Committing embryonic stem cells to early endocrine pancreas in vitro

A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothio-glycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin+ or C-peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of beta-cell specification and differentiation factors activin beta B, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.     

Intracellular survival of persistent group A streptococci in cultured epithelial cells

Group A streptococcus (GAS) is the principle etiologic agent of bacterial pharyngotonsillitis and a wide range of other diseases. Failure to eradicate GAS from patients has been documented in 5-30% of patients with pharyngotonsillitis, in spite of the continued sensitivity of GAS to penicillin and other beta-lactams. It was recently proposed that eradication failure might be attributed to the ability of GAS to maintain an intracellular reservoir during antibiotic treatment. We have previously shown that strains derived from patients with bacterial eradication failure, despite antibiotic treatment (persistent strains), adhered to and were internalized by cultured epithelial cells more efficiently than strains that were successfully eradicated. Since, penicillin and other beta-lactams do not penetrate well into mammalian cells, intracellular survival of GAS is crucial in order to persist during prolonged antibiotic treatment. In this study, we compared the survival of GAS strains from cases of eradication failure and eradication success, using an epithelial cell culture model. We found that persistent strains show significantly increased intracellular survival, compared to the 'eradication success' strains. This finding supports the idea that an intracellular reservoir of GAS plays a role in the etiology of antibiotic eradication failure.     

Differential Effects of Clostridium difficile Toxins on Tissue-Cultured Cells

Two immunologically distinct Clostridium difficile toxins elicited similar morphological changes on cultured cells, although there were differences in both toxin potency and cell sensitivities.     

Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors

CD1d-restricted invariant natural killer T cell (iNKT cells) have a limited T cell receptor (TCR) repertoire and share characteristics common to T cells and natural killer cells. While intrathymic selection facilitates the production of T cells carrying self major histocompatibility complex-restricted TCRs, natural killer cells carry an appropriate repertoire of self major histocompatibility complex-recognizing receptors to avoid self-reactivity. Here we show that chronic exposure to specific glycolipid antigen resulted in iNKT cell disappearance and thymus-dependent repopulation of iNKT cells with increased expression of inhibitory Ly-49 molecules that resulted in impaired responsiveness. Thymic selection of peripheral Ly-49-expressing iNKT cell repertoire inhibited cytokine production and other functions in vivo. These observations emphasize the acquisition of self-recognizing inhibitory receptors on NKT cells as a previously unknown mechanism of thymic tolerance after chronic antigen exposure.     

The role of fluid hydrostatic pressure in bone-implant interface load transfer

Permeability of the soft tissue-bone system surrounding artificial joints fixed in cancellous bone was measured in four adult dogs after implants had been in place 2 months. Fluid was forced through a cavity formed by removal of the implant, the cavity was capped with a stopper to allow for pressure generation. Surface permeability of the 2-month-old implant cavity was 45 times less than the permeability of freshly drilled holes in cancellous bone. A mathematical model of a rigid implant resting on a biphasic solid-fluid layer showed the fluid carried 90% of the load when the implant cavity permeability was assumed, but only 27% when the freshly drilled permeability was used. The results suggest caution in interpreting finite-element models with bonded interfaces and suggest a possible role of the fluid in biological response at the interface.     

Activity in deep intermediate layer collicular neurons during interrupted saccades

The activity of neurons located in the deep intermediate and adjacent deep layers (hereafter called just deep intermediate layer neurons) of the superior colliculus (SC) in monkeys was recorded during saccades interrupted by electrical stimulation of the brainstem omnipause neuron (OPN) region. The goal of the experiment was to determine if these neurons maintained their discharge during the saccadic interruption, and thus, could potentially provide a memory trace for the intended movement which ends accurately on target in spite of the perturbation. The collicular neurons recorded in the present study were located in the rostral three-fifths of the colliculus. Most of these cells tended to show considerable presaccadic activity during a delayed saccade paradigm, and, therefore, probably overlap with the population of SC cells called buildup neurons or prelude bursters in previous studies. The effect of electrical stimulation in the OPN region (which interrupted ongoing saccades) on the discharge of these neurons was measured by computing the percentage reduction in a cell's activity compared to that present during non-interrupted saccades. During saccade interruption about 70% of deep intermediate layer neurons experienced a major reduction (30% or greater) in their activity, but discharge recovered quickly after the termination of the stimulation as the eyes resumed their movement to finish the saccade on the target. Therefore, the pattern of activity recorded in most of the deep intermediate layer neurons during interrupted saccades qualitatively resembled that previously reported for the saccade-related burst neurons which tend to be located more dorsally in the intermediate layer. In contrast, some of our cells (30%) showed little or no perturbation in their activity caused by the saccade interrupting stimulation. Because all the more dorsally located burst neurons and the majority of our deep intermediate layer neurons show a total or major suppression in their discharge during interrupted saccades, it seems unlikely that the colliculus by itself could maintain an accurate memory of the desired saccadic goal or the remaining dynamic motor error required to account for the accuracy of the resumed movement which occurs following the interruption. However, it remains possible that the smaller proportion of our neurons whose activity was not perturbed during interrupted movements could play a role in the mechanisms underlying saccade accuracy in the interrupted saccade paradigm. Interrupted saccades have longer durations than normal saccades to the same target. Therefore, we investigated whether the discharge of our deeper collicular cells was also necessarily prolonged during interrupted saccades, and, if so, how the prolongation compared to the prolongation of the saccade. Sixty percent of our sample neurons showed a prolongation in discharge that was approximately the same as the prolongation in saccade duration (difference < 15 ms in magnitude). The, observation that temporal discharge in our neurons was perturbed to roughly match saccadic temporal perturbation suggests that dynamic feedback about ongoing saccadic motion is provided to the colliculus, but does not necessarily imply that this structure is the site responsible for the computation of dynamic motor error.     

Human eosinophils express and release IL-13 following CD28-dependent activation

Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)-4, a key cytokine in the pathogenesis of allergic inflammation, and that IL-4 and IL-13 share common biological functions, we investigated the possibility that IL-13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL-13. Granule localization was demonstrated after subcellular fractionation, and IL-13 immunoreactivity was localized to crystalloid, granule-enriched fractions. Furthermore, electron microscopic analyses specifically localized IL-13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL-13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL-13 secretion. Furthermore, eosinophil-derived IL-13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL-13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids.

Lipoteichoic acids (LTAs) from various bacterial species, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Listeria monocytogenes, were examined for the ability to induce secretory and cellular responses in a pure population of bone marrow-derived mononuclear phagocytes. Some of the highly purified LTAs, in particular LTAs from Bacillus subtilis, S. pyogenes, E. faecalis, and Enterococcus hirae, were able to affect each of the macrophage parameters measured, i.e., reductive capacity, secretion of tumor necrosis factor and nitrite, and tumoricidal activity. As after stimulation with whole organisms or other bacterial products, secretion of tumor necrosis factor induced by these LTAs reached its maximum within the first few hours of the interaction, while secretion of nitrite and tumoricidal activity required 24 to 36 h for full expression. Other purified LTAs, i.e., LTAs from Streptococcus sanguis, S. pneumoniae, and L. monocytogenes, as well as lipomannan from Micrococcus luteus affected only some of these parameters, while native LTA from S. aureus was inactive. There was no obvious correlation between biological activity and chain length, kind of glycosyl substituents, glycolipid structures, or fatty acid composition of LTAs. Deacylation of LTAs resulted in a complete loss of activity, and deacylated LTAs did not impair the activity of their acylated counterparts, suggesting that acyl chains may be essential for binding of LTA to the cell surface. The results demonstrate that some LTA species are potent inducers of macrophage secretory and cellular activities.