Granzyme B regulates antiviral CD8+ T cell responses

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.     

Relating Recombination,Density of States,and Device Performance in an Efficient Polymer:Fullerene Organic Solar Cell Blend

We explore the interrelation between density of states, recombination kinetics, and device performance in efficient poly[4,8‐bis‐(2‐ethylhexyloxy)‐benzo[1,2‐b:4,5‐b']dithiophene‐2,6‐diyl‐alt‐4‐(2‐ethylhexyloxy‐1‐one)thieno[3,4‐b]thiophene‐2,6‐diyl]:[6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PBDTTT‐C:PC₇₁BM) bulk‐heterojunction organic solar cells. We modulate the active‐layer density of states by varying the polymer:fullerene composition over a small range around the ratio that leads to the maximum solar cell efficiency (50–67 wt% PC₇₁BM). Using transient and steady‐state techniques, we find that nongeminate recombination limits the device efficiency and, moreover, that increasing the PC₇₁BM content simultaneously increases the carrier lifetime and drift mobility in contrast to the behavior expected for Langevin recombination. Changes in electronic properties with fullerene content are accompanied by a significant change in the magnitude or energetic separation of the density of localized states. Our comprehensive approach to understanding device performance represents significant progress in understanding what limits these high‐efficiency polymer:fullerene systems.     

Characterization of NLRP12 during the development of allergic airway disease in mice

Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp12(-/-) mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.     

Genotypic and phenotypic diversity of a baculovirus population within an individual insect host

It is becoming increasingly apparent that many pathogen populations, including those of insects, show high levels of genotypic variation. Baculoviruses are known to be highly variable, with isolates collected from the same species in different geographical locations frequently showing genetic variation and differences in their biology. More recent studies at smaller scales have also shown that virus DNA profiles from individual larvae can show polymorphisms within and between populations of the same species. Here, we investigate the genotypic and phenotypic variation of an insect baculovirus infection within a single insect host. Twenty four genotypically distinct nucleopolyhedrovirus (NPV) variants were isolated from an individual pine beauty moth, Panolis flammea, caterpillar by in vivo cloning techniques. No variant appeared to be dominant in the population. The PaflNPV variants have been mapped using three restriction endonucleases and shown to contain three hypervariable regions containing insertions of 70-750 bp. Comparison of seven of these variants in an alternative host, Mamestra brassicae, demonstrated that the variants differed significantly in both pathogenicity and speed of kill. The generation and maintenance of pathogen heterogeneity are discussed.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains

The effects of ceramides with varying saturated N-linked acyl chains (C2-C14) on cholesterol displacement from sphingomyelin-rich domains and on the stability of ordered domains were studied. The bilayers examined were made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), D-erythro-N-palmitoyl-sphingomyelin (PSM), D-erythro-N-acyl-sphingosine, and cholesterol (60:15:15:10 mol%, respectively). Cholestatrienol (CTL) or D-erythro-N-trans-parinoyl-sphingomyelin (tParSM) were used as reporter molecules (at 1 mol%) for the ordered domains, and 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphocholine (7SLPC) as a fluorescence quencher (30 mol%, replacing POPC) in the liquid-disordered phase. The results indicate that the ceramide had to have an N-linked acyl chain with at least 8 methylene units in order for it to displace cholesterol from the sphingomyelin-rich domains at the concentration used. The melting of the sphingomyelin-rich domain shifted to higher temperatures (compared to the ceramide-free control) with C2, C12 and longer chain ceramides, whereas C4-C10 ceramides led to domain melting at lower temperatures than control. This study shows that short-chain ceramides do not have the same effects on sterol- and sphingomyelin-rich domains as naturally occurring longer-chain ceramides have.     

Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.