmigration and activation of inflammatory- and immunocompetent cells.

Regulation of proinflammatory cytokines in seasonal allergic rhinitis.

BACKGROUND: Mediators and cytokines have been demonstrated to be released due to nasal allergen exposure in sensitized subjects, but little is known about the release of cytokines and their antagonists under natural conditions. METHODS: Mediators - histamine, eosinophilic cationic protein (ECP), leukotrienes (LT) C4/ D4/E4 - and cytokines - interleukin (IL)-1beta, IL-8, IL-1 receptor antagonist (ra) - were measured in nasal secretions throughout the grass pollen season (6 visits) and for 6 weeks thereafter (3 visits) in patients with seasonal allergic rhinitis (n = 13) and compared to controls (n = 12). A second study was performed comparing nasal secretions of 13 subjects allergic to house dust mite to 8 controls. RESULTS: Compared to controls, leukotrienes and ECP were significantly elevated at nearly all time points in and postseason in the allergic group. Whereas IL-1beta was significantly elevated throughout the study period, IL-1ra was significantly decreased from visit 1 to 3. IL-8 showed no increase compared to controls. Data from subjects with perennial allergic rhinitis supported these findings and additionally demonstrated decreased concentrations of IL-8 and myeloperoxidase in secretions compared to controls. CONCLUSION: Allergic rhinitis represents a persistent inflammation in terms of an activation of eosinophils and constant upregulation of the proinflammatory cytokine IL-1beta in the pollen season and thereafter. We additionally could demonstrate a dysfunction of the anti-inflammatory capacity, i.e. IL-1ra, a naturally occurring antagonist. Persistent inflammation may furthermore lead to the dysregulation of local cellular immunity by reducing the number and activity of neutrophils on the mucosal surface.

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals.

Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes.