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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.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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.

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.

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.

Objective: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts., Methods: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting., Results: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities., Conclusions: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.

Introduction: Since altered expression of gap junction proteins (connexins) in diseased myocardial tissue may lead to abnormal electrical coupling between cardiomyocytes and hence contribute to arrhythmogenesis, the expression of connexin(Cx)40 and Cx43 was studied in atrial appendage from goats in sinus rhythm (SR) and persistent atrial fibrillation (AF)., Methods and Results: Biopsies were taken from the left and right atrial appendages from goats in SR or after pacing-induced persistent AF. Analyses of Cx40 and Cx43 mRNA and protein levels, using quantitative (competitive) polymerase chain reaction and western blotting, respectively, revealed no significant changes in the overall expression of Cx40 and Cx43 as a result of persistent AF. At the cellular level, immunohistochemistry and confocal laser scanning microscopy showed a homogeneous distribution of either connexin in atrial sections taken during SR. After induction of AF, the distribution of Cx43 gap junctions was unchanged whereas the Cx40 pattern showed marked inhomogeneities with small areas (0.15 to 0.6 mm in diameter, 25% of section surface area) of low-density Cx40 located between larger areas of normal (unchanged) Cx40 density. Activation mapping (244 electrodes, spatial resolution 2.25 mm) of the right atrial wall did not reveal changes in atrial conduction velocity., Conclusion: Pacing-induced persistent AF in the goat gave rise to changes in the spatial organization of Cx40 gap junctions. Although the overall conduction velocity appeared not to have changed, microheterogeneities in conduction due to the local redistribution of Cx40 gap junctions might have contributed to the initiation and maintenance of AF.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.

Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.

For effective cardiac output, it is essential that electrical excitation spread rapidly throughout the atria and ventricles. This is effected by electrical coupling through gap junction channels at contact sites between myocytes. These channels form a low-resistance pathway between adjacent myocytes and consist of connexin proteins. The connexin family is a large multigene family, and the channels formed by different members of this family have distinct electrical and regulatory properties. We have studied gap junction channels between adult rabbit atrial and ventricular myocytes using immunocytochemical and electrophysiological methods. Gap junctions of ventricular myocytes were immunoreactive to antibodies directed against connexin43 (Cx43) and Cx45, but not to antibodies against Cx37 or Cx40. Gap junctions between atrial myocytes showed immunostaining with anti-Cx40, -Cx43, and -Cx45 antibodies, but not with anti-Cx37 antibody. Endocardial and endothelial tissue were labeled with both Cx37 and Cx40 antibodies. The conductance of rabbit myocardial gap junctions was measured using the double whole-cell voltage-clamp method. The average macroscopic junctional conductance, corrected for series resistance, of atrial and ventricular cell pairs did not differ significantly (169+/-146 and 175+/-147 nS, respectively), and both were at most only slightly sensitive to the applied transjunctional potential difference. The difference in connexin expression between atrial and ventricular myocytes was reflected in the distribution of single gap junction channel conductances. A single population of unitary channel conductances with an average of 100 pS was observed between ventricular myocyte pairs. In addition to this population, a population with an average conductance of 185 pS was present between atrial myocyte pairs. The observed difference in connexin expression between atrial and ventricular myocardium may enable differential regulation of conduction in these tissues.

The external rhinoplasty approach was used in nine patients for closure of septal perforations varying from 8 to 30 mm in diameter. Nasal obstruction was the most prominent symptom. An autologous graft was always inserted between the mucoperichondrial flaps. Seven perforations were closed successfully. Symptomatic improvement was achieved in all patients. The closure of nasal septal perforations via external rhinoplasty approach has a high percentage of success due to excellent exposure of the septum.

Objectives: Gap junctions have been demonstrated ultrastructurally in cardiac regions where connexin40 (Cx40) and connexin43 (Cx43) protein could not be detected immunohistochemically. We investigated therefore the distribution of their mRNAs with more sensitive techniques., Methods: In situ hybridizations with Cx40 and Cx43 cRNA probes were performed on sections of rat hearts from 9 embryonic days (ED 9) to adults., Results: From ED 13, Cx40 and Cx43 mRNA are detectable in atria and ventricles, but not in their flanking myocardium (inflow tract, atrioventricular canal and outflow tract). Even though Cx40 and Cx43 mRNA eventually become expressed in the inflow tract, they remain undetectable in the sinoatrial node, the atrioventricular canal (including atrioventricular node) and outflow tract. Expression of Cx40 is maximal in the fetal period and declines towards birth. Cx40 expression in the left and right ventricles evolves independently, its mRNA disappearing 4 days earlier from the right than from the left ventricle, and earlier from the free wall than from the trabeculations. Expression of Cx43 mRNA increases during development and changes postnatally from uniform to punctate. Prenatally, Cx43 mRNA was strongest in the subepicardial layer of the ventricular free wall. Nevertheless, we did not detect protein in this layer., Conclusions: Cardiac regions without detectable Cx40 or Cx43 mRNA either have extremely low levels of expression or express a different connexin. The temporally separate disappearance of Cx40 mRNA from the fetal ventricles implies that left and right ventricles mature independently with respect to gap-junctional communication. The division of the developing heart in compartments where Cx40 and Cx43 mRNA can and cannot be detected, implies pretranslationally regulated gene expression. The postnatally observed subcellular redistribution of Cx43 mRNA coincides with a reported increase in protein expression.

Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development.

Myocytes are electrically coupled by gap junctions, which are composed of low-resistance intercellular channels. The major cardiac gap junction protein is connexin43 (Cx43). The distribution of Cx43 has been studied by immunofluorescence to visualize the electrical coupling between atrial tissue and sinoatrial node. From modeling studies, this coupling was inferred to be gradual in order to shield the sinoatrial node from the atrial hyperpolarizing influence. The actual Cx43 labeling pattern did not show the expected gradient but instead a rather black and white staining in a striking pattern of strands of cells. We used an immunohistochemical marker (anti-alpha-smooth muscle actin [alpha SMA]) that specifically cross-reacts with guinea pig sinoatrial node cells together with Cx43 antibody to stain previously electrophysiologically mapped sinoatrial nodes. We found that in the guinea pig sinoatrial node the impulse originates in an alpha SMA-positive, virtually Cx43-negative, region (primary pacemaker region). The impulse then travels obliquely upward to the crista terminalis through a region where layers of alpha SMA-positive cells alternate with layers of Cx43-positive SMA-negative cells. The layers of Cx43-positive cells appear to become broader and thicker in the direction of the crista terminalis, whereas the layers of alpha SMA-positive cells become thinner and narrower. Lateral contacts between Cx43- and alpha SMA-positive cells were very sparse and only detected where the Cx43-positive strands ended (the region where alpha SMA-positive cells fill the whole space between endocardium and epicardium, ie, the putative primary pacemaker region). From these results, we conclude that the primary pacemaker is shielded from the hyperpolarizing influence of the atrium by a gradient in coupling brought about by tissue geometric factors rather than by a gradient of gap junction density.