Two Kinetic Patterns of Epitope-Specific CD8 T-Cell Responses following Murine Gammaherpesvirus 68 Infection

Murine gammaherpesvirus 68 (γHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6₄₈₇-specific cells predominate early in infection and then decline rapidly, whereas ORF61₅₂₄-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2^b-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that γHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.Murine gammaherpesvirus 68 (γHV68) is a mouse pathogen closely related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV). Intranasal infection of mice with γHV68 leads to an acute infection in lung epithelial cells that is ultimately cleared and the concurrent establishment of latency in B cells, dendritic cells, and macrophages that undergoes amplification in the spleen and is maintained lifelong (11, 12). Even though γHV68 has the capacity to downregulate major histocompatibility complex class I (MHC-I) molecules (36), CD8 T cells specific for γHV68 are generated and have been shown to proliferate in response to cognate antigen, protect naive mice from γHV68 infection, lyse peptide-pulsed target cells in vivo and in vitro, and maintain the ability to produce antiviral cytokines (5, 6, 13, 27, 35). Until recently, knowledge of the antiviral CD8 T-cell repertoire in C57BL/6 mice was largely limited to two well-characterized epitopes derived from ORF6 and ORF61. T-cell responses to these epitopes have been shown to progress with distinct kinetics, with ORF6₄₈₇-specific cells predominating early in infection and ORF61₅₂₄-specific cells continuing to expand through early latency before declining and then persisting at higher levels late in infection (33). The difference in response kinetics correlates with the differential presentation of the epitopes, with the ORF6₄₈₇ epitope being expressed only during lytic infection and the ORF61₅₂₄ epitope being expressed both during lytic infection and during the latency amplification phase (22, 28). Additionally, the latency amplification phase is associated with the expansion of CD8 T cells with a Vβ4 T-cell receptor (TCR) component in several mouse strains (17), presumably due to a superantigen-like effect of the γHV68 M1 protein (4, 9).To better understand the breadth of the anti-γHV68 T-cell response, we used an enzyme-linked immunospot (ELISpot) approach to identify new epitopes. We identified a large number of epitopes derived from 26 proteins that drive the acute CD8 T-cell response to γHV68, which then narrowed over time, resulting in a limited antiviral response during latency. We did not observe inflation of any of the responses, as has been demonstrated for some murine cytomegalovirus (MCMV)-specific responses (20, 26). There was no evidence for functional exhaustion, as all detectable CD8 T-cell responses maintained functionality, but the responses declined in numbers over time. The decline in responses occurred over a broad kinetic range, which segregated into two general groups that correlated precisely with those previously described for ORF6 and ORF61. Thus, some responses declined rapidly after the acute phase of infection, while others declined more slowly.We examined two epitope-specific responses from each of the two patterns in detail over time for functional and phenotypic characteristics and found the responses to be highly heterogeneous, differing in TCR affinity, functional avidity, and proliferation rates. Importantly, slowly declining responses were not maintained as efficiently after infection with a latency-deficient virus, consistent with a role for epitope expression in driving the heterogeneous rate of decline in cell number after the acute infection. The data show that the response kinetics seen for the ORF6₄₈₇ and ORF61₅₂₄ responses are broadly applicable to multiple CD8 T-cell epitopes.     

Genetic transformation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g⁻¹ dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g⁻¹ dry wt. and 9.97 mg g⁻¹ dry wt., respectively) comparable to those found in root tubers.     

Effects of Copper Supplementation on the Structure and Content of Elements in Kidneys of <Emphasis Type="Italic">Mosaic</Emphasis> Mutant Mice

Menkes disease is an effect of ATP7A gene mutation in humans, coding the Cu-ATP-ase which is essential in intestinal copper absorption and its subsequent transfer to circulation. This mutation results in a deficiency of copper in all tissues except the epithelia of intestine and kidney tubules. Subcutaneous injection of copper ions is the main therapy for Menkes patients. Mosaic (Atp7a^mo-ms) mice closely simulate the situation in Menkes disease. The aim of this study was to evaluate the changes in structure and element content in kidneys of mosaic mice after copper supplementation. Hematoxylin–eosin staining was used to analyze tissue morphology and atomic absorption spectrometry to estimate Cu and Zn content. X-ray microanalysis was performed to measure Na, Mg, P, Cl, and K content in the cells of the proximal and distal tubules. Copper administration lengthened the lifespan of the mutants but led to its high accumulation and results in severe kidney damage. Karyomegalia, necrosis of tubular and Bowman’s capsule epithelium, lesions, and atrophy of glomeruli were observed in the treated mutants. Copper treatment afterwards led to sclerosis of glomeruli and tubules enhanced proliferation of epithelial cells and formation of both polycystic and papillary carcinoma patterns in kidney. We suggest that copper excess may impair the activity of Na⁺/K⁺ ATP-ase in renal tubules of ms/− males. The content of Mg, P, and Cl in kidneys in mutants was also changed after copper administration.     

The effect of spermine concentration on the solution structure of complexes formed in copper(II)/adenosine 5′-triphosphate/phosphoserine system

Quaternary systems of copper(II) complexes with adenosine 5′-triphosphate, O-phospho l-serine and with equimolar or excessive amount of spermine have been investigated. The studies have been performed in aqueous solution. Types of complexes and the overall stability constants have been determined using the potentiometric method with computer analysis of the data. On the basis of the results of spectroscopic studies (nuclear magnetic resonance, visible, circular dichroism, Raman, infrared and electron paramagnetic resonance spectroscopies) as well as equilibrium studies, the mode of interactions has been proposed. The reaction centers in the systems studied are the phosphate, carboxyl and amine groups from phosphorylated serine, heterocyclic nitrogen atom from purine ring and phosphate groups from adenosine 5′-triphosphate as well as amine groups from polyamine. The influence of change in the concentration of the polyamine (spermine) on the mode of coordination is discussed. It has been shown that in the physiological conditions an increase in the polyamine concentration changes the mode of metal bonding in the CuH₃(ATP)(Ser-P)(Spm) complexes (isomer I — coordination {2 N,O_x}, isomer II — coordination {3 N,O_x} and significant differences in sites of interaction).     

Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation

In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.     

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.     

New aromatic monoesters of α-aminoaralkylphosphonic acids as inhibitors of aminopeptidase N/CD13

A series of new aromatic monoesters of α-aminoaralkylphosphonic acids were synthesized by selective hydrolysis of corresponding aromatic diesters of α-aminoaralkylphosphonic acids. New potential inhibitors of aminopeptidase N/CD13, an enzyme important in tumour angiogenesis, were developed. Some derivatives of the homophenylalanine and norleucine related monoaryl phosphonates displayed higher inhibition potency than corresponding α-aminoaralkylphosphonic acids toward aminopeptidase N/CD13. The effect of one of the new inhibitors on the growth of human PANC-1 and HT-1080 cell lines was examined, either alone or in combination with TNF-α.     

Karyological study of Amphisbaena ridleyi (Squamata, Amphisbaenidae), an endemic species of the Archipelago of Fernando de Noronha, Pernambuco, Brazil

The karyotype of Amphisbaena ridleyi, an endemic species of the archipelago of Fernando de Noronha, in State of Pernambuco, Brazil, is described after conventional staining, Ag-NOR impregnation and fluorescence in situ hybridization (FISH) with a telomeric probe. The diploid number is 46, with nine pairs of macrochromosomes (three metacentrics, four subtelocentrics and two acrocentrics) and 14 pairs of microchromosomes. The Ag-NOR is located in the telomeric region of the long arm of metacentric chromosome 2 and FISH revealed signals only in the telomeric region of all chromosomes. Further cytogenetic data on other amphisbaenians as well as a robust phylogenetic hypothesis of this clade is needed in order to understand the evolutionary changes on amphisbaenian karyotypes.     

N‐terminal interaction domain implicates PAK4 in translational regulation and reveals novel cellular localization signals

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, including regulation of cell morphology and motility, embryonic development, cell survival, response to infection, and oncogenic transformation. Consistently with its pro‐oncogenic features, PAK4 was found to be overexpressed in many cancer cell lines and tissues, and to be necessary to promote activation of survival pathways. PAK4, like other Paks, is now considered a promising target for specific therapy. Little is known on its modes of regulation, molecular partners, and substrates. Because the N‐terminal regulatory moiety plays important roles in PAK4 activity and functions, even independently of GTPase interactions, in this study we employed an affinity chromatography approach to identify N‐terminal domain binding partners. Within this protein region we identified a novel interaction domain involved in association with ribonucleoprotein (RNP) complexes, suggesting PAK4 implications in translational regulation. Indeed, we found that active PAK4 can affect (cap‐independent) translation from specific IRES sequences in vivo, and that the N‐terminal domain is critical for this regulation. Further, we could establish that within the RNP interacting sequence PAK4 regulatory domain contains targeting elements that drive cytoplasmic localization and act as nuclear export signal. Functional implication of endogenous PAK4 protein, which was found in both cytoplasmic and nuclear fractions, in IRES‐mediated translation further underlines the significance of the reported findings. Our data reveal novel means for PAK4 regulation of gene expression, and provide new elements to understand the molecular mechanisms that determine PAK4 cellular localization and functions. J. Cell. Physiol. 224: 722–733, 2010. © 2010 Wiley‐Liss, Inc.