Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle ( C _t) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Navβ1, Navβ3, Cav1.3, Cav3.1 and Cavα2δ2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cavβ2 and Cavγ4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvβ1, MinK and Cavγ7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.     

Beta2 integrins control the severity of murine Lyme carditis

Infection of C57BL/6 (B6) mice with the Lyme disease spirochete Borrelia burgdorferi can result in development of arthritis and carditis. B. burgdorferi induces expression of beta2/CD18 integrins, adhesion molecules that mediate the firm adhesion of leukocytes to the endothelium necessary for cellular extravasation during inflammation. The important role of beta2/CD18 integrins during extravasation suggests that these molecules play a role in the development of Lyme arthritis and carditis. The dependency of these inflammatory processes on the beta2 integrins was investigated in CD18 hypomorph mice, which express low levels of CD18. The results indicate that CD18 deficiency did not abrogate development of Lyme arthritis or carditis. Moreover, it resulted in increased severity of Lyme carditis. B. burgdorferi-infected CD18 hypomorph mice showed an increased macrophage infiltration of the heart, while they produced lower levels of borreliacidal anti-B. burgdorferi antibodies compared to wild-type mice. In accordance with these results, we demonstrate that dendritic cells from CD18 hypomorph mice secrete higher levels of monocyte/macrophage chemoattractant protein 1 (MCP-1/CCL2) in response to B. burgdorferi. Similarly, we show by real-time PCR that B. burgdorferi-infected hearts from CD18 hypomorph mice express increased levels of MCP-1 RNA compared to wild-type mice. Overall, our results indicate that beta2 integrin deficiency does not abrogate B. burgdorferi-induced inflammation; rather, it results in increased recruitment of macrophages into the B. burgdorferi-infected heart, likely due to the increased expression of MCP-1 in this tissue. Thus, beta2 integrins may play a regulatory role in B. burgdorferi-induced inflammation beyond mediating adhesion of leukocytes to the endothelium.     

Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes

Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.     

Transient requirement of the PrrA-PrrB two-component system for early intracellular multiplication of Mycobacterium tuberculosis

Adaptive regulation of gene expression in response to environmental changes is a general property of bacterial pathogens. By screening an ordered transposon mutagenesis library of Mycobacterium tuberculosis, we have identified three mutants containing a transposon in the coding sequence or in the 5' regions of genes coding for two-component signal transduction systems (trcS, regX3, prrA). The intracellular multiplication capacity of the three mutants was investigated in mouse bone marrow-derived macrophages. Only the prrA mutant showed a defect in intracellular growth during the early phase of infection, and this defect was fully reverted when the mutant was complemented with prrA-prrB wild-type copies. The mutant phenotype was transient, as after 1 week this strain recovered full growth capacity to reach levels similar to that of the wild type at day 9. Moreover, a transient induction of prrA promoter activity was observed during the initial phase of macrophage infection, as shown by a prrA promoter-gfp fusion in M. bovis BCG infecting the mouse macrophages. The concordant transience of the prrA mutant phenotype and prrA promoter activity indicates that the PrrA-PrrB two-component system is involved in the environmental adaptation of M. tuberculosis, specifically in an early phase of the intracellular growth, and that, similar to other facultative intracellular parasites, M. tuberculosis can use genes temporarily required at different stages in the course of macrophage infection.     

Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.     

Influenza virus infection of mouse lymphoblasts alters the binding affinity of anti-H-2 antibody: Requirement for viral neuraminidase

The requirement for histocompatibility in the T lymphocyte killing of virus-infected cells has led us to investigate the effect of influenza virus infection on mouse cell surface histocompatibility (H-2) antigens. Monoclonal anti-H-2 antibody made it possible to develop equilibrium binding conditions for the assay of H-2 antigen-antibody interactions on intact cells. Scatchard analysis of anti-H-2 binding with normal and virus-infected cells yielded linear curves indicating homogeneity of the interaction at varied concentrations of antibody through saturating levels. The estimated number of 2 × 10⁵ - 5 × 10⁵ H-2 antigenic sites per mouse lymphoblast does not appear to change during the course of influenza virus infection. However, the K_a (binding affinity constant) of anti-H-2 binding is rapidly elevated by virus infection (“0” time), continues to increase for 3 h post infection, then decreases. Control cells, treated with normal egg allantoic fluid, show no change in K_a during similar incubation. This change in K_a requires the presence of active viral neuraminidase. Thermal denaturation of the neuraminidase of the virus particles abolishes their ability to induce K_a alteration, even though hemagglutinin activity is retained. Treatment of cells with neuraminidase of bacterial origin led to an elevation of K_a, but did not mimic the viral effect in time dependence and magnitude of peak responses. The time-dependent lowering of K_a from peak values appeared to relate to virus replication, since UV light-inactivated virus-induced K_a elevation, but did not produce the typical K_a decline at 4-5 h post infection. The changes in K_a of anti-H-2 binding during influenza infection reflects a virus- induced alteration of the H-2 molecule or its environment in the host cell membrane. The molecular basis of this change and its relation to H-2-restricted recognition of influenza virus-infected cells by cytotoxic T cells requires further study.     

Assessing the effect of posture change on tactile inhibition-of-return

If a peripheral target follows an ipsilateral cue with a stimulus-onset-asynchrony (SOA) of 300 ms or more, its detection is delayed compared to a contralateral-cue condition. This phenomena, known as inhibition-of-return (IOR), affects responses to visual, auditory, and tactile stimuli, and is thought to provide an index of exogenous shifts of spatial attention. The present study investigated whether tactile IOR occurs in a somatotopic vs an allocentric frame of reference. In experiment 1, tactile cue and target stimuli were presented to the index and middle fingers of either hand, with the hands positioned in an uncrossed posture (SOA 500 or 1,000 ms). Speeded target detection responses were slowest for targets presented from the cued finger, and were also slower for targets presented to the adjacent finger on the cued hand than to either finger on the uncued hand. The same pattern of results was also reported when the index and middle fingers of the two hands were interleaved on the midline (experiment 2), suggesting that the gradient of tactile IOR surrounding a cued body site is modulated by the somatotopic rather than by the allocentric distance between cue and target.     

Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules

In many cancer cells, the p53 gene displays point mutations that result in stabilization and accumulation of the p53 protein. Therefore, p53 peptides could be presented to the immune system by tumor cells; thus, p53 might be a suitable target antigen for developing an immunotherapy against tumors using cytotoxic T lymphocytes (CTL). To map candidate CTL epitopes, we synthesized 150 peptides of 8–11 residues that contained putative anchor motifs required for binding to common HLA class I molecules. They were tested for their capacity to promote the assembly of purified and refolded HLA-A1, A2, B7 and B8 molecules. The following wild-type p53 peptides were found to be reactive with the HLA molecules tested: 196–205 and 226–234 bound moderately to HLA-A1; 25–35, 65–73, 129–137, 187–197, 263–272 and 264–272 bound strongly, and 187–195 and 256–264 moderately to HLA-A2; 26–35, 63–73, 189–197, 249–257 and 321–330 bound strongly to HLA-B7; and 135–143, 210–218 and 375–383 bound weakly to HLA-B8. We also analyzed the effects of p53 mutations occurring naturally in tumors on peptide/HLA assembly. We found substitutions that enhanced, diminished or had no effect on the peptide binding to HLA molecules. Polymorphism at position 72 mainly affected peptide/HLA-B7 binding, the proline allele P72 giving a less-reactive peptide (63–73) than the arginine allele R72. We have ranked potential p53 epitopes according to their reactivity for purified HLA molecules, allowing the selection of appropriate peptides and HLA molecules to attempt CTL induction in vitro.     

An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1–CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10⁻⁴ M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10⁻³ M to 5×10⁻⁴ M.