Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8⁺ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8⁺ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8⁺ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8⁺ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8⁺ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8⁺ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8⁺ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.     

Spatial and Temporal Gene Expression Differences in Core and Periinfarct Areas in Experimental Stroke: A Microarray Analysis

Background

A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke.

Methodology/Principal Findings

A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously.

Conclusions

This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research.     

Mexican Arnica Anti–Inflammatory Action: Plant Age is Correlated with the Concentration of Anti–Inflammatory Sesquiterpenes in the Medicinal Plant <Emphasis Type="Italic">Heterotheca inuloides</Emphasis> Cass. (Asteraceae)<Superscript>1</Superscript>

Mexican Arnica Anti–Inflammatory Action: Plant Age Is Correlated with the Concentration of Anti–inflammatory Sesquiterpenes in the Medicinal Plant Heterotheca inuloides Cass. (Asteraceae). Mexican árnica (Heterotheca inuloides Cass.) is a widely used anti–inflammatory medicinal plant in Mexican folk medicine. Although it has been suggested that plant age, fertilization, and harvesting regime influence the concentration of secondary compounds affecting the therapeutic activity of the plant, the effect of these variables on the concentration of the Mexican árnica anti–inflammatory compounds was not known. We quantified anti–inflammatory sesquiterpenes (caryolan–1, 9β–diol, cadalen–15–oic acid, 7–hydroxycadalene, 4–hydroxy–2–isopropyl–4, 7–dimethyl–1[4H] naftalinone, 7–hydroxy–4αH–3, 4–dihydrocadalene, β–caryophyllene, and β–caryophyllene epoxide) in Mexican árnica plants subjected to fertilization and successive harvests of flowering stems, conditions that mimic the cultivation and harvesting for árnica in México. Fertilization and successive harvesting and their interaction had no significant effect on the concentration of anti–inflammatory compounds. However, the concentrations of these compounds were 60% higher in flowering stems from 15–month–old plants than in those from 4– or 8–month–old plants and was independent of the number of harvests and fertilization regime applied.     

Structure-based functional study reveals multiple roles of transmembrane segment IX and loop VIII-IX in NhaA Na+/H+ antiporter of Escherichia coli at physiological pH

The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na(+)/H(+) antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu(241)-Phe(267)) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H(+)/Na(+) stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na(+), suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu(255) and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10-18 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K(m) for Na(+) of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.     

The promises and problems of linkage analysis by using the current canine genome map

There have been major advances in the canine genome map over recent years, with an agreed karyotype for all 38 pairs of autosomes and an integrated linkage-radiation-hybrid map now available. An individual dog breed represents a closed, isolated population, and many suffer genetic diseases. Some are homologs of human conditions, and dogs represent naturally occurring, potentially useful large mammal models. Other conditions may be unique to this species, but study of these offers new insights into metabolic pathways. Linkage analysis may identify a locus linked to a particular condition. Dog pedigrees facilitate these studies by being relatively more informative than human families, although planned breeding strategies to maximize the informativeness may be required. A study of a late-onset, acquired heart disease, dilated cardiomyopathy (DCM), in Newfoundland dogs highlights some of the problems. The disease occurs naturally in pedigrees within the UK in dogs kept as pets and breeding stock. Problems encountered include confirmation of the mode of inheritance and determination of phenotype. A genome-wide linkage analysis study with over 200 markers failed to detect linkage. The study indicates that more detailed linkage maps are required, and further synteny information between dog and human gained before study of diseases affecting naturally occurring families of dogs from inbred pedigrees may have maximal chance of success in order to utilize this model.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

High-resolution structure of a Na+/H+ antiporter dimer obtained by pulsed electron paramagnetic resonance distance measurements

Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na(+)/H(+) antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.     

An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.

We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.